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| <?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN" "JATS-archivearticle1.dtd"><article article-type="research-article" dtd-version="1.1d1" xmlns:xlink="http://www.w3.org/1999/xlink"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">00999</article-id><article-id pub-id-type="doi">10.7554/eLife.00999</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Developmental biology and stem cells</subject></subj-group></article-categories><title-group><article-title>Brahma is essential for <italic>Drosophila</italic> intestinal stem cell proliferation and regulated by Hippo signaling</article-title></title-group><contrib-group><contrib contrib-type="author" id="author-5794"><name><surname>Jin</surname><given-names>Yunyun</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-5765"><name><surname>Xu</surname><given-names>Jinjin</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-5766"><name><surname>Yin</surname><given-names>Meng-Xin</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-5795"><name><surname>Lu</surname><given-names>Yi</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-5796"><name><surname>Hu</surname><given-names>Lianxin</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-5769"><name><surname>Li</surname><given-names>Peixue</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-5770"><name><surname>Zhang</surname><given-names>Peng</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="con7"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-3710"><name><surname>Yuan</surname><given-names>Zengqiang</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="con8"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-5771"><name><surname>Ho</surname><given-names>Margaret S</given-names></name><xref ref-type="aff" rid="aff3"/><xref ref-type="fn" rid="con9"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-3696"><name><surname>Ji</surname><given-names>Hongbin</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con10"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-5788"><name><surname>Zhao</surname><given-names>Yun</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor2">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="other" rid="par-3"/><xref ref-type="fn" rid="con11"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-5527"><name><surname>Zhang</surname><given-names>Lei</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="other" rid="par-3"/><xref ref-type="other" rid="par-4"/><xref ref-type="fn" rid="con12"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><institution content-type="dept">State Key Laboratory of Cell Biology</institution>, <institution>Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences</institution>, <addr-line><named-content content-type="city">Shanghai</named-content></addr-line>, <country>China</country></aff><aff id="aff2"><institution content-type="dept">State Key Laboratory of Brain and Cognitive Science</institution>, <institution>Institute of Biophysics, Chinese Academy of Sciences</institution>, <addr-line><named-content content-type="city">Beijing</named-content></addr-line>, <country>China</country></aff><aff id="aff3"><institution content-type="dept">Department of Anatomy and Neurobiology</institution>, <institution>Tongji University School of Medicine</institution>, <addr-line><named-content content-type="city">Shanghai</named-content></addr-line>, <country>China</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Vijay Raghavan</surname><given-names>K</given-names></name><role>Reviewing editor</role><aff><institution>National Centre for Biological Sciences, Tata Institute of Fundamental Research</institution>, <country>India</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>rayzhang@sibcb.ac.cn</email> (LZ);</corresp><corresp id="cor2"><label>*</label>For correspondence: <email>yunzhao@sibcb.ac.cn</email> (YZ)</corresp></author-notes><pub-date date-type="pub" publication-format="electronic"><day>15</day><month>10</month><year>2013</year></pub-date><pub-date pub-type="collection"><year>2013</year></pub-date><volume>2</volume><elocation-id>e00999</elocation-id><history><date date-type="received"><day>27</day><month>05</month><year>2013</year></date><date date-type="accepted"><day>08</day><month>09</month><year>2013</year></date></history><permissions><copyright-statement>© 2013, Jin et al</copyright-statement><copyright-year>2013</copyright-year><copyright-holder>Jin et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/3.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/3.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife00999.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.00999.001</object-id><p>Chromatin remodeling processes are among the most important regulatory mechanisms in controlling cell proliferation and regeneration. <italic>Drosophila</italic> intestinal stem cells (ISCs) exhibit self-renewal potentials, maintain tissue homeostasis, and serve as an excellent model for studying cell growth and regeneration. In this study, we show that Brahma (Brm) chromatin-remodeling complex is required for ISC proliferation and damage-induced midgut regeneration in a lineage-specific manner. ISCs and enteroblasts exhibit high levels of Brm proteins; and without Brm, ISC proliferation and differentiation are impaired. Importantly, the Brm complex participates in ISC proliferation induced by the Scalloped–Yorkie transcriptional complex and that the Hippo (Hpo) signaling pathway directly restricted ISC proliferation by regulating Brm protein levels by inducing caspase-dependent cleavage of Brm. The cleavage resistant form of Brm protein promoted ISC proliferation. Our findings highlighted the importance of Hpo signaling in regulating epigenetic components such as Brm to control downstream transcription and hence ISC proliferation.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00999.001">http://dx.doi.org/10.7554/eLife.00999.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.00999.002</object-id><title>eLife digest</title><p>Most tissues can generate new cells to repair damage or replace worn-out cells. The new cells are often generated from stem cells—cells that can either reproduce themselves or mature into other types of cells. In the fruit-fly <italic>Drosophila</italic>, for example, intestinal stem cells in the midgut are capable of producing more stem cells or they can differentiate to produce immature cells called enteroblasts that go on to become either enterocytes (the cells that line the gut) or enteroendocrine cells (which secrete hormones).</p><p>Researchers have identified a number of signalling pathways that are involved in the proliferation and differentiation of intestinal stem cells in the midgut of fruit flies. These include the Hippo pathway, which is important for regulating both cell proliferation and programmed cell death (apoptosis). Activation of the Hippo protein triggers a cascade of signals that culminate in the regulation of many of the genes involved in cell proliferation, division and apoptosis.</p><p>Another process that is important for controlling the proliferation and differentiation of cells is chromatin remodelling. Chromatin is the ‘packaging’ that keeps DNA tightly wound within the cell nucleus, and remodelling refers to the structural changes that allow proteins called transcription factors to reach the genes and transcribe them into messenger RNA (which then leaves the nucleus to generate the protein).</p><p>Now, Jin et al. have explored how the Hippo pathway and chromatin remodelling work together to regulate of stem cells. Using a technique called RNA interference to block the expression of various genes in intestinal stem cells and enteroblasts, Jin et al. found that a protein called Brahma—which is an essential part of a chromatin-remodelling complex—must be present for the stem cells to multiply normally.</p><p>Jin et al. also showed how the Hippo signalling pathway interacts with chromatin remodelling. Activation of the Hippo pathway inhibits gene expression by preventing two other proteins, Yorkie and Scalloped, from forming a complex in the nucleus. The new work shows that Brahma interacts physically with the Yorkie and Scalloped proteins to regulate the proliferation of the intestinal stem cells. It also shows that the Hippo protein regulates the activity of the Brahma protein by inducing a process called caspase-dependent cleavage. Because many of the proteins involved in these pathways are evolutionarily conserved and expressed in a variety of tissues, these findings may have implications for stem cell function and tissue repair in many species.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00999.002">http://dx.doi.org/10.7554/eLife.00999.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>Hippo signaling</kwd><kwd>brahma</kwd><kwd>midgut</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd><italic>D. melanogaster</italic></kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution>National Basic Research Program of China (973 Program)</institution></institution-wrap></funding-source><award-id>2010CB912101, 2012CB945001, 2011CB943902</award-id><principal-award-recipient><name><surname>Zhang</surname><given-names>Lei</given-names></name><name><surname>Zhao</surname><given-names>Yun</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution>Strategic Priority Research Program of the Chinese Academy of Sciences</institution></institution-wrap></funding-source><award-id>XDA01010406, XDA01010405</award-id><principal-award-recipient><name><surname>Zhang</surname><given-names>Lei</given-names></name><name><surname>Zhao</surname><given-names>Yun</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution>National Natural Science Foundation of China</institution></institution-wrap></funding-source><award-id>31171394, 31371462, 31171414, 31371492</award-id><principal-award-recipient><name><surname>Zhang</surname><given-names>Lei</given-names></name><name><surname>Zhao</surname><given-names>Yun</given-names></name></principal-award-recipient></award-group><award-group id="par-4"><funding-source><institution-wrap><institution>Hundred Talents Program of the Chinese Academy of Sciences</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Zhang</surname><given-names>Lei</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>The Brahma chromatin remodelling complex interacts with the Hippo signalling pathway to regulate the proliferation and differentiation of stem cells in the <italic>Drosophila</italic> midgut.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>Central to the animal development is how chromatin assembly and regulation orchestrate cell-fate determination. Four epigenetic factors, DNA methylation, histone modifications, ATP-dependent chromatin remodeling, and the recently discovered non-coding RNAs play major roles in epigenetic regulation at the chromatin level. The SWI/SNF family is one of the most-studied families of ATP-dependent chromatin remodeling complexes, which regulate gene expression by destabilizing nucleosome structures to alter the DNA accessibility for transcription factors (<xref ref-type="bibr" rid="bib4">Cairns, 2007</xref>; <xref ref-type="bibr" rid="bib11">Hargreaves and Crabtree, 2011</xref>). Studies have implicated diverse roles for the mammalian SWI/SNF complexes in embryonic stem cell proliferation and differentiation. SWI/SNF complexes also function in neural, heart, and muscle development (<xref ref-type="bibr" rid="bib3">Bultman et al., 2000</xref>; <xref ref-type="bibr" rid="bib28">Lickert et al., 2004</xref>; <xref ref-type="bibr" rid="bib14">Ho et al., 2009</xref>; <xref ref-type="bibr" rid="bib13">Ho and Crabtree, 2010</xref>; <xref ref-type="bibr" rid="bib59">Zhan et al., 2011</xref>). In <italic>Drosophila</italic>, there are two SWI/SNF complexes, the Brahma (Brm)-associated proteins (BAP) complex and the polybromo-containing BAP (PBAP) complex. The BAP complex has a signature subunit Osa, while PBAP complex is defined by BAP170, Polybromo, and Syap (<xref ref-type="bibr" rid="bib8">Elfring et al., 1998</xref>; <xref ref-type="bibr" rid="bib6">Chalkley et al., 2008</xref>). Brm is a unique DNA-stimulated ATPase and common subunit for both BAP and PBAP complexes. Progress has been made in understanding the function of the Brm complex during <italic>Drosophila</italic> development (<xref ref-type="bibr" rid="bib48">Treisman et al., 1997</xref>; <xref ref-type="bibr" rid="bib7">Collins and Treisman, 2000</xref>; <xref ref-type="bibr" rid="bib17">Janody et al., 2004</xref>; <xref ref-type="bibr" rid="bib32">Moshkin et al., 2007</xref>; <xref ref-type="bibr" rid="bib5">Carrera et al., 2008</xref>; <xref ref-type="bibr" rid="bib47">Terriente-Felix and de Celis, 2009</xref>; <xref ref-type="bibr" rid="bib33">Neumuller et al., 2011</xref>), yet little is known about Brm complex functions in maintaining stem cell pluripotency of the epithelial tissues.</p><p>The simplicity of the structure and the multipotency of <italic>Drosophila</italic> posterior midgut make it an excellent model to study adult epithelial tissue homeostasis and regeneration (<xref ref-type="bibr" rid="bib30">Micchelli and Perrimon, 2006</xref>; <xref ref-type="bibr" rid="bib35">Ohlstein and Spradling, 2006</xref>). The midgut is composed of four cell types: enterocytes (ECs), enteroendocrine (ee) cells, intestinal stem cells (ISCs), and enteroblasts (EBs). The mature ECs are large polyploid cells of absorptive function and frame the midgut lining; ee and ISCs are the two types of diploid cells in the midgut that are less abundant. ISCs evenly locate at basal position underneath the ECs with a wedge-like morphology (<xref ref-type="bibr" rid="bib35">Ohlstein and Spradling, 2006</xref>, <xref ref-type="bibr" rid="bib36">2007</xref>) and are the only known cell type in the posterior midgut that proliferates. On cell division, ISCs undergo self-renewal or proliferation to become EBs, quiescent progenitor cells that ultimately differentiate to ECs or ee cells with the ratio 9:1 under the control of Delta (Dl) and Notch (<xref ref-type="bibr" rid="bib30">Micchelli and Perrimon, 2006</xref>; <xref ref-type="bibr" rid="bib35">Ohlstein and Spradling, 2006</xref>). Since the active Dl expression is retained in self-renewed ISCs and is lost in the newly generated EBs, antibody against the active Dl is used as the specific and the only known marker for <italic>Drosophila</italic> ISCs (<xref ref-type="bibr" rid="bib36">Ohlstein and Spradling, 2007</xref>). It has been demonstrated that the proliferation and differentiation of ISCs are tightly controlled by Notch, Janus kinase/signal transducer and activator of transcription (JAK/STAT), epidermal growth factor receptor/mitogen-activated protein kinase (EGFR), Hippo (Hpo), and Wingless signaling pathways (<xref ref-type="bibr" rid="bib19">Jiang and Edgar, 2011</xref>).</p><p>The evolutionarily conserved Hpo pathway controls organ size by regulating cell proliferation and apoptosis (<xref ref-type="bibr" rid="bib37">Pan, 2010</xref>; <xref ref-type="bibr" rid="bib10">Halder and Johnson, 2011</xref>; <xref ref-type="bibr" rid="bib57">Yin and Zhang, 2011</xref>; <xref ref-type="bibr" rid="bib16">Irvine, 2012</xref>). Hpo is a serine/threonine Ste20-like kinase (<xref ref-type="bibr" rid="bib12">Harvey et al., 2003</xref>; <xref ref-type="bibr" rid="bib18">Jia et al., 2003</xref>; <xref ref-type="bibr" rid="bib38">Pantalacci et al., 2003</xref>; <xref ref-type="bibr" rid="bib49">Udan et al., 2003</xref>; <xref ref-type="bibr" rid="bib54">Wu et al., 2003</xref>) that directly phosphorylates and activates downstream nuclear Dbf2-related (NDR) family protein kinase Warts (Wts). Wts activation mediated by Hpo requires scaffold proteins Salvador (Sav) (<xref ref-type="bibr" rid="bib22">Kango-Singh et al., 2002</xref>; <xref ref-type="bibr" rid="bib46">Tapon et al., 2002</xref>) and mob as tumor suppressor (Mats) (<xref ref-type="bibr" rid="bib24">Lai et al., 2005</xref>). Together, these proteins inhibit Yorkie (Yki) nuclear translocation. In the absence of Wts-mediated suppression, Yki forms a complex with transcription factor(s) such as Scalloped (Sd) (<xref ref-type="bibr" rid="bib9">Goulev et al., 2008</xref>; <xref ref-type="bibr" rid="bib55">Wu et al., 2008</xref>; <xref ref-type="bibr" rid="bib60">Zhang et al., 2008</xref>) in the nucleus to regulate the expression of a plethora of genes involved in cell proliferation, cell cycle progression, and apoptosis (<xref ref-type="bibr" rid="bib10">Halder and Johnson, 2011</xref>; <xref ref-type="bibr" rid="bib57">Yin and Zhang, 2011</xref>; <xref ref-type="bibr" rid="bib16">Irvine, 2012</xref>). In addition, the Hpo pathway maintains tissue homeostasis by regulating the balance between <italic>diap1</italic> expression and basal levels of activated caspases via the control of Dronc (<italic>Drosophila</italic> Nedd-2-like caspase orthologous to human Caspase 9) (<xref ref-type="bibr" rid="bib50">Verghese et al., 2012</xref>).</p><p>We present evidence that Brm is required for ISC proliferation in both normal and regenerating midguts, and it is required in ISCs for EC differentiation in normal midguts. In addition, we show that the Brm complex is physically associated with the Sd–Yki transcriptional complex in the nucleus and functions downstream of the Hpo pathway to regulate ISC proliferation. We also demonstrate that Brm is regulated by the Hpo pathway at the protein level through Hpo kinase-induced, caspase-dependent, cleavage of Brm at its D718 site. Altogether, as exemplified in the <italic>Drosophila</italic> ISCs, our study unravels a novel mechanism of the chromatin remodeling Brm complex in maintaining adult stem cell pluripotency of epithelial tissues.</p></sec><sec id="s2" sec-type="results"><title>Results</title><sec id="s2-1"><title>Brm is required for ISC proliferation in midguts</title><p>To gain insights on homeostasis and proliferation of <italic>Drosophila</italic> midguts, a small-scale screen searching for candidates that genetically alters the midgut regeneration and homeostasis was carried out. During the screen, <italic>escargot-Gal4</italic> (<italic>esg-Gal4</italic>) was used to drive RNAi expressions of different genes in ISCs and EBs in the presence of a temperature-sensitive Gal4 repressor, <italic>tubGal80</italic> (henceforth <italic>esg80</italic><sup><italic>ts</italic></sup>). Adult <italic>esg80</italic><sup><italic>ts</italic></sup> flies grown at the permissive temperature do not express GFP or RNAi in ISCs and EBs. Once shifted to the non-permissive temperature, RNAi expressions in ISCs and EBs are induced and simultaneously marked by <italic>esg-Gal4</italic>-driven GFP signals (<xref ref-type="bibr" rid="bib30">Micchelli and Perrimon, 2006</xref>). Interestingly, among the RNAi lines, VDRC (37720) and Bloomington (31712) abolished the expression of Brm, the energy providing subunit in <italic>Drosophila</italic> Brm complex (<xref ref-type="bibr" rid="bib33">Neumuller et al., 2011</xref>; <xref ref-type="bibr" rid="bib52">Waldholm et al., 2011</xref>). On Brm RNAi expression, the number of GFP positive (GFP<sup>+</sup>) cells in the adult posterior midgut decreased. Concomitantly, the number of phospho-histone3 positive (PH3<sup>+</sup>) cells also reduced, suggesting that ISC proliferation is affected (compare <xref ref-type="fig" rid="fig1">Figure 1B,B′</xref> with <xref ref-type="fig" rid="fig1">Figure 1A,A′</xref>, also <xref ref-type="fig" rid="fig1">Figure 1E</xref>). Immunostaining using an antibody against Brm 505–775 aa (<xref ref-type="bibr" rid="bib8">Elfring et al., 1998</xref>) confirmed that endogenous Brm protein can be efficiently knocked down in the cells of both wing imaginal discs and midguts that express Brm RNAi transgenes (compare <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1B–B′′,C–C′</xref> with <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1A</xref>; and compare <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1E–E′′′</xref> with <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1D–D′′′</xref>). In addition, GFP<sup>+</sup> cells exhibited a spherical shape in the absence of Brm compared with the angular shaped control cells (compare <xref ref-type="fig" rid="fig1">Figure 1B,B′</xref> with <xref ref-type="fig" rid="fig1">Figure 1A,A′</xref>), suggesting that the attachment of GFP<sup>+</sup> cells to surrounding cells is disrupted. We further tested whether knockdown of Brm in ISCs/EBs affects the division of ISCs. On Brm RNAi expression, EBs in the intestinal epithelium labeled with the expression of the Suppressor of Hairless reporter (Su(H)-LacZ, a specific marker for EBs) (<xref ref-type="bibr" rid="bib30">Micchelli and Perrimon, 2006</xref>) were detected (compare <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1G–G′′</xref> with <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1F–F′′</xref>). This piece of evidence suggests that EBs are still formed even when Brm expression is inhibited and ISC proliferation is blocked. Expression of Brm<sup>K804R</sup>, a dominant negative form of Brm defective for ATP hydrolysis activity without affecting the complex assembly (<xref ref-type="bibr" rid="bib8">Elfring et al., 1998</xref>), results in similar effects compared to Brm RNAi (<xref ref-type="fig" rid="fig1">Figure 1C,C′</xref> and <xref ref-type="fig" rid="fig1">Figure 1E</xref>). Of note, we observed a mild increase in the ISC/EB numbers on Brm overexpression, and the PH3<sup><italic>+</italic></sup> cell number was slightly increased (compare <xref ref-type="fig" rid="fig1">Figure 1D,D′</xref> with <xref ref-type="fig" rid="fig1">Figure 1A,A′,E</xref>).<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.00999.003</object-id><label>Figure 1.</label><caption><title>Brm is required for ISC proliferation in midguts.</title><p>(<bold>A</bold>–<bold>D′</bold>) Adult fly midguts expressing <italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Gal4/UAS-GFP</italic> (<italic>esg80</italic><sup><italic>ts</italic></sup>) (<bold>A</bold> and <bold>A′</bold>), <italic>Brm RNAi</italic> (<italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Brm RNAi</italic>) (<bold>B</bold> and <bold>B′</bold>), <italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Gal4/UAS-GFP-Brm</italic><sup><italic>K804R</italic></sup> (<italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Brm</italic><sup><italic>K804R</italic></sup>) (<bold>C</bold> and <bold>C′</bold>) or <italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Gal4/UAS-GFP-Brm</italic> (<italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Brm</italic>) (<bold>D</bold> and <bold>D′</bold>) were immunostained with DAPI (blue). ISCs and EBs were marked by <italic>esgGal4-driven</italic> GFP expression. (<bold>E</bold>) Quantification of PH3<sup><italic>+</italic></sup> cells of adult midguts of the indicated genotypes. The results represent the mean ± SEM, n = 10 for each genotype. (<bold>F</bold>–<bold>G</bold>′) Adult midguts containing nuclear localized GFP-labeled control MARCM clones (<bold>F</bold> and <bold>F′</bold>) or <italic>brm</italic> null allele <italic>brm</italic><sup><italic>2</italic></sup> clones (<bold>G</bold> and <bold>G′</bold>) were immunostained for DAPI (blue). Guts were dissected from the adult flies 72 hr after clone induction. (<bold>H</bold>) Quantification of the cell numbers of the control or mutant clones of the indicated genotypes. The results represent the mean ± SEM, n = 10 for each genotype. See also <xref ref-type="fig" rid="fig1s1 fig1s2">Figure 1—figure supplements 1 and 2</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00999.003">http://dx.doi.org/10.7554/eLife.00999.003</ext-link></p></caption><graphic xlink:href="elife00999f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00999.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>Brm is required for ISC proliferation.</title><p>(<bold>A</bold>–<bold>C′</bold>) The efficiency of Brm antibody was tested by immunostaining of the endogenous Brm in wild-type wing discs (<bold>A</bold>) or in the discs expressing Brm RNAi V37720 (<bold>B</bold>–<bold>B′′</bold>) and B31712 (<bold>C</bold> and <bold>C′</bold>) in the posterior compartment using <italic>hhGal4</italic> driver. Discs were immunostained for Brm (green) and Ci (blue). (<bold>D</bold>–<bold>E′′′</bold>) Adult flies expressing <italic>esg80</italic><sup><italic>ts</italic></sup> (<bold>D</bold>–<bold>D′′′</bold>), <italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Brm RNAi</italic> (<bold>E</bold>–<bold>E′′′</bold>) were cultured at 29°C for 7 days. Midguts were dissected and immunostained for Brm (red) and DAPI (blue). (<bold>F</bold>–<bold>G′′</bold>) Flies of <italic>Su(H)Z</italic> controls (<bold>F</bold>–<bold>F′′</bold>) or flies expressing Brm RNAi in the ISCs/EBs (<bold>G</bold>–<bold>G′′</bold>) were cultured at 29°C for 7 days. Dl is detected by immunostaining (red). Su(H)-lacZ staining identifies the EBs with elevated Notch signaling (green). Cells that retain ISC identity (small nuclei, Dl positive and lacZ-negative) are indicated by yellow arrows, and EBs are indicated by white arrows.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00999.004">http://dx.doi.org/10.7554/eLife.00999.004</ext-link></p></caption><graphic xlink:href="elife00999fs001"/></fig><fig id="fig1s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00999.005</object-id><label>Figure 1—figure supplement 2.</label><caption><title>Brm complex is required for ISC proliferation.</title><p>(<bold>A</bold>–<bold>B′</bold>) Adult midguts containing nuclear localized GFP-labeled control MARCM clones (<bold>A</bold> and <bold>A</bold>′) or <italic>brm</italic> null allele <italic>brm</italic><sup><italic>2</italic></sup> clones (<bold>B</bold> and <bold>B′</bold>) were immunostained with DAPI (blue). Guts were dissected from the adult flies 8 days after clone induction. (<bold>C</bold>) Quantification of the cell numbers of the indicated control clones or mutant clones. The results represent the mean ± SEM, n > 10 for each genotype. (<bold>D</bold>–<bold>J′</bold>) Subunits of Brm complex function in ISC proliferation. Adult midguts expressing Bap60 (<bold>E</bold> and <bold>E′</bold>), Bap60 RNAi (NIG 4303R-1, <bold>F</bold> and <bold>F′</bold>), Mor (<bold>G</bold> and <bold>G′</bold>), Mor RNAi (VDRC 6969, <bold>H</bold> and <bold>H′</bold>), Osa (<bold>I</bold> and <bold>I′</bold>) and Osa RNAi (VDRC 7810, <bold>J</bold> and <bold>J′</bold>) with <italic>esg80</italic><sup><italic>ts</italic></sup> driver were immunostained with DAPI (blue).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00999.005">http://dx.doi.org/10.7554/eLife.00999.005</ext-link></p></caption><graphic xlink:href="elife00999fs002"/></fig></fig-group></p><p>Interestingly, ISC/EB reduction induced by the loss of Brm might be due to an alternation in the rate of proliferation and differentiation. We hypothesized that the loss of Brm might result in an inhibition of ISC proliferation, precocious ISC differentiation, or a blockage of ISC differentiation. To test these possible mechanisms, the Mosaic analysis with a repressible cell marker (MARCM) approach (<xref ref-type="bibr" rid="bib25">Lee and Luo, 2001</xref>) was used to generate <italic>brm</italic> null allele (<italic>brm</italic><sup><italic>2</italic></sup>) clones, and its impact on midgut proliferation was analyzed (<xref ref-type="bibr" rid="bib30">Micchelli and Perrimon, 2006</xref>; <xref ref-type="bibr" rid="bib35">Ohlstein and Spradling, 2006</xref>). The wild-type MARCM stem cell clones divided indefinitely, their sizes increased linearly, and contained several or all midgut cell types (<xref ref-type="bibr" rid="bib30">Micchelli and Perrimon, 2006</xref>; <xref ref-type="bibr" rid="bib35">Ohlstein and Spradling, 2006</xref>). If Brm is important for ISC proliferation, the <italic>brm</italic><sup><italic>2</italic></sup> clones will be retained in a limited size; if Brm is necessary for EC differentiation, the <italic>brm</italic><sup><italic>2</italic></sup> clones should mostly contain the small nuclear ISCs/EBs. Compared with the control clones that contain an average of five cells including both large nuclear cells and small nuclear cells within each clone, 3-day <italic>brm</italic><sup><italic>2</italic></sup> clones contain only one or two cells, which are all small nuclear cells (<xref ref-type="fig" rid="fig1">Figure 1H</xref>, and compare <xref ref-type="fig" rid="fig1">Figure 1G,G′</xref> with <xref ref-type="fig" rid="fig1">Figure 1F,F′</xref>). In addition, 8-day <italic>brm</italic><sup><italic>2</italic></sup> clones contain only one or two cells (<xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2A,A′,B,B′,C</xref>). These results suggest that both proliferation of these clones and the EC differentiation are affected, suggesting that Brm is indispensable for ISC proliferation and EC differentiation in midguts.</p><p>We further tested the function of other subunits of the Brm complex in ISC proliferation. We found that the knockdown of other components in the Brm complex, including Bap60, Mor, and Osa by RNAi respectively under the control of <italic>esg80</italic><sup><italic>ts</italic></sup> inhibited ISC proliferation to different extents and the GFP signal intensities were reduced simultaneously (compare <xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2F,F′,H,H′,J,J′</xref> with <xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2D,D′</xref>). Similar to Brm overexpression, overexpression of other Brm complex components induced only a mild enhancement on midgut ISC proliferation (compare <xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2E,E′,G,G′,I,I′</xref> with <xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2D,D′</xref>). In toto, these findings indicate that the maintenance of ISCs and EBs requires the presence of Brm complex.</p></sec><sec id="s2-2"><title>Brm is required for EC differentiation in normal midguts</title><p>Our results indicated that <italic>brm</italic><sup><italic>2</italic></sup> clones only contained small nuclear cells (<xref ref-type="fig" rid="fig1">Figure 1G,G′</xref>), suggesting that Brm plays a role during ISC differentiation in addition to ISC proliferation. We first analyzed the expression pattern of Brm during ISC cell maturation using <italic>Myo1AGal4-GFP</italic> (<xref ref-type="bibr" rid="bib31">Morgan et al., 1994</xref>). <italic>Myo1AGal4</italic> is an enhancer trap in the gut-specific brush border <italic>myosin 1A</italic> gene that combined <italic>tubGal80</italic><sup><italic>ts</italic></sup> with the <italic>Myo1AGal4</italic> driver and <italic>UAS-GFP</italic> (together referred to as <italic>Myo1A-GFP</italic>). Interestingly, Brm antibody staining detected a high level of endogenous Brm proteins in ISCs/EBs (GFP<sup>−</sup> cells in <xref ref-type="fig" rid="fig2">Figure 2A–A′′′</xref> and GFP<sup>+</sup> cells in <xref ref-type="fig" rid="fig2">Figure 2B–B′′′</xref>), and some ee cells (co-labeled by <italic>prospero</italic>, a conserved homodomain transcription factor), whereas a relatively low level of Brm protein was detected in ECs (GFP<sup>+</sup> cells in <xref ref-type="fig" rid="fig2">Figure 2A–A′′′</xref>).<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.00999.006</object-id><label>Figure 2.</label><caption><title>Brm is required for EC differentiation.</title><p>(<bold>A</bold>–<bold>B′′′</bold>) Adult guts of wild-type <italic>Myo1A-Gal4/UAS-GFP;tubGal80</italic><sup><italic>ts</italic></sup> (<bold>A</bold>–<bold>A′′′</bold>) and <italic>esgGal4/UAS-GFP</italic> (<bold>B</bold>–<bold>B′′′</bold>) were immunostained with Brm antibody (indicated with arrows) to show the endogenous Brm protein level in the different cell types. (<bold>C</bold>–<bold>H′</bold>) Adult female midguts differentiation measured via the <italic>esg</italic><sup><italic>ts</italic></sup> <italic>F/O</italic> system. Transgenes were induced for 2 days (<bold>C</bold>–<bold>E′</bold>) or 5 days (<bold>F</bold>–<bold>H′</bold>). <italic>esg</italic><sup><italic>ts</italic></sup> <italic>F/O</italic>-<italic>Brm</italic> (<bold>D</bold>, <bold>D′</bold> and <bold>G</bold>, <bold>G′</bold>) promoted the formation of ECs, while <italic>esg</italic><sup><italic>ts</italic></sup> <italic>F/O</italic>-<italic>Brm RNAi</italic> (<bold>E</bold>, <bold>E′</bold> and <bold>H</bold>, <bold>H′</bold>) blocked the EC differentiation. ECs are marked by PDM-1 (red) and arrows. (<bold>I</bold>) Female posterior midguts were scored for GFP<sup><italic>+</italic></sup> and PDM-1<sup>+</sup> EC cells in the same region near the Malpighian tubules. The results represent the mean ± SEM, n = 10 for each genotype. (<bold>J</bold>) A schematic diagram of the regulation of Brm activity in intestinal homeostasis. ISCs divide asymmetrically to an EB and an ISC. EBs then differentiate into ECs or ee cells. Cell-type-specific markers are indicated. In normal state (left side), Brm is expressed at a high level in nuclei of ISCs, EBs, and some ee cells, and at a low level in nuclei of ECs. The different Brm protein levels in nuclei are marked by red (ISCs, EBs, and ee cells) or pink (ECs). Decrease of Brm protein level in ISCs reduces the ISC proliferative ability and inhibits EC differentiation (right). See also <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00999.006">http://dx.doi.org/10.7554/eLife.00999.006</ext-link></p></caption><graphic xlink:href="elife00999f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00999.007</object-id><label>Figure 2—figure supplement 1.</label><caption><title>Brm is required for EC differentiation.</title><p>(<bold>A</bold>–<bold>C′</bold>) The differentiation of adult female midguts was analyzed using the <italic>esg</italic><sup><italic>ts</italic></sup> <italic>F/O</italic> system. Transgenes were induced in the midgut for 13 days. <italic>esg</italic><sup><italic>ts</italic></sup> <italic>F/O</italic>-<italic>Brm</italic> (<bold>B</bold> and <bold>B′</bold>) promoted EC formation, while <italic>esg</italic><sup><italic>ts</italic></sup> <italic>F/O</italic>-<italic>Brm RNAi</italic> (<bold>C</bold> and <bold>C′</bold>) blocked the EC differentiation. PDM-1 marked the EC cells (red).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00999.007">http://dx.doi.org/10.7554/eLife.00999.007</ext-link></p></caption><graphic xlink:href="elife00999fs003"/></fig></fig-group></p><p>On the basis of these findings, we examined the role of Brm in ISC differentiation by overexpression or knockdown of Brm in the ISCs using a lineage induction system, <italic>esg</italic><sup><italic>ts</italic></sup> <italic>F/O.</italic> In this lineage tracing system, progenitor cells and their newborn progenies express <italic>Gal4</italic> and UAS-linked Gal4 targets, including the <italic>UAS-GFP</italic> marker (<xref ref-type="bibr" rid="bib20">Jiang et al., 2009</xref>). PDM-1, a marker for fully differentiated ECs, is used to identify ECs (<xref ref-type="bibr" rid="bib56">Xu et al., 2011</xref>). Overexpressing Brm for 2 days generated new EC-like GFP<sup>+</sup> cells with large nuclei (<xref ref-type="fig" rid="fig2">Figure 2D,D′</xref>), whereas the wild type control group and the Brm RNAi group only contained GFP<sup>+</sup> cells with small nuclei (<xref ref-type="fig" rid="fig2">Figure 2C,C′,E,E′,I</xref>). It is implicated that high levels of Brm lead to precocious differentiation of ISCs. After 5-day or even 13-day induction, large mature ECs were formed in wild-type midguts, while Brm RNAi suppressed ISC proliferation and EC differentiation in experimental midguts (compare <xref ref-type="fig" rid="fig2">Figure 2H,H′</xref> with <xref ref-type="fig" rid="fig2">Figure 2F,F′,I</xref>, and <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1A–C′</xref>), suggesting that Brm is essential for ISCs and EBs to differentiate into ECs. In summary, the knockdown of Brm by RNAi blocks ISC proliferation and EC differentiation.</p></sec><sec id="s2-3"><title>Brm is required for midgut regeneration</title><p>Interestingly, in addition to its role in ISC proliferation under normal physiological context, Brm is also required for damage-induced midgut regeneration. Previous studies have reported that the feeding of dextran sulphate sodium (DSS) causes midgut cell proliferation via the disruption of basement membrane organization and increases in the intestinal stem cell division without affecting the final EB differentiation (<xref ref-type="bibr" rid="bib1">Amcheslavsky et al., 2009</xref>). It is plausible to think that Brm also exerts an effect on DSS-induced midgut cell proliferation, as it is required for midgut cell proliferation. Indeed, when Brm RNAi was expressed, DSS-induced increase of GFP<sup>+</sup> cells was blocked (compare <xref ref-type="fig" rid="fig3">Figure 3C,C′,D,D′</xref> with <xref ref-type="fig" rid="fig3">Figure 3A,A’,B,B′</xref>), suggesting that Brm is required in ISCs for DSS-induced proliferation. Of note, we did not observe dramatic change in ISC proliferation when overexpressing Brm in these GFP<sup>+</sup> cells with or without DSS treatment (compare <xref ref-type="fig" rid="fig3">Figure 3E,E′,F,F′</xref> with <xref ref-type="fig" rid="fig3">Figure 3A,A′,B,B′</xref>).<fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.00999.008</object-id><label>Figure 3.</label><caption><title>Brm was required for midgut regeneration.</title><p>(<bold>A</bold>–<bold>F′</bold>) Adult flies expressing <italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Gal4/UAS-GFP</italic> (<italic>esg80</italic><sup><italic>ts</italic></sup>) (<bold>A</bold>–<bold>B′</bold>), <italic>Brm RNAi</italic> (<italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Brm RNAi</italic>) (<bold>C</bold>–<bold>D′</bold>) or <italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Gal4/UAS-GFP-Brm</italic> (<italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Brm</italic>) (<bold>E</bold>–<bold>F′</bold>) were treated with glucose or DSS. Glucose solution with 3% DSS was fed to the flies (<bold>B</bold>–<bold>B′</bold>, <bold>D</bold>–<bold>D′</bold>, and <bold>F</bold>–<bold>F′</bold>) for 3 days before guts dissection.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00999.008">http://dx.doi.org/10.7554/eLife.00999.008</ext-link></p></caption><graphic xlink:href="elife00999f003"/></fig></p></sec><sec id="s2-4"><title>Brm interacts with the Hpo pathway effector Yki–Sd transcriptional complex</title><p>Previous studies implicated that the Hpo pathway effector Yki functions as a driver of proliferation in both ECs and ISCs and damage-induced ISC proliferation via both cell-autonomous and non-cell-autonomous mechanisms (<xref ref-type="bibr" rid="bib23">Karpowicz et al., 2010</xref>; <xref ref-type="bibr" rid="bib42">Ren et al., 2010</xref>; <xref ref-type="bibr" rid="bib44">Shaw et al., 2010</xref>). Considering that Brm is involved in DSS-induced ISC proliferation (<xref ref-type="fig" rid="fig3">Figure 3</xref>), we tested whether there is a functional link between Brm and Yki–Sd transcriptional complex. To this end, mass spectrum (MS) analysis was first performed. Co-immunoprecipitation (Co-IP) experiments were performed in S2 cells to pull down the endogenous Yki or Sd protein using antibodies, and the pull-down products were sent for MS analysis. Several Brm complex components were found in the MS results, including Brm, Osa, Bap60, Bap55, and Mor (<xref ref-type="table" rid="tbl1">Table 1</xref>, see <xref ref-type="table" rid="tbl1">Table 1</xref> for MS details). Consistent with the results of Yki MS analysis (<xref ref-type="table" rid="tbl1">Table 1</xref>), we found that Yki and Brm coimmunoprecipitated with each other when Myc-tagged Yki (Myc–Yki) and V5-tagged Brm (Brm–V5) were coexpressed in S2 cells (<xref ref-type="fig" rid="fig4">Figure 4A</xref>). We also verified the interaction between Brm and Sd using Co-IP in S2 cells. Results showed that overexpressed HA-tagged Sd (HA–Sd) interacted with the endogenous Brm (<xref ref-type="fig" rid="fig4">Figure 4B</xref>). Sd also coimmunoprecipitated with Mor and Osa but not Bap60 when they were coexpressed in S2 cells (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1A</xref>). In addition, we checked the cellular localization of these proteins in S2 cells. The majority of overexpressed Brm and overexpressed Sd were located in the nucleus (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1B–D′′′</xref>), whereas cytoplasmic–nuclear localization of Yki was not affected by Brm coexpression (data not shown), implicating that Brm complex does not promote the nuclear localization of Yki to influence the transcriptional activity of Yki–Sd complex.<table-wrap id="tbl1" position="float"><object-id pub-id-type="doi">10.7554/eLife.00999.009</object-id><label>Table 1.</label><caption><p>Mass spectrum analysis results</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00999.009">http://dx.doi.org/10.7554/eLife.00999.009</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th>Protein description</th><th>Molecular function</th><th>Pep count</th><th>Unique Pep count</th></tr></thead><tbody><tr><td>Yki mass spectrum</td><td/><td/><td/></tr><tr><td> Brahma (Brm)</td><td>ATP-dependent helicase</td><td align="char" char=".">11</td><td align="char" char=".">5</td></tr><tr><td> Osa</td><td>DNA binding</td><td align="char" char=".">5</td><td align="char" char=".">4</td></tr><tr><td>Sd mass spectrum</td><td/><td/><td/></tr><tr><td> Brahma associated protein 55kD (Bap55)</td><td>Structural constituent of cytoskeleton</td><td align="char" char=".">8</td><td align="char" char=".">5</td></tr><tr><td> Brahma associated protein 60kD (Bap60)</td><td>Protein binding</td><td align="char" char=".">5</td><td align="char" char=".">3</td></tr><tr><td> Brahma (Brm)</td><td>ATP-dependent helicase</td><td align="char" char=".">4</td><td align="char" char=".">4</td></tr><tr><td> Brahma associated protein 155 kDa (Mor)</td><td>Protein binding</td><td align="char" char=".">1</td><td align="char" char=".">1</td></tr></tbody></table><table-wrap-foot><fn><p>To determine whether there are physical interactions between Yki/Sd transcriptional complex and Brm complex and gain further understanding of the regulation mechanism of Brm in regulating ISC proliferation, we immunoprecipitated endogenous Sd or Yki protein in S2 cells using generated rabbit anti-Sd or anti-Yki antibodies, respectively, followed by mass spectrometry (MS) analysis. The corresponding proteins of Brm complex identified in association with Yki (Yki mass spectrum) or Sd (Sd mass spectrum) are listed with the number of peptides identified by mass spectrometry.</p></fn></table-wrap-foot></table-wrap><fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.00999.010</object-id><label>Figure 4.</label><caption><title>Sd and Yki interact with Brm complex components.</title><p>(<bold>A</bold>) Interaction between overexpressed Myc–Yki and Brm–V5 was detected in S2 cells. Myc–Yki or Brm–V5 was immunoprecipitated with anti-Myc or anti-V5 antibodies. (<bold>B</bold>) Association between HA–Sd and endogenous Brm in vitro. S2 cells were transfected with the HA–Sd. The arrow indicated HA–Sd coimmunoprecipitated with endogenous Brm. (<bold>C</bold>–<bold>H</bold>) Wild-type male wings (<bold>C</bold>) or hemizygous male wings of null allele <italic>brm</italic><sup><italic>2</italic></sup><italic>/+</italic> (<bold>D</bold>), or hypomorphic allele <italic>sd</italic><sup><italic>1</italic></sup><italic>/Y</italic> (<bold>E</bold>), or double-mutant combinations of <italic>sd</italic><sup><italic>1</italic></sup><italic>/Y; brm</italic><sup><italic>2</italic></sup><italic>/+</italic> (<bold>F</bold>), or hypomorphic allele <italic>osa</italic><sup><italic>2</italic></sup><italic>/+</italic> (<bold>G</bold>), or combinations of <italic>sd</italic><sup><italic>1</italic></sup><italic>/Y; osa</italic><sup><italic>2</italic></sup><italic>/+</italic> (<bold>H</bold>). See also <xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00999.010">http://dx.doi.org/10.7554/eLife.00999.010</ext-link></p></caption><graphic xlink:href="elife00999f004"/></fig><fig id="fig4s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00999.011</object-id><label>Figure 4—figure supplement 1.</label><caption><title>Brm complex associates with Sd.</title><p>(<bold>A</bold>) Sd interacted with Brm, Osa in one direction while in both directions with Mor. The interaction between Sd and Bap60 was not detected. The asterisk marked the band of heavy chain of IgG and the arrow marked the band of Osa. (<bold>B</bold>–<bold>D′′′</bold>) Overexpressed Sd and Brm localized in the nuclei of S2 cells. Cells were immunostained with indicated antibodies, HA (red), Flag (green) and DAPI (blue).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00999.011">http://dx.doi.org/10.7554/eLife.00999.011</ext-link></p></caption><graphic xlink:href="elife00999fs004"/></fig></fig-group></p></sec><sec id="s2-5"><title>brm interacts with sd genetically</title><p>The genetic interaction between <italic>brm</italic> mutant (<italic>brm</italic><sup><italic>2</italic></sup>) and <italic>sd</italic> hypomorphic allele (<italic>sd</italic><sup><italic>1</italic></sup>) in adult fly wings was examined. Strong mutations in <italic>sd</italic> cause lethality, while hypomorphic mutant <italic>sd</italic><sup><italic>1</italic></sup> flies are viable with a scalloped wing phenotype (compare <xref ref-type="fig" rid="fig4">Figure 4E</xref> with <xref ref-type="fig" rid="fig4">Figure 4C</xref>). Single-mutant <italic>brm</italic><sup><italic>2</italic></sup> fly wings are normal (<xref ref-type="fig" rid="fig4">Figure 4D</xref>). Interestingly, the double-mutant combination of <italic>sd</italic><sup><italic>1</italic></sup> and <italic>brm</italic><sup><italic>2</italic></sup> shows a strong enhancement of the <italic>sd</italic><sup><italic>1</italic></sup> phenotype (compare <xref ref-type="fig" rid="fig4">Figure 4F</xref> with <xref ref-type="fig" rid="fig4">Figure 4E</xref>). A similar enhanced phenotype was also found in the flies with <italic>sd</italic><sup><italic>1</italic></sup> and <italic>osa</italic><sup><italic>2</italic></sup>, a hypomorphic allele of a Brm complex subunit (compare <xref ref-type="fig" rid="fig4">Figure 4H</xref> with <xref ref-type="fig" rid="fig4">Figure 4E,G</xref>). These observations indicate that <italic>brm</italic> and <italic>osa</italic> genetically interact with <italic>sd</italic> and contribute to the wing vein alternation phenotype. Together with the biochemical results, these results suggest that Brm complex plays a crucial role in Yki–Sd mediated function.</p></sec><sec id="s2-6"><title>Brm functions downstream of Yki–Sd to maintain ISC proliferative ability</title><p>To further test whether Yki-mediated ISC proliferation depends on Brm, we examined the requirement of Brm activity during Yki–Sd induced ISC proliferation. Overexpression of either Yki or SdGA, an active form of Sd (<xref ref-type="bibr" rid="bib60">Zhang et al., 2008</xref>), under the control of <italic>esg80</italic><sup><italic>ts</italic></sup> resulted in an increase in GFP<sup>+</sup> and PH3<sup><italic>+</italic></sup> cell numbers (compared <xref ref-type="fig" rid="fig5">Figure 5C,C′′,E,E′′</xref> with <xref ref-type="fig" rid="fig5">Figure 5A,A′′,K</xref>), suggesting an enhancement of ISC proliferation. Interestingly, Yki overexpression resulted in pronounced hyperplasia of intestine with a thicker intestinal epithelium composed of a multi-layer of tightly packed cells (<xref ref-type="bibr" rid="bib45">Staley and Irvine, 2010</xref>) (compare <xref ref-type="fig" rid="fig5">Figure 5H</xref> with <xref ref-type="fig" rid="fig5">Figure 5G</xref>), whereas SdGA expression did not induce such a phenomenon (compare <xref ref-type="fig" rid="fig5">Figure 5J</xref> with <xref ref-type="fig" rid="fig5">Figure 5G</xref>), suggesting that Yki and Sd may have distinct mechanisms in regulating ISC proliferation. When Brm was knocked down, ISC proliferation was greatly suppressed (<xref ref-type="fig" rid="fig5">Figure 5K</xref>, and compare <xref ref-type="fig" rid="fig5">Figure 5B,B′′,D,D′′,F,F′′</xref> with <xref ref-type="fig" rid="fig5">Figure 5A,A′′,C,C′′,E,E′′</xref>) with a decreased Dl signal intensity (<xref ref-type="fig" rid="fig5">Figure 5B′,D′</xref>), and the formation of thicker intestinal epithelium induced by Yki overexpression was inhibited (<xref ref-type="fig" rid="fig5">Figure 5I</xref>). Moreover, similar results were obtained by MARCM analysis of <italic>brm</italic><sup><italic>2</italic></sup>. Overexpression of Yki in control MARCM clones resulted in a significant increase in the cell numbers and in the formation of large clones (<xref ref-type="fig" rid="fig5">Figure 5L,L′,N</xref>), whereas this Yki-induced proliferation was completely blocked in the <italic>brm</italic><sup><italic>2</italic></sup> clones (<xref ref-type="fig" rid="fig5">Figure 5M,M′,N</xref>). Taken these results together, the depletion of Brm compromised Yki or SdGA overexpression induced ISC proliferation, indicating that Brm functions downstream of Yki–Sd to maintain ISC proliferative ability.<fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.00999.012</object-id><label>Figure 5.</label><caption><title>Knockdown of Brm blocks Yki/SdGA-induced ISC proliferation.</title><p>(<bold>A</bold>–<bold>F′′</bold>) Adult flies expressing <italic>esg80</italic><sup><italic>ts</italic></sup> (<bold>A</bold>–<bold>A′′</bold>), <italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Brm RNAi</italic> (<bold>B</bold>–<bold>B′′</bold>), <italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Yki</italic> (<bold>C</bold>–<bold>C′′</bold>), <italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Yki+Brm RNAi</italic> (<bold>D</bold>–<bold>D′′</bold>), <italic>esg80</italic><sup><italic>ts</italic></sup><italic>-SdGA</italic> (<bold>E</bold>–<bold>E′′</bold>), <italic>esg80</italic><sup><italic>ts</italic></sup><italic>-SdGA +Brm RNAi</italic> (<bold>F</bold>–<bold>F′′</bold>) were cultured at 29°C for 8–9 days. Midguts were dissected and immunostained for Dl (red) and DAPI (blue). White arrows indicated the EBs, and yellow arrowheads indicated the ISCs. (<bold>G</bold>–<bold>J</bold>) Images show an optical cross-section through the center of the intestine, DAPI (green). (<bold>K</bold>) Quantification of PH3 positive mitotic cells of the indicated guts. The results represent the mean ± SEM, n = 10 for each genotype. (<bold>L</bold>–<bold>M′</bold>) Adult midguts containing nuclear localized GFP-labeled control non-tagged form of Yki overexpressed clones (<bold>L</bold> and <bold>L′</bold>) or Yki plus <italic>brm</italic><sup><italic>2</italic></sup> clones (<bold>M</bold> and <bold>M′</bold>) were immunostained for Yki (red) and DAPI (blue). Guts were dissected from the adult flies 72 hr after clone induction. (<bold>N</bold>) Quantification of the cell number of <italic>Yki</italic> or <italic>Yki+brm</italic><sup><italic>2</italic></sup> clones. 10 guts were counted for each genotype. (<bold>O</bold>–<bold>P′′′</bold>) Adult guts of <italic>Myo1A-Gal4 UAS-GFP;tubGal80</italic><sup><italic>ts</italic></sup> control (<bold>O</bold>–<bold>O′′′</bold>) or expressing <italic>Myo1A-Gal4 UAS-GFP;tubGal80</italic><sup><italic>ts</italic></sup>-<italic>Yki</italic> (<bold>P</bold>–<bold>P′′′</bold>) were immunostained for Brm (red), Dl (purple), and DAPI (blue). Arrows indicated ISCs with a high endogenous Brm protein level.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00999.012">http://dx.doi.org/10.7554/eLife.00999.012</ext-link></p></caption><graphic xlink:href="elife00999f005"/></fig></p><p>Interestingly, when Yki was expressed in ECs using <italic>Myo1A-Gal4</italic> to induce non-autonomous ISC proliferation, the number of ISCs/EBs was increased with high levels of Brm in the nucleus (compare <xref ref-type="fig" rid="fig5">Figure 5P–P′′′</xref> with <xref ref-type="fig" rid="fig5">Figure 5O–O′′′</xref>), suggesting that Yki-induced non-autonomous ISC proliferation also induces high levels of Brm in nuclei of ISCs and EBs.</p></sec><sec id="s2-7"><title>The Hpo signaling regulates Brm protein cleavage</title><p>Given that Brm physically interacts with Yki–Sd complex and that the function of Yki–Sd in ISC proliferation requires Brm activity, we sought to determine the underlying mechanism by which the Hpo pathway and Brm regulate ISC proliferation. Interestingly, cotransfection of Brm and Hpo in S2 cells resulted in a lower Brm protein level, suggesting that Brm was destabilized in the presence of Hpo (<xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1A</xref>). This result raised the concern that the Hpo pathway might regulate Brm activity by controlling its protein stability. Furthermore, Brm cleavage event in which a small protein band at about 100 kD was detected (<xref ref-type="fig" rid="fig6">Figure 6A</xref>) in the presence of Hpo upon MG132 treatment. To detect whether this small band represents a cleaved Brm fragment, we generated a Brm construct with a Flag tag at the N-terminus and a V5 tag at the C-terminus (referred to as Flag-Brm-V5). Using this construct, we were able to identify a Flag-tagged N-terminal cleavage product about 100 kD and a V5-tagged C-terminal cleavage product about 130 kD in the presence of Hpo (<xref ref-type="fig" rid="fig6">Figure 6A</xref>). Considering that the molecular weight of full length Brm is about 230 kD, it is possible that Hpo regulates Brm stability by inducing Brm cleavage at only one site. We also found that this cleavage action depended on Hpo protein in a dose-dependent manner, since increasing the dose of Hpo plasmids resulted in an accumulation of the cleaved Brm product and a decrease in the full length Brm protein (<xref ref-type="fig" rid="fig6">Figure 6B</xref>). A truncation of Hpo without kinase activity (Hpo-C) did not induce such cleavage (<xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1B</xref>, lanes 1 and 2), indicating that Hpo kinase domain but not C-terminal regulatory domain induces Brm cleavage. In addition, Hpo-induced Brm cleavage was blocked in the presence of Yki–Sd (<xref ref-type="fig" rid="fig6">Figure 6C</xref>), suggesting that it was regulated by downstream events of the Hpo signaling pathway.<fig-group><fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.00999.013</object-id><label>Figure 6.</label><caption><title>Brm is cleaved at the D718 site by Hpo-induced caspase.</title><p>(<bold>A</bold>) Flag-Brm-V5 was transfected with or without Myc-Hpo. Western blots (anti-Flag or anti-V5) of IP samples were performed to detect the N- or C-terminus of Brm. MG132 was treated 6 hr before harvesting the cells. Arrows indicated the full length Brm (top) and the N- (bottom), C- (middle) terminal cleaved product of Brm. (<bold>B</bold>) 3 μg of Flag-Brm was cotransfected with different dosages of Hpo plasmids in S2 cells, MG132 was treated 6 hr before harvesting the cells. (<bold>C</bold>) Cotransfected Flag-Brm and Myc-Hpo together with Sd/Yki or in the presence of caspase inhibitor Z-VAD-FMK, the cleaved Brm fragments were unable to be detected. Z-VAD-FMK was added to a final concentration of 10 mM for 6 hr. (<bold>D</bold>) S2 cells were transfected with Myc-Hpo and Flag-Brm with HA-Diap1. (<bold>E</bold>) Flag-Brm and Myc-Hpo were cotransfected in S2 cells to induce the cleavage of Brm. Inhibitors of Caspase 3, 8, 9, 10 were added to block the cleavage in a final concentration of 10 mM for 6 hr. Asterisk indicates IgG bands (loading control). (<bold>F</bold>) Brm<sup>D718A</sup> mutation blocked Hpo-induced Brm cleavage in S2 cells. (<bold>G</bold>) A schematic representation of Brm deletions and mutations. Brm-D1 to D4 were the deletions that were used to map the cleavages site of Brm. Brm-D718A/D726A/D728A/D731A/D740,741A were the mutants generated for mapping the cleavage sites. The novel caspase recognition motif (DATD) in Brm is indicated by a single blue underline including D718 residue. See also <xref ref-type="fig" rid="fig6">Figure 6—figure supplement 1</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00999.013">http://dx.doi.org/10.7554/eLife.00999.013</ext-link></p></caption><graphic xlink:href="elife00999f006"/></fig><fig id="fig6s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00999.014</object-id><label>Figure 6—figure supplement 1.</label><caption><title>Brm is cleaved by Hpo-induced caspase.</title><p>(<bold>A</bold>) The protein level of Brm was decreased when cotransfected with Myc-Hpo (lane two), but not Myc-Yki (lane three). (<bold>B</bold>) Hpo N-terminal kinase domain 1–342 aa (Hpo-N) induced Brm cleavage, but not Hpo C-terminal regulatory domain 343 aa-end (Hpo-C) (lane one and lane two). Brm deletions, D1 and D2, failed to produce the 100 KD band when Hpo existed (lane three and lane four). Brm deletions were immunoprecipitated with anti-Flag antibody. (<bold>C</bold>) Brm deletion D3 still was cleaved when Hpo was cotransfected, but the D4 could not. The full length Brm and the N-terminal cleaved fragment of Brm are marked by arrows. (<bold>D</bold>–<bold>E′′′</bold>) Overexpressing Hpo rather than Hpo kinase dead (Hpo-KD) induced the activated caspase 3 signal. Wing discs expressing wild-type <italic>UAS- Hpo</italic> (<bold>D</bold>–<bold>D′′′</bold>) or <italic>UAS-Hpo-KD</italic> (<bold>E</bold>–<bold>E′′′</bold>) under control of <italic>hhGal4</italic> driver were immunostained with activated casepase 3 antibody (Cas3, green), Flag antibody (red) and Ci antibody (blue).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00999.014">http://dx.doi.org/10.7554/eLife.00999.014</ext-link></p></caption><graphic xlink:href="elife00999fs005"/></fig></fig-group></p></sec><sec id="s2-8"><title>Brm cleavage is mediated by Hpo-induced caspase activation</title><p>On the basis of above observations, it is feasible to consider that Hpo activates downstream caspases to cleave Brm. In fact, previous studies have implicated a role of the Hpo signaling pathway in caspase activation and cell apoptosis (<xref ref-type="bibr" rid="bib50">Verghese et al., 2012</xref>). This function of Hpo was verified by detecting active caspase 3 expression in wing discs overexpressing Hpo or Hpo-KD (a kinase dead form of Hpo). As shown in <xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1D–E′′′</xref>, the cleaved caspase 3, a functional read-out of initiator caspase activity, was detected only in Hpo overexpressed discs but not in the Hpo-KD overexpressed discs. Taken together, we speculated that Hpo-induced caspase activity might regulate the protein level of Brm.</p><p>To verify this idea, we used the unspecific caspase inhibitor, Z-VAD-FMK, to test whether the activities of caspases are required for Hpo-induced Brm cleavage. We found that the 100 kD cleaved Brm product disappeared on the addition of Z-VAD-FMK (<xref ref-type="fig" rid="fig6">Figure 6C</xref>), suggesting that the inhibition of caspase activities blocks Brm cleavage. Furthermore, it is known that the <italic>Drosophila</italic> inhibitor of apoptosis protein, Diap1, which is a transcription product of the Hpo pathway target genes (<xref ref-type="bibr" rid="bib60">Zhang et al., 2008</xref>), inhibits caspase activity. Diap1 was cotransfected with Brm and Hpo in S2 cells to inhibit caspase activity. Interestingly, we found that Diap1 cotransfection inhibited Hpo-induced Brm cleavage (<xref ref-type="fig" rid="fig6">Figure 6D</xref>), indicating that the Hpo regulates Brm cleavage by inducing caspase activity.</p><p>To further study the function of caspases during Brm cleavage in details, Hpo and Brm were cotransfected in S2 cells in the presence of inhibitors of mammalian caspase 3, 8, 9, 10, respectively (no commercial <italic>Drosophila</italic> caspase inhibitors were available). As shown in <xref ref-type="fig" rid="fig6">Figure 6E</xref>, the addition of inhibitor of caspase 3 or caspase 10 completely abolished Hpo-induced Brm cleavage, whereas the addition of other caspase inhibitors only partially affected the cleavage reaction as revealed by the presence of the 100 kD Brm protein fragment. Caspase 10 is an initiator in the extrinsic death-receptor-mediated cell death (<xref ref-type="bibr" rid="bib51">Wachmann et al., 2010</xref>), and caspase 3 is the effector caspase generally believed to carry out the cleavage of nuclear protein substrates. These results suggest that <italic>Drosophila</italic> homologs of caspase 3 and caspase 10 play important roles in Hpo-induced Brm cleavage.</p></sec><sec id="s2-9"><title>D718 site is required for Hpo-induced, caspase-dependent, Brm cleavage</title><p>In an attempt to identify the cleavage site of Brm, two Brm deletion forms, D1 (Δ601–800 aa) and D2 (Δ694–768 aa), were generated based on previous observations of N- and C-terminal cleavage products (<xref ref-type="fig" rid="fig6">Figure 6G</xref>). No cleavage reaction was detected for these two Brm deletion forms (<xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1B</xref>). Mapping of the D2 form using two other deletion forms of Brm, D3 (Δ694–711 aa) and D4 (Δ712–729 aa), indicated that D3 was cleaved but not D4 (<xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1C</xref>), suggesting that the cleavage site locates within the region of amino acid 712–729. Although no canonical caspase 3 tetra-peptide cleavage site DEVD was found in this region, several aspartic acids that potentially serve as the caspase cleavage sites were identified. To validate these sites, individual aspartic acids were mutated to alanine separately. Interestingly, Brm mutant carrying aspartic acids to alanine mutation at D718 site (Brm<sup>D718A</sup>) does not undergo cleavage (<xref ref-type="fig" rid="fig6">Figure 6F</xref>). In conclusion, Brm protein stability was regulated by Hpo-induced caspase-dependent cleavage at the D718 site.</p></sec><sec id="s2-10"><title>The cleavage resistant Brm mutant Brm<sup>D718A</sup> promotes ISC proliferation</title><p>Given the finding that Brm<sup>D718A</sup> was a cleavage resistant Brm mutant (<xref ref-type="fig" rid="fig6">Figure 6F</xref>), we wondered whether Brm<sup>D718A</sup> is an active form of Brm. To test the function of Brm<sup>D718A</sup>, we expressed Brm<sup>D718A</sup> under the control of <italic>esg80</italic><sup><italic>ts</italic></sup> in ISCs/EBs. An upregulation of ISC/EB (GFP<sup>+</sup>) and PH3<sup>+</sup> cell numbers was detected in guts expressing Brm<sup>D718A</sup> mutant (compare <xref ref-type="fig" rid="fig7">Figure 7B–B′</xref> with <xref ref-type="fig" rid="fig7">Figure 7A–A′,J</xref>), whereas expressing wild-type Brm induced a mild increase in the ISC/EB numbers and PH3<sup>+</sup> cell numbers (<xref ref-type="fig" rid="fig7">Figure 7G,G′,J</xref>). On coexpression of Brm<sup>D718A</sup> and Yki, the number of PH3<sup>+</sup> cells was further increased, suggesting that ISC proliferation is promoted (<xref ref-type="fig" rid="fig7">Figure 7C–D′,J</xref>). Furthermore, analysis of 5-bromodeosyuridine (BrdU) incorporation in midguts showed that Brm<sup>D718A</sup> overexpression greatly enhanced BrdU ectopic expression, whereas Brm RNAi resulted in a lower proliferative activity (<xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1A–D′</xref>). Altogether, these results indicate that Brm<sup>D718A</sup> promotes ISC proliferation.<fig-group><fig id="fig7" position="float"><object-id pub-id-type="doi">10.7554/eLife.00999.015</object-id><label>Figure 7.</label><caption><title>The cleavage resistant mutant Brm<sup>D718A</sup> promotes ISC proliferation with antagonistic ability against Hpo activity.</title><p>(<bold>A</bold>–<bold>H′</bold>) Adult guts of <italic>esg80</italic><sup><italic>ts</italic></sup> control (<bold>A</bold> and <bold>A′</bold>), <italic>esg80</italic><sup><italic>ts</italic></sup>-<italic>Brm</italic><sup><italic>D718A</italic></sup> (<bold>B</bold> and <bold>B′</bold>), <italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Yki</italic> (<bold>C</bold> and <bold>C′</bold>), <italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Yki+Brm</italic><sup><italic>D718A</italic></sup> (<bold>D</bold> and <bold>D′</bold>), <italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Hpo</italic> (<bold>E</bold> and <bold>E′</bold>), <italic>esg80</italic><sup><italic>ts</italic></sup><italic>- Brm</italic><sup><italic>D718A</italic></sup> <italic>+Hpo</italic> (<bold>F</bold> and <bold>F′</bold>), <italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Brm</italic> (<bold>G</bold> and <bold>G′</bold>) and <italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Brm+Hpo</italic> (<bold>H</bold> and <bold>H′</bold>) were immunostained for DAPI (blue). (<bold>I</bold>–<bold>I′′′</bold>) Adult midguts containing GFP-labeled MARCM clones of <italic>hpo</italic> null allele (<italic>BF33(16)</italic>). White arrows indicate the ECs in the <italic>BF33(16)</italic> clones, and yellow arrowheads indicate the ECs outside the clones. (<bold>J</bold>) Quantification of PH3 positive mitotic cells of the indicated guts. The results represent the mean ± SEM, n > 10 for each genotype. (<bold>K</bold>) A model of the regulation of Brm protein stability by the Hpo pathway. The Hpo pathway restricts Brm protein level by inducing the activation of caspase to cleave Brm and/or by inhibiting the expression of Yki–Sd target genes, especially <italic>diap1</italic> that inhibits the caspase activity. See also <xref ref-type="fig" rid="fig7s4">Figure 7—figure supplements 1–4</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00999.015">http://dx.doi.org/10.7554/eLife.00999.015</ext-link></p></caption><graphic xlink:href="elife00999f007"/></fig><fig id="fig7s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00999.016</object-id><label>Figure 7—figure supplement 1.</label><caption><title>The cleavage resistance mutant Brm<sup>D718A</sup> promotes ISCs proliferation.</title><p>(<bold>A</bold>–<bold>D′</bold>) Adult guts of <italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Gal4</italic> control (<bold>A</bold> and <bold>A′</bold>), <italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Brm</italic> (<bold>B</bold> and <bold>B′</bold>), <italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Brm RNAi</italic> (<bold>C</bold> and <bold>C′</bold>), <italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Brm</italic><sup><italic>D718A</italic></sup> (<bold>D</bold> and <bold>D′</bold>) were immunostained for BrdU (red). Note that Brm<sup>D718A</sup> increased the BrdU number. (<bold>E</bold> and <bold>F′</bold>) Adult midguts containing GFP-labeled MARCM clones of <italic>hpo</italic> null allele <italic>BF33(16)+ Brm</italic> (<bold>E</bold>–<bold>E′</bold>) or <italic>hpo</italic> null allele <italic>BF33(16)+ Brm RNAi</italic> (<bold>F</bold>–<bold>F′</bold>). Brm RNAi blocked the cell proliferation in <italic>hpo</italic> null allele clones.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00999.016">http://dx.doi.org/10.7554/eLife.00999.016</ext-link></p></caption><graphic xlink:href="elife00999fs006"/></fig><fig id="fig7s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00999.017</object-id><label>Figure 7—figure supplement 2.</label><caption><title>The cleavage products of Brm have low activity in promoting ISC proliferation.</title><p>(<bold>A</bold>–<bold>J</bold>) Adult guts of <italic>esg80</italic><sup><italic>ts</italic></sup> control (<bold>A</bold>), <italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Brm</italic> (<bold>B</bold>), <italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Brm</italic><sup><italic>D718A</italic></sup> (<bold>C</bold>), <italic>esg80</italic><sup><italic>ts</italic></sup><italic>- esg80</italic><sup><italic>ts</italic></sup><italic>-Brm-N</italic> (<bold>D</bold>), <italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Brm-C</italic> (<bold>E</bold>), <italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Yki</italic> (<bold>F</bold>), <italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Yki+Brm</italic> (<bold>G</bold>), <italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Yki+Brm</italic><sup><italic>D718A</italic></sup> (<bold>H</bold>), <italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Yki+Brm-N</italic> (<bold>I</bold>) and <italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Yki+Brm-C</italic> (<bold>J</bold>) were immunostained for PH3 (red) and DAPI (blue). (<bold>K</bold>) Quantification of PH3 positive mitotic cells of the indicated guts. The results represent the mean ± SEM, n > 10 for each genotype. (<bold>L</bold>) Quantification of the cell number of the MARCM clones of the indicated genotypes. Guts were divided into two groups after clone induction: 3 days and 10 days. The results represent the mean ± SEM, n > 10 for each group. (<bold>M</bold>–<bold>R</bold>) Adult midguts containing nuclear localized GFP-labeled wild-type control clones (<bold>M</bold>), <italic>brm</italic> null allele <italic>brm</italic><sup><italic>2</italic></sup> clones (<bold>N</bold>), <italic>Brm+brm</italic><sup><italic>2</italic></sup> clones (<bold>O</bold>), <italic>Brm</italic><sup><italic>D718A</italic></sup><italic>+brm</italic><sup><italic>2</italic></sup> clones (<bold>P</bold>), <italic>Brm-N+brm</italic><sup><italic>2</italic></sup> clones (<bold>Q</bold>) and <italic>Brm-C+brm</italic><sup><italic>2</italic></sup> clones (<bold>R</bold>) were immunostained to show the DAPI (blue). Guts were dissected from the adult flies 10 days after clone induction.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00999.017">http://dx.doi.org/10.7554/eLife.00999.017</ext-link></p></caption><graphic xlink:href="elife00999fs007"/></fig><fig id="fig7s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00999.018</object-id><label>Figure 7—figure supplement 3.</label><caption><title>Genetic interaction assays between Brm and Yki/Sd in Drosophila eyes.</title><p>(<bold>A</bold>–<bold>F′</bold>) Genetic interaction assays between Brm and Yki in Drosophila eyes. Brm<sup>D718A</sup> further increased Yki-induced eye overgrowth. Adult eyes of <italic>GMR</italic> (<bold>A</bold> and <bold>A′</bold>), <italic>GMR-Brm</italic> (<bold>B</bold> and <bold>B′</bold>), <italic>GMR- Brm</italic><sup><italic>D718A</italic></sup> (<bold>C</bold> and <bold>C′</bold>), <italic>GMR-Gal4/UAS-Yki</italic> (<bold>D</bold> and <bold>D′</bold>), <italic>GMR-Gal4/UAS-Yki+wild type Brm</italic> (<bold>E</bold> and <bold>E′</bold>), <italic>GMR-Gal4/UAS-Yki+Brm</italic><sup><italic>D718A</italic></sup> (<bold>F</bold> and <bold>F′</bold>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00999.018">http://dx.doi.org/10.7554/eLife.00999.018</ext-link></p></caption><graphic xlink:href="elife00999fs008"/></fig><fig id="fig7s4" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00999.019</object-id><label>Figure 7—figure supplement 4.</label><caption><title>Brm regulates Hpo pathway target genes in wing discs.</title><p>(<bold>A</bold>–<bold>D′′</bold>) Wing discs of <italic>hh-Gal4</italic> control (<bold>A</bold>–<bold>A′′</bold>) or expressing <italic>UAS-Flag-Brm</italic> (<bold>B</bold>–<bold>B′′</bold>) or <italic>UAS-Flag-Brm</italic><sup><italic>D718A</italic></sup> (<bold>C</bold>–<bold>C′′</bold>) or <italic>UAS-Flag-Brm</italic><sup><italic>K804R</italic></sup> (<bold>D</bold>–<bold>D′′</bold>) were immunostained to show the expression of flag (red), and Diap1 (green). P-compartment of the wing discs was marked by arrows. Of note, Brm<sup>K804R</sup> always shows a weak expression in wing discs. (<bold>E</bold>–<bold>F′</bold>) Bantam sensor upregulated in P-compartment of the wing discs when Brm<sup>K804R</sup> was expressed, which stands for decreased Bantam level in the P-compartment. Wing discs of <italic>hhBanGFP</italic> control (<bold>E</bold> and <bold>E′</bold>) or expressing <italic>UAS-Flag-Brm</italic><sup><italic>K804R</italic></sup> (<bold>F</bold> and <bold>F′</bold>) were immunostained to show the expression of Bantam sensor (BanGFP, green) and Ci (blue).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00999.019">http://dx.doi.org/10.7554/eLife.00999.019</ext-link></p></caption><graphic xlink:href="elife00999fs009"/></fig></fig-group></p><p>To further investigate the function of Brm in ISC proliferation, we overexpressed the truncated form of Brm-N (1–717 aa) or Brm-C (718–1639 aa) in ISCs/EBs under the control of <italic>esg80</italic><sup><italic>ts</italic></sup>. Compared with the wild-type Brm or Brm<sup>D718A</sup>, Brm-C exhibited a weak influence on ISC proliferation while Brm-N did not show any obvious effect (<xref ref-type="fig" rid="fig7s2">Figure 7—figure supplement 2A–E,K</xref>). On coexpression with Yki, both Brm-N and Brm-C further promoted Yki-induced ISC proliferation, but not as dramatic as Brm or Brm<sup>D718A</sup> (<xref ref-type="fig" rid="fig7s2">Figure 7—figure supplement 2F–J,K</xref>). To better understand the impact of Brm cleavage on ISC proliferation, rescue experiments were carried out using MARCM approach. We overexpressed Brm, Brm<sup>D718A</sup>, Brm-N and Brm-C in <italic>brm</italic><sup><italic>2</italic></sup> MARCM clones and found that all of them were able to partially rescue the growth defect of <italic>brm</italic><sup><italic>2</italic></sup> clones to different degrees. Brm<sup>D718A</sup> possessed the strongest rescue ability, as it generated bigger clones that contain more cells (<xref ref-type="fig" rid="fig7s2">Figure 7—figure supplement 2P,L</xref>), while Brm-N and Brm-C only showed weak rescue phenotypes (<xref ref-type="fig" rid="fig7s2">Figure 7—figure supplement 2Q,R,L</xref>). These results indicate that Brm cleavage is important for controlling the stability and activity of Brm during ISC proliferation.</p></sec><sec id="s2-11"><title>Brm<sup>D718A</sup> rescues Hpo-restricted ISC proliferation more efficiently than Brm</title><p>According to the in vivo observations above, Brm<sup>D718A</sup> promotes ISC proliferation and exhibits higher activity than wild-type Brm. We speculated that higher activity of Brm<sup>D718A</sup> might be due to the resistance of Brm<sup>D718A</sup> against Hpo signaling regulated cleavage. To verify this hypothesis, we coexpressed Hpo and Brm<sup>D718A</sup> under the control of <italic>esg80</italic><sup><italic>ts</italic></sup> and found that Brm<sup>D718A</sup> completely rescued the impairment of ISC proliferation induced by Hpo overexpression. In comparison with the decrease of ISC/EB numbers induced by Hpo overexpression, coexpression of Hpo and Brm<sup>D718A</sup> exhibited a dramatic increase of ISCs/EBs as well as PH3<sup><italic>+</italic></sup> cells in midguts (compare <xref ref-type="fig" rid="fig7">Figure 7F,F′</xref> with <xref ref-type="fig" rid="fig7">Figure 7E,E′,J</xref>). On the other hand, coexpression of wild-type Brm and Hpo only slightly rescued Hpo-induced decrease of ISCs/EBs (compare <xref ref-type="fig" rid="fig7">Figure 7H,H′</xref> with <xref ref-type="fig" rid="fig7">Figure 7E,E′</xref>). In addition, PH3<sup><italic>+</italic></sup> cell number was increased when Brm and Hpo were coexpressed (<xref ref-type="fig" rid="fig7">Figure 7J</xref>), a phenomenon that might be due to an unidentified feedback mechanism of homeostasis in response to Hpo-induced impairment.</p><p>To determine whether loss of Hpo expression regulates Brm protein level in midguts, we generated <italic>hpo</italic> null allele <italic>BF33(16)</italic> (<xref ref-type="bibr" rid="bib21">Jin et al., 2012</xref>) MARCM clones in midguts. ECs within the clone regions obtained higher Brm protein levels than ECs outside the clones (<xref ref-type="fig" rid="fig7">Figure 7I–I′′′</xref>), suggesting that Hpo also restricts Brm protein level in ECs. We next expressed Brm or Brm RNAi in <italic>BF33(16)</italic> MARCM clones (<xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1E–F′</xref>). The growth of the <italic>hpo</italic> null allele clones was not affected by Brm overexpression. Yet, it was suppressed by the knockdown of Brm using RNAi (<xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1E–F′</xref>), indicating that Brm is required in the loss-of-Hpo-induced intestinal proliferation. Taken together, these results indicate that Brm protein level is restricted by Hpo activity. Brm<sup>D718A</sup>, as an uncleavable form of Brm, bypasses the Hpo restriction to retain its ability to promote ISC proliferation.</p><p>To further understand how Brm<sup>D718A</sup> functions to promote proliferation in other tissues, we investigated the genetic relationship of Brm and Yki in adult eyes under the control of <italic>GMR-Gal4</italic> driver. Overexpression of <italic>UAS-Yki</italic> posterior to the morphogenetic furrow using the <italic>GMR-Gal4</italic> (referred to as <italic>GMR-Yki</italic>) resulted in dramatic eye overgrowth (compare <xref ref-type="fig" rid="fig7s3">Figure 7—figure supplement 3D,D′</xref> with <xref ref-type="fig" rid="fig7s3">Figure 7—figure supplement 3A,A′</xref>). Consistent with the findings in midguts, expressing wild-type Brm did not significantly affect Yki overexpression induced eye overgrowth (<xref ref-type="fig" rid="fig7s3">Figure 7—figure supplement 3E,E′</xref>), yet expressing Brm<sup>D718A</sup> clearly enhanced <italic>GMR-Yki</italic> induced overgrowth (<xref ref-type="fig" rid="fig7s3">Figure 7—figure supplement 3F,F′</xref>). In addition, overexpression of Brm<sup>D718A</sup> using <italic>hhGal4</italic> caused an upregulation of Diap1 protein levels in the posterior region of the wing discs (compare <xref ref-type="fig" rid="fig7s4">Figure 7—figure supplement 4C–C′′</xref> with <xref ref-type="fig" rid="fig7s4">Figure 7—figure supplement 4A–A′′</xref>). Conversely, overexpression of the dominant-negative form of Brm, Brm<sup>K804R</sup>, resulted in a reduction in Diap1 and Bantam levels (<xref ref-type="fig" rid="fig7s4">Figure 7—figure supplement 4D–D′′</xref> and <xref ref-type="fig" rid="fig7s4">Figure 7—figure supplement 4E–F′</xref>). These assays indicate that activated Brm promoted the expression of the Hpo pathway target genes, such as <italic>diap1.</italic></p></sec></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><p>SWI/SNF complex subunits regulate the chromatin structure by shutting off or turning on the gene expression during differentiation (<xref ref-type="bibr" rid="bib43">Roberts and Orkin, 2004</xref>). Recently, the findings from several research reports based on the stem cell system reveal important roles of chromatin remodeling complex in stem cell state maintenance (<xref ref-type="bibr" rid="bib26">Lessard et al., 2007</xref>; <xref ref-type="bibr" rid="bib14">Ho et al., 2009</xref>). Our study suggested that the chromatin remodeling activity of Brm complex was required for the proliferation and differentiation of <italic>Drosophila</italic> ISCs. Based on our findings, we propose that Brm is critical for maintaining <italic>Drosophila</italic> intestinal homeostasis (<xref ref-type="fig" rid="fig2">Figure 2J</xref>). High levels of Brm in the ISC nucleus represent high proliferative ability and are essential for EC differentiation; low levels of Brm in the EC nucleus may be a response for homeostasis. Changes in Brm protein levels resulted in the disruption of differentiation and deregulation of cell proliferation. In line with previous findings in human, the cell-type-specific expression of <italic>Drosophila</italic> homologs BRG1 and BRM were also detected in adult tissues (<xref ref-type="bibr" rid="bib41">Reisman et al., 2005</xref>). BRG1 is mainly expressed in cell types that constantly undergo proliferation or self-renewal, whereas BRM is expressed in other cell types (<xref ref-type="bibr" rid="bib29">Marenda et al., 2004</xref>; <xref ref-type="bibr" rid="bib41">Reisman et al., 2005</xref>). These observations indicate that Brm may act similarly as BRG1 and BRM in controlling proliferation and differentiation.</p><p>It is known that the Hpo pathway restricts cell proliferation and promotes cell death at least in two ways: inhibiting the transcriptional co-activator Yki (<xref ref-type="bibr" rid="bib15">Huang et al., 2005</xref>; <xref ref-type="bibr" rid="bib60">Zhang et al., 2008</xref>), and inducing activation of pro-apoptotic genes such as caspases directly (<xref ref-type="bibr" rid="bib50">Verghese et al., 2012</xref>). In our study, we identified a novel regulatory mechanism of the Hpo pathway in maintaining intestinal homeostasis. In this scenario, Brm activity is regulated by the Hpo pathway. In normal physiological conditions, under the control of Hpo signaling, the function of Yki–Sd to promote ISC proliferation is restricted and the pro-proliferation of target genes such as <italic>diap1</italic> that inhibits Hpo-induced caspase activity cannot be further activated (<xref ref-type="fig" rid="fig7">Figure 7K</xref>). Therefore, Hpo signaling normally functions to restrict cell numbers in the midgut by keeping ISC proliferation at low levels. Yki is enriched in ISCs, but predominantly inactivated in cytoplasm by the Hpo pathway (<xref ref-type="bibr" rid="bib23">Karpowicz et al., 2010</xref>; <xref ref-type="bibr" rid="bib45">Staley and Irvine, 2010</xref>). The knockdown of Yki in ISCs did not cause any phenotype in the midgut (<xref ref-type="bibr" rid="bib23">Karpowicz et al., 2010</xref>), suggesting that Yki is inactivated in ISCs under normal homeostasis. During an injury, Hpo signaling is suppressed or disrupted, Yki translocates into the nuclei to form a complex with Sd (<xref ref-type="bibr" rid="bib23">Karpowicz et al., 2010</xref>; <xref ref-type="bibr" rid="bib42">Ren et al., 2010</xref>; <xref ref-type="bibr" rid="bib44">Shaw et al., 2010</xref>), which may allow Yki–Sd to interact with Brm complex in the nucleus to activate transcriptional targets. Of note, the loss-of-function of Brm resulted in growth defect of ISCs, suggesting that Brm is required for ISC homeostasis and possessing a different role of Brm from Yki in the regulation of ISCs. It is possible that the function of Brm on ISC homeostasis is regulated via other signaling pathways by recruiting other factors. Therefore, different phenotypes induced by the loss-of-function of Brm and Yki in midgut might be due to different regulatory mechanisms. Despite its unique function cooperating with Yki in midgut, that Brm complex is essential for Yki-mediated transcription might be a general requirement for cell proliferation. While this manuscript was under preparation, Irvine lab reported a genome-wide association of Yki with chromatin and chromatin-remodeling complexes (<xref ref-type="bibr" rid="bib34">Oh et al., 2013</xref>). These results support our model.</p><p>Our results also suggest that the interaction between Brm and Yki–Sd transcriptional complex is under tight regulation. The loss of Hpo signaling stabilizes Brm protein, whereas the active Hpo pathway restricts Brm levels by activating <italic>Drosophila</italic> caspases to cleave Brm at the D718 site and inhibiting downstream target gene <italic>diap1</italic> transcription simultaneously. In addition, overexpression of Brm complex components induces only a mild enhancement on midgut proliferation (<xref ref-type="fig" rid="fig1">Figure 1D,D′</xref> and <xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2E,E′,G,G′,I,I′</xref>). One possibility is that overexpressing only one of the Brm complex components does not provide full activation of the whole complex; the other possibility is that due to the restriction of the Hpo signaling, as overexpressing Brm<sup>D718A</sup> mutant protein in ISCs/EBs exhibits a stronger phenotype than expressing the wild-type Brm (<xref ref-type="fig" rid="fig7">Figure 7B,G</xref>) and coexpression of Brm<sup>D718A</sup> completely rescues the impairment of Hpo-induced ISC proliferation (<xref ref-type="fig" rid="fig7">Figure 7F</xref>). D718A mutation blocks the caspase-dependent Brm cleavage and exhibits high activity in promoting ISC proliferation. We have defined a previously unknown, yet essential epigenetic mechanism underlying the role of the Hpo pathway in regulating Brm activity.</p><p>It is a novel finding that Brm protein level is regulated by the caspase-dependent cleavage. To focus on the function of Brm cleavage in the presence of cell death signals, we tried to examine the activities of the cleaved Brm fragments. Although in vivo experiments did not show strong activity of Brm N- and C-cleavage products in promoting proliferation of ISCs, the C-terminal fragment of Brm that contains the ATPase domain exhibits a relative higher activity than the N-terminal fragment in ISCs (<xref ref-type="fig" rid="fig7s2">Figure 7—figure supplement 2D,E,K</xref>). The cleavage might induce faster degradation of Brm N- and C-terminus, since it was difficult to detect N- or C-fragments of Brm by Western blot analysis without MG132 treatment. It reveals that the degradation events of Brm including both ubiquitination and cleavage at D718 site can be important for Brm functional regulation under different conditions. To this end, the intrinsic signaling(s) may balance the activity of Brm complex through degradation of some important components, such as Brm, to maintain tissue homeostasis. Of note, the cleavage of Brm at D718 is occurred at a novel DATD sequence that is not conserved in human Brm. It has been reported that Cathepsin G, not caspase, cut hBrm during apoptosis (<xref ref-type="bibr" rid="bib2">Biggs et al., 2001</xref>), suggesting that the cleavage regulatory mechanism of Brm is relatively conserved between <italic>Drosophila</italic> and mammals.</p><p>In this study, we provide evidence that the Brm complex plays an important role in <italic>Drosophila</italic> ISC proliferation and differentiation and is regulated by multi-levels of Hpo signaling. Our findings indicate that Hpo signaling not only exhibits regulatory roles in organ size control during development but also directly regulates epigenetics through a control of the protein level of epigenetic regulatory component Brm. In mammals, it is known that Hpo signaling and SWI/SNF complex-mediated chromatin remodeling processes play critical roles in tissue development. Malfunction of the Hpo signaling pathway and aberrant expressions of SWI/SNF chromatin-remodeling proteins BRM and BRG1 have been documented in a wide variety of human cancers including colorectal carcinoma (<xref ref-type="bibr" rid="bib40">Reisman et al., 2009</xref>; <xref ref-type="bibr" rid="bib37">Pan, 2010</xref>; <xref ref-type="bibr" rid="bib53">Watanabe et al., 2011</xref>). Thus, our study that implicated a functional link between Hpo signaling pathway and SWI/SNF activity may provide new strategies to develop biomarkers or therapeutic targets.</p></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title><italic>Drosophila</italic> stocks and genetics</title><p>The following fly stocks were used: <italic>UAS-yki</italic> (<xref ref-type="bibr" rid="bib60">Zhang et al., 2008</xref>), <italic>UAS-HA-Sd</italic> (<xref ref-type="bibr" rid="bib60">Zhang et al., 2008</xref>), <italic>UAS-HA-SdGA</italic> (<xref ref-type="bibr" rid="bib60">Zhang et al., 2008</xref>), <italic>BF33(16)</italic> (<xref ref-type="bibr" rid="bib21">Jin et al., 2012</xref>), <italic>UAS-Flag-Hpo</italic> (<xref ref-type="bibr" rid="bib21">Jin et al., 2012</xref>), <italic>esg-Gal4/UAS-GFP</italic> (<xref ref-type="bibr" rid="bib30">Micchelli and Perrimon, 2006</xref>), <italic>esg-Gal4/UAS-GFP;TubGal80</italic><sup><italic>ts</italic></sup> (<xref ref-type="bibr" rid="bib30">Micchelli and Perrimon, 2006</xref>), <italic>Myo1A-Gal4/UAS-GFP;TubGal80</italic><sup><italic>ts</italic></sup> (<xref ref-type="bibr" rid="bib30">Micchelli and Perrimon, 2006</xref>)<italic>, w;esgGal4 tubGal80</italic><sup><italic>ts</italic></sup> <italic>UAS-GFP; UAS-flp Act>CD2>Gal4</italic> (<italic>esg</italic><sup><italic>ts</italic></sup><italic>F/O,</italic> a gift from Dr Huaqi Jiang), <italic>esg</italic><sup><italic>ts</italic></sup><italic>Su(H)Z</italic>, <italic>brm</italic><sup><italic>2</italic></sup> (Bloomington 3619), <italic>FRT80 brm</italic><sup><italic>2</italic></sup>, <italic>sd</italic> hypomorphic allele (<italic>sd</italic><sup><italic>1</italic></sup>) (<xref ref-type="bibr" rid="bib60">Zhang et al., 2008</xref>), <italic>osa</italic><sup><italic>2</italic></sup> (a gift from Professor Asgar Klebes)<italic>, UAS-Brm RNAi</italic> (VDRC 37720, VDRC 37721, and Bloomington 31712), <italic>UAS-Bap60 RNAi</italic> (NIG4303R-1), <italic>UAS-Mor RNAi</italic> (VDRC 6969), <italic>UAS-Osa RNAi</italic> (VDRC 7810), <italic>hhBanGFP</italic>, <italic>UAS-Bap60</italic>, <italic>UAS-Mor</italic>, <italic>UAS-Osa</italic>, <italic>UAS-Brm-N</italic>, <italic>and UAS-Brm-C</italic>. Bap60, Mor, Osa were cloned from the <italic>Drosophila</italic> cDNA. <italic>UAS-Flag-Brm, UAS-Flag-Brm</italic><sup><italic>K804R</italic></sup><italic>, UAS-Flag-Brm</italic><sup><italic>D718A</italic></sup><italic>,</italic> Brm point mutations and deletions were generated by PCR-based site directed mutagenesis. These cDNA fragments were cloned into the <italic>pUAST</italic> vector. A <italic>pUAST</italic> vector with <italic>attB</italic> sequence inserted upstream of the UAS-binding sites was used to make <italic>pUAST-attB-Brm</italic> and Brm mutants constructs. All plasmids were verified by DNA sequencing. Transgenic flies carrying these constructs were generated.</p></sec><sec id="s4-2"><title>MARCM clone analysis</title><p>Mutant clones were made using the MARCM system (<xref ref-type="bibr" rid="bib25">Lee and Luo, 2001</xref>). Genotypes for making <italic>brm</italic> mutant clone: <italic>hsflp, tub-Gal4, UAS-GFPnls; tubGal80 FRT80B/FTR80B brm</italic><sup><italic>2</italic></sup>. <italic>brm</italic> mutant clone expressing Yki transgene: <italic>hsflp, tub-Gal4, UAS-GFPnls;UAS-Yki/+;tubGal80 FRT80B/FTR80B brm</italic><sup><italic>2</italic></sup>. Hpo mutant clone expressing Brm transgene: <italic>yw UAS-GFP hsflp;FRT42D hpo</italic><sup><italic>BF33</italic></sup><italic>/FRT42D tub-Gal80; tubulin-Gal4/Brm</italic>. Hpo clone expressing Brm RNAi transgene: <italic>yw UAS-GFP hsflp; FRT42D hpo</italic><sup><italic>BF33</italic></sup><italic>/FRT42D tub-Gal80; tubulin-Gal4/UAS-Brm RNAi</italic>. Flies were cultured at 25°C. F1 adult flies with appropriate genotypes were subjected to heat shock at 37°C for 1 hr to induce clone at 5-day-old flies. Then, flies were raised at 25°C for 3 or 8 days before dissection. Clones of more than 10 midguts were scored in each group.</p></sec><sec id="s4-3"><title>Temperature-controlled expression</title><p>The experiment using <italic>esgGal4 UAS-GFP; tubGal80</italic><sup><italic>ts</italic></sup> was cultured under 18°C to restrict Gal4 activity. 3-day-old F1 adult flies with appropriate genotypes were then shifted to 29°C for a 7-day incubation to allow inactivation of <italic>Gal80</italic><sup><italic>ts</italic></sup> and expression of the UAS transgenes or RNAi. 20 female adults with correct genotypes were dissected and subjected to immunostaining. For intestinal stem cell lineage tracing experiment, we used the inducible lineage tracing <italic>esg 80</italic><sup><italic>ts</italic></sup> <italic>F/O</italic> system. 2- to 5-day-old F1 adult flies with correct genotypes cultured at 18°C were then shifted to 29°C to induce the expression of transgenes.</p></sec><sec id="s4-4"><title>DSS feeding experiments</title><p>Female adult flies (5/6-day-old) were used to perform DSS-treated feeding experiments. Flies were cultured in an empty vial containing a piece of 9 cm<sup>2</sup> chromatography paper wet with 3% dextran sulfate sodium (MP Biomedicals, Santa Ana, California, United States) in 5% glucose solution for 3 days at 25°C or 29°C.</p></sec><sec id="s4-5"><title>Cell culture, transfection and Western blotting</title><p>S2 cells were cultured in <italic>Drosophila</italic> Schneider’s Medium (Invitrogen, Carlsbad, California, United States) with 10% fetal bovine serum, 100 U/ml of penicillin, and 100 mg/ml of Streptomycin. Plasmid transfection was carried out using LipofectAMINE (Invitrogen) according to manufacturer’s instructions. A construct of ubiquitin-Gal4 was cotransfected with <italic>pUAST</italic> expression vectors for all transfection experiments. Immunoprecipitation and Western blot analyses were performed according to standard protocols as previously described (<xref ref-type="bibr" rid="bib21">Jin et al., 2012</xref>). Antibodies used were as follows: mouse anti-Myc (1:5000; Sigma, St. Louis, Missouri, United States), mouse anti-Flag (1:5000; Sigma), mouse anti-V5 (1:5000; Invitrogen), mouse anti-HA (Sigma), rabbit anti-Sd (produced by immunizing rabbits with the peptide of Sd amino acids 208–440), and rabbits anti-Brm (produced by immunizing rabbits with the peptide of Brm amino acids 505–775). The proteasome inhibitor MG132 (Sigma) was solubilized in DMSO and added to a final concentration of 50 μM for 6 hr. Z-VAD-FMK and caspase inhibitors (R&D systems, Minneapolis, Minnesota, United States) were added to a final concentration of 10 μM.</p></sec><sec id="s4-6"><title>Immunofluorescence staining</title><p>Immunostaining of intestine and S2 cells were carried out as described (<xref ref-type="bibr" rid="bib42">Ren et al., 2010</xref>; <xref ref-type="bibr" rid="bib21">Jin et al., 2012</xref>). Primary antibodies used in this study include mouse anti-Delta (DSHB), mouse anti-Prospero (DSHB, Iowa City, Iowa, United States), rat anti-Ci (1:500), rabbit anti-PH3 (Millipore), rabbit anti-Yki (1:50, produced by immunizing rabbits with the peptide of Yki amino acids 180–418), rabbits anti-Brm (this study), mouse anti-Flag (1:500; Sigma), mouse anti-HA (1:500; Sigma), mouse anti-GFP (1:1000; Santa Cruz, Dallas, Texas, United States), rabbit anti-PDM-1 (1:2000; a gift from Xiaohang Yang, Zhejiang University, Hang Zhou, China).</p></sec><sec id="s4-7"><title>Microscopy and data analysis</title><p>Fluorescent microscopy was performed on a Leica LAS SP5 confocal microscope; confocal images were obtained using the Leica AF Lite system. Images were processed in Photoshop CS. The GFP<sup>+</sup> EC cells in <italic>esg80</italic><sup><italic>ts</italic></sup><italic>F/O</italic> gut were counted in one field of view of the posterior midgut near the Malpighian tubules using a 40× objective.</p></sec><sec id="s4-8"><title>BrdU incorporation</title><p>Adult flies of 7–9 days on food containing BrdU (200 μg/ml in PBS) were mixed into the upper layer and dissected 3 days later. The guts were treated with DNase I for 30 min at 37°C.</p></sec><sec id="s4-9"><title>MS samples preparation</title><p>Thirty 10-cm dishes of S2 cells were collected and washed twice with cold PBS. The cells were equally divided into two samples and lysed in 2.5 ml lysis buffer (Tris HCL pH 8.0, 50 mM, NaCl 100 mM, NaF 10 mM, Na<sub>3</sub>VO<sub>4</sub> 1 mM, EDTA 1.5 mM, NP-40 1%, glycerol 10% supplied with protease inhibitor cocktail [Sigma]), and centrifuged at 14,000 rpm for 15 min. Supernatant was transferred to and mixed with 150 μl protein A/G beads (Santa Cruz Biotechnology) at 4°C for 1 hr on a rolling mixer. Then, the mixture was centrifuged at 14,000 rpm for 1 min. Cell supernatant was transferred to a new tube and stored at 4°C.</p><p>0.5 mg of Brm antibody was mixed with 0.25 ml of wet beads (use the appropriate antibody/protein A/G combination) in room temperature for 1 hr on a rolling mixer, using serum mixed with beads as control. The beads were washed using 10 vol of Borate Buffer (Sodium Borate pH 9.0, 0.2 mM) twice. 10 μl aliquot of beads was stored on ice (sample 1). The rest of the beads were mixed with solid Dimethyl Pimelimidate Dihydrochloride (final concentration is 20 mM) on the rolling mixer for 30 min at room temperature. Another 10 μl aliquot of beads was collected as sample 2. The rest of the beads were washed twice by equal volume of Ethanolamine after discarding the supernatant. Equal volume of Ethanolamine was added and incubated at room temperature for 2 hr on the rolling mixer, then washed by PBS twice and mixed with the cell supernatant at 4°C for 1 hr on a rolling mixer respectively. The mixture was washed by lysis buffer for three times and stored at −20°C for left experiment.</p><p>The coupling of antibody to beads was checked by analyzing the sample 1 and 2 on a SDS gel. After checking the coupling efficiency, for Sd MS, two total samples (serum control and Sd IP sample) were sent for MS directly. For Yki MS, we ran an SDS-PAGE to separate the proteins of Yki IP sample, and then specific bands, which were absent in IgG IP control, were selected for further MS analysis.</p></sec><sec id="s4-10"><title>In-gel digestion</title><p>For MS fingerprinting, the gel slices were cut out of the preparative Coomassie blue-stained gels, destained with100 mmol/l NH4HCO<sub>3</sub>/30% ACN, and then dried completely by centrifugal lyophilization. The dried gel slices were rehydrated with a total of 25 ng of sequencing grade, modified trypsin (Promega, Madison, Wisconsin, United States) in 100 mmol/l ammonium bicarbonate at 4°C for 2 hr. After 20 ml of 50 mmol/l NH<sub>4</sub>HCO<sub>3</sub>, pH 8.3 was supplied, the gel slices were incubated at 37°C for 20 hr. The digest buffer was removed and saved. The gel pieces were then extracted with 200 ml of 60% ACN/0.1%TFA for 15 min with sonication, and the supernatant was removed. The extraction was repeated twice. The three extracts plus the first saved digest buffer were pooled and dried completely by centrifugal lyophilization. This in-gel digestion method was mainly performed according to the method described previously (<xref ref-type="bibr" rid="bib58">Yu et al., 2000</xref>; <xref ref-type="bibr" rid="bib27">Li et al., 2005</xref>) with some modifications as described above.</p></sec><sec id="s4-11"><title>MS and protein identification</title><p>Peptide mixtures of each gel slice were redissolved in 0.1%TFA, then desalted and concentrated using Stage Tips as reported (<xref ref-type="bibr" rid="bib39">Rappsilber et al., 2007</xref>). Peptide solution was measured using a LTQ Deca XP system (Thermo Finnigan, San Jose, California, United States). HPLC separation was performed with a capillary LC pump. The flow rate of the pump was at 250 μl/min and was about 2 μl/min after split. The mobile phases used for reverse phase were A: 0.1% formic acid in water, pH 3.0, B: 0.1% formic acid in ACN. Peptides were eluted using a 2–35%, 35–90% stepped linear gradient of solvent B in 60 min, 90 min following 90% solvent B in 10 min, and 2% solvent B in 30 min for balance. An ESIIT mass spectrometer (LTQ Deca XP; Thermo Finnigan) was used for peptide detection. The positive ion mode was employed, and the spray voltage was set at 3.4 KV. The spray temperature was set at 200°C for peptides. Collision energy is automatically set by the LTQ Deca XP system. After acquisition of a full scan mass spectrum, 10 MS/MS scans were acquired for the next 10 most intense ions using dynamic exclusion. Peptides and proteins were identified using Turbo Sequest software (Thermo Finnigan), which uses the MS and MS/MS spectra of peptide ions to search against the publicly available Uniport fly database (Version 2011-05-26). The protein identification criteria that we used were based on Delta (CN ≥ 0.1) and Xcorr (one charge ≥ 1.9, two charges ≥ 2.2, three charges ≥ 3.75).</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We thank Dr Yingzi Yang, Dr Wei Du, and Dr Dangsheng Li for helpful comments on an earlier version of the manuscript. We also thank Jin Jiang, Dahua Chen, the Bloomington and Vienna Stock Centres, the DSHB, and DGRC for fly stocks.</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>The authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>YJ, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con2"><p>JX, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con3"><p>M-XY, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con4"><p>YL, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con5"><p>LH, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con6"><p>PL, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con7"><p>PZ, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con8"><p>ZY, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con9"><p>MSH, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con10"><p>HJ, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con11"><p>YZ, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con12"><p>LZ, Conception and design, Analysis and interpretation of data, Drafting or revising the 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content-type="section"><contrib contrib-type="editor"><name><surname>Vijay Raghavan</surname><given-names>K</given-names></name><role>Reviewing editor</role><aff><institution>National Centre for Biological Sciences, Tata Institute of Fundamental Research</institution>, <country>India</country></aff></contrib></contrib-group></front-stub><body><boxed-text><p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elife.elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “Brahma is essential for <italic>Drosophila</italic> intestinal stem cell proliferation and regulated by Hippo signaling” for consideration at eLife. While your article has been favorably peer reviewed by a Senior editor (K VijayRaghavan) and another reviewer, it will be essential for you to respond in full to the comments below as they point to substantive requirements for examining a resubmission.</p><p>The Senior editor and the other reviewer discussed their comments before we reached this decision, and the Senior editor has assembled the following comments to help you prepare a revised submission.</p><p>Zhang et al. set out to show that “Brahma is essential for <italic>Drosophila</italic> intestinal stem cell proliferation and regulated by Hippo signaling.” There are many features of this paper that make it interesting, suggesting novel mechanisms underlying ISC proliferation. Aspects of this study are thorough with some of the major conclusions well supported by the data. Yet, in other parts, it falls short. A complete resubmission with substantial, robust, and (reasonably rapidly doable) additional experiments could make for an acceptable new submission, which will be examined to see if it retains novelty and whether the hypotheses stand up after examination of results from the experiments suggested.</p><p>The principal (potential) novelty and scientific importance of the current study come from the demonstration of the role of Brahma in intestinal stem cell (ISC) regulation and of mechanisms by which its action is regulated. SWI/SNF and Brahma's role has been studied in several other contexts including a variety of stem cell ones. The added value of this study will be if it shows how Brahma mechanistically functions downstream of specific signaling pathways in the ISC context.</p><p>The authors start by examining the role of Brm in ISC proliferation. They do this by using an Brm-RNAi under the control of a <italic>escargot-Gal4</italic> driver, conditionally expressed in the adult using a <italic>Gal80</italic><sup><italic>ts</italic></sup> system (the temperature shift timings should be clearly stated in the results and better detailed in the Materials and methods). The RNAi knockdown is stated in the text as being confirmed by antibody staining, but the relevant figure shows that the RNAi works in the imaginal disc: how do we know that the <italic>escargot-Gal4</italic> is as effective in the adult as the <italic>hh-Gal4</italic> is in the third instar disc? The RNAi phenotype is also averred to be similar to a dominant-negative phenotype: the figures presented do suggest a similarity, though how deep this is not evident from what is presented. The reduction of ISC cells is seen better in the MARCM experiment and this suggests that the RNAi could be effective as assumed. In sum, the conclusion of a role for Brm in ISC proliferation would have been stronger if an independent ISC antibody marker were used to verify the RNAi result. This important point needs to be addressed. As it stands, the principal assertion of the first figure is weak, thereby weakening the rest that follows.</p><p>The authors next go on to examine the role of Brm in damage induced midgut regeneration. A couple of points here are puzzling. First, the <italic>Gal80</italic><sup><italic>ts</italic></sup> does not seem to be used here. Presumably then, the RNAi will be active throughout development. Does this not alter the possibilities for interpreting the data? Figure 1O and <xref ref-type="fig" rid="fig1">Figure 1B</xref> do not look similar. Can the authors address this? Could it not be that the ISCs are very few (as shown earlier) and that the absence of proliferation of these few ISC upon DSS induction is due to an earlier role for Brm rather than an additional later role on damage induced midgut regeneration?</p><p>Next, the authors examine if Brm is required for EC differentiation. They are led to this by the determination that in <italic>brm</italic><sup><italic>2</italic></sup> mutant clones they see “that <italic>brm</italic><sup><italic>2</italic></sup> clones only contained one small nuclear cell (Figure 1G–G').” Brm is expressed at low levels in ECs. The lineage trace method is not well explained anywhere in the text. Presumably they imply that this method allows the removal of even this low-level of Brm in early-ECs (<xref ref-type="bibr" rid="bib35">Ohlstein and Spradling, 2006</xref>) and they then examine the effect on EC differentiation. This point needs to be clarified and clearly demonstrated. If, on the other hand (and this seems to be the case), they suggest that the ISC that are present (without Brm) do not proliferate and therefore there are fewer ECs as seen by the absence of PDM-1, then this suggests a role for Brm in ISCs and not necessarily in EC differentiation too. A clean way to examine the role of Brm in EC differentiation will be to down-regulate Brm specifically in early-ECs but not in ISCs. The authors could examine if this is feasible using conditional <italic>Gal4</italic> drivers.</p><p>The authors then go on to examine the interaction of Brm with the Yki-Sd complex. This is done in S2 cells using pull-down of endogenously expressed Yki or Sd proteins followed by MS. They also use over-expressed tagged proteins to show co-immunoprecipitation. This section, with respect to the main theme of the paper, raises concern. So far, we have epistasis showing that Brm is downstream of Yki-Sd. Now we are shown physical interaction in another very different context (S2) cells (similar results have also been recently published by the Ken Irvine Lab). These S2 results are also interpreted quantitatively and this interpretation transferred to an in vivo context: it is not clear how one can do this, coming as it does from a rather artificial and experimentally highly-manipulated cell-culture context. If the authors do not show in vivo interaction by high-resolution imaging methods they are not adding anything too much to what we have already from the Irvine lab paper. Just reporting this as an independent conformation of the Irvine lab results is confirmatory, but that can be stated in a less elaborate way. Similarly, the genetic interactions of Brm with Sd are very interesting from a genetic perspective, but other that hinting tantalizingly to a similar interaction in ISCs we do not know if this wing-blade interaction applies in this context. Indeed does the mild phenotype from Sd over-expression not suggest that Sd may not have a major role in ISC differentiation, although Yki does?</p><p>Again, the fact that the EGFR gain- and Notch-RNAi phenotypes are not altered by Brm loss of function does not necessarily mean that the EGFR and Notch pathways function independent of Brm. Several more gain and loss of function studies and colocalization (or otherwise) of effectors will be needed before any conclusion is reached. Indeed, by bringing in this section too the authors do not help themselves. Leaving it out will not change the main point they are trying to make.</p><p>Finally, the result that the Hippo-kinase activity regulates a caspase, which cleaves Brm and that the Cleavage resistant Brm illuminates a regulatory role for Brm is very interesting. Indeed this is the most valuable part of the paper. Unfortunately, it is not shown as being substantively related to the ISC context. This is a fundamental gap that needs to be filled. The authors need further evidence to address the significance of the Brm cleavage during ISC proliferation. The activity of Brm N and C terminus was mentioned as “data not shown”. It will be important to show them clearly and interpret them. The activity, in ISCs, of both cleaved products of Brm should be analyzed in wild-type, Brm null and Yki over-expression backgrounds. The Hippo dependence of cleavage in ISCs also needs demonstration. As presented, while these last sets of results (on Brm cleavage) are quite interesting from a Brm regulation viewpoint, they are not fundamental to the claims the paper starts out to make as they are not substantively demonstrated in ISCs.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.00999.021</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>The authors start by examining the role of Brm in ISC proliferation. They do this by using an Brm-RNAi under the control of a</italic> escargot-Gal4 <italic>driver, conditionally expressed in the adult using a</italic> Gal80<sup>ts</sup> <italic>system (The temperature shift timings should be clearly stated in the Results and better detailed in the Materials and methods). The RNAi knockdown is stated in the text as being confirmed by antibody staining, but the relevant figure shows that the RNAi works in the imaginal disc: how do we know that the</italic> escargot<italic>-</italic>GaL4 <italic>is as effective in the adult as the</italic> hh-Gal4 <italic>is in the third instar disc? The RNAi phenotype is also averred to be similar to a dominant-negative phenotype: The figures presented do suggest a similarity, though how deep this is not evident from what is presented. The reduction of ISC cells is seen better in the MARCM experiment and this suggests that the RNAi could be effective as assumed. In sum, the conclusion of a role for Brm in ISC proliferation would have been stronger if an independent ISC antibody marker were used to verify the RNAi result. This important point needs to be addressed. As it stands, the principal assertion of the first figure is weak, thereby weakening the rest that follows</italic>.</p><p>We now added a description of the <italic>Gal80</italic><sup><italic>ts</italic></sup> system in the main text and a detailed experimental protocol in the Materials and methods section. To clarify the point that the Brm RNAi works efficiently in the ISCs/EBs, we stained the endogenous Brm protein in the control wild type <italic>esg80</italic><sup><italic>ts</italic></sup> gut and <italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Brm</italic> RNAi guts (<xref ref-type="fig" rid="fig1s1">Figure 1–figure supplement 1 D-E”’</xref>). Compared with the control (<xref ref-type="fig" rid="fig1s1">Figure 1–figure supplement 1 D-D”’</xref>), Brm levels in the ISCs/EBs were efficiently reduced by Brm RNAi (<xref ref-type="fig" rid="fig1s1">Figure 1–figure supplement 1 E-E”’</xref>). Of note, the weak signal of Brm in <xref ref-type="fig" rid="fig1s1">Figure 1–figure supplement 1E’</xref> is from the large nuclear ECs. Brm dominant-negative form K804R does not possess its ATPase activity, yet does not affect the complex assembly. We predict that it will have a weaker function than Brm RNAi. To verify the RNAi results, we had used Delta (Dl), which is a unique ISC marker, to mark ISC in <italic>esg80</italic><sup><italic>ts</italic></sup><italic>-Brm</italic> RNAi experiments (<xref ref-type="fig" rid="fig5">Figure 5A’, B’</xref>). Brm RNAi expression decreased the Dl<sup>+</sup> cell number in <xref ref-type="fig" rid="fig5">Figure 5B</xref>’.</p><p><italic>The authors next go on to examine the role of Brm in damage induced midgut regeneration. A couple of points here are puzzling. First, the</italic> Gal80<sup>ts</sup> <italic>does not seem to be used here. Presumably then, the RNAi will be active throughout development. Does this not alter the possibilities for interpreting the data?</italic> <xref ref-type="fig" rid="fig1"><italic>Figure 1O</italic></xref> <italic>and</italic> <xref ref-type="fig" rid="fig1"><italic>Figure 1B</italic></xref> <italic>do not look similar. Can the authors address this? Could it not be that the ISCs are very few (as shown earlier) and that the absence of proliferation of these few ISC upon DSS induction is due to an earlier role for Brm rather than an additional later role on damage induced midgut regeneration</italic>?</p><p>In fact, we used both <italic>esgGal4</italic> and <italic>esg80</italic><sup><italic>ts</italic></sup> to examine the role of Brm in damage induced midgut regeneration in the experiment. Both gave similar results. In the previous manuscript, we showed the <italic>esgGal4</italic> data; to avoid misinterpretation of developmental difference, we now replaced the <italic>esgGal4</italic> data with the <italic>esg80</italic><sup><italic>ts</italic></sup> data (<xref ref-type="fig" rid="fig3">Figure 3A-F’</xref>). The old <xref ref-type="fig" rid="fig1">Figure 1O</xref> and <xref ref-type="fig" rid="fig1">Figure 1B</xref> do not look similar due to: 1) the difference in GFP protein distribution for <italic>esgGal4</italic>-GFP (in both nucleus and cytoplasm) and <italic>esg80</italic><sup><italic>ts</italic></sup>-GFP (mainly in the cytoplasm) and 2) different culture conditions and food conditions of these two experiments. The flies used for <xref ref-type="fig" rid="fig1">Figure 1B</xref> were fed with normal food, but the flies used for DSS-treated experiments (<xref ref-type="fig" rid="fig3">Figure 3C-D’</xref>) were fed with glucose and water in order to make the final concentration of DSS steady at 3%. During the experimental operation, we noticed that the guts expressing Brm RNAi from the DSS-treated experiments (<xref ref-type="fig" rid="fig3">Figure 3C-C’</xref>) were more fragile and much thinner than the guts from the experiments of <xref ref-type="fig" rid="fig1">Figure 1B-B’</xref> using the same esg80<sup>ts</sup> driver. Therefore, based on these results, we believe that the DSS-induced midgut regeneration can be blocked by the loss of Brm, but not due to the earlier role of Brm knockdown in development.</p><p><italic>Next, the authors examine if Brm is required for EC differentiation. They are led to this by the determination that in</italic> brm<sup>2</sup> <italic>mutant clones they see “that</italic> brm<sup>2</sup> <italic>clones only contained one small nuclear cell (</italic><xref ref-type="fig" rid="fig1"><italic>Figure 1G-G'</italic></xref><italic>).” Brm is expressed at low levels in ECs. The lineage trace method is not well explained anywhere in the text. Presumably they imply that this method allows the removal of even this low-level of Brm in early-ECs (</italic><xref ref-type="bibr" rid="bib35">Ohlstein and Spradling, 2006</xref><italic>) and they then examine the effect on EC differentiation. This point needs to be clarified and clearly demonstrated. If, on the other hand (and this seems to be the case) they suggest that the ISC that are present (without Brm) do not proliferate and therefore there are fewer ECs as seen by the absence of PDM-1, then this suggests a role for Brm in ISCs and not necessarily in EC differentiation too. A clean way to examine the role of Brm in EC differentiation will be to down-regulate Brm specifically in early-ECs but not in ISCs. The authors could examine if this is feasible using conditional</italic> Gal4 <italic>drivers</italic>.</p><p>As suggested, we gave a description of the lineage trace method in the revised manuscript. To better understand the role of Brm in EC differentiation, we tried to find probable drivers for lineage tracing of early-ECs, but unfortunately, no suitable early-ECs specific conditional <italic>Gal4</italic> drivers is reported yet (Myo1A driver is also expressed in mature ECs (Jiang and Edgar, 2009)). Instead, we analyzed the function of Brm in ISCs division by detecting the Su(H)lacZ, a marker of EB cells. We found that Brm RNAi did not block EBs formation, and added these data to <xref ref-type="fig" rid="fig1s1">Figure 1–figure supplement 1F-G”</xref> with a description in the revised manuscript. Together with our esgF/O lineage tracing data, we speculated that, if Brm do not affect the differentiation from EBs to ECs, EBs expressing esgF/O-Brm RNAi will finally differentiate into ECs like control group (<xref ref-type="fig" rid="fig2">Figure 2F-F’</xref>, <xref ref-type="fig" rid="fig2s1">Figure 2–figure supplement 1A-A’</xref>). However, we observed very few ECs in the Brm RNAi guts (<xref ref-type="fig" rid="fig2">Figure 2E-E’, H-H’</xref> and <xref ref-type="fig" rid="fig2s1">Figure 2–figure supplement 1C-C’</xref>) even though relatively a large number of EB cells was observed (<xref ref-type="fig" rid="fig1s1">Figure 1–figure supplement 1F-G”</xref>), indicating that the differentiation from EBs to ECs was affected. The other evidence that supports the conclusion is that Brm overexpression promotes precocious differentiation of ECs at day 2 after heat shock (<xref ref-type="fig" rid="fig2">Figure 2D-D’</xref> compared with <xref ref-type="fig" rid="fig2">Figure 2C-C’</xref>), while the GFP<sup>+</sup> cells in control group do not form EC at this time point.</p><p><italic>The authors then go on to examine the interaction of Brm with the Yki-Sd complex. This is done in S2 cells using pull-down of endogenously expressed Yki or Sd proteins followed by MS. They also use over-expressed tagged proteins to show co-immunoprecipitation. This section, with respect to the main theme of the paper, raises concern. So far, we have epistasis showing that Brm is downstream of Yki-Sd. Now we are shown physical interaction in another very different context (S2) cells (similar results have also been recently published by the Ken Irvine Lab). These S2 results are also interpreted quantitatively and this interpretation transferred to an in vivo context: it is not clear how one can do this, coming as it does from a rather artificial and experimentally highly-manipulated cell-culture context. If the authors do not show in vivo interaction by high-resolution imaging methods they are not adding anything too much to what we have already from the Irvine lab paper. Just reporting this as an independent conformation of the Irvine lab results is confirmatory, but that can be stated in a less elaborate way. Similarly, the genetic interactions of Brm with Sd are very interesting from a genetic perspective, but other that hinting tantalizingly to a similar interaction in ISCs we do not know if this wing-blade interaction applies in this context. Indeed does the mild phenotype from Sd over-expression not suggest that Sd may not have a major role in ISC differentiation, although Yki does</italic>?</p><p>We agree with reviewers’ opinions. As suggested, we weakened this part in the revised manuscript. The function of Brm on cell proliferation may be a general requirement even though the regulation is cell type specific. In tissues other than guts, such as wing discs (<xref ref-type="bibr" rid="bib34">Oh et al., 2013</xref>), knockdown of Brm inhibits Yki-induced proliferation, suggesting that Brm is generally required for Yki activity. Knockdown of Yki in ISCs does not lead to any obvious phenotypes even though Yki protein is enriched in ISCs (<xref ref-type="bibr" rid="bib23">Karpowicz et al., 2010</xref>); knockdown of Yki in tissues, like wing discs, blocks the growth of the cells (<xref ref-type="bibr" rid="bib15">Huang et al., 2005</xref>). This piece of evidence suggests that distinct regulatory mechanisms exist in the ISCs to control Yki’s activity.</p><p>The genetic interactions of <italic>brm</italic> with <italic>sd</italic> in wings together with their physical interactions also suggested that Sd activity is generally regulated by Brm, which is novel. In midguts, when we generated <italic>sd</italic> null MARCM clones in guts, we found that, after 14 days of heat shock, the size of <italic>sd</italic> mutant clones were larger than the wild type control clones, suggesting that loss of Sd promotes proliferation of the gut cells in normal condition, a phenomenon not similar to that by Yki knockdown. When we overexpressed Yki in the <italic>sd</italic> MARCM clones, the clone size is much smaller than the Yki overexpressed clones. These data suggested that, Sd is involved in hyper-active Yki induced proliferation of ISCs, but it is also a default transcriptional repressor in normal condition, which is consistent with the findings recently published by DJ Pan’s lab (Koontz et al., 2013) and by our lab (Guo et al., 2013). We observed that SdGA (an active form of Sd) over-expression in ISCs/EBs had a mild phenotype (mono-layer) than Yki over-expression, suggesting that Yki plays a more extensive role in ISC proliferation through cooperation with other factors except Sd.</p><p><italic>Again, the fact that the EGFR gain- and Notch-RNAi phenotypes are not altered by Brm loss of function does not necessarily mean that the EGFR and Notch pathways function independent of Brm. Several more gain and loss of function studies and colocalization (or otherwise) of effectors will be needed before any conclusion is reached. Indeed, by bringing in this section too the authors do not help themselves. Leaving it out will not change the main point they are trying to make</italic>.</p><p>We agree with the reviewers’ opinions. To put less emphasis on this point, we have deleted this part.</p><p><italic>Finally, the result that the Hippo-kinase activity regulates a caspase, which cleaves Brm and that the Cleavage resistant Brm illuminates a regulatory role for Brm is very interesting. Indeed this is the most valuable part of the paper. Unfortunately, it is not shown as being substantively related to the ISC context. This is a fundamental gap that needs to be filled. The authors need further evidence to address the significance of the Brm cleavage during ISC proliferation. The activity of Brm N and C terminus was mentioned as “data not shown”. It will be important to show them clearly and interpret them. The activity, in ISCs, of both cleaved products of Brm should be analyzed in wild-type, Brm null and Yki over-expression backgrounds. The Hippo dependence of cleavage in ISCs also needs demonstration. As presented, while these last set of results (on Brm cleavage) are quite interesting from a Brm regulation viewpoint, they are not fundamental to the claims the paper starts out to make as they are not substantively demonstrated in ISCs</italic>.</p><p>We thank the reviewers for these suggestions. As suggested, we comprehensively analyzed the activity of the cleaved products of Brm in wild-type, Yki overexpression, and Brm null backgrounds, and added these data to <xref ref-type="fig" rid="fig7s2">Figure 7–figure supplement 2</xref>. In wild-type background, Brm-C induces ISC proliferation by increasing the number of ISCs/EBs and PH3<sup>+</sup> cells, but not as strong as Brm<sup>D718A</sup>, while Brm-N didn’t exhibit any obvious change (<xref ref-type="fig" rid="fig7s2">Figure 7–figure supplement 2A-E, 2K</xref>).</p><p>Similar results were also observed in Yki overexpression backgrounds. Compared with Yki overexpression in ISCs/EBs (<xref ref-type="fig" rid="fig7s2">Figure 7–figure supplement 2F</xref>), overexpressing wild type Brm or Brm<sup>D718A</sup> together with Yki further promoted the proliferation of ISCs and increased the PH3<sup>+</sup> cell number dramatically (<xref ref-type="fig" rid="fig7s2">Figure 7–figure supplement 2 G, 2H, 2K</xref>). Brm-N and Brm-C were able to promote the Yki induced ISCs proliferation mildly (<xref ref-type="fig" rid="fig7s2">Figure 7–figure supplement 2I, 2J, 2K</xref>).</p><p>We overexpressed Brm, Brm<sup>D718A</sup>, Brm-N and Brm-C in <italic>brm</italic> null MARCM clones. After induction for 3 or 10 days, all of them were able to partially rescue the growth defect of <italic>brm</italic> null clones in different degrees, and formed larger clones containing more cells than the <italic>brm</italic><sup><italic>2</italic></sup> clones. Brm<sup>D718A</sup> exhibited the strongest activity as shown by forming bigger clones (<xref ref-type="fig" rid="fig7s2">Figure 7–figure supplement 2P, 2L</xref>), and Brm cleavage products, Brm-N or C, only had weak rescue ability (<xref ref-type="fig" rid="fig7s2">Figure 7–figure supplement 2Q, 2R, 2L</xref>).</p><p>We also tried to check the cleavage event of Brm in ISCs and EBs in midgut. We dissected more than 100 guts each to detect the cleavage of endogenous Brm or the overexpressed Flag-Brm/Flag- Brm<sup>D718A</sup> in ISCs/EBs with or without Hpo co-expression by Western blot. Unfortunately, we failed to detect any Brm signal. It may be due to the fact that Brm protein levels are high in ISCs and EBs, yet the gut only contains a small number of ISCs and EBs, and most of the gut cells are ECs with low Brm protein levels.</p></body></sub-article></article> |