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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">02981</article-id><article-id pub-id-type="doi">10.7554/eLife.02981</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Human biology and medicine</subject></subj-group><subj-group subj-group-type="heading"><subject>Neuroscience</subject></subj-group></article-categories><title-group><article-title>Nuclear envelope protein MAN1 regulates clock through <italic>BMAL1</italic></article-title></title-group><contrib-group><contrib contrib-type="author" equal-contrib="yes" id="author-12921"><name><surname>Lin</surname><given-names>Shu-Ting</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" equal-contrib="yes" id="author-12891"><name><surname>Zhang</surname><given-names>Luoying</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" equal-contrib="yes" id="author-12890"><name><surname>Lin</surname><given-names>Xiaoyan</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="fn" rid="pa1">‡</xref><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-12892"><name><surname>Zhang</surname><given-names>Linda Chen</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-12893"><name><surname>Garcia</surname><given-names>Valentina Elizabeth</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-12894"><name><surname>Tsai</surname><given-names>Chen-Wei</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-1168"><name><surname>Ptáček</surname><given-names>Louis</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><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="con7"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-12720"><name><surname>Fu</surname><given-names>Ying-Hui</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="fn" rid="con8"/><xref ref-type="fn" rid="conf2"/></contrib><aff id="aff1"><institution content-type="dept">Department of Neurology</institution>, <institution>University of California, San Francisco</institution>, <addr-line><named-content content-type="city">San Francisco</named-content></addr-line>, <country>United States</country></aff><aff id="aff2"><institution>Howard Hughes Medical Institute, University of California, San Francisco</institution>, <addr-line><named-content content-type="city">San Francisco</named-content></addr-line>, <country>United States</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Griffith</surname><given-names>Leslie C</given-names></name><role>Reviewing editor</role><aff><institution>Brandeis University</institution>, <country>United States</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>ying-hui.fu@ucsf.edu</email></corresp><fn fn-type="con" id="equal-contrib"><label>†</label><p>These authors contributed equally to this work</p></fn><fn fn-type="present-address" id="pa1"><label>‡</label><p>Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, Bethesda, United States</p></fn></author-notes><pub-date date-type="pub" publication-format="electronic"><day>02</day><month>09</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e02981</elocation-id><history><date date-type="received"><day>01</day><month>04</month><year>2014</year></date><date date-type="accepted"><day>10</day><month>08</month><year>2014</year></date></history><permissions><copyright-statement>© 2014, Lin et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Lin et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">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="elife02981.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.02981.001</object-id><p>Circadian clocks serve as internal pacemakers that influence many basic homeostatic processes; consequently, the expression and function of their components are tightly regulated by intricate networks of feedback loops that fine-tune circadian processes. Our knowledge of these components and pathways is far from exhaustive. In recent decades, the nuclear envelope has emerged as a global gene regulatory machine, although its role in circadian regulation has not been explored. We report that transcription of the core clock component <italic>BMAL1</italic> is positively modulated by the inner nuclear membrane protein MAN1, which directly binds the <italic>BMAL1</italic> promoter and enhances its transcription. Our results establish a novel connection between the nuclear periphery and circadian rhythmicity, therefore bridging two global regulatory systems that modulate all aspects of bodily functions.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02981.001">http://dx.doi.org/10.7554/eLife.02981.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.02981.002</object-id><title>eLife digest</title><p>If rodents, or indeed humans, are kept in constant darkness for a number of days, they continue to show patterns of sleep and wakefulness that repeat roughly every 24 hr. This internal ‘circadian rhythm’ controls many aspects of animal physiology, including body temperature, blood pressure, and hormone levels. It does so by regulating the expression of key genes: this means that the activity of the proteins encoded by these genes also varies in accordance with the circadian rhythm.</p><p>A second mechanism used by the body to coordinate gene expression on a large scale entails making adjustments to the membrane that surrounds the cell nucleus. This ‘nuclear envelope’ consists mainly of lipids, but it also contains proteins that bind DNA. These proteins regulate gene expression by controlling how easy it is for other proteins that activate or repress genes to gain access to specific DNA sequences.</p><p>Lin et al. now reveal that these mechanisms work together. The first evidence for this was the discovery that the levels of three specific nuclear envelope proteins influence, and are influenced by, circadian rhythms. In particular, two of these proteins control the activity of the third, which is known as MAN1. This protein in turn triggers the expression of a gene called <italic>BMAL1</italic>, which is one of the small number of ‘clock genes’ that are responsible for generating the internal circadian rhythm.</p><p>As well as adding to our knowledge of circadian biology and the nuclear envelope, this study reveals a mechanism by which cells can orchestrate the expression of large numbers of genes. Such mechanisms allow a wide range of physiological and behavioral processes to be co-ordinated.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02981.002">http://dx.doi.org/10.7554/eLife.02981.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>nuclear envelope</kwd><kwd>circadian rhythm</kwd><kwd>MAN1</kwd><kwd>BMAL1</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd><italic>D. melanogaster</italic></kwd><kwd>mouse</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>HL059596</award-id><principal-award-recipient><name><surname>Ptáček</surname><given-names>Louis</given-names></name><name><surname>Fu</surname><given-names>Ying-Hui</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>GM079180</award-id><principal-award-recipient><name><surname>Ptáček</surname><given-names>Louis</given-names></name><name><surname>Fu</surname><given-names>Ying-Hui</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>NS072360</award-id><principal-award-recipient><name><surname>Ptáček</surname><given-names>Louis</given-names></name><name><surname>Fu</surname><given-names>Ying-Hui</given-names></name></principal-award-recipient></award-group><award-group id="par-4"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000011</institution-id><institution>Howard Hughes Medical Institute</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Ptáček</surname><given-names>Louis</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>A protein within the nuclear membrane, MAN1, controls the expression of the circadian clock gene, BMAL1, in an example of cross-talk between two major gene regulatory pathways.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>Most organisms, ranging from cyanobacteria to humans, are governed by their circadian rhythms: endogenous and self-sustained oscillations with a period of roughly 24 hr, which manifest in diverse metabolic, physiological, and behavioral processes (<xref ref-type="bibr" rid="bib40">Ueda et al., 2005</xref>). This internal pacemaker is charged with two important roles: to perpetuate its own rhythms and to regulate the expression of genes that are under circadian control. In mammals, this internal pacemaker consists of a complex network of transcriptional regulations, at the core of which is transcription activators BMAL1 (also known as ARNTL1 or MOP3) and CLOCK, which form heterodimers and regulate gene expression. Up to 15% of the organism's genome is regulated in a circadian manner (<xref ref-type="bibr" rid="bib30">Panda et al., 2002</xref>; <xref ref-type="bibr" rid="bib9">Emery and Reppert, 2004</xref>; <xref ref-type="bibr" rid="bib48">Zhang and Kay, 2010</xref>). Well-studied examples include the transcription repressors (PERIODs and CRYPTOCRHOMEs) that bind to CLOCK/BMAL1 and suppress their own transcription, thereby forming a feedback loop. Since the identification and cloning of the first mammalian clock gene, <italic>CLOCK</italic>, two decades ago (<xref ref-type="bibr" rid="bib43">Vitaterna et al., 1994</xref>), the field of chronobiology has uncovered many additional players that regulate circadian rhythms on a transcriptional and/or post-transcriptional level, and many more such candidates are currently being evaluated.</p><p>Recently, mutations in nuclear envelope (NE) proteins have been shown to cause a surprisingly broad range of inherited diseases, thus shedding light on roles played by the NE in global regulations at cellular and organismal levels (<xref ref-type="bibr" rid="bib28">Padiath et al., 2006</xref>; <xref ref-type="bibr" rid="bib8">Dauer and Worman, 2009</xref>). These diseases (often referred to as nuclear envelopathies or laminopathies) can impact muscle, nerve, fat metabolism, bone formation, and others. NE consists of outer and inner nuclear membranes (connected by nuclear pore complexes) and nuclear lamina. The inner nuclear membrane proteins (such as MAN1, LBR, LAP2, etc) include approximately 60 putative transmembrane proteins specifically retained in the inner nuclear membrane and most of them are poorly characterized (<xref ref-type="bibr" rid="bib36">Schirmer et al., 2005</xref>). In metazoan, nuclear lamina is a protein mesh-like structure composed of type V intermediate filament proteins lamins (including A, C, B1, B2, and B3 types) and sits primarily underneath the inner nuclear membrane (<xref ref-type="bibr" rid="bib49">Zwerger and Medalia, 2013</xref>). The idea of nuclear envelope components as transcription regulators in mammals is relatively new, conceived from the observation that gene-rich chromosomes are generally located in more internal nuclear regions, whereas gene-poor chromosomes are relegated to the periphery (<xref ref-type="bibr" rid="bib38">Spector, 2003</xref>). Many NE components such as inner nuclear membrane proteins, nuclear lamina, and the nuclear pore complex, harbor DNA-binding domains that are involved in anchoring chromatin to the periphery (<xref ref-type="bibr" rid="bib41">Ulbert et al., 2006</xref>; <xref ref-type="bibr" rid="bib25">Mekhail and Moazed, 2010</xref>). Functional relevance of these positional distinctions became apparent as studies with yeast and flies revealed that the NE can sequester factors that affect gene transcription in both repressive and, surprisingly, activating manners (<xref ref-type="bibr" rid="bib3">Akhtar and Gasser, 2007</xref>). Although recent findings highlight the important functions of the nuclear periphery, its relationship with the circadian clock has not been probed. Given the increasing awareness of the global roles that these two systems play in myriad pathways, we set out to investigate the possibility that these seemingly separate pathways are connected and can work synergistically in regulating diverse functions.</p></sec><sec id="s2" sec-type="results"><title>Results</title><sec id="s2-1"><title>Nuclear envelope participates in circadian regulation</title><p>In order to investigate whether NE proteins are involved in circadian regulation, we began by focusing on lamin B1 since it has been shown to play a role in transcriptional regulation (<xref ref-type="bibr" rid="bib16">Hutchison, 2002</xref>; <xref ref-type="bibr" rid="bib37">Shevelyov et al., 2009</xref>). In vivo oscillation of lamin B1 (<italic>Lmnb1</italic>) expression patterns (both RNA and protein levels) were confirmed using mouse tissues from suprachiasmatic nuclei (SCN), kidney, and liver (<xref ref-type="fig" rid="fig1">Figure 1A,B</xref>). To test whether the level of lamin B1 affects the molecular clock, we examined the protein expression patterns for the core clock gene PERIOD2 (PER2) using <italic>Lmnb1</italic> heterozygous knock out (homozygosity is lethal) and <italic>LMNB1</italic> wild-type BAC transgenic mice (<xref ref-type="bibr" rid="bib42">Vergnes et al., 2004</xref>; <xref ref-type="bibr" rid="bib14">Heng et al., 2013</xref>). Oscillating PER2 expression patterns were phase delayed in <italic>Lmnb1</italic> heterozygous knock out mice and phase advanced in <italic>LMNB1</italic> BAC transgenic mice (overexpression) when compared to wild-type control mice (<xref ref-type="fig" rid="fig1">Figure 1C</xref>), suggesting that the level of lamin B1 can modulate circadian clock. However, neither <italic>Lmnb1</italic> heterozygous knock out mice nor <italic>LMNB1</italic> BAC transgenic mice demonstrated significant output behavioral change (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>). To expand the investigation, we chose to include two additional NE proteins that are known to associate with lamin B1, LBR, and MAN1. We found that LBR and MAN1 expression also oscillate, albeit mildly for MAN1 (<xref ref-type="fig" rid="fig1">Figure 1D</xref>).<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.02981.003</object-id><label>Figure 1.</label><caption><title>Lamin b1 regulates the circadian clock.</title><p>Expression levels of lamin b1 from SCN, kidney, and liver extracts in C57BL/6J mice (<bold>A</bold> and <bold>B</bold>). (<bold>A</bold>) mRNA levels of <italic>Lmnb1</italic> and <italic>Gapdh</italic> were assayed at indicated circadian times (CT, n = 4). (<bold>B</bold>) Representative immunoblots show the levels of LMNB1, GAPDH, and β-ACTIN. (<bold>C</bold>) Representative immunoblots show PER2 (with intensity values indicated at the bottom) and LMNB1 abundance in <italic>Lmnb1</italic><sup>+/Δ</sup>, wild-type and <italic>LMNB1</italic><sup>BAC</sup> liver extracts. (<bold>D</bold>) Expression patterns of LMNB1, LBR, and MAN1 in C57BL/6 mouse livers at indicated Zeitgeber times (ZT) (n = 3). Quantifications (top panels) of Western blots (bottom panel) were obtained by using GAPDH as a loading control. Data represent means ± SD.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02981.003">http://dx.doi.org/10.7554/eLife.02981.003</ext-link></p></caption><graphic xlink:href="elife02981f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02981.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>Mice with altered LMNB1 levels do not exhibit altered behavioral rhythms.</title><p>The period (<bold>A</bold>), activity level (<bold>B</bold>), and amplitude (<bold>C</bold>) of wheel-running rhythms under DD condition (n = 9–27, Student's <italic>t</italic> test, *p < 0.05). Error bars represent SEM.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02981.004">http://dx.doi.org/10.7554/eLife.02981.004</ext-link></p></caption><graphic xlink:href="elife02981fs001"/></fig></fig-group></p><p>To determine if these NE genes passively receive cues from the core clock apparatus or if their protein products also actively play a role in maintaining circadian rhythms, we altered their protein levels in human osteosarcoma U2OS cells that express a luciferase reporter gene under the control of mouse <italic>Bmal1</italic> promoter (<italic>Bmal1</italic>-Luc) and examined circadian period in cell culture (<xref ref-type="bibr" rid="bib45">Vollmers et al., 2008</xref>). siRNA-induced reduction of <italic>LMNB1</italic>, <italic>LBR</italic>, and <italic>MAN1</italic> in this cell-based system resulted in a longer circadian period (τ) (<xref ref-type="fig" rid="fig2">Figure 2A</xref>), whereas the over-expression of all three led to a shorter τ (<xref ref-type="fig" rid="fig2">Figure 2B</xref>). Cells transfected with <italic>LBR</italic>, <italic>LMNB1</italic>, and <italic>MAN1</italic> siRNA lengthened τ by 54–69 min (n ≥ 4, *p < 0.05), when compared with control siRNA (τ = 27.39 ± 0.22 hr, n = 8) (<xref ref-type="fig" rid="fig2">Figure 2C</xref>). On the other hand, overexpression of FLAG-tagged LBR, LMNB1, or MAN1 shortened τ by 25.8–37.8 min (n ≥ 4, *p < 0.05) compared to empty vector controls (τ = 27.7 ± 0.15, <xref ref-type="fig" rid="fig2">Figure 2D</xref>). These changes in τ together with the altered phase of <italic>Lmnb1</italic> heterozygous knock out mice and over-expressing mice suggest that these NE proteins participate in modulating circadian clock and therefore could impose significant impacts on downstream biological pathways.<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.02981.005</object-id><label>Figure 2.</label><caption><title>LBR, LMNB1, and MAN1 are necessary for normal circadian rhythms.</title><p>Two representative traces of real-time bioluminescence analyses are shown for each, and Western blot verification of down-regulation or over-expression is demonstrated in the inset images. (<bold>A</bold>) Period was lengthened when <italic>LBR</italic>, <italic>LMNB1</italic>, or <italic>MAN1</italic> was knocked down. (<bold>B</bold>) Over-expression of FLAG-tagged LBR (F-LBR), LMNB1 (F-LMNB1), or MAN1 (F-MAN1) shortened period compared to cells transfected with empty vector (ctrl). (<bold>C</bold> and <bold>D</bold>) Summary of period in (<bold>A</bold> and <bold>B</bold>). <italic>CRYPTOCRHOME2</italic> (<italic>CRY2</italic>) and <italic>PER2</italic> siRNA knockdowns served as positive controls. Data represent means ± SD.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02981.005">http://dx.doi.org/10.7554/eLife.02981.005</ext-link></p></caption><graphic xlink:href="elife02981f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02981.006</object-id><label>Figure 2—figure supplement 1.</label><caption><title>Over-expressing or knocking down nuclear envelope components alters circadian rhythms in flies.</title><p>(<bold>A</bold>) Normalized locomotor activity profiles of flies over-expressing <italic>MAN1</italic>, <italic>Lam</italic>, or <italic>LBR</italic> during LD for 1 day followed by 4 days of DD. (<bold>B</bold>) Normalized locomotor activity profiles of flies with <italic>MAN1</italic>, <italic>Lam</italic>, or <italic>LBR</italic> knocked down by RNAi. White box indicates light period, black box indicates dark period, and gray box indicates subjective light period. Error bars represent SEM (n = 13–76).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02981.006">http://dx.doi.org/10.7554/eLife.02981.006</ext-link></p></caption><graphic xlink:href="elife02981fs002"/></fig><fig id="fig2s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02981.007</object-id><label>Figure 2—figure supplement 2.</label><caption><title>The mRNA levels of nuclear envelope genes are reduced in the corresponding knockdown flies.</title><p>Plots of relative mRNA abundance for <italic>MAN1, Lam,</italic> and <italic>LBR</italic> from whole head extracts of <italic>tim</italic>GAL4/+;UAS<italic>dicer2</italic>/+, <italic>tim</italic>GAL4/UAS<italic>MAN1RNAi</italic>;UAS<italic>dicer2</italic>/+, <italic>tim</italic>GAL4/+;UAS<italic>dicer2</italic>/UAS<italic>LamRNAi</italic> and <italic>tim</italic>GAL4/UAS<italic>LBRRNAi</italic>;UAS<italic>dicer2</italic>/+ flies determined by qRT-PCR. Error bars represent SEM (n = 3–6). Significant differences indicated by asterisks (Student's <italic>t</italic> test, *p < 0.05, **p < 0.01, ***p < 0.001). The value of <italic>tim</italic>GAL4/+;UAS<italic>dicer2</italic>/+ for one experiment was set to 1.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02981.007">http://dx.doi.org/10.7554/eLife.02981.007</ext-link></p></caption><graphic xlink:href="elife02981fs003"/></fig></fig-group></p><p>The effects of NE proteins on <italic>Drosophila</italic> circadian clock were also examined. Consistent with the mammalian data, over-expressing d<italic>Lamin</italic> (d<italic>Lam</italic>) (<xref ref-type="bibr" rid="bib28">Padiath et al., 2006</xref>) in circadian neurons with <italic>cryptochrome</italic> (<italic>cry</italic>)GAL4-39 and <italic>cry</italic>GAL4-16 (<xref ref-type="bibr" rid="bib11">Emery et al., 2000</xref>) in vivo resulted in substantially shortened periods of behavioral rhythms in constant darkness compared to GAL4 controls (but not to UAS<italic>LMNB1</italic>/+, <xref ref-type="table" rid="tbl1">Table 1</xref>; <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1A</xref>). Knocking down d<italic>Lam</italic> in circadian neurons lengthened the period (<xref ref-type="table" rid="tbl2">Table 2</xref>; <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1B</xref>). On the other hand, over-expressing d<italic>MAN1</italic> and d<italic>LBR</italic> lengthened the period (<xref ref-type="table" rid="tbl1">Table 1</xref>; <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1A</xref>), while knocking down d<italic>MAN1</italic> also lengthened period (<xref ref-type="table" rid="tbl2">Table 2</xref>; <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1B</xref>). Besides altering the period, most of these manipulations reduced the amplitude of behavioral rhythms as indicated by the reduced power values. In addition, we have assessed the mRNA levels of d<italic>MAN1</italic>, d<italic>Lam</italic>, and d<italic>LBR</italic> to confirm knockdown (<xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2</xref>). Taken together, these results indicate that NE proteins also participate in the regulation of fly clock.<table-wrap id="tbl1" position="float"><object-id pub-id-type="doi">10.7554/eLife.02981.008</object-id><label>Table 1.</label><caption><p>Over-expressing NE genes alters the behavioral period in flies</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02981.008">http://dx.doi.org/10.7554/eLife.02981.008</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th>Genotype</th><th>Period (hr)</th><th>Power</th><th>Rhythmic%</th><th>N</th></tr></thead><tbody><tr><td>UAS<italic>MAN1</italic>/+</td><td>23.8 ± 0.0</td><td align="char" char="plusmn">90 ± 5</td><td align="char" char=".">96</td><td align="char" char=".">76</td></tr><tr><td><italic>cry</italic>GAL4-39/UAS<italic>MAN1</italic></td><td>26.4 ± 0.3<xref ref-type="table-fn" rid="tblfn1">*</xref>,<xref ref-type="table-fn" rid="tblfn2">†</xref></td><td align="char" char="plusmn">31 ± 6<xref ref-type="table-fn" rid="tblfn1">*</xref>,<xref ref-type="table-fn" rid="tblfn2">†</xref></td><td align="char" char=".">64</td><td align="char" char=".">28</td></tr><tr><td>UAS<italic>MAN1</italic>/+; <italic>cry</italic>GAL4-16/+</td><td>26.8 ± 0.2<xref ref-type="table-fn" rid="tblfn1">*</xref>,<xref ref-type="table-fn" rid="tblfn2">†</xref></td><td align="char" char="plusmn">40 ± 7<xref ref-type="table-fn" rid="tblfn1">*</xref>,<xref ref-type="table-fn" rid="tblfn2">†</xref></td><td align="char" char=".">66</td><td align="char" char=".">32</td></tr><tr><td>UAS<italic>Lam</italic>/+</td><td>23.9 ± 0.0</td><td align="char" char="plusmn">87 ± 6</td><td align="char" char=".">93</td><td align="char" char=".">55</td></tr><tr><td><italic>cry</italic>GAL4-39/UAS<italic>Lam</italic></td><td>23.8 ± 0.2<xref ref-type="table-fn" rid="tblfn2">†</xref></td><td align="char" char="plusmn">33 ± 7<xref ref-type="table-fn" rid="tblfn1">*</xref>,<xref ref-type="table-fn" rid="tblfn2">†</xref></td><td align="char" char=".">73</td><td align="char" char=".">22</td></tr><tr><td>UAS<italic>LMNB1</italic>/+; <italic>cry</italic>GAL4-16/+</td><td>24.4 ± 0.2<xref ref-type="table-fn" rid="tblfn2">†</xref></td><td align="char" char="plusmn">5 ± 2<xref ref-type="table-fn" rid="tblfn1">*</xref>,<xref ref-type="table-fn" rid="tblfn2">†</xref></td><td align="char" char=".">26</td><td align="char" char=".">31</td></tr><tr><td>UAS<italic>LBR</italic>/+</td><td>23.7 ± 0.0</td><td align="char" char="plusmn">59 ± 5</td><td align="char" char=".">79</td><td align="char" char=".">67</td></tr><tr><td><italic>cry</italic>GAL4-39/UAS<italic>LBR</italic></td><td>25.6 ± 0.1<xref ref-type="table-fn" rid="tblfn1">*</xref>,<xref ref-type="table-fn" rid="tblfn2">†</xref></td><td align="char" char="plusmn">44 ± 6<xref ref-type="table-fn" rid="tblfn2">†</xref></td><td align="char" char=".">79</td><td align="char" char=".">33</td></tr><tr><td>UAS<italic>LBR</italic>/+; <italic>cry</italic>GAL4-16/+</td><td>N/A</td><td align="char" char="plusmn">1 ± 0<xref ref-type="table-fn" rid="tblfn1">*</xref>,<xref ref-type="table-fn" rid="tblfn2">†</xref></td><td align="char" char=".">0</td><td align="char" char=".">32</td></tr><tr><td><italic>cry</italic>GAL4-39/+</td><td>24.8 ± 0.1</td><td align="char" char="plusmn">86 ± 5</td><td align="char" char=".">93</td><td align="char" char=".">57</td></tr><tr><td><italic>cry</italic>GAL4-16/+</td><td>25.6 ± 0.1</td><td align="char" char="plusmn">86 ± 5</td><td align="char" char=".">94</td><td align="char" char=".">64</td></tr></tbody></table><table-wrap-foot><fn id="tblfn1"><label>*</label><p>One-way ANOVA compared to UAS control lines, p < 0.001.</p></fn><fn id="tblfn2"><label>†</label><p>One-way ANOVA compared to GAL4 control lines, p < 0.001.</p></fn></table-wrap-foot></table-wrap><table-wrap id="tbl2" position="float"><object-id pub-id-type="doi">10.7554/eLife.02981.009</object-id><label>Table 2.</label><caption><p>Knocking-down NE genes lengthens the behavioral period in flies</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02981.009">http://dx.doi.org/10.7554/eLife.02981.009</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th>Genotype</th><th>Period (hr)</th><th>Power</th><th>%Rhythmic</th><th>N</th></tr></thead><tbody><tr><td>UAS<italic>MAN1RNAi</italic></td><td align="char" char="plusmn">24.4 ± 0.1</td><td align="char" char="plusmn">110 ± 10</td><td>100</td><td>14</td></tr><tr><td>UAS<italic>MAN1RNAi</italic>;<italic>cry</italic>GAL4-39/+; UAS<italic>dcr2</italic>/+</td><td align="char" char="plusmn">26.3 ± 0.3<xref ref-type="table-fn" rid="tblfn3">*</xref>,<xref ref-type="table-fn" rid="tblfn4">†</xref></td><td align="char" char="plusmn">29 ± 6<xref ref-type="table-fn" rid="tblfn3">*</xref>,<xref ref-type="table-fn" rid="tblfn4">†</xref></td><td>80</td><td>15</td></tr><tr><td>UAS<italic>MAN1RNAi</italic>; UAS<italic>dcr2</italic>/+;<italic>cry</italic>GAL4-16/+</td><td align="char" char="plusmn">27.9 ± 0.4<xref ref-type="table-fn" rid="tblfn3">*</xref>,<xref ref-type="table-fn" rid="tblfn4">†</xref></td><td align="char" char="plusmn">39 ± 8<xref ref-type="table-fn" rid="tblfn3">*</xref>,<xref ref-type="table-fn" rid="tblfn4">†</xref></td><td>69</td><td>16</td></tr><tr><td>UAS<italic>LamRNAi</italic>/+</td><td align="char" char="plusmn">23.6 ± 0.1</td><td align="char" char="plusmn">129 ± 12</td><td>100</td><td>16</td></tr><tr><td><italic>cry</italic>GAL4-39/+; UAS<italic>LamRNAi</italic>/UAS<italic>dcr2</italic></td><td align="char" char="plusmn">25.1 ± 0.4<xref ref-type="table-fn" rid="tblfn3">*</xref></td><td align="char" char="plusmn">26 ± 17<xref ref-type="table-fn" rid="tblfn3">*</xref>,<xref ref-type="table-fn" rid="tblfn4">†</xref></td><td>50</td><td>14</td></tr><tr><td>UAS<italic>LamRNAi</italic>/UAS<italic>dcr2</italic>; <italic>cry</italic>GAL4-16/+</td><td align="char" char="plusmn">26.8 ± 0.1<xref ref-type="table-fn" rid="tblfn3">*</xref>,<xref ref-type="table-fn" rid="tblfn4">†</xref></td><td align="char" char="plusmn">126 ± 17</td><td>100</td><td>13</td></tr><tr><td>UAS<italic>LBRRNAi</italic>/+</td><td align="char" char="plusmn">23.5 ± 0.0</td><td align="char" char="plusmn">112 ± 14</td><td>100</td><td>15</td></tr><tr><td><italic>cry</italic>GAL4-39/UAS<italic>LBRRNAi</italic>; UAS<italic>dcr2</italic>/+</td><td align="char" char="plusmn">24.9 ± 0.1</td><td align="char" char="plusmn">61 ± 12<xref ref-type="table-fn" rid="tblfn3">*</xref></td><td>80</td><td>15</td></tr><tr><td>UAS<italic>LBRRNAi</italic>/UAS<italic>dcr2</italic>; <italic>cry</italic>GAL4-16/+</td><td align="char" char="plusmn">24.6 ± 1.7</td><td align="char" char="plusmn">12 ± 4<xref ref-type="table-fn" rid="tblfn3">*</xref>,<xref ref-type="table-fn" rid="tblfn4">†</xref></td><td>44</td><td>16</td></tr><tr><td><italic>cry</italic>GAL4-39/+; UAS<italic>dcr2</italic>/+</td><td align="char" char="plusmn">24.9 ± 0.1</td><td align="char" char="plusmn">69 ± 12</td><td>88</td><td>16</td></tr><tr><td>UAS<italic>dcr2</italic>/+;<italic>cry</italic>GAL4-16/+</td><td align="char" char="plusmn">26.1 ± 0.1</td><td align="char" char="plusmn">87 ± 10</td><td>94</td><td>16</td></tr></tbody></table><table-wrap-foot><fn id="tblfn3"><label>*</label><p>One-way ANOVA compared to UAS<italic>RNAi</italic> control lines, p < 0.05.</p></fn><fn id="tblfn4"><label>†</label><p>One-way ANOVA compared to control lines with GAL4 and UAS<italic>dcr2</italic>, p < 0.05.</p></fn><fn><p><italic>dicer2</italic> (<italic>dcr2</italic>) is co-expressed to enhance the effects of RNAi.</p></fn></table-wrap-foot></table-wrap></p></sec><sec id="s2-2"><title>Lamin B1 and LBR likely act through MAN1</title><p>We next explored the relationship of <italic>LBR</italic>, <italic>LMNB1</italic>, and <italic>MAN1</italic> by examining mRNA and protein levels while knocking them down one at a time. Both <italic>LBR</italic> and <italic>LMNB1</italic> knockdown significantly decreased the transcript level of <italic>MAN1</italic> (by 15% and 40%, respectively) (<xref ref-type="fig" rid="fig3">Figure 3A</xref>). The effects of <italic>LBR</italic> or <italic>LMNB1</italic> knockdown on MAN1 expression are even more dramatic at the protein level, with 54% and 44% reductions, respectively (<xref ref-type="fig" rid="fig3">Figure 3B</xref>). Moreover, knockdown of <italic>LBR</italic> expression reduces the amount of LMNB1 protein by 32%, which is consistent with the observation that reduction of <italic>LBR</italic> expression in the fibroblasts of patients harboring a heterozygous <italic>LBR</italic> mutation results in the abolition of LMNB1 protein (<xref ref-type="bibr" rid="bib12">Gaudy-Marqueste et al., 2010</xref>), whereas a decrease in <italic>LMNB1</italic> does not significantly affect <italic>LBR</italic> expression. <italic>MAN1</italic> knockdown also does not change the expression of <italic>LBR</italic> and <italic>LMNB1</italic>, either at the mRNA or protein level (<xref ref-type="fig" rid="fig3">Figure 3</xref>). These results suggest that <italic>MAN1</italic> is modulated by LBR and LMNB1, and thus the effects of LBR and LMNB1 on the clock are at least partially through MAN1. Therefore, we further investigated the effects of MAN1 on the molecular clock.<fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.02981.010</object-id><label>Figure 3.</label><caption><title>Knocking down <italic>LBR</italic>/<italic>LMNB1</italic> reduces <italic>MAN1</italic> mRNA and protein levels but not vice versa.</title><p>Assessing mRNA (<bold>A</bold>) and protein (<bold>B</bold>) levels of <italic>LBR</italic>, <italic>LMNB1</italic>, and <italic>MAN1</italic> while knocking them down one at a time in U2OS cells via RNAi. (<bold>A</bold>) mRNA levels of <italic>LBR</italic>, <italic>LMNB1</italic>, and <italic>MAN1</italic> in each of the three knockdown conditions were quantified using qRT-PCR (n = 14, *p < 0.05). (<bold>B</bold>) MAN1 was significantly down-regulated when <italic>LBR</italic> or <italic>LMNB1</italic> was knocked down (n = 14 *p < 0.001). The error bars represent SEM (left panel). Representative immunoblots show the protein levels of LBR, LMNB1 and MAN1 (right panel).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02981.010">http://dx.doi.org/10.7554/eLife.02981.010</ext-link></p></caption><graphic xlink:href="elife02981f003"/></fig></p></sec><sec id="s2-3"><title>MAN1 regulates the clock by promoting <italic>BMAL1</italic> expression</title><p>A lengthened period due to decreased MAN1 may arise from altered regulation of core clock proteins and/or altered transcription of core clock genes. Either of these effects would result in disruptions of the stoichiometry and temporal control of the dynamics of the core circadian feedback loops. Given what is known regarding the role of NE proteins in transcriptional regulation, we tested whether reductions in MAN1 expression would affect the transcription of clock genes. We examined the circadian oscillation of core clock genes at the mRNA level and found that only <italic>BMAL1</italic> showed a clear difference wherein overall mRNA levels were down-regulated to half the levels of controls (<xref ref-type="fig" rid="fig4">Figure 4</xref>). Western blots also showed lower expression of BMAL1 when MAN1 was knocked down (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>). The non-oscillatory <italic>CLOCK</italic> showed no significant change of either transcript or protein levels (<xref ref-type="fig" rid="fig4">Figure 4</xref>, <xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>). The conserved mRNA levels of <italic>REV-ERBα and RORα</italic> in MAN1 knockdown cells suggest that the reduced <italic>BMAL1</italic> expression is not caused by altered transcriptional activation of <italic>REV-ERBα</italic>, a <italic>BMAL1</italic> repressor, or transcriptional repression of <italic>RORα</italic>, a <italic>BMAL1</italic> activator.<fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.02981.011</object-id><label>Figure 4.</label><caption><title>Knocking down <italic>MAN1</italic> reduces the levels of <italic>BMAL1</italic> mRNA.</title><p>Each graph shows cells transfected with <italic>MAN1</italic> siRNA vs ctrl siRNA. Time 0 indicates the moment that U2OS cells were treated with dexamethasone (100 nM). Data are presented as means ± SEM, n = 3.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02981.011">http://dx.doi.org/10.7554/eLife.02981.011</ext-link></p></caption><graphic xlink:href="elife02981f004"/></fig><fig id="fig4s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02981.012</object-id><label>Figure 4—figure supplement 1.</label><caption><title>Knocking down <italic>MAN1</italic> reduces BMAL1 protein levels.</title><p>(<bold>A</bold>) Time course evaluations of U2OS cells transfected with <italic>MAN1</italic> siRNA showed robust reduction of BMAL1 protein levels compared to ctrl siRNA. CLOCK levels were unaffected by <italic>MAN1</italic> abundance (n = 3). (<bold>B</bold>) The quantification results of (<bold>A</bold>) were graphed (n = 14). Error bars indicate SEM.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02981.012">http://dx.doi.org/10.7554/eLife.02981.012</ext-link></p></caption><graphic xlink:href="elife02981fs004"/></fig><fig id="fig4s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02981.013</object-id><label>Figure 4—figure supplement 2.</label><caption><title>Over-expressing <italic>Bmal1</italic> suppresses the period lengthening effect of <italic>MAN1</italic> knockdown.</title><p>Periods of <italic>Bmal1</italic>-Luc U2OS cells transfected with <italic>MAN1</italic> siRNA or control (ctrl) siRNA together with either empty vector or varying amounts of <italic>Bmal1</italic>. Error bars represent SEM (n = 2–4, Student's <italic>t</italic> test).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02981.013">http://dx.doi.org/10.7554/eLife.02981.013</ext-link></p></caption><graphic xlink:href="elife02981fs005"/></fig></fig-group></p><p>To confirm MAN1 regulates the clock by targeting <italic>BMAL1</italic>, we over-expressed <italic>Bmal1</italic> in <italic>MAN1</italic> knockdown U2OS <italic>Bmal1</italic>-Luc cells. Knocking down <italic>MAN1</italic> lengthened the period in control cells as described above (<xref ref-type="fig" rid="fig2">Figure 2</xref>), whereas cells over-expressing sufficient <italic>Bmal1</italic> did not demonstrate period lengthening compared to cells without <italic>MAN1</italic> knockdown (<xref ref-type="fig" rid="fig4s2">Figure 4—figure supplement 2</xref>), suggesting that the lengthened period caused by <italic>MAN1</italic> deficiency is due to reduction of BMAL1. Together, these results indicate that MAN1 functions to promote <italic>BMAL1</italic> expression, and thus exerting effects on the clock.</p><p>Consistent with the cell culture data, over-expressing MAN1 in all clock cells in flies using a <italic>timeless</italic> (<italic>tim</italic>) GAL4 driver (<xref ref-type="bibr" rid="bib10">Emery et al., 1998</xref>) resulted in a significantly increased level of <italic>cycle</italic> (<italic>cyc</italic>) mRNA, the <italic>Drosophila BMAL1</italic> homologue (<xref ref-type="bibr" rid="bib35">Rutila et al., 1998</xref>; <xref ref-type="fig" rid="fig5">Figure 5A</xref>). The mRNA level of core clock gene <italic>tim</italic> was also significantly elevated. In addition, we have assessed the levels of <italic>MAN1</italic> mRNA to confirm over-expression (<xref ref-type="fig" rid="fig5">Figure 5B</xref>). We also examined the effect of knocking down <italic>MAN1</italic> in clock cells but did not observe altered <italic>cyc</italic> mRNA levels (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1</xref>).<fig-group><fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.02981.014</object-id><label>Figure 5.</label><caption><title>MAN1 increases <italic>cyc</italic> mRNA levels.</title><p>(<bold>A</bold>) Plots of relative mRNA abundance vs CT for clock genes from whole head extracts of <italic>tim</italic>GAL4/+ and <italic>tim</italic>GAL4/UAS<italic>MAN1</italic> flies during the first day of DD determined by qRT-PCR (n = 2). Gray box indicates subjective light period and black box indicates dark period. Significant effect of genotypes (Two-way ANOVA) were found for <italic>cyc</italic> (p = 0.0278) and <italic>tim</italic> (p = 0.0161). For each time series, the value of the lowest time point was set to 1. (<bold>B</bold>) Plot of relative mRNA abundance for <italic>MAN1</italic> from whole head extracts of <italic>tim</italic>GAL4/+ and <italic>tim</italic>GAL4/UAS<italic>MAN1</italic> flies determined by qRT-PCR (n = 6, Student's <italic>t</italic> test, ***p < 0.001). The value of <italic>tim</italic>GAL4/+ in one experiment was set to 1. Error bars represent SEM.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02981.014">http://dx.doi.org/10.7554/eLife.02981.014</ext-link></p></caption><graphic xlink:href="elife02981f005"/></fig><fig id="fig5s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02981.015</object-id><label>Figure 5—figure supplement 1.</label><caption><title><italic>cyc</italic> transcript level is not altered in <italic>MAN1</italic> knock-down flies.</title><p>Plot of relative mRNA abundance for <italic>cyc</italic> from whole head extracts of <italic>tim</italic>GAL4/+;UAS<italic>dicer2</italic>/+ and <italic>tim</italic>GAL4/UAS<italic>MAN1RNAi</italic>;UAS<italic>dicer2</italic>/+ flies determined by qRT-PCR. Error bars represent SEM (n = 6). The value of <italic>tim</italic>GAL4/+;UAS<italic>dicer2</italic>/+ for one experiment was set to 1.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02981.015">http://dx.doi.org/10.7554/eLife.02981.015</ext-link></p></caption><graphic xlink:href="elife02981fs006"/></fig></fig-group></p></sec><sec id="s2-4"><title>MAN1 enhances <italic>BMAL1</italic> transcription</title><p>A luciferase reporter assay using HEK293 cells was used to further investigate the effect of MAN1 on <italic>BMAL1</italic> transcription. <italic>MAN1</italic> knockdown decreased <italic>Bmal1</italic>-Luc activity by 72% (<xref ref-type="fig" rid="fig6">Figure 6A</xref>), whereas overexpression of FLAG-MAN1 increased the luciferase activity by more than twofold vs cells transfected with empty vector (<xref ref-type="fig" rid="fig6">Figure 6B</xref>). Similar results were obtained with a longer, human <italic>BMAL1</italic> promoter (<xref ref-type="fig" rid="fig6">Figure 6C</xref>). These data indicate that MAN1 may play a role in circadian regulation by activating the promoter of <italic>BMAL1</italic>.<fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.02981.016</object-id><label>Figure 6.</label><caption><title>MAN1 promotes <italic>BMAL1</italic> transcriptional activity.</title><p>(<bold>A</bold>) Reduction of <italic>MAN1</italic> transcripts (13 nM siRNA) reduced <italic>Bmal1</italic> promoter activity (n = 3, *p < 0.001). (<bold>B</bold>) Over-expression of FLAG-tagged MAN1 (F-MAN1) enhanced <italic>Bmal1</italic>-Luc activity (n = 3, *p < 0.001). (<bold>C</bold>) Over-expression of FLAG-tagged MAN1 (F-MAN1) enhanced luciferase activities driven by m<italic>Bmal1</italic> promoter (530 bp) or h<italic>BMAL1</italic> promoter (3.4 kb) (n = 3, *p < 0.05). Error bars represent SD.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02981.016">http://dx.doi.org/10.7554/eLife.02981.016</ext-link></p></caption><graphic xlink:href="elife02981f006"/></fig></p><p>Previously, MAN1 has been shown to exert antagonistic regulatory functions on signal transduction through its binding to R-SMADs (<xref ref-type="bibr" rid="bib27">Osada et al., 2003</xref>; <xref ref-type="bibr" rid="bib33">Raju et al., 2003</xref>; <xref ref-type="bibr" rid="bib13">Hellemans et al., 2004</xref>; <xref ref-type="bibr" rid="bib21">Lin et al., 2005</xref>; <xref ref-type="bibr" rid="bib29">Pan et al., 2005</xref>; <xref ref-type="bibr" rid="bib7">Cohen et al., 2007</xref>) and two types of R-SMADs are found in mammals: TGFβ-responsive (SMAD2 and SMAD3) and BMP-responsive (SMAD1, SMAD5, and SMAD8). To determine whether R-SMADs have an effect on <italic>BMAL1</italic> transcription, we first expressed R-SMADs individually in HEK293 cells transfected with <italic>BMAL1</italic>-Luc to determine which R-SMAD/s is/are involved in regulating <italic>BMAL1</italic> transcription. Expressing SMAD1, SMAD5, SMAD8, and SMAD3 had no significant effect on <italic>BMAL1</italic> transcription but SMAD2 showed significant enhancing effect, suggesting a possible regulatory function by SMAD2 in <italic>BMAL1</italic> regulation (<xref ref-type="fig" rid="fig7">Figure 7A</xref>). The enhancing action of SMAD2 was then examined together with MAN1 to determine whether there is interplay between MAN1 and SMAD2 on <italic>BMAL1</italic> promoter activity. Intriguingly, MAN1 further augmented the enhancing effect of SMAD2 on <italic>BMAL1</italic> in an additive manner, indicating that the positive regulatory function of MAN1 and SMAD2 on <italic>BMAL1</italic> might be independent of each other (<xref ref-type="fig" rid="fig7">Figure 7B</xref>).<fig id="fig7" position="float"><object-id pub-id-type="doi">10.7554/eLife.02981.017</object-id><label>Figure 7.</label><caption><title>MAN1 and SMAD2 enhance <italic>BMAL1</italic> transcription.</title><p>(<bold>A</bold>–<bold>D</bold>) Luciferase reporter activities in transfected HEK293 cells. Cells transfected with indicated constructs in the presence of the 3.4 kb h<italic>BMAL1</italic> promoter for 48 hr and relative luciferase activities were measured in extracts and normalized to <italic>Renilla</italic> luciferase activities. Relative luciferase activities were shown on the y-axis. Values are means ± SEM, n = 3, **p < 0.01 compared to control. †p < 0.05, ††p < 0.01 compared to MAN1 0, one-way ANOVA with Newman–Keuls test.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02981.017">http://dx.doi.org/10.7554/eLife.02981.017</ext-link></p></caption><graphic xlink:href="elife02981f007"/></fig></p><p>Since <italic>BMAL1</italic> transcription is regulated by RORα and REV-ERBα, we wondered whether the effect of MAN1 on <italic>BMAL1</italic> is influenced by RORα/REV-ERBα. MAN1 increases <italic>BMAL1</italic> transcription in the HEK293 luciferase reporter assay but this effect was overshadowed by the presence of either RORα or REV-ERBα, and the impact of RORα and REV-ERBα on <italic>BMAL1</italic> was not influenced by the presence of MAN1 (<xref ref-type="fig" rid="fig7">Figure 7C</xref>). In addition, the effect of MAN1 was also not significantly altered by mutating the RORE sequence (+3 ∼ +13 and +39 ∼ +48) in the <italic>BMAL1</italic> promoter (<xref ref-type="fig" rid="fig7">Figure 7D</xref>), which serves as the DNA binding target of RORα and REV-ERBα. Together, these data suggest that the effect of MAN1 on <italic>BMAL1</italic> transcription is not through the RORE and does not require or impact RORα and REV-ERBα.</p></sec><sec id="s2-5"><title>MAN1 binds to the <italic>BMAL1</italic> promoter</title><p>Since MAN1 does not execute its function through RORE, we investigated the promoter region of <italic>BMAL1</italic> to determine what is necessary for the enhancing effect of MAN1. A series of deletion constructs of the <italic>BMAL1</italic> promoter were generated for luciferase assays and a 900 bp region (−795 ∼ +106) was identified to be the region harboring the necessary DNA sequence for the regulatory effect of MAN1 on <italic>BMAL1</italic> (<xref ref-type="fig" rid="fig8">Figure 8A</xref>, <xref ref-type="fig" rid="fig8s1">Figure 8—figure supplement 1A</xref>).<fig-group><fig id="fig8" position="float"><object-id pub-id-type="doi">10.7554/eLife.02981.018</object-id><label>Figure 8.</label><caption><title>MAN1 binds to the <italic>BMAL1</italic> promoter to enhance its transcription.</title><p>(<bold>A</bold>) Luciferase activities of deleted h<italic>BMAL1</italic>-promoter constructs in the absence or presence of MAN1 expression vectors. n = 3, Student's <italic>t</italic> test, **p < 0.01, ***p < 0.001. (<bold>B</bold> and <bold>C</bold>) Luciferase activities of the 3.4 Kb h<italic>BMAL1-Luc</italic> in the presence of MAN1 constructs as indicated. n = 3, **p < 0.01, ***p < 0.001 compared to control; †p < 0.05; ††p < 0.001 compared to WT MAN1. (<bold>D</bold>) ChIP analysis of MAN1 (WT or DNA binding truncation) for 14 segments of h<italic>BMAL1</italic> promoter region. Data represent pull-down relative to input. n = 6, †p < 0.05, compared to WT MAN1. One-way ANOVA with Newman–Keuls test. All data are presented as ratio of means ± SEM.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02981.018">http://dx.doi.org/10.7554/eLife.02981.018</ext-link></p></caption><graphic xlink:href="elife02981f008"/></fig><fig id="fig8s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02981.019</object-id><label>Figure 8—figure supplement 1.</label><caption><title>Domain-specific interactions between MAN1 and the h<italic>BMAL1</italic> promoter.</title><p>(<bold>A</bold>) Schematic representation of the indicated constructs generated from the 3.4 kb h<italic>BMAL1</italic> promoter and cloned into the luciferase reporter vector pGL3. Histogram of luciferase activity in HEK293 cells transfected with the deleted h<italic>BMAL1</italic> promoter constructs in the absence or presence of MAN1 expression vectors. Cells transfected for 48 hr and relative luciferase activities measured in extracts and normalized to <italic>Renilla</italic> luciferase activities. Activities (relative luciferase activity) are shown on the y-axis. Values are means ± SEM, n = 3, Student's <italic>t</italic> test, **p < 0.01, ***p < 0.001 compared to control. (<bold>B</bold>) Schematic representation of the deletion constructs generated from the MAN1 expressing construct. (<bold>C</bold>) Sequences of constructs with point mutations generated from the MAN1 expressing construct.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02981.019">http://dx.doi.org/10.7554/eLife.02981.019</ext-link></p></caption><graphic xlink:href="elife02981fs007"/></fig></fig-group></p><p>MAN1 has an N-terminal <underline>L</underline>AP2-<underline>E</underline>merin-<underline>M</underline>AN1 (LEM) domain, two transmembrane segments in the middle, a unique DNA binding domain, and a C-terminal RNA recognition motif (RRM) that is required for its binding with R-SMADs (<xref ref-type="bibr" rid="bib5">Caputo et al., 2006</xref>). We next examined the domain of MAN1 necessary for its effect on <italic>BMAL1</italic>. Two truncation constructs of MAN1 were generated, one without the DNA binding domain (amino acids 707–725) and the other without the RRM domain (amino acids 760–911) (<xref ref-type="bibr" rid="bib29">Pan et al., 2005</xref>; <xref ref-type="fig" rid="fig8s1">Figure 8—figure supplement 1B</xref>). In addition, we utilized a substitution mutant of MAN1 (YV-DD) (<xref ref-type="bibr" rid="bib29">Pan et al., 2005</xref>), containing two amino acid alterations in the RRM that nullify the ability of MAN1 to antagonize R-SMADs. These constructs were then used to test their transcriptional enhancing effect on <italic>BMAL1</italic> promoters (either full length 3.4 kb or 2.4 kb [−2300 ∼ +105]). Intriguingly, the RRM truncation mutation lost the enhancing effect on the <italic>BMAL1</italic> promoter (<xref ref-type="fig" rid="fig8">Figure 8B</xref>). The DNA binding domain truncation mutation also lost the activating effect on <italic>BMAL1</italic>, and this effect can be produced by simply mutating three positively charged amino acids (RKK) at amino acids 709–711 (<xref ref-type="fig" rid="fig8">Figure 8C</xref>, <xref ref-type="fig" rid="fig8s1">Figure 8—figure supplement 1C</xref>). These results imply that the effect of MAN1 on <italic>BMAL1</italic> transcription requires potential DNA binding ability of MAN1 as well as interaction with a protein partner (possibly SMAD2) through RRM.</p><p>Since the DNA binding domain is required for the effect of MAN1 on the <italic>BMAL1</italic> promoter, we next tested whether MAN1 directly binds to the <italic>BMAL1</italic> promoter. Chromatin immunoprecipitation (ChIP) analysis revealed that the region −237 bp to +45 bp from transcriptional start site was pulled down by MAN1 indicating a direct interaction (<xref ref-type="fig" rid="fig8">Figure 8D</xref>). All together, these data suggest that MAN1 binds to the <italic>BMAL1</italic> promoter region (−237 bp to +45 bp) to enhance its transcription.</p></sec></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><p>The nuclear envelope plays essential roles in diverse cellular functions including global regulation of gene expression. Interestingly, another global regulatory mechanism is the molecular clock that modulates our body and cellular daily rhythm. We sought to see whether there is cross talk between these two regulatory mechanisms. Our studies revealed that some components of nuclear envelope do show daily oscillations, indicating that nuclear envelope is subject to clock control. On the other hand, we found that one of the major transcription activators of the molecular clock, <italic>BMAL1</italic>, is regulated by one of the nuclear envelope proteins, MAN1. Thus, there is reciprocity between these two global regulatory mechanisms.</p><p>The nuclear envelope physically separates genomic DNA from the cytoplasm and functions as a signaling control center. An increasing number of human diseases are recognized to be caused by mutations in genes encoding nuclear envelope proteins and hence, termed ‘envelopathies’ (<xref ref-type="bibr" rid="bib8">Dauer and Worman, 2009</xref>). Several inner nuclear membrane proteins are known to regulate critical signaling pathways and act as intranuclear regulators of signaling pathways that receive and transduce signals from extracellular cues. The nuclear lamina provides structural support for the nucleus and the nuclear envelope; however, lamins and their associated proteins are also involved in most of the nuclear processes. Lamin B1 is essential for brain development and is required for the integrity of the nuclear lamina (<xref ref-type="bibr" rid="bib28">Padiath et al., 2006</xref>; <xref ref-type="bibr" rid="bib6">Coffinier et al., 2011</xref>). Interestingly, no abnormality has been reported for the heterozygous <italic>Lmnb1</italic> knock out mice (<xref ref-type="bibr" rid="bib39">Stewart et al., 2007</xref>; <xref ref-type="bibr" rid="bib6">Coffinier et al., 2011</xref>), and it is estimated that these heterozygous mice still express 70% of lamin b1 (SG Young, personal communication). Consistent with these reports, we did not find significant phenotype including circadian behavior change in these mice despite the clear phase shift on PER2 oscillation in the liver. It is possible that the oscillation of one or more additional core clock components are also altered by reduced lamin b1 level and these additional alterations can compensate the effect of PER2 phase shift on output behavior. Alternatively, it is also possible that the SCN clock is resilient to perturbations caused by reduced lamin b1 level, hence we can only observe alterations in peripheral clocks. Further investigation is necessary to reveal the mechanism leading to the findings we report here.</p><p>MAN1 belongs to the inner nuclear membrane LEM protein family (<xref ref-type="bibr" rid="bib46">Worman, 2006</xref>; <xref ref-type="bibr" rid="bib4">Bengtsson, 2007</xref>). LEM domains mediate the interaction with a chromatin-binding protein, barrier-to-autointegration factor (BAF), which has the ability to bind dsDNA, chromatin, histones, lamin binding proteins, and various transcription factors (<xref ref-type="bibr" rid="bib22">Liu et al., 2003</xref>; <xref ref-type="bibr" rid="bib46">Worman, 2006</xref>; <xref ref-type="bibr" rid="bib4">Bengtsson, 2007</xref>). Therefore, LEM proteins have roles in gene regulation, chromatin organization, regulation of transcription factor activity at the nuclear periphery, and regulation of specific signal pathways. Both amino and carboxyl termini of MAN1 are nucleoplasmic domains. The amino-terminal nucleoplasmic region of MAN1 (including LEM) binds to the nuclear lamins and emerin in addition to BAF. It is also necessary for efficient localization of MAN1 to the inner nuclear membrane (<xref ref-type="bibr" rid="bib24">Mansharamani and Wilson, 2005</xref>). The carboxyl-terminal nucleoplasmic region (residues 655–911) exhibits two globular domains (<xref ref-type="bibr" rid="bib29">Pan et al., 2005</xref>; <xref ref-type="bibr" rid="bib5">Caputo et al., 2006</xref>). The first globular domain contains a winged helix (including the sequence RKKMKKVWDR) which is mainly used for DNA binding and recognition. The second domain (amino acids 782–911) is an RRM-like protein interaction domain where it can interact with R-SMADs. The entire carboxy-terminal region of MAN1 was shown to participate in DNA binding, and this interaction is synergistic to the binding of MAN1 to different transcriptional regulators, including R-SMADs (<xref ref-type="bibr" rid="bib27">Osada et al., 2003</xref>; <xref ref-type="bibr" rid="bib33">Raju et al., 2003</xref>; <xref ref-type="bibr" rid="bib21">Lin et al., 2005</xref>; <xref ref-type="bibr" rid="bib29">Pan et al., 2005</xref>). Consistently, we found that both RRM and DNA binding domains are required for the activation of the <italic>BMAL1</italic> promoter by MAN1. Also congruent with previous findings, we found that mutating three of the highly positively charged and conserved amino acids within the winged helix region is sufficient to dampen the activation efficiency of MAN1. In contrast to previous reports, we found that MAN1 further augments (but not antagonizes) the positive effect of SMAD2 on <italic>BMAL1.</italic> Consistent with this result, we also found that the substitution mutation YV-DD of MAN1 does not influence its effect on <italic>BMAL1</italic> (though RRM domain is required). Collectively, these results suggest that the effects of MAN1 and SMAD2 on <italic>BMAL1</italic> might not be completely independent to each other and that MAN1 might interact with SMAD2 in more than one way (presumably through other protein partners) to modulate transcription of target genes.</p><p>Many of our body functions manifest a daily rhythm which is maintained by the rhythmic regulation of approximately 15% of genes by the core molecular clock (<xref ref-type="bibr" rid="bib44">Vollmers et al., 2009</xref>; <xref ref-type="bibr" rid="bib26">Menet et al., 2012</xref>). However, cells must be flexible enough to allow for responses to exogenous and endogenous stimuli. This regulation is likely to be mediated not solely by the molecular clock, but also by many additional global and local mechanisms including at the level of chromatin and genome organization (<xref ref-type="bibr" rid="bib1">Aguilar-Arnal et al., 2013</xref>; <xref ref-type="bibr" rid="bib15">Hubner et al., 2013</xref>). Genetic loci associated with the nuclear lamina through large regions of chromatin (lamin associated domains–LADs) are associated with changes in transcriptional status (<xref ref-type="bibr" rid="bib31">Peric-Hupkes et al., 2010</xref>). Circadian genes or genes involved in rhythmic processes display robust rhythmic expression patterns at the level of nascent mRNA and mRNA (<xref ref-type="bibr" rid="bib26">Menet et al., 2012</xref>; <xref ref-type="bibr" rid="bib34">Rodriguez et al., 2012</xref>), suggesting prominent contribution of transcriptional regulation to clock gene expression. Interestingly, genes harboring this expression pattern are dramatically enriched for specific function in transcriptional regulation and chromatin organization (<xref ref-type="bibr" rid="bib26">Menet et al., 2012</xref>). These unbiased genome-wide transcriptome reports raised the possibility that components of nuclear envelope may modulate oscillation of clock genes at transcriptional levels. A recent study has also demonstrated that the molecular clock drives circadian changes in spatial and temporal chromosomal organization (<xref ref-type="bibr" rid="bib1">Aguilar-Arnal et al., 2013</xref>). Indeed, our study links the nuclear periphery with circadian regulation via the regulatory effects of MAN1 on <italic>BMAL1</italic> through transcription.</p><p>This effect is evident not only in mammalian systems, but also in flies, as over-expressing <italic>MAN1</italic> resulted in significantly increased levels of <italic>cyc</italic> mRNA. MAN1 over-expression also increased <italic>tim</italic> mRNA levels, which may be at least partially due to increased <italic>cyc</italic>. Knocking down <italic>MAN1</italic> did not alter <italic>cyc</italic> levels, possibly because the residual MAN1 is still sufficient to maintain normal <italic>cyc</italic> levels. However, this manipulation lengthened behavioral period, suggesting that MAN1 may target other clock components in addition to <italic>cyc</italic>. Over-expressing <italic>MAN1</italic> and <italic>LBR</italic> in flies led to a lengthened behavioral period, while in U2OS cells, these manipulations resulted in moderate shortening of the period. On the other hand, over-expressing and knocking down <italic>Lam</italic> in flies shortened and lengthened the period, respectively, which is consistent with the mammalian data. These discrepancies reflect the differences between mammalian and insect clock, which has been implied in previous work as well (<xref ref-type="bibr" rid="bib23">Lowrey et al., 2000</xref>; <xref ref-type="bibr" rid="bib32">Preuss et al., 2004</xref>; <xref ref-type="bibr" rid="bib47">Xu et al., 2005</xref>). Nevertheless, our results indicate that there is a conserved role for NE components in setting the clock in organisms ranging from invertebrates to humans.</p><p>Although only 22–30% of cycling mRNA is driven transcriptionally (<xref ref-type="bibr" rid="bib18">Koike et al., 2012</xref>; <xref ref-type="bibr" rid="bib26">Menet et al., 2012</xref>), demonstration of involvement of the nuclear envelope in regulation of the molecular circadian clock suggests one pathway through which the nuclear envelope may globally and temporally regulate large numbers of genes. Interestingly, expression of the genes for many nuclear envelope proteins also oscillates. This finding sheds new light on the interconnectedness of these biological processes and provides further insight into the mechanism whereby cellular, metabolic, physiological, and behavioral processes that oscillate are modulated in a highly coordinated manner.</p></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Plasmids</title><p>To generate FLAG-tagged proteins, human <italic>LBR</italic>, <italic>LMNB1</italic>, and <italic>MAN1</italic> genes were subcloned into pCMV-Tag2A (Stratagene; La Jolla, CA). HA-tagged SMAD1, FLAG-MAN1 (YV-DD), and FLAG-MAN1 (1–759) were gifts from Dr Kunxin Luo (<xref ref-type="bibr" rid="bib29">Pan et al., 2005</xref>). m<italic>Bmal1</italic>-Luc was generated in Dr Satchidananda Panda's lab and kindly provided by Dr John Hogenesch (<xref ref-type="bibr" rid="bib45">Vollmers et al., 2008</xref>), while the human <italic>BMAL1</italic>-luc construct was a generous gift from Dr Toru Takumi (<xref ref-type="bibr" rid="bib2">Akashi and Takumi, 2005</xref>). Syrian hamster <italic>Bmal1</italic> in pcDNA3.1 vector was provided by Dr David Weaver (<xref ref-type="bibr" rid="bib19">Kume et al., 1999</xref>). pRL-TK was purchased from Promega (Madison, WI). Mutations of expression constructs were introduced by PCR, and all constructs used in this study were verified by sequencing.</p></sec><sec id="s4-2"><title>LumiCycle analysis</title><p>A stable U2OS-B6 cell line that expresses a destabilized firefly luciferase gene under the control of the m<italic>Bmal1</italic> promoter was obtained from Dr Satchidananda Panda (<xref ref-type="bibr" rid="bib45">Vollmers et al., 2008</xref>). siRNAs targeted to <italic>LBR</italic>, <italic>LMNB1</italic>, <italic>MAN1</italic>, or <italic>SMAD1</italic> (10 nM, Invitrogen; Carlsbad, CA; see <xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1A</xref>) were individually transfected into 35-mm culture dishes using Lipofectamine RNAiMAX (Invitrogen). For overexpression of FLAG-tagged constructs, plasmid (2 µg) was distributed into each well along with FuGENE HD (4 μl, Roche; Switzerland). For co-transfection of <italic>MAN1</italic> siRNA and <italic>Bmal1</italic>, Lipofectamine 2000 (Invitrogen) was used. 24 hr after transfection, cells were synchronized with 100 nM dexamethasone in serum-free DMEM containing 10 mM HEPES (pH 7.5) at 37°C for 2 hr. Following synchronization, the media were replaced with phenol red-free DMEM supplemented with 10 mM HEPES and 40 µM Luciferin-EF (Promega). Cells sealed with coverslips were incubated in a 32-channel LumiCycle (Actimetrics; Evanston, IL) to monitor real-time bioluminescence for 5 days. Data were analyzed using Lumicycle Analysis (Actimetrics).</p></sec><sec id="s4-3"><title>Fly behavior experiments and analysis</title><p>To over-express NE, <italic>cry</italic>GAL4-39 and <italic>cry</italic>GAL4-16 (<xref ref-type="bibr" rid="bib11">Emery et al., 2000</xref>) were crossed to <italic>MAN1</italic><sup><italic>GS2297</italic></sup> (Kyoto Stock Center; Japan), UAS<italic>Lam</italic> (<xref ref-type="bibr" rid="bib28">Padiath et al., 2006</xref>) and <italic>LBR</italic><sup><italic>GS2162</italic></sup> (Kyoto Stock Center). To knock down NE, <italic>cry</italic>GAL4-39;UAS<italic>dcr2</italic> and UAS<italic>dcr2</italic>;<italic>cry</italic>GAL4-16 were crossed to UAS<italic>MAN1RNAi</italic> (3167R-1, NIG; Japan), UAS<italic>LamRNAi</italic> (45,635, VDRC; Vienna) and UAS<italic>LBRRNAi</italic> (KK110508, VDRC). For controls, the UAS and GAL4 lines were crossed to <italic>w</italic><sup><italic>1118</italic></sup> or <italic>yw</italic> strains. Male progenies were assayed for behavior.</p><p>Locomotor activity levels of flies were monitored using Trikinetics Activity Monitors (Waltham, MA) for 7 days of 12 hr light-12 hr dark (LD) conditions followed by 7 days of constant darkness (DD). For DD rhythmicity, chi-squared periodogram analyses were performed using Clocklab (Actimetrics). Rhythmic flies were defined as those in which the chi-squared power was ≥10 above the significance line. Period calculations also considered all flies with rhythmic power ≥10.</p></sec><sec id="s4-4"><title>Quantitative real-time PCR (qRT-PCR)</title><p>RNeasy Mini Kit (Qiagen) was used to isolate total RNAs from synchronized U2OS-B6 cells that were collected at interval of 4 hr over the course of 48 hr. Purified RNA (2 µg) was applied in 20-μl reactions for RT primed with Oligo(dT)<sub>20</sub> using Super-Script III First-Strand Synthesis System (Invitrogen). All qPCR reactions were carried out on a Rotor-Gene RG-3000 (Corbett Research; Netherland)/or 7900HT Fast Real-Time PCR System (Life technologies; Carlsbad, CA) using FastStart SYBR Green Master (Rox) (Roche). The templates were denatured at 95°C for 10 min, followed by forty cycles with 15 s at 95°C, 10 s at 58°C (Rotor-Gene) or 60 s at 60°C (HT7900 system and data acquisition at the end of this step), or 40 s at 72°C, and an additional 2 s for data acquisition (Rotor-Gene). The standard curve and delta–delta CT methods were used for quantification (Applied Biosystems; Carlsbad, CA). Primers used for expression analysis are listed in <xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1B</xref>. Primers used for ChIP assay are labeled as the nucleotide distance from the transcriptional start site (TSS) and +1 indicates the starting of TSS.</p><p>Fly heads were isolated at the indicated time points and total RNA was isolated with TRIzol reagent (Invitrogen). After the removal of contaminating genomic DNA by RQ1 DNase (Promega) digestion, total RNA was directly amplified with the QuantiTect SYBR green RT-PCR kit (QIAGEN). The following primers were used: for <italic>cyc</italic>, cyc_110.f 5′-GAGGTCTTCGTCGGAAAGG-3′ and cyc_347.r 5′-AAAGCACATGGGAATCATGG-3′; for <italic>tim</italic>, tim.f 5′-CTGGGGAGTGACCATGG-3′ and tim.r 5′-GCTGGAATCGCCACTG-3′; for d<italic>MAN1</italic>, dMAN1_148.f 5′-ATTTTGGCCTGTGACACTGC-3′ and dMAN1_303.r 5′-GAAGCCGCTCTGGATTAGC-3′; for d<italic>Lam</italic>, dLam_446.f 5′-CGAGGAGCTCAAGAACAAGC-3′ and dLam_675_r. 5′-GCGACAGTGTCTCCTGTTCC-3′; for d<italic>LBR</italic>, dLBR_645.f 5′-CATTGACCACCAACACATCC-3′ and dLBR_825.r 5′-GTTATGCGTTTGCGAATGG-3′; for d<italic>Actin</italic>, dActin.f1 5′-CTAACCTCGCCCTCTCCTCT-3′ and dActin.r1 5′-GCAGCCAAGTGTGAGTGTGT-3′. All other primers used for fly tissues are previously published (<xref ref-type="bibr" rid="bib20">Lim et al., 2007</xref>; <xref ref-type="bibr" rid="bib17">Kilman et al., 2009</xref>).</p></sec><sec id="s4-5"><title>Mouse behavior experiment and analyses</title><p><italic>Lmnb1</italic><sup><bold><italic>+</italic></bold><italic>/Δ</italic></sup> mice with a targeted disruption of the <italic>Lmnb1</italic> gene (<xref ref-type="bibr" rid="bib42">Vergnes et al., 2004</xref>) or <italic>LMNB1</italic><sup>BAC</sup> mouse model overexpressing lamin B1 (<xref ref-type="bibr" rid="bib14">Heng et al., 2013</xref>) was generated as previous described. The animals used here were derived from these mice and have been backcrossed to a C57BL/6J background for at least 10 generations. Wild-type littermates were used in pairs for subsequent experiments. Mice housed in light-tight, sound-attenuated cabinets were entrained to LD cycle for 14 days and then released into DD. Wheel-running activity of mice were monitored using Clocklab (Actimetrics). For DD rhythmicity, chi-squared periodogram analyses were performed using Clocklab. Experiments were approved by the Institutional Animal Care and Use Committee at University of California, San Francisco.</p></sec><sec id="s4-6"><title>Western blot</title><p>Brains or livers were collected from mice that were entrained in LD cycle for 14 days and were then released into DD. Total cellular proteins were extracted from mouse brain or liver using RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 20 mM Tris, pH 7.5, and 5 mM EDTA). Protein lysates from cells were prepared in SDS-PAGE loading buffer. Equal amounts of protein were resolved on 8% SDS-PAGE gels and then transferred to nitrocellulose membrane. After incubation with primary antibody at 4°C overnight, membranes were incubated with secondary antibodies at room temperature for 1 hr. The primary antibodies were anti-LBR rabbit polyclonal antibody (1:500; Abcam), anti-LMNB1 rabbit polyclonal antibody (1:1000; Abcam; England), anti-MAN1 rabbit polyclonal antibody (1:3000; from Dr Kunxin Luo) (<xref ref-type="bibr" rid="bib29">Pan et al., 2005</xref>), anti-mPER2 rabbit polyclonal antibody (1:500; Alpha Diagnostic International; San Antonio, TX), anti-GAPDH mouse monoclonal antibody (1:5000; Chemicon; Billerica, MA), anti-BMAL1 goat polyclonal antibody (1:500; Santa Cruz; Dallas, TX), anti-CLOCK rabbit polyclonal antibody (1:1000; Santa Cruz), and anti-FLAG M2 antibody (1:5000; Sigma; St Louis, MO). The MAN1 antibody was generated by immunizing rabbits with C-terminal peptide (SHLRLRTGLTNSQGSS) of human MAN1 (1:1000; Covance and Agbio, Inc; Princeton, NJ). Secondary antibodies were conjugated either with IRDye 680 or IRDye 800 (LI-COR Biosciences; Lincoln, NE) and visualized with an Odyssey Infrared Imaging System (LI-COR Biosciences).</p></sec><sec id="s4-7"><title>Luciferase assays</title><p>HEK293 cells were cultured in 24-well plates in DMEM containing 10% fetal bovine serum 24 hr prior to transient transfection with FuGENE HD (Roche) for overexpression (50–200 ng cDNA constructs), or Lipofectamine 2000 (Invitrogen) for siRNA knockdown (8–13 pmol, Invitrogen). All transfection mixtures included a <italic>Renilla</italic> luciferase plasmid (pRL-TK; 0.7 ng), as well as a reporter construct consisting of firefly luciferase driven by mouse <italic>Bmal1</italic> or human <italic>BMAL1</italic> promoter (50 ng). We assayed the <italic>Bmal1</italic>/<italic>BMAL1</italic> promoter luciferase activity using the Dual-Luciferase Reporter Assay System (Promega), modifying the protocol to use 30 μl of luciferase substrate and Stop-n-Glo/substrate mix for each reaction. The luciferase activity was quantified with a TECAN GENios Pro Microplate Reader (TECAN; Switzerland) 48 hr after the initial transfection. Luciferase reporter vector used is pGL3-basic.</p></sec><sec id="s4-8"><title>Chromatin immunoprecipitation (ChIP) assay</title><p>We performed ChIP assays using Millipore's EZ-ChIP assay kit (cat. # 17–371; Millipore; Billerica, MA) and protein-G sepharose. In brief, HEK293 cells were transfected with h<italic>BMAL1</italic>-luciferase (3.4 Kb) plus vector, FLAG-tagged WT or truncated DNA-binding constructs of MAN1 as indicated. Cell lysates were sonicated on ice using Branson digital sonifier #250 and 1% of cell lysate was taken as input sample. After incubation with FLAG M2 antibody (Sigma), antibody-loaded protein G agarose beads were washed with cold wash buffer six times followed by low-salt buffer, high salt wash buffer, LiCl wash buffer, and then once with TE (10 mM Tris–HCl at pH 8.0, 1 mM EDTA at pH 8.0). After washing, the beads were re-suspended in 100 μl of ChIP elution buffer supplemented with proteinase K and incubated for 2 hr at 65°C followed by 10 min at 95°C. The beads were spun down and the supernatant was saved. DNA was recovered from the spin column and resuspended in 50 μl of TE, and a 1 μl portion was used for qRT-PCR. The PCR products were analyzed by qRT-PCR and quantitated using 7900HT Fast Real-Time PCR System (Life technologies).</p></sec><sec id="s4-9"><title>Statistics</title><p>Statistical analyses were performed using the unpaired Student's <italic>t</italic> test, one-way ANOVA with Newman–Keuls test, or two-way ANOVA (Prism5, GraphPad; La Jolla, CA). Data are presented as Mean ± SEM or SD. Significant differences (p < 0.05) are marked with asterisks in figures.</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We thank Drs S Panda and J Hogenesch for m<italic>Bmal1</italic>-luc construct; Dr T Takumi for h<italic>BMAL1</italic>-luc construct; Dr D Weaver for pcDNA3.1-<italic>Bmal1</italic> construct; and Dr K Luo for MAN1 constructs, HA-SMAD1 construct, and MAN1 antibody.</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>LP: Reviewing editor, <italic>eLife</italic>.</p></fn><fn fn-type="conflict" id="conf2"><p>The other 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>S-TL, 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>LZ, 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="con3"><p>XL, Conception and design, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con4"><p>LCZ, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con5"><p>VEG, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con6"><p>C-WT, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con7"><p>LP, Conception and design, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con8"><p>Y-HF, Conception and design, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn></fn-group><fn-group content-type="ethics-information"><title>Ethics</title><fn fn-type="other"><p>Animal experimentation: All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by Institutional Animal Care and Use Committee of University of California San Francisco. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of California San Francisco (AN089663-01).</p></fn></fn-group></sec><sec sec-type="supplementary-material"><title>Additional files</title><supplementary-material id="SD1-data"><object-id pub-id-type="doi">10.7554/eLife.02981.020</object-id><label>Supplementary file 1.</label><caption><p>(<bold>A</bold>) Sequences of siRNAs used in the study. 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person-group-type="author"><name><surname>Zwerger</surname><given-names>M</given-names></name><name><surname>Medalia</surname><given-names>O</given-names></name></person-group><year>2013</year><article-title>From lamins to lamina: a structural perspective</article-title><source>Histochemistry and Cell Biology</source><volume>140</volume><fpage>3</fpage><lpage>12</lpage><pub-id pub-id-type="doi">10.1007/s00418-013-1104-y</pub-id></element-citation></ref></ref-list></back><sub-article article-type="article-commentary" id="SA1"><front-stub><article-id pub-id-type="doi">10.7554/eLife.02981.021</article-id><title-group><article-title>Decision letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Griffith</surname><given-names>Leslie C</given-names></name><role>Reviewing editor</role><aff><institution>Brandeis University</institution>, <country>United States</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://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 “Nuclear envelope protein MAN1 regulates clock through <italic>BMAL1</italic>” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by a Senior editor and 3 reviewers, one of whom is a member of our Board of Reviewing Editors.</p><p>The following individuals responsible for the peer review of your submission have agreed to reveal their identity: Leslie Griffith, BRE; Satchidananda Panda, reviewer.</p><p>The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.</p><p>This paper provides a link between the nuclear envelope and the circadian clock and posits that the link is evolutionarily conserved, by suggesting that regulation of the clock by NE proteins, in particular MAN1, is present in both rodents and flies. The novelty of the study is high and the data are mostly clear and compelling. There are, however, a few weak links in the story which, if strengthened would make it quite compelling. The major issues are:</p><p>1) At the organismal/cellular level: The fly data generally are not completely convincing. It’s very easy to get RNAi phenotypes and a lot of times these don't map to the gene targeted. Phenotypes based on single RNAis, with no mutant data or experiments mapping the effect to the relevant gene, are not very convincing. In addition, the data in <xref ref-type="table" rid="tbl1">Table 1</xref> for human LMB1 do not support what is stated in the text- there appears to be no change in period. Some genetic evidence that the RNAi phenotypes are actually due to changes in the relevant genes is required.</p><p>2) At the molecular mechanism level: The cell assay data are quite subtle. If the authors could show suppression of the long period of the NE knockdown cells (or knockout mouse fibroblasts) this would provide very strong evidence of the linkage the authors wish to make.</p><p><italic>Reviewer #1:</italic></p><p>This is a very interesting and novel study which suggests that NE proteins can directly modulate the circadian clock in both mammals and insects. Gene regulation by the nuclear envelope is a very new concept and connecting it to the clock is a potentially very important observation.</p><p>For the most part the data are very clear and consistent. The only piece of evidence that is less than clear is in <xref ref-type="fig" rid="fig1">Figure 1D</xref>. How do the authors draw the conclusion that MAN1 is cycling? It looks dead flat to me. Is there any evidence to suggest that the very small difference statistically significant? Ditto for LMNB1 and LBR. They are by eye a bit more convincing, but a quantitative treatment is required if they authors want to claim the protein cycles (which I am not sure they need to given the strength of the functional evidence- so what if the protein does not cycle in an extract of liver?).</p><p><italic>Reviewer #2:</italic></p><p>The manuscript demonstrates that nuclear envelope proteins contribute to the determination of circadian period. It focuses in particular on the MAN1 protein, showing that this is a positive regulator of BMAl1 transcription. Knockdown or overexpression of MAN1 or other nuclear envelope (NE) proteins, Lamin B1 and LBR, produces a small effect on circadian period in mammalian cultured cells. A more robust phenotype is seen with some of these manipulations in <italic>Drosophila</italic> although the directionality of the effect (short or long period) or the mechanism is not easily explained. Microarray studies have reported cyclic expression of nucleoporins, and not surprisingly, the authors report cyclic expression for a couple of other NE proteins. Overall, these are interesting observations although they do not quite provide a picture of how the nuclear envelope Figure in the clock mechanism. Additional comments are below:</p><p><xref ref-type="fig" rid="fig1">Figure 1</xref> should show quantification of multiple blots for PER2 cycling in the LaminB1 mutant and overexpression backgrounds (<xref ref-type="fig" rid="fig1">Figure 1C</xref>). Also, what happens to other clock proteins in these backgrounds?</p><p>Effects of knockdown and over-expression of NE proteins in mammalian cells are subtle. In mice heterozygous for a Lamin B1 mutation, or overexpressing LaminB1, PER2 oscillations appear to be altered, although this needs to be better documented, and is not linked to any overt rhythm. Period lengthening by overexpression of human MAN1 or LBR in <italic>Drosophila</italic> has a more robust effect, but then knockdown of MAN1 has the same effect. Given that the molecular mechanism is not known (see below for the inconclusive data regarding the effect on cyc), and mutant data are not shown (only a single RNAi for each gene, without any mapping of the RNAi effect) it is difficult to assess the significance of the fly data.</p><p>The text indicates that over-expression of human LMNB1 shortens the period in <italic>Drosophila</italic>, but this is not supported by the data in <xref ref-type="table" rid="tbl1">Table 1</xref>.</p><p>Given that knocking down MAN1 has no effect on cyc, the effect of overexpression could be non-physiological. Also, increased expression of cyc (through MAN1 overexpression) should not lengthen period as shown here, so it is unlikely that the effect on circadian period is going through cyc. Along the same lines, it would be good to know if the effects on circadian period in mammalian cells are going through BMal1. Does overexpression of BMal1 rescue the long period produced by knockdown of NE proteins?</p><p>Additive effects of SMAD and MAN1 suggest that they are acting independently, and not as part of the same pathway.</p><p>The studies mapping MAN1 interactions with the BMAL1 promoter are good, and indicate direct interaction.</p><p><italic>Reviewer #3:</italic></p><p>This manuscript offers a novel link between nuclear envelope components and circadian clock that is conserved from insects to mammals. The experiments are straightforward and well described. However, some gaps in data presentation need to be addressed.</p><p>Since the authors have access to the lamin mutant mice and flies with specific perturbation of Ne components, figure (a) showing the circadian behavior phenotype of these animals will substantially strengthen the manuscript. If the het mice lack any appreciable defect in free running circadian period, activity consolidation, phase angle of entrainment, such lack of phenotype may be explained in the discussion section. Circadian behavior phenotype of the flies should be shown in the main figures to strengthen the manuscript.</p><p>siRNA knockdown experiments on Bmal1:Luc expression should ideally have another promoter:Luc construct to rule out the possibility that the down regulation of Bmal1:luc signal is not due to general defect in nuclear integrity or overall transcription.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.02981.022</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>1) At the organismal/cellular level: The fly data generally are not completely convincing. It’s very easy to get RNAi phenotypes and a lot of times these don't map to the gene targeted. Phenotypes based on single RNAis, with no mutant data or experiments mapping the effect to the relevant gene, are not very convincing. In addition, the data in</italic> <xref ref-type="table" rid="tbl1"><italic>Table 1</italic></xref> <italic>for human LMB1 do not support what is stated in the text- there appears to be no change in period. Some genetic evidence that the RNAi phenotypes are actually due to changes in the relevant genes is required</italic>.</p><p>In our original submission, we showed that MAN1 mRNA level is reduced in flies expressing MAN1-RNAi. Here we have included additional data demonstrating that Lam and LBR mRNA levels are reduced in flies expressing Lam-RNAi and LBR-RNAi, respectively (<xref ref-type="fig" rid="fig2s2">Figure 2–figure supplement 2</xref>). This does not eliminate the possibility that the RNAi is also disrupting expression of other genes. However, given that we also observe circadian phenotypes when over-expressing these genes in flies, and that deficiency of these genes lead to disrupted circadian clock in the mammalian system, we believe it is highly likely that the circadian phenotypes associated with expressing RNAi constructs of MAN1/Lam/LBR in flies are due to reduction in the levels of these genes.</p><p>As for Lam over-expressing flies, the behavioral period is shorter than the GAL4 controls, which have long periods, but not shorter than UASLam control, which has a ∼24hr period. It is possible that there is a limit to how much Lam over-expression can shorten the period in flies. It may be able to shorten period to ∼24hr but cannot further shorten period to less than 24hr.</p><p><italic>2) At the molecular mechanism level: The cell assay data are quite subtle. If the authors could show suppression of the long period of the NE knockdown cells (or knockout mouse fibroblasts) this would provide very strong evidence of the linkage the authors wish to make</italic>.</p><p>Thank you for the suggestion. We have added new data demonstrating that Bmal1 over-expression suppresses the effect of MAN1 knockdown on circadian period in U2OS cells (<xref ref-type="fig" rid="fig4s2">Figure 4–figure supplement 2</xref>).</p><p>Reviewer #1:</p><p><italic>This is a very interesting and novel study which suggests that NE proteins</italic> can <italic>directly modulate the circadian clock in both mammals and insects. Gene regulation by the nuclear envelope is a very new concept and connecting it to the clock is a potentially very important observation</italic>.</p><p><italic>For the most part the data are very clear and consistent. The only piece of evidence that is less than clear is in</italic> <xref ref-type="fig" rid="fig1"><italic>Figure 1</italic>D</xref><italic>. How do the authors draw the conclusion that MAN1 is cycling? It looks dead flat to me. Is there any evidence to suggest that the very small difference statistically significant? Ditto for LMNB1 and LBR. They are by eye a bit more convincing, but a quantitative treatment is required if they authors want to claim the protein cycles (which I am not sure they need to given the strength of the functional evidence- so what if the protein does not cycle in an extract of liver?)</italic>.</p><p>Quantification of the Western blot is shown in <xref ref-type="fig" rid="fig1">Figure 1D</xref> top panel. The reviewer is correct that the oscillation for MAN1 is mild. However, the subsequent functional data strengthened our original idea that MAN1 could be the link between NE and the molecular clock.</p><p>Reviewer #2:</p><p><italic>The manuscript demonstrates that nuclear envelope proteins contribute to the determination of circadian period. It focuses in particular on the MAN1 protein, showing that this is a positive regulator of BMAl1 transcription. Knockdown or overexpression of MAN1 or other nuclear envelope (NE) proteins, Lamin</italic> B1 <italic>and LBR, produces a small effect on circadian period in mammalian cultured cells. A more robust phenotype is seen with some of these manipulations in Drosophila although the directionality of the effect (short or long period) or the mechanism is not easily explained. Microarray studies have reported cyclic expression of nucleoporins, and not surprisingly, the authors report cyclic expression for a couple of other NE proteins. Overall, these are interesting observations although they do not quite provide a picture of how the nuclear envelope Figure in the clock mechanism. Additional comments are below:</italic></p><p><xref ref-type="fig" rid="fig1"><italic>Figure 1</italic></xref> <italic>should show quantification of multiple blots for PER2 cycling in the LaminB1 mutant and overexpression backgrounds (</italic><xref ref-type="fig" rid="fig1"><italic>Figure 1</italic>C</xref><italic>). Also, what happens to other clock proteins in these backgrounds?</italic></p><p>We have included quantification of PER2 Western in <xref ref-type="fig" rid="fig1">Figure 1C</xref>. We did not examine other clock proteins at the time of experimentation and thought that PER2 was the most logical representative clock protein for us to test. Unfortunately, the tissue samples were lost in a freezer breakdown in the lab and the mouse lines have been cryopreserved. Although we would like to examine the other clock proteins as well, it would take tremendous amount of time and effort to re-derive these animals and perform the experiments</p><p><italic>Effects of knockdown and over-expression of NE proteins in mammalian cells are subtle. In mice heterozygous for a Lamin</italic> B1 <italic>mutation, or overexpressing LaminB1, PER2 oscillations appear to be altered, although this needs to be better documented, and is not linked to any overt rhythm</italic>.</p><p>From the Western blot in <xref ref-type="fig" rid="fig1">Figure 1C</xref>, the peak PER2 oscillation time for heterozygous Lmnb1 is CT 0 vs CT 20 for WT, and the peak time for LMNB<sup>BAC</sup> is clearly CT 16. Either way, there is a 4 hr phase shift compared to control. Although we did not show the same shift in wheel-running rhythm, we reason that this may be due to the resilience of the SCN clock to perturbations. Therefore we can only observe alterations in peripheral clocks. It is also possible that other molecular changes in addition to PER2 can somehow neutralize the behavior rhythm. We have added this discussion in the text.</p><p><italic>Period lengthening by overexpression of human MAN1 or LBR in Drosophila has a more robust effect, but then knockdown of MAN1 has the same effect. Given that the molecular mechanism is not known (see below for the inconclusive data regarding the effect on cyc), and mutant data are not shown (only a single RNAi for each gene, without any mapping of the RNAi effect) it is difficult to assess the significance of the fly data</italic>.</p><p>Please see point 1 (General) above. As addressed earlier, we have added additional data validating that Lam and LBR are knocked down by RNAi. It is true that not all changes are consistent between mammals and fly, and there is still need for future investigation to understand the differences between these two systems. We have added discussion in the text regarding this point.</p><p><italic>The text indicates that over-expression of human LMNB1 shortens the period in Drosophila, but this is not supported by the data in</italic> <xref ref-type="table" rid="tbl1"><italic>Table 1</italic></xref>.</p><p>We apologize for not making this clear in the original submission. When comparing cryGAL-39/UASLam to cryGAL4-39/+, or UASLam/+;cryGAL4-16/+ to cryGAL4-16/+, there are shortened periods. We have modified the text to clarify this.</p><p><italic>Given that knocking down MAN1 has no effect on cyc, the effect of overexpression could be non-physiological. Also, increased expression of cyc (through MAN1 overexpression) should not lengthen period as shown here, so it is unlikely that the effect on circadian period is going through cyc</italic>.</p><p>We agree that over-expression of MAN1 in flies drives MAN1 much higher than the endogenous level, which may be “non-physiological”, but regardless, it demonstrates in this in vivo system, that increasing MAN1 results in increased cyc expression, consistent with what we demonstrate in cell culture, i.e. increasing MAN1 leads to increased Bmal1 expression. We were not able to demonstrate that knocking down MAN1 reduces cyc in vivo as we showed for Bmal1 in vitro, but we believe this is probably because in vivo knockdown is not as efficient as in vitro, and/or that the in vivo system is more resilient to perturbations compared to in vitro. Thus we do believe that Bmal1/cyc is a target of MAN1 in the clock, although it may not be the only target. When MAN1 is over-expressed in flies, the period is lengthened and cyc appears to be the clock gene that exhibits the largest changes, leading us to suspect that under the condition of MAN1 over-expression, cyc is the primary target of MAN1 in the clock. However, it is certainly possible that MAN1 is also targeting other clock genes. When MAN1 is knocked down in flies, cyc is not affected while period is still lengthened, suggesting that under MAN1 deficient condition, some other clock genes are altered which contribute to the period phenotype. Therefore, we can only draw the conclusion that in MAN1 knock-down flies, the period phenotype is not going through cyc, but we cannot draw the same conclusion for MAN1 over-expressing flies.</p><p><italic>Along the same lines, it would be good to know if the effects on circadian period in mammalian cells are going through BMal1. Does overexpression of BMal1 rescue the long period produced by knockdown of NE proteins?</italic></p><p>Thank you for the suggestion. As mentioned above in point 2 (General), we have included new data to address this question (<xref ref-type="fig" rid="fig4s2">Figure 4–figure supplement 2</xref>).</p><p><italic>Additive effects of SMAD and MAN1 suggest that they are acting independently, and not as part of the same pathway</italic>.</p><p>We agree with the reviewer that this would seem to be the case at first. However, since MAN1’s enhancing effect on BMAL1 requires its RRM domain which is the SMAD interacting domain and the only SMAD that has effect on BMAL1 is SMAD2. For these reasons, MAN1 and SMAD2 may not be acting completely independently.</p><p><italic>The studies mapping MAN1 interactions with the BMAL1 promoter are good, and indicate direct interaction</italic>.</p><p>Reviewer #3:</p><p><italic>This manuscript offers a novel link between nuclear envelope components and circadian clock that is conserved from insects to mammals. The experiments are straightforward and well described. However, some gaps in data presentation need to be addressed</italic>.</p><p><italic>Since the authors have access to the lamin mutant mice and flies with specific perturbation of Ne components, figure (a) showing the circadian behavior phenotype of these animals will substantially strengthen the manuscript. If the het mice lack any appreciable defect in free running circadian period, activity consolidation, phase angle of entrainment, such lack of phenotype may be explained in the discussion section</italic>.</p><p>Neither Lamin b1 heterozygous mutant nor Lamin B1 over-expressing mice show altered wheel-running rhythms (<xref ref-type="fig" rid="fig1s1">Figure 1–figure supplement 1</xref>). We have included these data along with relevant discussion in the revised manuscript.</p><p><italic>Circadian behavior phenotype of the flies should be shown in the main figures to strengthen the manuscript</italic>.</p><p>We have added a new figure to demonstrate the behavioral profiles of flies with NE over-expressed or knocked-down (<xref ref-type="fig" rid="fig2s1">Figure 2–figure supplement 1</xref>).</p><p><italic>siRNA knockdown experiments on Bmal1:Luc expression should ideally have another promoter:Luc construct to rule out the possibility that the down regulation of Bmal1:luc signal is not due to general defect in nuclear integrity or overall transcription</italic>.</p><p>For in vitro transcription assay, Renilla-luc was always used as internal control. Since Renilla-luc was not affected by siRNA, we believe that the effect we observed for Bmal-luc is not due to the general defect in nuclear integrity or overall transcription. In addition, the new data of Bmal1 overexpression rescuing the period change in U2OS cells further demonstrates that the down regulation of Bmal1:luc signal is not due to general defect in nuclear integrity or overall transcription.</p></body></sub-article></article> |