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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">02811</article-id><article-id pub-id-type="doi">10.7554/eLife.02811</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Short report</subject></subj-group><subj-group subj-group-type="heading"><subject>Developmental biology and stem cells</subject></subj-group></article-categories><title-group><article-title>TAF7L modulates brown adipose tissue formation</article-title></title-group><contrib-group><contrib contrib-type="author" id="author-1936"><name><surname>Zhou</surname><given-names>Haiying</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="fn" rid="con1"/><xref ref-type="fn" rid="conf2"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" id="author-12306"><name><surname>Wan</surname><given-names>Bo</given-names></name><xref ref-type="aff" rid="aff3"/><xref ref-type="aff" rid="aff4"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf2"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" id="author-1963"><name><surname>Grubisic</surname><given-names>Ivan</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="fn" rid="con4"/><xref ref-type="fn" rid="conf2"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" id="author-1961"><name><surname>Kaplan</surname><given-names>Tommy</given-names></name><xref ref-type="aff" rid="aff5"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf2"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-1967"><name><surname>Tjian</surname><given-names>Robert</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/></contrib><aff id="aff1"><institution content-type="dept">Department of Molecular and Cell Biology</institution>, <institution>Howard Hughes Medical Institute, University of California, Berkeley</institution>, <addr-line><named-content content-type="city">Berkeley</named-content></addr-line>, <country>United States</country></aff><aff id="aff2"><institution>CIRM Center of Excellence, Li Ka Shing Center For Biomedical and Health Sciences, University of California</institution>, <addr-line><named-content content-type="city">Berkeley</named-content></addr-line>, <country>United States</country></aff><aff id="aff3"><institution content-type="dept">State Key Laboratory of Genetic Engineering, School of Life Sciences</institution>, <institution>Fudan University</institution>, <addr-line><named-content content-type="city">Shanghai</named-content></addr-line>, <country>China</country></aff><aff id="aff4"><institution content-type="dept">California Institute of Quantitative Biosciences</institution>, <institution>University of California, Berkeley</institution>, <addr-line><named-content content-type="city">Berkeley</named-content></addr-line>, <country>United States</country></aff><aff id="aff5"><institution content-type="dept">School of Computer Science and Engineering</institution>, <institution>The Hebrew University of Jerusalem</institution>, <addr-line><named-content content-type="city">Jerusalem</named-content></addr-line>, <country>Israel</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Kadonaga</surname><given-names>James T</given-names></name><role>Reviewing editor</role><aff><institution>University of California, San Diego</institution>, <country>United States</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>tjianr@hhmi.org</email></corresp></author-notes><pub-date date-type="pub" publication-format="electronic"><day>29</day><month>05</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e02811</elocation-id><history><date date-type="received"><day>16</day><month>03</month><year>2014</year></date><date date-type="accepted"><day>25</day><month>05</month><year>2014</year></date></history><permissions><copyright-statement>© 2014, Zhou et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Zhou et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/3.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/3.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife02811.pdf"/><related-article ext-link-type="doi" id="ra1" related-article-type="article-reference" xlink:href="10.7554/eLife.00170"/><related-article ext-link-type="doi" id="ra2" related-article-type="article-reference" xlink:href="10.7554/eLife.02559"/><related-article ext-link-type="doi" id="ra3" related-article-type="commentary" xlink:href="10.7554/eLife.03575"/><abstract><object-id pub-id-type="doi">10.7554/eLife.02811.001</object-id><p>Brown adipose tissue (BAT) plays an essential role in metabolic homeostasis by dissipating energy via thermogenesis through uncoupling protein 1 (UCP1). Previously, we reported that the TATA-binding protein associated factor 7L (TAF7L) is an important regulator of white adipose tissue (WAT) differentiation. In this study, we show that TAF7L also serves as a molecular switch between brown fat and muscle lineages in vivo and in vitro. In adipose tissue, TAF7L-containing TFIID complexes associate with PPARγ to mediate DNA looping between distal enhancers and core promoter elements. Our findings suggest that the presence of the tissue-specific TAF7L subunit in TFIID functions to promote long-range chromatin interactions during BAT lineage specification.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02811.001">http://dx.doi.org/10.7554/eLife.02811.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.02811.002</object-id><title>eLife digest</title><p>Mammals produce two distinct types of adipose tissue: white adipose tissue (white fat) is the more common type and is used to store energy; brown adipose tissue (brown fat) is mostly found in young animals and infants, and it plays an important role in dissipating energy as heat rather than storing it in fat for future use. In adults, higher levels of brown fat are associated with lower levels of fat overall, so there is considerable interest in learning more about this form of fat to help address rising levels of obesity in the world.</p><p>Building on previous work in which they showed that a gene control protein called TAF7L has a central role in the development of the cells that make up white adipose tissue, Zhou et al. now show that this protein also helps to regulate the development of brown adipose tissue.</p><p>Mice lacking the gene for this protein developed embryos with 40% less brown fat than wild-type mice with the gene. Moreover, these mice developed muscle-like cells in the regions that should have contained brown fat. Gene expression analysis revealed that ‘knocking out’ the gene for TAF7L changed the expression of more than a thousand genes in these mice.</p><p>Zhou et al. suggest that TAF7L works as a ‘molecular switch’ that determines whether certain precursor cells (called mesenchymal stem cells) go on to become brown fat cells or muscle cells. A future challenge will be to devise interventions to regulate the activity or levels of TAF7L as a potential means of modulating brown fat depots in animals and humans.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02811.002">http://dx.doi.org/10.7554/eLife.02811.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>PPARγ</kwd><kwd>DNA looping</kwd><kwd>UCP1</kwd><kwd>BAT</kwd><kwd>obesity</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><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/10.13039/100000011</institution-id><institution>Howard Hughes Medical Institute (HHMI)</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Zhou</surname><given-names>Haiying</given-names></name><name><surname>Grubisic</surname><given-names>Ivan</given-names></name><name><surname>Tjian</surname><given-names>Robert</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>TAF7L acts as a molecular switch that regulates the development of mesenchymal stem cells.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><p>Recent studies found that adult humans retain active BAT depots capable of sustaining elevated basal metabolic rates compared to WAT and those higher levels of BAT correlate with lower body mass index (<xref ref-type="bibr" rid="bib4">Carey and Kingwell, 2013</xref>; <xref ref-type="bibr" rid="bib7">Harms and Seale, 2013</xref>; <xref ref-type="bibr" rid="bib5">Chechi et al., 2014</xref>; <xref ref-type="bibr" rid="bib8">Kajimura and Saito, 2014</xref>). These findings inspired heightened efforts to better understand the formation of BAT during mammalian development. Previously, we showed that TAF7L, an orphan TBP-associated factor cooperates with PPARγ in directing WAT gene regulation (<xref ref-type="bibr" rid="bib23">Zhou et al., 2013a</xref>). Given the central role of PPARγ in both WAT and BAT development (<xref ref-type="bibr" rid="bib3">Cannon and Nedergaard, 2004</xref>; <xref ref-type="bibr" rid="bib10">Kajimura et al., 2010</xref>), we wondered whether TAF7L might also serve as a co-activator regulating the formation of BAT.</p><p>To test this hypothesis and assess the functional requirement for TAF7L during BAT development, we performed haematoxylin & eosin (H&E) staining and immunostaining using FABP4 and UCP1 antibodies to delineate regions of BAT in wild-type (WT) and <italic>Taf7l</italic> knockout (KO) embryos. As expected, FABP4 stains both WAT and BAT, while UCP1 only stains BAT (<xref ref-type="bibr" rid="bib14">Pedersen et al., 2001</xref>), which is filled with multilocular lipid droplets (<xref ref-type="fig" rid="fig1">Figure 1A</xref>). We found that UCP1<sup>+</sup> BAT in <italic>Taf7l</italic> KO mice become largely disorganized, is significantly reduced in size and contains decreased lipid levels (<xref ref-type="fig" rid="fig1">Figure 1A</xref>, <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1A–C</xref>). Measurement of dissected BAT pads showed a ∼40% weight reduction of BAT in <italic>Taf7l</italic> KO animals compared to WT embryos (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1B</xref>). Intriguingly, skeletal muscle-like tissue emerges and invades regions that normally contain BAT in <italic>Taf7l</italic> KO mice (<xref ref-type="fig" rid="fig1">Figure 1A</xref>, <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1D</xref>), as revealed by a more pronounced red color of <italic>Taf7l</italic> KO BAT and the staining of pan-skeletal muscle marker myosin heavy chain (MYHC) (<xref ref-type="bibr" rid="bib2">Beylkin et al., 2006</xref>; <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1C,E</xref>). Our data suggest that loss of TAF7L substantially alters the relative proportion of BAT and muscle lineages in vivo.<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.02811.003</object-id><label>Figure 1.</label><caption><title>TAF7L is required for proper mouse brown adipose tissue (BAT) formation.</title><p>(<bold>A</bold>) Wild type (WT) and <italic>Taf7l</italic> knockout (<italic>Taf7l</italic> KO) interscapular BAT from E18.5 embryos was stained with haematoxylin and eosin (H&E), FABP4, and UCP1 (25X magnification shown). Rightmost panel shows FABP4 staining at 200X magnification. (<bold>B</bold>) Left panel: scatterplot shows gene expression profile in WT versus <italic>Taf7l</italic> KO BAT tissue; red dots represent genes up-regulated in <italic>Taf7l</italic> KO, green dots represent down-regulated genes in <italic>Taf7l</italic> KO. Right panel: gene ontology analysis shows functional groups of genes changed by at least threefold. (<bold>C</bold>) Difference in expression of either muscle-specific (top) or brown fat (bottom) genes between WT and <italic>Taf7l</italic> KO BAT with progressive red and green shades showing degrees of up- and down-regulation, respectively.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02811.003">http://dx.doi.org/10.7554/eLife.02811.003</ext-link></p></caption><graphic xlink:href="elife02811f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02811.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>Depletion of TAF7L shifts BAT to muscle lineage.</title><p>(<bold>A</bold>) UCP1-stained BAT samples as in <xref ref-type="fig" rid="fig1">Figure 1A</xref> for three additional animals (2WT and 1 TAF7L KO). (<bold>B</bold>) Average BAT weight from WT (n = 10) and <italic>Taf7l</italic> KO embryos (n = 9). (<bold>C</bold>) Photographs of whole BAT from 1- or 4-month-old WT and <italic>Taf7l</italic> KO mice. (<bold>D</bold>) High-magnification images (200X) of similar areas of WT and <italic>Taf7l</italic> KO embryonic BAT stained with FABP4 antibody. (<bold>E</bold>) Haematoxylin and eosin (H&E) and myosin heavy chain (MYHC) staining of <italic>Taf7l</italic> KO embryos shows muscle–tissue structures along the BAT at high magnification (200X).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02811.004">http://dx.doi.org/10.7554/eLife.02811.004</ext-link></p></caption><graphic xlink:href="elife02811fs001"/></fig><fig id="fig1s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02811.005</object-id><label>Figure 1—figure supplement 2.</label><caption><title>RT-qPCR comparison of gene expression in WT and <italic>Taf7l</italic> KO BAT.</title><p>Expression levels of BAT-specific genes <italic>Ucp1</italic>, <italic>Cidea</italic>, <italic>Pgc1a</italic>, and <italic>Scd1</italic>, muscle-specific genes <italic>Myhc</italic>, <italic>Myh1, Myh4</italic>, and <italic>Myot</italic>. Common fat marker gene <italic>Adipoq</italic>, adipocyte progenitor marker <italic>Dlk1</italic>, <italic>Taf7l</italic> and <italic>Cyclophilin</italic> were used as control. mRNA levels in WT BAT was assigned to 1, and mRNA levels of each gene in <italic>Taf7l</italic> KO BAT was compared to WT BAT. RT-qPCR was normalized to the levels of <italic>Cyclophilin</italic> (L). *p<0.05, data is mean and SEM is from triplicates.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02811.005">http://dx.doi.org/10.7554/eLife.02811.005</ext-link></p></caption><graphic xlink:href="elife02811fs002"/></fig></fig-group></p><p>Next, we analyzed global gene expression profiles in WT and <italic>Taf7l</italic> KO BAT by RNA-sequencing (mRNA-seq) (<xref ref-type="fig" rid="fig1">Figure 1B</xref>). Over a thousand genes exhibited altered expression levels by >threefold upon <italic>Taf7l</italic> KO. In particular, BAT-selective genes such as <italic>Ucp1</italic>, <italic>Pgc1α</italic>, <italic>Cidea</italic>, and <italic>Scd1</italic> involved in brown fat differentiation, thermogenesis, and mitochondrial function became significantly down-regulated (<xref ref-type="fig" rid="fig1">Figure 1C</xref>, <xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2</xref>; <xref ref-type="bibr" rid="bib7">Harms and Seale, 2013</xref>; <xref ref-type="bibr" rid="bib13">Ohno et al., 2013</xref>). Consistent with the observed enhanced skeletal muscle morphological phenotype (<xref ref-type="fig" rid="fig1">Figure 1A</xref>), we observed a concomitant up-regulation of skeletal muscle genes including <italic>Myh1-8</italic>, <italic>Myf5</italic>, and <italic>Pax3</italic> (<xref ref-type="fig" rid="fig1">Figure 1C</xref>, <xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2</xref>; <xref ref-type="bibr" rid="bib1">Abe et al., 2013</xref>). Loss of TAF7L also led to activation of select genes in the formation of cartilage and bone development, but the majority of up-regulated genes appear to be involved in skeletal muscle development and function. We speculate that because BAT and muscle share common MYF5<sup><italic>+</italic></sup> dermotomal precursors (<xref ref-type="bibr" rid="bib9">Kajimura et al., 2009</xref>; <xref ref-type="bibr" rid="bib15">Seale and Lazar, 2009</xref>; <xref ref-type="bibr" rid="bib16">Seale et al., 2011</xref>), this relationship might favor the switch from BAT to skeletal muscle as shown in <xref ref-type="fig" rid="fig1">Figure 1A</xref>. Our findings suggest that TAF7L tips the balance in favor of BAT development at the expense of skeletal muscle.</p><p>To examine the effects of TAF7L loss on brown adipocyte differentiation in vitro, we used C3H10T1/2 mesenchymal stem cells which is able to form multiple cell lineages including adipocytes, muscle, cartilage, and bone. We first depleted TAF7L levels in C3H10T1/2 cells by RNA interference using previously described shTAF7L and control shGFP constructs followed by induction of BAT differentiation (<xref ref-type="bibr" rid="bib23">Zhou et al., 2013a</xref>). As expected, we observed efficient formation of round fat cells peaking at day 2 post-induction in C3H10T1/2 cells treated with shGFP controls (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). By contrast, TAF7L-depleted C3H10T1/2 cells formed elongated muscle-like cells rather than round fat laden adipocytes (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). mRNA analysis of post-induction cells (day2) revealed that specific muscle markers (<italic>Myf5</italic>, <italic>Myod1</italic>, and <italic>Mef2c</italic>) become activated in cells treated with shTAF7L, in some cases reaching up to ∼30% of their expression level in myotubes (<xref ref-type="fig" rid="fig2">Figure 2B</xref>). At the same time, protein (PPARγ and UCP1) and mRNA (<italic>Pgc1α</italic>, <italic>Ucp1</italic>, and <italic>Cidea</italic>) levels of brown adipocyte markers become significantly reduced 5 days post-induction, suggesting an efficient blockade of brown fat differentiation (<xref ref-type="fig" rid="fig2">Figure 2C,D</xref>). We confirmed these results using primary WT and <italic>Taf7l</italic> KO brown adipocytes (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>). Our findings suggest that loss of TAF7L shifts mesenchymal stem cells in culture from adopting an adipocyte fate toward formation of muscle-like cells.<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.02811.006</object-id><label>Figure 2.</label><caption><title>TAF7L bidirectionally regulates BAT/muscle lineages.</title><p>(<bold>A</bold>) 2 days (2D) and 5 days (5D) post-induced control (shCNTL) and TAF7L knockdown (shTAF7L) C3H10T1/2 cells stained with Oil Red O. (<bold>B</bold>) mRNA levels of myoblast genes <italic>Myf5</italic>, <italic>Myod1</italic>, <italic>Mef2c</italic>, <italic>Myhc</italic>, and <italic>Mlc</italic> in 2D post-induced shCNTL and shTAF7L C3H10T1/2 cells, compared to C2C12-induced myotubes. (<bold>C</bold>) Protein levels of TAF7L, PPARγ<italic>,</italic> and UCP1 in 5D post-induced shCNTL and shTAF7L cells; Pol II was used as loading control. (<bold>D</bold>) mRNA levels of brown fat genes (<italic>Pgc1a, Ucp1, and Cidea</italic>) in 5D post-induced control and shTAF7L cells. (<bold>E</bold>) 5D post-induced control (C2C12.CNTL) and TAF7L-expressing C2C12 (C2C12.TAF7L) cells were stained with Oil Red O. (<bold>F</bold>) mRNA levels of myoblast genes <italic>Myf5</italic> and <italic>Myod1</italic> in pre-induction cells. (<bold>G</bold>) mRNA levels of BAT-specific genes (<italic>Ucp1, Cidea, Cox8b, Pgc1a, and Pparα)</italic> 5D post-differentiation. *p<0.05, data is mean and SEM is from triplicates.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02811.006">http://dx.doi.org/10.7554/eLife.02811.006</ext-link></p></caption><graphic xlink:href="elife02811f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02811.007</object-id><label>Figure 2—figure supplement 1.</label><caption><title>TAF7L is required for brown fat cell differentiation from primary brown adipocytes.</title><p>(<bold>A</bold>) Isolated brown adipose progenitor cells from WT (TAF7L+/Y) and <italic>Taf7l</italic> KO (TAF7L-/Y) mice was induced with brown adipocyte differentiation regime for 5 days and then stained with Oil Red O. (<bold>B</bold> and <bold>C</bold>) mRNA levels of fat-selective genes (<bold>B</bold>) and muscle-selective genes (<bold>C</bold>) pre-differentiation. *p<0.05, data is mean and SEM is from triplicates. (<bold>D</bold>) Expression levels of <italic>Taf7l</italic> and brown adipocyte marker genes on cells from <bold>A</bold>, the expression levels of genes in 5D post-induced <italic>Taf7l</italic> knockout cells were compared to WT cells, whose levels were assigned to 1.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02811.007">http://dx.doi.org/10.7554/eLife.02811.007</ext-link></p></caption><graphic xlink:href="elife02811fs003"/></fig><fig id="fig2s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02811.008</object-id><label>Figure 2—figure supplement 2.</label><caption><title>Gene expression analysis of control (CNTL) and TAF7L-expressing (TAF7L) C2C12 cells from 0 (0D) to 5 days (5D) post BAT-induction.</title><p>mRNA levels of <italic>Ucp1</italic>, <italic>Prdm16</italic>, <italic>Cidea</italic>, <italic>Pgc1a</italic>, <italic>Taf7l</italic>, and <italic>Myod1</italic> are plotted as relative value to 0D CNTL cells, data is mean and SEM is from triplicates.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02811.008">http://dx.doi.org/10.7554/eLife.02811.008</ext-link></p></caption><graphic xlink:href="elife02811fs004"/></fig></fig-group></p><p>We next examined TAF7L gain-of-function in vitro using C2C12 myoblasts that can efficiently form myotubes in vitro. Instead of treating C2C12 cells with the normal muscle-inducing protocol, we applied the brown fat differentiation regime to either control C2C12 cells (C2C12.CNTL) or myoblasts ectopically expressing TAF7L (C2C12.TAF7L) and found that only C2C12.TAF7L cells form multilocular brown fat cells (<xref ref-type="fig" rid="fig2">Figure 2E</xref>; <xref ref-type="bibr" rid="bib23">Zhou et al., 2013a</xref>). RNA analysis showed that TAF7L in C2C12 cells represses the expression of myoblast genes <italic>Myf5</italic> and <italic>Myod</italic>1 while significantly increasing the expression of brown fat-selective genes (<italic>Ucp1, Cidea, Pgc1α,</italic> and <italic>Pparα)</italic> post-differentiation (<xref ref-type="fig" rid="fig2">Figure 2F,G</xref>, <xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2</xref>), suggesting that TAF7L can drive myoblasts toward the brown fat lineage (<xref ref-type="bibr" rid="bib17">Seale et al., 2008</xref>). These gain of function in vitro results are consistent with the switch from BAT to muscle we observed in <italic>Taf7l</italic> KO animals in vivo.</p><p>Previously, we showed that TAF7L associates with PPARγ when overexpressed in 293T cells (<xref ref-type="bibr" rid="bib23">Zhou et al., 2013a</xref>). Here, we wanted to confirm this interaction in C3H10T1/2 cells upon brown fat induction. By using sequential immunoprecipitations of doubly tagged TAF7L (FLAG&V5), followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and western blotting, we found that TAF7L pulls down a subset of TAFs normally associated with the prototypical TFIID complex. Although this coIPed complex contained a number of canonical TFIID TAF subunits, we nevertheless suspect that it may represent a fat-specific and structurally distinct complex (Fat-TFIID) because it behaved in a manner distinct from canonical TFIID by size exclusion chromatography (data not shown). These immunoprecipitation assays also revealed that PPARγ co-purifies with TAF7L in the Fat-TFIID complex from differentiated C3H10T1/2 cells but not from control FLAG-V5-GFP cells (<xref ref-type="fig" rid="fig3">Figure 3A,B</xref>) nor with canonical TFIID lacking TAF7L (data not shown). Although we failed to detect PRDM16 in these affinity purification experiments, we did find that ectopically expressed TAF7L and PRDM16 associate with each other in 293T cells (<xref ref-type="fig" rid="fig3">Figure 3C</xref>). These data suggest that TAF7L-containing Fat-TFIID has gained the ability to bind endogenous PPARγ, which we speculate might facilitate its association with distinct cofactors such as PRDM16 in BAT or TLE3 in WAT to differentially regulate brown and white adipocyte formation (<xref ref-type="bibr" rid="bib20">Villanueva et al., 2011</xref>, <xref ref-type="bibr" rid="bib21">2013</xref>).<fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.02811.009</object-id><label>Figure 3.</label><caption><title>TAF7L within a Fat-TFIID associates with PPARγ and facilitates DNA looping formation.</title><p>(<bold>A</bold>) Silver staining shows co-immunoprecipitated proteins in FLAG-V5-GFP-expressing (GFP, lane 1) and FLAG-V5-TAF7L-expressing (TAF7L, lane 2) C3H10T1/2 differentiated fat cells. Comparative LC-MS/MS analysis identified peptides matching with TFIID subunits (TAF1, TAF4, TBP, TAF5, TAF6, and TAF10) and PPARγ in TAF7L-expressing but not in GFP-expressing cells. FKBP15 and ACSL1 are representative non-specific associated proteins. (<bold>B</bold>) Western blot analyzing input and immunoprecipitated protein levels of PPARγ, TAF4, TAF7L, and TBP in samples from <bold>A</bold>. (<bold>C</bold>) HA-tagged TAF7L and FLAG-tagged PRDM16 were overexpressed in 293T cells, immunoprecipitations were performed on both FLAG and HA antibodies and followed by Western blotting with FLAG and HA antibodies. (<bold>D</bold>) Upper panel, schematic picture shown the distance between distal enhancer (D) and core promoter (P) of <italic>Cidea</italic> gene; middle panel, read accumulation of TAF7L, TBP, and PPARγ on <italic>Cidea</italic> locus in differentiated fat cells from ChIP-seq analysis; bottom panel, 3C experiments assess long-range DNA interactions between the TAF7L/PPARγ binding distal enhancer (D) and core promoter (P) sites of <italic>Cidea</italic> in WT and <italic>Taf7l</italic> KO BAT. ▲, anchor point. Also see <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1C</xref>. (<bold>E</bold>) Model shows TAF7L-mediating regulatory DNA looping to specify BAT differentiation from mesenchymal stem cells (MSC).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02811.009">http://dx.doi.org/10.7554/eLife.02811.009</ext-link></p></caption><graphic xlink:href="elife02811f003"/></fig><fig id="fig3s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02811.010</object-id><label>Figure 3—figure supplement 1.</label><caption><title>TAF7L colocalizes with PPARγ on core promoters and enhancers of BAT-specific genes.</title><p>ChIP-seq read accumulation for TAF7L and PPARγ in both pre- and post-differentiated C3H10T1/2 cells on <italic>Ajap1</italic>/<italic>Prdm16</italic> locus (<bold>A</bold>) and <italic>Ucp1</italic>/<italic>Bmod2</italic> locus (<bold>B</bold>). (<bold>C</bold>) 3C experiments assess long-range interactions between the TAF7L/PPAR<italic>γ</italic> binding distal enhancer (D) and core promoter (P) sites of <italic>Scd1</italic> in WT and <italic>Taf7l</italic> KO BAT. ▲, anchor point.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02811.010">http://dx.doi.org/10.7554/eLife.02811.010</ext-link></p></caption><graphic xlink:href="elife02811fs005"/></fig></fig-group></p><p>To probe the potential functional relevance of these interactions, we next examined the genome-wide co-occupancy of TAF7L and PPARγ at BAT-selective genes in differentiated C3H101/2 adipocytes. Due to the presence of rosiglitazone in the differentiation regime, our previous ChIP-seq analysis of C3H10T1/2-derived fat cells represented a mixture of white and brown fat cells, as manifested by enhanced <italic>Cidea</italic>, <italic>Elovl3</italic>, and <italic>Ucp1</italic> expression (<xref ref-type="bibr" rid="bib7">Harms and Seale, 2013</xref>). Here, we have re-analyzed the binding site data for TAF7L, TBP, and PPARγ focusing on loci of activated BAT-selective genes (<italic>Cidea</italic>, <italic>Prdm16</italic>, and <italic>Ucp1)</italic> (<xref ref-type="fig" rid="fig3">Figure 3D</xref>, <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>). As shown in <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1A, B</xref>, TAF7L and PPARγ co-occupy overlapping regions at the <italic>Prdm16</italic> and <italic>Ucp1</italic> loci in post-differentiated (-post) cells, but not at control genes (<italic>Ajap1</italic> and <italic>Bmod2)</italic> nor to the same genes in pre-differentiated cells (-pre). These findings suggest that PPARγ and TAF7L likely function as a coupled activator–coactivator pair that regulates BAT-specific gene transcription much as we previously described for WAT differentiation.</p><p>To extend our analysis of TAF7L mechanisms in regulating fat-specific gene transcription, we next employed chromatin conformation capture (3C) to assess its participation in transcription factor-mediated long distance DNA looping at two TAF7L-activated genes <italic>Cidea</italic> and <italic>Scd1</italic> (<xref ref-type="fig" rid="fig3">Figure 3D</xref>, <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>; <xref ref-type="bibr" rid="bib12">Liu et al., 2011</xref>). These two target genes are bound by TAF7L and PPARγ both at their core promoters as well as at their distal enhancers located ∼10 kb away. In contrast, TBP only binds at the core promoters of these genes. Our 3C results revealed the likely formation of DNA looping between core promoters and distal enhancers in WT BAT for both loci while significantly reduced looping was seen in BAT lacking TAF7L (<xref ref-type="fig" rid="fig3">Figure 3D</xref>, <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1C</xref>). These data suggest that the presence/incorporation of TAF7L into the putative Fat-TFIID complex likely mediates or enhances long-range chromatin transactions that influence BAT cell fate.</p><p>Taken in aggregate, our findings strongly suggest that TAF7L may serve as a key component of an alternative TFIID complex that favors brown fat formation vs muscle lineages. We do not fully understand the molecular mechanisms driving the enhanced formation of skeletal muscle upon loss of TAF7L, but we speculate that TAF7L depletion decreases the transcription of BAT-selective genes, which might indirectly de-repress muscle-selective genes thereby switching precursor cells toward a skeletal muscle lineage. Moreover, the presence of TAF7L potentiates the efficient participation of a specialized Fat-TFIID complex in long-range chromatin interactions. Such a DNA looping mechanism is likely mediated through association with the fat-specific transcription factor PPARγ (<xref ref-type="fig" rid="fig3">Figure 3E</xref>). These new findings and our previous studies support the notion that TAF7L functions as a common cofactor regulator required for both WAT and BAT development.</p><sec id="s1" sec-type="materials|methods"><title>Materials and methods</title><sec id="s1-1"><title>Vectors and plasmids</title><p>Full-length green fluorescent protein (GFP) or <italic>Taf7l</italic> cDNAs were inserted into p3XFLAG-CMV-10 vector to construct pCMV-FLAG-V5-GFP/TAF7L or into pCS2+ vector to construct pCS2+HA-TAF7L; full-length PRDM16 cDNA was inserted into p3XFLAG-CMV-10 vector to construct pCMV-FLAG-PRDM16.</p></sec><sec id="s1-2"><title>Antibodies</title><p>TAF4 (612054; BD, San Jose, CA), TBP (62126; abcam, Cambridge, MA), V5 (R960-25; Invitrogen, Carlsbad, CA), HA (9110; abcam), FLAG (F31655; Sigma, Saint Louis, MI) MYHC (05-716; Millipore, Billerica, MA), FABP4 (66682; abcam), UCP1(10983; abcam), TAF7L (prepared in-house, Covance, Denver, PA), Pol II (monoclonal 8WG16, protein-A purified), PPARγ (sc-7196), ANTI-FLAG M2 Affinity Gel (A2220; Sigma), Anti-V5-agarose affinity gel (A7345; Sigma).</p></sec><sec id="s1-3"><title>Cells culture, stable cell line establishment</title><p>C3H10T1/2 and C2C12 cells were cultured in high glucose DMEM with 10% fetal bovine serum at 5% CO<sub>2</sub>.</p><p>C3H10T1/2 cells stably expressing FLAG-V5-GFP/TAF7L were established by transfection of the pCMV-FLAG-V5-GFP/TAF7L plasmids followed by 2 weeks of 1 µg/µl neomycin selection.</p></sec><sec id="s1-4"><title>Brown adipocyte differentiation, Oil red O staining, and C2C12 myogenesis</title><p>For adipogenesis, C3H10T1/2, control and FLAG-TAF7L-expressing C2C12, and FLAG-V5-GFP/TAF7L-expressing C3H10T1/2 cells were grown in high glucose DMEM supplemented with 10% fetal bovine serum. At confluence, cells were exposed to induction medium containing dexamethasone (1 μM) (265005, CalBiochem, San Diego, CA), isobutylmethylxanthine (IBMX, 0.5 mM) (I-5879, Sigma), Indomethacin (0.125 mM) (I-7378, Sigma), and 10% FBS. 3 days later, cells were further cultured in high glucose DMEM-containing insulin (5 μg/ml) (19278, Sigma), T3 (0.1 μM) (T-2877, Sigma), and rosiglitazone (5 μM) (71740, Cayman, Ann Arbor, MI) until they were ready for harvest.</p><p>For Oil red O staining, pre- and post-differentiated C3H10T1/2 cells, shGFP and shTAF7L-treated C3H10T1/2 cells, C2C12.CNTL and C2C12.TAF7L cells were washed once in PBS and fixed with freshly prepared 4% formaldehyde in 1 × PBS for 30 min, followed by standard Oil red O staining method described previously (<xref ref-type="bibr" rid="bib23">Zhou et al., 2013a</xref>).</p><p>C2C12 myogenesis procedure followed a previous study (<xref ref-type="bibr" rid="bib23">Zhou et al., 2013a</xref>).</p></sec><sec id="s1-5"><title>RNA isolation and real-time PCR analysis</title><p>Total RNA from cultured cells or mouse tissues was isolated using QIAGEN RNeasy Plus mini columns according to the manufacturer's instructions (Qiagen, Germany). For RT-qPCR analysis, 1 μg total RNA was reverse transcribed using cDNA reverse transcription kit (Invitrogen). PCR reactions using the SYBR Green PCR Master Mix (Applied Biosystems, Grand Island, NY) were performed according to the manufacturer's instruction using an ABI 7300 real time PCR machine (Applied Biosystems). Relative expression of mRNA was determined after normalization to cyclophilin gene. Student's <italic>t</italic> test was used to evaluate statistical significance (<xref ref-type="bibr" rid="bib24">Zhou et al., 2013b</xref>).</p></sec><sec id="s1-6"><title>Western blot analysis, immunoprecipitation, and silver staining</title><p>Whole cell extracts were prepared from cells by homogenization in lysis buffer containing 50 mM Tris-Cl, pH 8.0, 500 mM NaCl, and 0.1% Triton X-100, 10% glycerol, and 1 mM EDTA, supplemented with protease inhibitor cocktail (Roche, Indianapolis, IN) and phenylmethylsulphonyl fluoride (PMSF). 15 μg of whole-cell lysates were separated by SDS-PAGE and transferred to nitrocellulose membrane. For immunoblotting, membranes were blocked in 10% milk, 0.1% Tween-20 in TBS for 30 min, and then incubated with TAF7L, UCP1, PPARγ, and POL II antibodies for 2 hr at room temperature; detailed Western blotting procedure was performed as previously described (<xref ref-type="bibr" rid="bib22">Zhou et al., 2006</xref>).</p><p>3 mg whole-cell extracts from FLAG-V5-GFP/TAF7L post-differentiated adipocytes were immunoprecipitated with 100 µl of ANTI-FLAG M2 Affinity Gel under the conditions of 0.3 M NaCl, 0.2% NP-40 at 4°C overnight. After extensive washing with buffer containing 0.3 M NaCl and 0.1% NP-40, proteins were eluted from the affinity gel with 100 µg/ml FLAG peptide in 0.1 M NaCl Tris buffer. Elutions from both samples were subsequently immunoprecipitated with 40 µl Anti-V5-agarose affinity gel antibody with a similar procedure as above. After extensive washes, proteins were eluted with 40 µl pH2.5 0.1 M glycine for three times and immediately neutralized with 12 µl of pH9.0 2 M Tris-Cl. 10 µl of eluted samples were subjected to 10% SDS-PAGE and followed by standard silver staining or by western blotting analysis with V5, PPARγ, TAF4, and TBP antibodies to detect tagged-proteins in the inputs and associated proteins as previously described (<xref ref-type="bibr" rid="bib7">Harms and Seale, 2013</xref>). The remaining samples were precipitated by 20% trichloroacetic acid, and the precipitates were sent to liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect peptides derived from proteins pulled-down by FLAG-V5-GFP/TAF7L.</p><p>500 μg whole-cell extracts from 293T cells transfected with HA-TAF7L and FLAG-PRDM16 were immunoprecipitated with FLAG or HA antibodies at 4°C for overnight under the conditions of 0.3 M NaCl and 0.2% NP-40, 30 μl protein A/G beads were added and incubated for additional 2 hr at 4°C, after extensive washing with buffer containing 0.3 M NaCl and 0.1% NP-40, remaining beads were subjected to 10% SDS-PAGE and followed by western blotting analysis with FLAG and HA antibodies to detect tagged-proteins in the inputs and IPs as described previously (<xref ref-type="bibr" rid="bib23">Zhou et al., 2013a</xref>).</p></sec><sec id="s1-7"><title>Animals and genotype analysis</title><p>The derivation of <italic>Taf7l</italic> KO mice has been previously described (<xref ref-type="bibr" rid="bib9">Kajimura et al., 2009</xref>). All animal experiments were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to animal use protocols (#R007) approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, Berkeley. Mice were maintained on a standard rodent chow diet with 12 hr light and dark cycles. <italic>Taf7l</italic> KO mouse line was maintained on a C57BL/6J background. Genotyping was performed by PCR as previously described (<xref ref-type="bibr" rid="bib6">Cheng et al., 2007</xref>).</p></sec><sec id="s1-8"><title>Immunohistochemistry</title><p>For histological analysis on interscapular BAT of E18.5 embryos from WT and <italic>Taf7l</italic> KO mice, freshly-harvested mouse embryos were genotyped and the interscapular regions of embryos were transversally dissected and then fixed in 10% formaldehyde for 24 hr at 4°C; tissue was embedded in paraffin using the microwave method as described and then sectioned into 8–10 μm to mount on slides (<xref ref-type="bibr" rid="bib24">Zhou et al., 2013b</xref>). The following immunohistochemistry by haematoxylin and eosin (H&E) staining and FABP4, UCP1, and MYHC immunostaining were performed using the method described previously (<xref ref-type="bibr" rid="bib23">Zhou et al., 2013a</xref>).</p></sec><sec id="s1-9"><title>Preparation of primary brown adipocytes and brown fat differentiation</title><p>Fresh interscapular brown adipose tissues were removed from 3-week-old euthanized WT and <italic>Taf7l</italic> KO mice and finely minced, digested with 0.25% trypsin for 30 min at 37°C, and centrifuged for 5 min at 2,000×<italic>g</italic> to get rid of debris. The pellet was resuspended in culture media before plated on gelatin coated plates. Cells were cultured at 37°C in high glucose DMEM supplemented with 20% FBS. Brown adipocyte differentiation and staining were followed the same procedure as C3H10T1/2 cells.</p></sec><sec id="s1-10"><title>mRNA-seq libraries preparation and deep sequencing</title><p>Total RNA was extracted from BAT, carefully excised to get rid of surrounding tissue based on the texture and color, of 6 WT and 6 <italic>Taf7l</italic> KO mice. RNA was extracted separately for each mouse by RNeasy Plus Mini Kit (Qiagen) and then pooled for WT or <italic>Taf7l</italic> KO samples; 4 μg of each RNA pool was used to purify mRNA using oligo (dT) and subsequently converted into multiplexed mRNA-seq libraries using mRNA-Seq Trueseq Kit (Illumina, San Diego, CA). Samples were multiplexed and sequenced in one lane on an Illumina HiSeq 2000 sequencer (QB3 Vincent J Coates Genomics Sequencing Library, University of California, Berkeley, CA). 50 bp single-end reads were used for both samples; each sample produced over 30 million reads.</p></sec><sec id="s1-11"><title>Digital gene expression of mRNA-seq and gene ontology analysis</title><p>Reads were mapped to the mouse transcriptome (mm10), using TopHat (<xref ref-type="bibr" rid="bib11">Langmead et al., 2009</xref>; <xref ref-type="bibr" rid="bib18">Trapnell et al., 2009</xref>), version v1.4.0., with default parameters. We then applied cufflinks (<xref ref-type="bibr" rid="bib19">Trapnell et al., 2010</xref>), version v1.3.0, using the default parameters except: --max-mle-iterations 1, to estimate the digital expression levels at each transcript. Gene ontology analysis was done using DAVID Bioinformatics Resources 6.7.</p></sec><sec id="s1-12"><title>Chromosome conformation capture (3C)</title><p>3C analysis was performed as previously described on WT and <italic>Taf7l</italic> KO BAT (<xref ref-type="bibr" rid="bib12">Liu et al., 2011</xref>), which was carefully excised to get rid of all other possible tissue based on the brown color and the tissue texture. 2 mg of freshly dissected interscapular BAT from 6 WT or 6 <italic>Taf7l</italic> KO mice were minced, homogenized extensively to nearly single cells and washed, crosslinked with 1% formaldehyde for 15 min at 4°C and then quenched with 0.125 M glycine for 5 min.</p><p>Crosslinked BATs were lysed and the chromatin was digested with 8 U HaeIII (NEB) for the <italic>Cidea</italic> and <italic>Scd1</italic> loci. Digested fragments were cleaned and subsequently ligated with 60 units T4 DNA ligase (Invitrogen) for 4 hr at 16°C. Following proteinase K digestion and decrosslinking at 65°C overnight, DNA fragments was recovered by phenol–chloroform extraction.</p><p>Control templates were generated using individual BAC clones covering <italic>Cidea</italic> or <italic>Scd1</italic> locus (Bacpac). 10 µg of BAC DNA was digested with 20 units HaeIII and then randomly ligated with 10 units T4 DNA ligase in 50 µl volume.</p><p>3C primers were designed upstream and downstream of the core promoter site (P). Primers annealing to distal enhancers (D) corresponding to TAF7L and PPARγ binding sites on either <italic>Cidea</italic> or <italic>Scd1</italic> were used as anchor points. 3C analysis was done by qPCR using as a primer pair the anchor point primer and one annealing to region under investigation. Each data point in WT and <italic>Taf7l</italic> KO BAT was normalized by the BAC control template and presented as interaction frequency.</p></sec><sec id="s1-13"><title>Data availability</title><p>Raw and mapped sequencing reads are available from the National Center for Biotechnology Information's GEO database (<ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/geo/">http://www.ncbi.nlm.nih.gov/geo/</ext-link>) under accession number GSE55797. Primer sequences are listed in <xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1</xref>.</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We thank PJ Wang for providing <italic>Taf7l</italic> KO mice; S Kajimura for protocols and suggestions; R Steven and D Schichness for help with immunochemistry; G Dailey, S Zheng, M Haggart, C Cattoglio, SS Teves, SE Torigoe, and FL Xie for their kind help and useful discussions. H Zhou is a research associate of the Howard Hughes Medical Institute. T Kaplan is a member of the Israeli Center of Excellence (I-CORE) for Gene Regulation in Complex Human Diseases (no. 41/11), and the Israeli Center of Excellence (I-CORE) for Chromatin and RNA in Gene Regulation (1796/12). R Tjian is an investigator of the Howard Hughes Medical Institutes.</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>RT: Robert Tjian is President of the Howard Hughes Medical Institute (2009-present), one of the three founding funders of <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>HZ, 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>BW, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con3"><p>RT, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con4"><p>IG, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con5"><p>TK, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn></fn-group><fn-group content-type="ethics-information"><title>Ethics</title><fn fn-type="other"><p>Animal experimentation: All animal experiments were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to animal use protocols (#R007) approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, Berkeley.</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.02811.011</object-id><label>Supplementary file 1.</label><caption><p>Primer sequences for RT-qPCR experiments (upper panel) and 3C experiments (lower panels).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02811.011">http://dx.doi.org/10.7554/eLife.02811.011</ext-link></p></caption><media mime-subtype="tif" mimetype="image" xlink:href="elife02811s001.tif"/></supplementary-material><sec sec-type="datasets"><title>Major datasets</title><p>The following dataset was generated:</p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro1"><name><surname>Zhou</surname><given-names>H</given-names></name>, <name><surname>Wan</surname><given-names>B</given-names></name>, <name><surname>Grubisic</surname><given-names>I</given-names></name>, <name><surname>Kaplan</surname><given-names>T</given-names></name>, <name><surname>Tjian</surname><given-names>R</given-names></name>, <year>2014</year><x>, </x><source>Taf7l Modulates Brown Adipose Tissue Formation</source><x>, </x><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE55797">http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE55797</ext-link><x>, </x><comment>Publicly available at NCBI Gene Expression Omnibus.</comment></related-object></p><p><bold>Reporting standards:</bold> Standard used to collect data: The data was followed NCBI standards for uploading megadata set.</p><p>The following previously published dataset was used:</p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro2"><name><surname>Zhou</surname><given-names>H</given-names></name>, <name><surname>Kaplan</surname><given-names>T</given-names></name>, <name><surname>Li</surname><given-names>Y</given-names></name>, <name><surname>Grubisic</surname><given-names>I</given-names></name>, <name><surname>Zhang</surname><given-names>Z</given-names></name>, <name><surname>Wang</surname><given-names>PJ</given-names></name>, <name><surname>Eisen</surname><given-names>MB</given-names></name>, <name><surname>Tjian</surname><given-names>R</given-names></name>, <year>2012</year><x>, </x><source>Dual Functions of TAF7L in Adipocyte Differentiation</source><x>, </x><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE41937">http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE41937</ext-link><x>, </x><comment>Publicly available at NCBI Gene Expression Omnibus.</comment></related-object></p></sec></sec><ref-list><title>References</title><ref id="bib1"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Abe</surname><given-names>T</given-names></name><name><surname>Hirasaka</surname><given-names>K</given-names></name><name><surname>Kagawa</surname><given-names>S</given-names></name><name><surname>Kohno</surname><given-names>S</given-names></name><name><surname>Ochi</surname><given-names>A</given-names></name><name><surname>Utsunomiya</surname><given-names>K</given-names></name><name><surname>Sakai</surname><given-names>A</given-names></name><name><surname>Ohno</surname><given-names>A</given-names></name><name><surname>Teshima-Kondo</surname><given-names>S</given-names></name><name><surname>Okumura</surname><given-names>Y</given-names></name><name><surname>Oarada</surname><given-names>M</given-names></name><name><surname>Maekawa</surname><given-names>Y</given-names></name><name><surname>Terao</surname><given-names>J</given-names></name><name><surname>Mills</surname><given-names>EM</given-names></name><name><surname>Nikawa</surname><given-names>T</given-names></name></person-group><year>2013</year><article-title>Cbl-b is a critical regulator of macrophage activation associated with obesity-induced insulin resistance in mice</article-title><source>Diabetes</source><volume>62</volume><fpage>1957</fpage><lpage>1969</lpage><pub-id pub-id-type="doi">10.2337/db12-0677</pub-id></element-citation></ref><ref id="bib2"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Beylkin</surname><given-names>DH</given-names></name><name><surname>Allen</surname><given-names>DL</given-names></name><name><surname>Leinwand</surname><given-names>LA</given-names></name></person-group><year>2006</year><article-title>MyoD, Myf5, and the calcineurin pathway activate the developmental myosin heavy chain genes</article-title><source>Developmental Biology</source><volume>294</volume><fpage>541</fpage><lpage>553</lpage><pub-id pub-id-type="doi">10.1016/j.ydbio.2006.02.049</pub-id></element-citation></ref><ref id="bib3"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cannon</surname><given-names>B</given-names></name><name><surname>Nedergaard</surname><given-names>J</given-names></name></person-group><year>2004</year><article-title>Brown adipose tissue: function and physiological significance</article-title><source>Physiological Reviews</source><volume>84</volume><fpage>277</fpage><lpage>359</lpage><pub-id pub-id-type="doi">10.1152/physrev.00015.2003</pub-id></element-citation></ref><ref id="bib4"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Carey</surname><given-names>AL</given-names></name><name><surname>Kingwell</surname><given-names>BA</given-names></name></person-group><year>2013</year><article-title>Brown adipose tissue in humans: therapeutic potential to combat obesity</article-title><source>Pharmacology & Therapeutics</source><volume>140</volume><fpage>26</fpage><lpage>33</lpage><pub-id pub-id-type="doi">10.1016/j.pharmthera.2013.05.009</pub-id></element-citation></ref><ref id="bib5"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chechi</surname><given-names>K</given-names></name><name><surname>Nedergaard</surname><given-names>J</given-names></name><name><surname>Richard</surname><given-names>D</given-names></name></person-group><year>2014</year><article-title>Brown adipose tissue as an anti-obesity tissue in humans</article-title><source>Obesity Reviews</source><volume>15</volume><fpage>92</fpage><lpage>106</lpage><pub-id pub-id-type="doi">10.1111/obr.12116</pub-id></element-citation></ref><ref id="bib6"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cheng</surname><given-names>Y</given-names></name><name><surname>Buffone</surname><given-names>MG</given-names></name><name><surname>Kouadio</surname><given-names>M</given-names></name><name><surname>Goodheart</surname><given-names>M</given-names></name><name><surname>Page</surname><given-names>DC</given-names></name><name><surname>Gerton</surname><given-names>GL</given-names></name><name><surname>Davidson</surname><given-names>I</given-names></name><name><surname>Wang</surname><given-names>PJ</given-names></name></person-group><year>2007</year><article-title>Abnormal sperm in mice lacking the Taf7l gene</article-title><source>Molecular and Cellular Biology</source><volume>27</volume><fpage>2582</fpage><lpage>2589</lpage><pub-id pub-id-type="doi">10.1128/MCB.01722-06</pub-id></element-citation></ref><ref id="bib7"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Harms</surname><given-names>M</given-names></name><name><surname>Seale</surname><given-names>P</given-names></name></person-group><year>2013</year><article-title>Brown and beige fat: development, function and therapeutic potential</article-title><source>Nature Medicine</source><volume>19</volume><fpage>1252</fpage><lpage>1263</lpage><pub-id pub-id-type="doi">10.1038/nm.3361</pub-id></element-citation></ref><ref id="bib8"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kajimura</surname><given-names>S</given-names></name><name><surname>Saito</surname><given-names>M</given-names></name></person-group><year>2014</year><article-title>A new era in brown adipose tissue biology: molecular control of brown fat development and energy homeostasis</article-title><source>Annual Review of Physiology</source><volume>76</volume><fpage>225</fpage><lpage>249</lpage><pub-id pub-id-type="doi">10.1146/annurev-physiol-021113-170252</pub-id></element-citation></ref><ref id="bib9"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kajimura</surname><given-names>S</given-names></name><name><surname>Seale</surname><given-names>P</given-names></name><name><surname>Kubota</surname><given-names>K</given-names></name><name><surname>Lunsford</surname><given-names>E</given-names></name><name><surname>Frangioni</surname><given-names>JV</given-names></name><name><surname>Gygi</surname><given-names>SP</given-names></name><name><surname>Spiegelman</surname><given-names>BM</given-names></name></person-group><year>2009</year><article-title>Initiation of myoblast to brown fat switch by a PRDM16-C/EBP-beta transcriptional complex</article-title><source>Nature</source><volume>460</volume><fpage>1154</fpage><lpage>1158</lpage><pub-id pub-id-type="doi">10.1038/nature08262</pub-id></element-citation></ref><ref id="bib10"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kajimura</surname><given-names>S</given-names></name><name><surname>Seale</surname><given-names>P</given-names></name><name><surname>Spiegelman</surname><given-names>BM</given-names></name></person-group><year>2010</year><article-title>Transcriptional control of brown fat development</article-title><source>Cell Metabolism</source><volume>11</volume><fpage>257</fpage><lpage>262</lpage><pub-id pub-id-type="doi">10.1016/j.cmet.2010.03.005</pub-id></element-citation></ref><ref id="bib11"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Langmead</surname><given-names>B</given-names></name><name><surname>Trapnell</surname><given-names>C</given-names></name><name><surname>Pop</surname><given-names>M</given-names></name><name><surname>Salzberg</surname><given-names>SL</given-names></name></person-group><year>2009</year><article-title>Ultrafast and memory-efficient alignment of short DNA sequences to the human genome</article-title><source>Genome Biology</source><volume>10</volume><fpage>R25</fpage><pub-id pub-id-type="doi">10.1186/gb-2009-10-3-r25</pub-id></element-citation></ref><ref id="bib12"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Liu</surname><given-names>Z</given-names></name><name><surname>Scannell</surname><given-names>DR</given-names></name><name><surname>Eisen</surname><given-names>MB</given-names></name><name><surname>Tjian</surname><given-names>R</given-names></name></person-group><year>2011</year><article-title>Control of embryonic stem cell lineage commitment by core promoter factor, TAF3</article-title><source>Cell</source><volume>146</volume><fpage>720</fpage><lpage>731</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2011.08.005</pub-id></element-citation></ref><ref id="bib13"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ohno</surname><given-names>H</given-names></name><name><surname>Shinoda</surname><given-names>K</given-names></name><name><surname>Ohyama</surname><given-names>K</given-names></name><name><surname>Sharp</surname><given-names>LZ</given-names></name><name><surname>Kajimura</surname><given-names>S</given-names></name></person-group><year>2013</year><article-title>EHMT1 controls brown adipose cell fate and thermogenesis through the PRDM16 complex</article-title><source>Nature</source><volume>504</volume><fpage>163</fpage><lpage>167</lpage><pub-id pub-id-type="doi">10.1038/nature12652</pub-id></element-citation></ref><ref id="bib14"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pedersen</surname><given-names>SB</given-names></name><name><surname>Bruun</surname><given-names>JM</given-names></name><name><surname>Kristensen</surname><given-names>K</given-names></name><name><surname>Richelsen</surname><given-names>B</given-names></name></person-group><year>2001</year><article-title>Regulation of UCP1, UCP2, and UCP3 mRNA expression in brown adipose tissue, white adipose tissue, and skeletal muscle in rats by estrogen</article-title><source>Biochemical and Biophysical Research Communications</source><volume>288</volume><fpage>191</fpage><lpage>197</lpage><pub-id pub-id-type="doi">10.1006/bbrc.2001.5763</pub-id></element-citation></ref><ref id="bib17"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Seale</surname><given-names>P</given-names></name><name><surname>Bjork</surname><given-names>B</given-names></name><name><surname>Yang</surname><given-names>W</given-names></name><name><surname>Kajimura</surname><given-names>S</given-names></name><name><surname>Chin</surname><given-names>S</given-names></name><name><surname>Kuang</surname><given-names>S</given-names></name><name><surname>Scimè</surname><given-names>A</given-names></name><name><surname>Devarakonda</surname><given-names>S</given-names></name><name><surname>Conroe</surname><given-names>HM</given-names></name><name><surname>Erdjument-Bromage</surname><given-names>H</given-names></name><name><surname>Tempst</surname><given-names>P</given-names></name><name><surname>Rudnicki</surname><given-names>MA</given-names></name><name><surname>Beier</surname><given-names>DR</given-names></name><name><surname>Spiegelman</surname><given-names>BM</given-names></name></person-group><year>2008</year><article-title>PRDM16 controls a brown fat/skeletal muscle switch</article-title><source>Nature</source><volume>454</volume><fpage>961</fpage><lpage>967</lpage><pub-id pub-id-type="doi">10.1038/nature07182</pub-id></element-citation></ref><ref id="bib16"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Seale</surname><given-names>P</given-names></name><name><surname>Conroe</surname><given-names>HM</given-names></name><name><surname>Estall</surname><given-names>J</given-names></name><name><surname>Kajimura</surname><given-names>S</given-names></name><name><surname>Frontini</surname><given-names>A</given-names></name><name><surname>Ishibashi</surname><given-names>J</given-names></name><name><surname>Cohen</surname><given-names>P</given-names></name><name><surname>Cinti</surname><given-names>S</given-names></name><name><surname>Spiegelman</surname><given-names>BM</given-names></name></person-group><year>2011</year><article-title>Prdm16 determines the thermogenic program of subcutaneous white adipose tissue in mice</article-title><source>The Journal of Clinical Investigation</source><volume>121</volume><fpage>96</fpage><lpage>105</lpage><pub-id pub-id-type="doi">10.1172/JCI44271</pub-id></element-citation></ref><ref id="bib15"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Seale</surname><given-names>P</given-names></name><name><surname>Lazar</surname><given-names>MA</given-names></name></person-group><year>2009</year><article-title>Brown fat in humans: turning up the heat on obesity</article-title><source>Diabetes</source><volume>58</volume><fpage>1482</fpage><lpage>1484</lpage><pub-id pub-id-type="doi">10.2337/db09-0622</pub-id></element-citation></ref><ref id="bib18"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Trapnell</surname><given-names>C</given-names></name><name><surname>Pachter</surname><given-names>L</given-names></name><name><surname>Salzberg</surname><given-names>SL</given-names></name></person-group><year>2009</year><article-title>TopHat: discovering splice junctions with RNA-Seq</article-title><source>Bioinformatics</source><volume>25</volume><fpage>1105</fpage><lpage>1111</lpage><pub-id pub-id-type="doi">10.1093/bioinformatics/btp120</pub-id></element-citation></ref><ref id="bib19"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Trapnell</surname><given-names>C</given-names></name><name><surname>Williams</surname><given-names>BA</given-names></name><name><surname>Pertea</surname><given-names>G</given-names></name><name><surname>Mortazavi</surname><given-names>A</given-names></name><name><surname>Kwan</surname><given-names>G</given-names></name><name><surname>van Baren</surname><given-names>MJ</given-names></name><name><surname>Salzberg</surname><given-names>SL</given-names></name><name><surname>Wold</surname><given-names>BJ</given-names></name><name><surname>Pachter</surname><given-names>L</given-names></name></person-group><year>2010</year><article-title>Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation</article-title><source>Nature Biotechnology</source><volume>28</volume><fpage>511</fpage><lpage>515</lpage><pub-id pub-id-type="doi">10.1038/nbt.1621</pub-id></element-citation></ref><ref id="bib21"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Villanueva</surname><given-names>CJ</given-names></name><name><surname>Vergnes</surname><given-names>L</given-names></name><name><surname>Wang</surname><given-names>J</given-names></name><name><surname>Drew</surname><given-names>BG</given-names></name><name><surname>Hong</surname><given-names>C</given-names></name><name><surname>Tu</surname><given-names>Y</given-names></name><name><surname>Hu</surname><given-names>Y</given-names></name><name><surname>Peng</surname><given-names>X</given-names></name><name><surname>Xu</surname><given-names>F</given-names></name><name><surname>Saez</surname><given-names>E</given-names></name><name><surname>Wroblewski</surname><given-names>K</given-names></name><name><surname>Hevener</surname><given-names>AL</given-names></name><name><surname>Reue</surname><given-names>K</given-names></name><name><surname>Fong</surname><given-names>LG</given-names></name><name><surname>Young</surname><given-names>SG</given-names></name><name><surname>Tontonoz</surname><given-names>P</given-names></name></person-group><year>2013</year><article-title>Adipose subtype-selective recruitment of TLE3 or Prdm16 by PPARgamma specifies lipid storage versus thermogenic gene programs</article-title><source>Cell Metabolism</source><volume>17</volume><fpage>423</fpage><lpage>435</lpage><pub-id pub-id-type="doi">10.1016/j.cmet.2013.01.016</pub-id></element-citation></ref><ref id="bib20"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Villanueva</surname><given-names>CJ</given-names></name><name><surname>Waki</surname><given-names>H</given-names></name><name><surname>Godio</surname><given-names>C</given-names></name><name><surname>Nielsen</surname><given-names>R</given-names></name><name><surname>Chou</surname><given-names>WL</given-names></name><name><surname>Vargas</surname><given-names>L</given-names></name><name><surname>Wroblewski</surname><given-names>K</given-names></name><name><surname>Schmedt</surname><given-names>C</given-names></name><name><surname>Chao</surname><given-names>LC</given-names></name><name><surname>Boyadjian</surname><given-names>R</given-names></name><name><surname>Mandrup</surname><given-names>S</given-names></name><name><surname>Hevener</surname><given-names>A</given-names></name><name><surname>Saez</surname><given-names>E</given-names></name><name><surname>Tontonoz</surname><given-names>P</given-names></name></person-group><year>2011</year><article-title>TLE3 is a dual-function transcriptional coregulator of adipogenesis</article-title><source>Cell Metabolism</source><volume>13</volume><fpage>413</fpage><lpage>427</lpage><pub-id pub-id-type="doi">10.1016/j.cmet.2011.02.014</pub-id></element-citation></ref><ref id="bib22"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zhou</surname><given-names>H</given-names></name><name><surname>Kim</surname><given-names>S</given-names></name><name><surname>Ishii</surname><given-names>S</given-names></name><name><surname>Boyer</surname><given-names>TG</given-names></name></person-group><year>2006</year><article-title>Mediator modulates Gli3-dependent Sonic hedgehog signaling</article-title><source>Molecular and Cellular Biology</source><volume>26</volume><fpage>8667</fpage><lpage>8682</lpage><pub-id pub-id-type="doi">10.1128/MCB.00443-06</pub-id></element-citation></ref><ref id="bib23"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zhou</surname><given-names>H</given-names></name><name><surname>Kaplan</surname><given-names>T</given-names></name><name><surname>Li</surname><given-names>Y</given-names></name><name><surname>Grubisic</surname><given-names>I</given-names></name><name><surname>Zhang</surname><given-names>Z</given-names></name><name><surname>Wang</surname><given-names>PJ</given-names></name><name><surname>Eisen</surname><given-names>MB</given-names></name><name><surname>Tjian</surname><given-names>R</given-names></name></person-group><year>2013a</year><article-title>Dual functions of TAF7L in adipocyte differentiation</article-title><source>eLife</source><volume>2</volume><fpage>e00170</fpage><pub-id pub-id-type="doi">10.7554/eLife.00170</pub-id></element-citation></ref><ref id="bib24"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zhou</surname><given-names>H</given-names></name><name><surname>Grubisic</surname><given-names>I</given-names></name><name><surname>Zheng</surname><given-names>K</given-names></name><name><surname>He</surname><given-names>Y</given-names></name><name><surname>Wang</surname><given-names>PJ</given-names></name><name><surname>Kaplan</surname><given-names>T</given-names></name><name><surname>Tjian</surname><given-names>R</given-names></name></person-group><year>2013b</year><article-title>Taf7l cooperates with Trf2 to regulate spermiogenesis</article-title><source>Proceedings of the National Academy of Sciences of the United States of America</source><volume>110</volume><fpage>16886</fpage><lpage>16891</lpage><pub-id pub-id-type="doi">10.1073/pnas.1317034110</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.02811.012</article-id><title-group><article-title>Decision letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Kadonaga</surname><given-names>James T</given-names></name><role>Reviewing editor</role><aff><institution>University of California, San Diego</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 “TAF7L Modulates Brown Adipose Tissue Formation” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by Fiona Watt (Senior editor) and 3 reviewers, one of whom, Jim Kadonaga, is a member of our Board of Reviewing Editors.</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>The manuscript “TAF7L Modulates Brown Adipose Tissue Formation” by Zhou et al. provides new evidence for the role of TAF7L in brown fat development. Brown and white fat are now appreciated to come from different developmental lineages, and the transcriptional control of their development is an area of active research. The work presented in this paper suggests an important role for a new molecular player, TAF7L. While the data shown are quite exciting, additional experiments, particularly BAT-specific studies, should be performed to support the conclusions of this manuscript.</p><p>Major points:</p><p>1) In <xref ref-type="fig" rid="fig3">Figure 3</xref>, the authors used 10T1/2 cells to study the mechanism whereby TAF7L modulates brown fat development. TAF7L was overexpressed, and the complex containing this protein was purified and shown to contain PPARg. In a previous paper, the authors had observed that these two proteins interact in 293T cells. Since PPARg is required for white and brown adipocyte development, it is not evident how this interaction specifically modulates brown adipogenesis. To address this point, the authors performed ChIP-Seq and found that TAF7L and PPARg co-occupy overlapping regions of PRDM16 and Ucp1 in post-differentiated cells. To clarify this line of investigation, the authors should address the following questions.</p><p>a) What other proteins were in the TAF7L complex? Were there any brown-fat enriched proteins that TAF7L interacts with such as PRDM16, PGC1a, or EHMT1?</p><p>b) Might TAF7L modulate PPARg selectivity for PRDM16 (and brown fat) vs. TLE3 (and white fat)?</p><p>2) In their previous paper (Zhou et al., <italic>eLife</italic>, 2013), the authors showed that white fat development (in vitro and in vivo) is disrupted in the absence of TAF7L. This finding argues for a broader role of TAF7L in adipogenesis (or perhaps even more broadly in development). As highlighted in point 1, the authors should address how TAF7L is specifically involved in brown fat development. In other words, the role of TAF7L in the browning of WAT should be addressed.</p><p>3) Photographs of whole BAT from young and adult WT and TAF7L KO mice should be provided to see the gross morphology of BAT.</p><p>4) Cold exposure experiments should be performed in TAF7L KO mice to understand the physiological consequence of lack of TAF7L in BAT.</p><p>5) The mechanism of how TAF7L suppresses muscle-specific genes is unclear. This point should be further investigated or minimally speculated on and discussed.</p><p>6) Differentiation assays using primary brown adipocytes (SVF differentiated to adipocytes) from WT and TAF7L KO mice should be performed.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.02811.013</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p>We thank the reviewers for a thoughtful and comprehensive review of our manuscript “TAF7L Modulates Brown Adipose Tissue Formation”, which we here, resubmit as a Short report to <italic>eLife</italic>. As a significant advancement of our original finding that TAF7L ablation disrupts white fat tissue development (Zhou et al., <italic>eLife</italic>, 2013), we report that TAF7L forms a key component of an alternative Fat-TFIID complex that works in conjunction with PPARγ via a DNA looping mechanism to modulate brown adipose tissue (BAT) formation.</p><p>A note from the editors appended below outlines the critical changes to our manuscript that were recommended by the BRE and reviewers:</p><p><italic>- Two points (Major Points 1a, 3) involve data that the authors probably already have</italic>.</p><p><italic>- Several issues (Major Points 1b, 2, 5) could be addressed simply by discussing them in the text</italic>.</p><p><italic>- Major Points 4 and 6 would make it a better paper, but it could be argued that such experiments are beyond the scope of this study. It's up to the authors whether they want to strengthen this work, particularly with regard to brown fat development and physiology</italic>.</p><p>We appreciate these many useful suggestions from reviewers, which has further strengthened and broadened this study. In response to the reviewers’ comments, we have now included new data to address major points 1a, 3, and 6. Below, we also address points 1b, 2, 4 and 5, which includes suggestions that we believe are beyond the scope of this study.</p><p><italic>Major</italic> <italic>points:</italic></p><p><italic>1) In</italic> <xref ref-type="fig" rid="fig3"><italic>Figure 3</italic></xref><italic>, the authors used 10T1/2 cells to study the mechanism whereby TAF7L modulates brown fat development. TAF7L was overexpressed, and the complex containing this protein was purified and shown to contain PPARg. In a previous paper, the authors had observed that these two proteins interact in 293T cells. Since PPARg is required for white and brown adipocyte development, it is not evident how this interaction specifically modulates brown adipogenesis. To address this point, the authors performed ChIP-Seq and found that TAF7L and PPARg co-occupy overlapping regions of PRDM16 and Ucp1 in post-differentiated cells. To clarify this line of investigation, the authors should address the following questions</italic>.</p><p><italic>a) What other proteins were in the TAF7L complex? Were there any brown-fat enriched proteins</italic> <italic>that TAF7L interacts with such as PRDM16, PGC1a, or EHMT1?</italic></p><p>Although we made multiple attempts, we were unable to recover sufficient levels of protein from the IP experiments using adipocytes to perform unambiguous protein identification. However, we did find that TAF7L can interact with PRDM16 and be coIP'd when they are expressed in 293T cells. We have now included this new data in <xref ref-type="fig" rid="fig3">Figure 3C</xref>.</p><p><italic>b) Might TAF7L modulate PPARg selectivity for</italic> <italic>PRDM16 (and brown fat) vs. TLE3 (and white fat)?</italic></p><p>We have not tested the association between TLE3 with TAF7L in WAT, but we did find that TAF7L can associate with PRDM16 when ectopically expressed in 293T cells. We have now revised the text to read: Although we failed to detect PRDM16 in these affinity purification experiments, we did find that ectopically expressed TAF7L and PRDM16 associate with each other in 293T cells (<xref ref-type="fig" rid="fig3">Figure 3C</xref>). These data suggest that TAF7L-containing Fat-TFIID has gained the ability to bind endogenous PPARγ we speculate which might facilitate its association with distinct cofactors such as PRDM16 in BAT or TLE3 in WAT to differentially regulate brown and white adipocyte formation.</p><p><italic>2) In their previous paper (Zhou et al.,</italic> eLife, <italic>2013), the authors showed that white fat development (in vitro and in vivo) is disrupted in the absence of TAF7L. This finding argues for a broader role of TAF7L in adipogenesis (or perhaps even more broadly in development). As highlighted in point 1, the authors should address how TAF7L is specifically involved in brown fat development. In other words, the role of TAF7L in the browning of WAT should be addressed.</italic></p><p>We agree that this is a reasonable scenario since <italic>Taf7l</italic> KO mice showed changes consistent with the browning of WAT and changes in the expression of WAT genes in <italic>Taf7l</italic> KO mice compared to WT mice. However, we believe a thorough and rigorous test of this hypothesis is well beyond the scope of this current study. We are particularly concerned that fully dissecting the browning of WAT will require a careful analysis of beige cells which are likely not the same as canonical BAT.</p><p><italic>3) Photographs of whole BAT from young and adult WT and TAF7L KO mice should be provided to see the gross morphology of BAT</italic>.</p><p>We have now included photographs of whole BAT from one-month and four-month old WT and TAF7L KO mice in <xref ref-type="fig" rid="fig1s1">Figure 1–figure supplement 1C</xref>.</p><p><italic>4) Cold exposure experiments should be performed in TAF7L KO mice to understand the physiological consequence of lack of TAF7L in BAT</italic>.</p><p>In this Short report we chose to focus primarily on the role of TAF7L with respect to BAT developmental. In addition, our preliminary physiological studies showed mixed phenotypes because TAF7L KO resulted in differential changes in WAT versus BAT development. We felt that it would be least ambiguous to study the physiological consequences of environmental parameters such as cold exposure using BAT or WAT-specific TAF7L KO mice in future studies.</p><p><italic>5) The mechanism of how TAF7L suppresses muscle-specific genes is unclear. This point should be further investigated or minimally speculated on and discussed</italic>.</p><p>Although the mechanisms of how TAF7L suppresses muscle-specific genes is unclear at this point, we speculate that it might be an indirect effect, since our ChIP-seq data showed that TAF7L is not targeted to muscle gene promoters. We therefore favor the idea that a de-repression of muscle-genes might result from a reduction in certain fat genes caused by loss of TAF7L, which we establish here to be functioning as an activator of fat cell lineages. We have now included this speculation in the revised text.</p><p><italic>6) Differentiation assays using primary brown adipocytes (SVF differentiated to adipocytes) from WT and TAF7L KO mice should be performed</italic>.</p><p>We have now included this new data in a revised <xref ref-type="fig" rid="fig2s1">Figure 2–figure supplement 1</xref>.</p></body></sub-article></article> |