Permalink
Cannot retrieve contributors at this time
Fetching contributors…
Cannot retrieve contributors at this time
| <?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">02833</article-id><article-id pub-id-type="doi">10.7554/eLife.02833</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Developmental biology and stem cells</subject></subj-group><subj-group subj-group-type="heading"><subject>Genes and chromosomes</subject></subj-group></article-categories><title-group><article-title>H3K27 modifications define segmental regulatory domains in the <italic>Drosophila</italic> bithorax complex</article-title></title-group><contrib-group><contrib contrib-type="author" equal-contrib="yes" id="author-12260"><name><surname>Bowman</surname><given-names>Sarah K</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="other" rid="par-4"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" equal-contrib="yes" id="author-12492"><name><surname>Deaton</surname><given-names>Aimee M</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" id="author-12493"><name><surname>Domingues</surname><given-names>Heber</given-names></name><xref ref-type="aff" rid="aff3"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" id="author-12494"><name><surname>Wang</surname><given-names>Peggy I</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" id="author-12495"><name><surname>Sadreyev</surname><given-names>Ruslan I</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff4"/><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-1379"><name><surname>Kingston</surname><given-names>Robert E</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="other" rid="par-3"/><xref ref-type="fn" rid="con7"/><xref ref-type="fn" rid="conf1"/><xref ref-type="other" rid="dataro1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-12496"><name><surname>Bender</surname><given-names>Welcome</given-names></name><xref ref-type="aff" rid="aff3"/><xref ref-type="corresp" rid="cor2">*</xref><xref ref-type="other" rid="par-2"/><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 Biology</institution>, <institution>Massachusetts General Hospital</institution>, <addr-line><named-content content-type="city">Boston</named-content></addr-line>, <country>United States</country></aff><aff id="aff2"><institution content-type="dept">Department of Genetics</institution>, <institution>Harvard Medical School</institution>, <addr-line><named-content content-type="city">Boston</named-content></addr-line>, <country>United States</country></aff><aff id="aff3"><institution content-type="dept">Department of Biological Chemistry and Molecular Pharmacology</institution>, <institution>Harvard Medical School</institution>, <addr-line><named-content content-type="city">Boston</named-content></addr-line>, <country>United States</country></aff><aff id="aff4"><institution content-type="dept">Department of Pathology</institution>, <institution>Harvard Medical School</institution>, <addr-line><named-content content-type="city">Boston</named-content></addr-line>, <country>United States</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Reinberg</surname><given-names>Danny</given-names></name><role>Reviewing editor</role><aff><institution>Howard Hughes Medical Institute, New York University School of Medicine</institution>, <country>United States</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>kingston@molbio.mgh.harvard.edu</email> (REK);</corresp><corresp id="cor2"><label>*</label>For correspondence: <phone>(+1) 617-432-1906</phone> (WB)</corresp><fn fn-type="con" id="equal-contrib"><label>†</label><p>These authors contributed equally to this work</p></fn></author-notes><pub-date date-type="pub" publication-format="electronic"><day>31</day><month>07</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e02833</elocation-id><history><date date-type="received"><day>23</day><month>03</month><year>2014</year></date><date date-type="accepted"><day>30</day><month>07</month><year>2014</year></date></history><permissions><copyright-statement>© 2014, Bowman et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Bowman 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="elife02833.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.02833.001</object-id><p>The bithorax complex (BX-C) in <italic>Drosophila melanogaster</italic> is a cluster of homeotic genes that determine body segment identity. Expression of these genes is governed by cis-regulatory domains, one for each parasegment. Stable repression of these domains depends on Polycomb Group (PcG) functions, which include trimethylation of lysine 27 of histone H3 (H3K27me3). To search for parasegment-specific signatures that reflect PcG function, chromatin from single parasegments was isolated and profiled. The H3K27me3 profiles across the BX-C in successive parasegments showed a ‘stairstep’ pattern that revealed sharp boundaries of the BX-C regulatory domains. Acetylated H3K27 was broadly enriched across active domains, in a pattern complementary to H3K27me3. The CCCTC-binding protein (CTCF) bound the borders between H3K27 modification domains; it was retained even in parasegments where adjacent domains lack H3K27me3. These findings provide a molecular definition of the homeotic domains, and implicate precisely positioned H3K27 modifications as a central determinant of segment identity.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02833.001">http://dx.doi.org/10.7554/eLife.02833.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.02833.002</object-id><title>eLife digest</title><p>Like other insects, the body of the fruit fly is divided into three main parts—the head, the thorax and the abdomen—and each part, in turn, is made up of several smaller segments. The bithorax complex is a cluster of three genes that together control the identity of the segments that make up the back half of the fruit fly's body. This gene cluster has been studied for several decades and these studies have helped to further our understanding of how genetic information is accessed and used to make an animal’s body plan.</p><p>Early on in a fruit fly embryo, stretches of DNA within the bithorax complex regulate where the complex's genes are switched on, and where they are switched off. Proteins called Polycomb group proteins then keep the silenced genes off, in part by adding small chemical marks to other proteins called histones. Most DNA in a cell is wrapped around histones, and the addition of such chemical marks causes the DNA to become more tightly packed. This prevents the bithorax complex genes from being accessed and switched on. It had previously been suggested that each segment might have a unique pattern of chemical marks on the bithorax complex histones, but evidence to support this idea was lacking.</p><p>Bowman et al. have now undertaken the technically challenging task of purifying the DNA and its histones from individual segments of fruit fly embryos. This revealed that segments closer to the embryo's head contain larger stretches of bithorax complex DNA covered with histones marked by the Polycomb group proteins. Bowman et al. also found that the coverage of chemical marks on the histones changed dramatically when one segment was compared to its neighboring segments. These sharp boundaries clearly outline which regulatory regions of the DNA are switched on and which are switch off; however the same pattern is not seen for the Polycomb group proteins themselves. Instead, within the bithorax complex, the pattern of these proteins is almost identical in different segments.</p><p>The challenge now is to understand how the chemical marks and the Polycomb group proteins work together to restrict access to DNA in such precise patterns. Also—since similar gene clusters control the development of the body plans of mammals—this, in turn, might help us to understand how the Polycomb group proteins perform similar functions in human development and disease.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02833.002">http://dx.doi.org/10.7554/eLife.02833.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>chromatin</kwd><kwd>Polycomb</kwd><kwd>bithorax complex</kwd><kwd>CTCF</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd><italic>D. melanogaster</italic></kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution-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>GM43901</award-id><principal-award-recipient><name><surname>Kingston</surname><given-names>Robert E</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>GM28630</award-id><principal-award-recipient><name><surname>Bender</surname><given-names>Welcome</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/10.13039/100000057</institution-id><institution>National Institute of General Medical Sciences</institution></institution-wrap></funding-source><award-id>R37 GM048405</award-id><principal-award-recipient><name><surname>Kingston</surname><given-names>Robert E</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/10.13039/100001021</institution-id><institution>Damon Runyon Cancer Research Foundation</institution></institution-wrap></funding-source><award-id>1995-08</award-id><principal-award-recipient><name><surname>Bowman</surname><given-names>Sarah K</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>Segment-specific boundaries of histone modifications reveal the regulatory logic of a classic developmental control region.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>Cells in higher organisms choose among developmental pathways in response to transitory cues. The choice must be remembered; this is often described as changing the epigenetic state. One prominent mechanism to fix such choices relies on the genes of the Polycomb Group (PcG). The members of this family have been well defined by genetic and biochemical studies (<xref ref-type="bibr" rid="bib43">Simon and Kingston, 2013</xref>). The PcG proteins are present in all cells, but they impose long-term repression on different target genes in different lineages. PcG proteins form distinct complexes with distinct functions. These include the PRC1 family of complexes, which compact chromatin and ubiquitylate histone H2A, and the PRC2 complexes, which methylate histone H3 on lysine 27. The molecular details of how PcG functions are coordinated to lead to repression are poorly understood.</p><p>The prototypic targets of PcG action are the <italic>Drosophila</italic> homeotic genes that control segment identity. Indeed, a mutation in <italic>Polycomb</italic>, the founding member of the PcG, was discovered for its segmental transformations (<xref ref-type="bibr" rid="bib25">Lewis, 1947</xref>). In <italic>Drosophila melanogaster</italic>, the homeotics lie in two clusters, the Antennapedia complex (Antp-C) and the bithorax complex (BX-C). The BX-C is the better-studied cluster, due largely to the effort of EB Lewis. Lewis showed that mutations that transform specific segments align on the genetic map in the order of the body segments that they affect (<xref ref-type="bibr" rid="bib23">Lewis, 1978</xref>). The evolutionary conservation of this gene order, including that in the mammalian HOX complexes, suggests that chromosomal position dictates the expression pattern. Lewis used nested deletions to show that successively more distal genetic functions of the BX-C are activated in successively more posterior segments along the body axis (<xref ref-type="bibr" rid="bib24">Lewis, 1981</xref>). The genetic functions are now thought to encompass nine regulatory domains, each for a different parasegment. The regulatory domains control three homeobox-containing genes of the complex, <italic>Ultrabithorax</italic> (<italic>Ubx</italic>), <italic>abdominal-A</italic> (<italic>abd-A</italic>), and <italic>Abdominal-B</italic> (<italic>Abd-B</italic>) (<xref ref-type="bibr" rid="bib26">Maeda and Karch, 2009</xref>). The regulatory domains have been roughly defined by mutant lesions (<xref ref-type="bibr" rid="bib23">Lewis, 1978</xref>), and by enhancer traps in the BX-C, which show different anterior limits of expression (<xref ref-type="bibr" rid="bib2">Bender and Hudson, 2000</xref>). The molecular features that define these regions, as well as the precise locations of their boundaries, have not been defined.</p><p>Both PRC1 and PRC2 are required to maintain repression of homeotic genes in appropriate parasegments, so these complexes may generate segment-specific chromatin features in the regulatory domains of the BX-C. We initially evaluated H3K27me3 patterns because they reflect the central function of PRC2. The entire BX-C is heavily marked with H3K27me3 in whole embryos (<xref ref-type="bibr" rid="bib41">Schuettengruber et al., 2009</xref>; <xref ref-type="bibr" rid="bib30">Nègre et al., 2011</xref>). This histone modification is necessary for maintaining repression of BX-C genes (<xref ref-type="bibr" rid="bib38">Pengelly et al., 2013</xref>), yet these genes are not repressed throughout the body. It seemed likely that parasegment-specific patterns of histone modification might be obscured when all the parasegments are pooled for analysis. Indeed, <xref ref-type="bibr" rid="bib35">Papp and Müller (2006)</xref> observed loss of the K27me3 mark across the <italic>Ubx</italic> transcription unit in haltere and third leg imaginal discs, tissues where <italic>Ubx</italic> is transcribed. These discs include cells of two parasegments (PS5&6), and so it was not clear how the K27me3-free regions might correlate with transcription units or with the genetic regulatory domains. The issue could be resolved if H3K27me3 patterns could be studied in single parasegments.</p></sec><sec id="s2" sec-type="results"><title>Results</title><p>The chromatin features of the BX-C have not been studied in individual parasegments because of the technical challenges of cell isolation and molecular analysis on small samples. To address this, we marked single parasegments with a combination of Gal4 and Gal80 drivers, and developed a nuclear sorting-chromatin immunoprecipitation-sequencing protocol. Gal4, a transcriptional activator, was expressed in a series of parasegments with a defined anterior boundary, and Gal80 was expressed in a pattern shifted one parasegment more posterior. Gal80 binds to and inactivates Gal4, leaving Gal4 activity in a single parasegment (<xref ref-type="fig" rid="fig1">Figure 1A</xref>). Gal4 and Gal80 expression domains were established either by enhancer trapping or by selecting an enhancer to achieve the desired expression pattern (<xref ref-type="fig" rid="fig1s1">Figure1—figure supplement 1</xref>). Various combinations of Gal4 and Gal80 drivers were combined genetically to limit Gal4 activity to each of parasegments 4, 5, 6, and 7, which approximately correspond to the second thoracic through the second abdominal segments (<xref ref-type="fig" rid="fig1">Figure 1B</xref>, <xref ref-type="fig" rid="fig1s1 fig1s2">Figure1—figure supplements 1 and 2</xref>; ‘Materials and methods’ for details).<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.02833.003</object-id><label>Figure 1.</label><caption><title>Marking single parasegments.</title><p>(<bold>A</bold>) Drivers for the Gal4 activator and the Gal80 repressor, each with a different anterior limit, are combined genetically. Gal4 activity is thus limited to a single parasegment, and is used to activate transcription of a fluorescent nuclear envelope protein. (<bold>B</bold>) Expression patterns used for isolation of parasegment nuclei are shown, visualized with antibody to the FLAG epitope on the INTACT fusion protein. Each panel shows embryos at about 6, 8, 10, 12, 14, and 16 hr after fertilization, with the stained cells marking the indicated parasegments. Embryos between 5 hr and 13 hr old (or between 4 hr and 10 hr for PS6) were harvested for analysis.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02833.003">http://dx.doi.org/10.7554/eLife.02833.003</ext-link></p></caption><graphic xlink:href="elife02833f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02833.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>Tools for parasegment-specific ChIP-seq.</title><p>(<bold>A</bold>) A P element was constructed that initially expresses Gal4 from the P promoter, and transgenes were moved into the homeotic gene complexes by P element swapping. Recombination between the FRT sites flanking Gal4 convert the transgene into one expressing Gal80. (<bold>B</bold>) A second P element expressed Gal4 or Gal80, driven by enhancers from the BX-C. The enhancers were positioned upstream of a promoter and a PRE from the <italic>engrailed</italic> locus. The PRE helps to maintain the restricted expression by the enhancer in older embryos. A cluster of binding sites for the <italic>suppressor of Hairy-wing</italic> protein flanks the enhancer, to block potential position effects at random chromosomal locations. (<bold>C</bold>) Nuclei were sorted using Hoechst 33,342 (to select only single nuclei) and mCherry fluorescence. Nuclei from wild type (Oregon R, OR) embryos were used to set the fluorescence threshold. (<bold>D</bold>) The flow chart from embryos to sequencing libraries is diagramed; the procedure could be paused at either freezing step.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02833.004">http://dx.doi.org/10.7554/eLife.02833.004</ext-link></p></caption><graphic xlink:href="elife02833fs001"/></fig><fig id="fig1s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02833.005</object-id><label>Figure 1—figure supplement 2.</label><caption><title>Close-up of parasegment-specific expression patterns.</title><p>Embryos expressing the INTACT marker in parasegments 4–7 were obtained as in <xref ref-type="fig" rid="fig1">Figure 1B</xref>. All are at ∼10 hr of development, prior to dorsal closure; they were dissected along the dorsal midline, and flattened to display the epidermis in one focal plane. The last panel shows an embryo stained for UBX (in blue) and ABD-A (in brown) (<xref ref-type="bibr" rid="bib19">Karch et al., 1990</xref>), to illustrate the expression patterns of these two proteins in parasegments 4–7.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02833.005">http://dx.doi.org/10.7554/eLife.02833.005</ext-link></p></caption><graphic xlink:href="elife02833fs002"/></fig></fig-group></p><p>For isolation of marked nuclei, we used Gal4 to drive an mCherry-RanGAP fusion protein, from the INTACT system (<xref ref-type="bibr" rid="bib13">Deal and Henikoff, 2011</xref>; <xref ref-type="bibr" rid="bib46">Steiner et al., 2012</xref>). Staged embryos with single parasegment stripes of mCherry were formaldehyde-fixed and disrupted, and their nuclei were sorted on a FACS instrument (<xref ref-type="fig" rid="fig1s1">Figure1—figure supplement 1C</xref>). DNA enriched by chromatin immunoprecipitation (ChIP) was prepared for paired-end sequencing, using a picogram-scale library protocol (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1D</xref>; <xref ref-type="bibr" rid="bib6">Bowman et al., 2013</xref>).</p><p>The largest differences in H3K27me3 enrichment between parasegments occurred at the BX-C (<xref ref-type="fig" rid="fig2">Figure 2A</xref>, <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>). While the H3K27me3 profiles in our four adjacent parasegments were very similar elsewhere in the genome (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>), the BX-C exhibited a striking ‘stairstep’ pattern. Large expanses of H3K27me3 enrichment were lost as we moved from anterior to posterior along the body axis (<xref ref-type="fig" rid="fig2">Figure 2C–F</xref>), revealing the locations of the regulatory domains. This is reminiscent of the temporal activation pattern of the Hox-D cluster in mouse, which correlates with loss of H3K27me3 (<xref ref-type="bibr" rid="bib45">Soshnikova and Duboule, 2009</xref>). Our analysis of single parasegments locates distinct domain borders where K27me3 status changes abruptly. The contrast between the whole embryo profile (<xref ref-type="fig" rid="fig2">Figure 2B</xref>) and the single segment profiles (<xref ref-type="fig" rid="fig2">Figure 2C–F</xref>) shows how isolated cell types can identify chromatin changes linked to cell identity.<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.02833.006</object-id><label>Figure 2.</label><caption><title>H3K27me3, CTCF, and CP190 profiles.</title><p>(<bold>A</bold>) ChIP-seq profiles across 27 Mb of chromosome 3R are virtually identical, except at the BX-C, in the middle of the chromosome arm. (<bold>B</bold>–<bold>F</bold>) H3K27me3 profiles across 380 kb encompassing the BX-C. (<bold>G</bold>–<bold>H</bold>) CTCF binding sites. (<bold>I</bold> and <bold>J</bold>) CP190 binding sites. Panels <bold>B</bold>, <bold>G</bold>, and <bold>I</bold> were prepared from unsorted nuclei; parasegment-specific nuclei were used for the other panels, as indicated. Transcription units of coding genes are shown below the profiles. The Drosophila reference sequence includes a 6.1 kb Diver retroposon insertion at the indicated position; it was not present in the strains used for this analysis. At the bottom are shown the three regulatory domains defined by this analysis.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02833.006">http://dx.doi.org/10.7554/eLife.02833.006</ext-link></p></caption><graphic xlink:href="elife02833f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02833.007</object-id><label>Figure 2—figure supplement 1.</label><caption><title>Genome-wide comparisons of H3K27me3 patterns.</title><p>The most striking differences in H3K27me3 enrichment across different parasegments occur at the BX-C. Each plot compares the density of H3K27me3 between two parasegments over all H3K27me3 peaks (see supplementary methods for additional details). A linear line of fit was drawn. H3K27me3 density across the entire genome was largely unchanging between parasegments, with the exception of a few points corresponding to the BX-C that are markedly far off from the line of fit. Within each plot, the comparison of five BX-C segments (labeled a–e) are indicated with pink or red circles, the color of which corresponds to the Z-score (number of observed standard deviations from the line of fit).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02833.007">http://dx.doi.org/10.7554/eLife.02833.007</ext-link></p></caption><graphic xlink:href="elife02833fs003"/></fig><fig id="fig2s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02833.008</object-id><label>Figure 2—figure supplement 2.</label><caption><title>H3K27me3, CTCF, and CP190 profiles in PS7 for the Antennapedia complex, illustrated as in <xref ref-type="fig" rid="fig2">Figure 2</xref>.</title><p>The H3K27me3 profiles in parasegments 4 through 7 are very similar. ANTP is expressed at high levels in PS4 and PS5 in wild type embryos, and in the absence of the BX-C, parasegments 4–12 all show similar expression patterns (<xref ref-type="bibr" rid="bib8">Carroll et al., 1986</xref>). By this criterion, <italic>Antp</italic> appears to be free of Pc-G repression in PS4–12, which is consistent with the lack of H3K27 methylation over its transcription unit in PS7, as shown. CTCF and CP190 profiles look identical between mixed and PS7 nuclei, as they are in the BX-C. CP190 marks several potential domain boundaries in the Antennapedia complex that lack apparent CTCF binding.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02833.008">http://dx.doi.org/10.7554/eLife.02833.008</ext-link></p></caption><graphic xlink:href="elife02833fs004"/></fig></fig-group></p><p>In each isolated parasegment, the extent of K27me3 coverage was largely consistent with genetic studies indicating the location of BX-C active and repressed regulatory domains. The PS4 H3K27me3 pattern is mesa-like across the complex (<xref ref-type="fig" rid="fig2">Figure 2C</xref>), with only a few narrow gaps at nucleosome-free regions (<xref ref-type="bibr" rid="bib27">Mito et al., 2005</xref>). The BX-C is almost completely repressed in PS4, with only a few neuronal cells on the midline showing UBX expression in embryos. The PS5 regulatory domain, associated with the <italic>bithorax</italic> (<italic>bx</italic>) mutant lesions, is defined by the low level of H3K27me3 modification across the leftmost (proximal) 92 kb of the complex (<xref ref-type="fig" rid="fig2">Figure 2D</xref>). There is a sharp transition to full H3K27me3 coverage at a position upstream of the <italic>Ubx</italic> transcription start site. The low residual level of H3K27me3 marks in the 92 kb PS5 domain is likely due to partial contamination from more anterior parasegments in our preparation of PS5 nuclei (<xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2</xref>). In PS6, the leftmost 137 kb of the BX-C is clear of the K27me3 mark (<xref ref-type="fig" rid="fig2">Figure 2E</xref>); the additional K27me3-clear region reveals the PS6 domain. The PS6 regulatory domain is the one best defined by genetic analysis, with a series of rearrangement breakpoints giving <italic>bithoraxoid</italic> (<italic>bxd</italic>) phenotypes (<xref ref-type="bibr" rid="bib3">Bender and Lucas, 2013</xref>). The transition point to full K27 methylation coincides with the genetically defined <italic>Fub</italic> border between the PS6 and the PS7 regulatory domains (<xref ref-type="bibr" rid="bib3">Bender and Lucas, 2013</xref>). In PS7, an additional 36 kb was depleted of H3K27me3 (<xref ref-type="fig" rid="fig2">Figure 2F</xref>). This region includes the <italic>abd-A</italic> transcription unit, and is associated with the <italic>infraabdominal-2</italic> (<italic>iab-2</italic>) class of genetic mutations. We observed a reproducible mound of H3K27me3 in PS7 nuclei close to the <italic>Ubx</italic> promoter (arrow in <xref ref-type="fig" rid="fig2">Figure 2F</xref>). This may reflect repression of <italic>Ubx</italic> by <italic>abd-A</italic> in this parasegment (<xref ref-type="bibr" rid="bib19">Karch et al., 1990</xref>), mediated by the PcG.</p><p>The regulatory domains defined by the sharp transitions in K27me3 enrichment do not correspond in every detail to expectations from genetic studies. Prior mapping of domains has relied upon random insertion of transgenic reporter elements into the BX-C to interrogate local enhancer activity. These enhancer traps show sharp anterior edges to their expression patterns, with the anterior-most labeled parasegment corresponding to the domain in which the insertion resides (<xref ref-type="bibr" rid="bib2">Bender and Hudson, 2000</xref>). In the current study, the border between the PS5 and PS6 domains, defined by the discontinuity in K27me3 enrichment in PS5 nuclei, lies 14 kb upstream of the <italic>Ubx</italic> promoter. However, transgenic reporter elements inserted between the <italic>Ubx</italic> promoter and the K27me3-defined border (presumably part of the PS5 regulatory domain) show patterns with a PS6 anterior limit. Perhaps the promoters of these reporters interact with the <italic>Ubx</italic> promoter in distinctive ways. There was also prior evidence of an active BX-C domain in PS4. An enhancer trap 3 kb downstream of the <italic>Ubx</italic> start site shows a PS4 anterior border (<xref ref-type="bibr" rid="bib9">Casares et al., 1997</xref>), and a ncRNA with a PS4 anterior expression limit is transcribed from the region 5–15 kb downstream of the <italic>Ubx</italic> start (<xref ref-type="bibr" rid="bib36">Pease et al., 2013</xref>). Despite the activity of these regulatory regions in PS4 cells, we did not detect a decrease in K27me3 at these locations. The PS4 active state may be early and transient, limited to a subset of PS4 cells, or resistant to H3K27 methylation.</p><p>We have profiled additional histone modifications and several chromosomal proteins in single parasegments to understand better how domains are established and bounded. The sharp discontinuities in K27 methylation coincide with binding sites of the CCCTC-binding factor (CTCF), which is thought to impose boundaries on regulatory domains in many systems (<xref ref-type="bibr" rid="bib32">Ohlsson et al., 2010</xref>). <xref ref-type="fig" rid="fig2">Figure 2G</xref> shows the CTCF ChIP pattern in unsorted nuclei, similar to that previously reported (<xref ref-type="bibr" rid="bib31">Nègre et al., 2010</xref>). CTCF in PS7 nuclei gives a pattern virtually identical to the whole embryo pattern (<xref ref-type="fig" rid="fig2">Figure 2H</xref>). Apparently, the activation state of a domain is not coupled to the presence of CTCF at its borders. We note that our experimental conditions do not detect robust CTCF enrichment in some BX-C locations that have been reported by others (<xref ref-type="bibr" rid="bib31">Nègre et al., 2010</xref>), notably at the border between regulatory regions for PS7 and PS8, and proximal to the 3′ end of the <italic>Ubx</italic> transcription unit.</p><p>CTCF is present in a protein complex with Centrosomal Protein 190 (CP190) (<xref ref-type="bibr" rid="bib28">Mohan et al., 2007</xref>), and the binding sites of these proteins often overlap (<xref ref-type="bibr" rid="bib31">Nègre et al., 2010</xref>). The CP190 sites largely coincide with CTCF sites in the BX-C, and, as with CTCF, there is little difference between the CP190 pattern in unsorted nuclei (<xref ref-type="fig" rid="fig2">Figure 2I</xref>; <xref ref-type="bibr" rid="bib31">Nègre et al., 2010</xref>) and in PS7 nuclei (<xref ref-type="fig" rid="fig2">Figure 2J</xref>). There is a noteworthy CP190 site immediately proximal to the 3′ end of the <italic>Ubx</italic> transcription unit, separating <italic>Ubx</italic> from the adjacent gene (<italic>modular serine protease</italic>). In the Antennapedia complex, the other homeotic gene complex in flies, CP190 peaks often appear without coincident CTCF peaks (<xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2</xref>).</p><p>Acetylation of H3K27 (H3K27ac) is correlated with active enhancer regions (<xref ref-type="bibr" rid="bib17">Heintzman et al., 2009</xref>; <xref ref-type="bibr" rid="bib20">Kharchenko et al., 2011</xref>), and is anti-correlated with PcG function (<xref ref-type="bibr" rid="bib49">Tie et al., 2009</xref>), prompting us to examine its pattern in the BX-C. H3K27ac showed the opposite pattern to H3K27me3. There is relatively little H3K27ac across the BX-C in unsorted nuclei, but in PS7, the active domains show broad enrichment for H3K27ac (<xref ref-type="fig" rid="fig3">Figure 3B,C</xref>). The H3K27ac levels differ in the three active domains, for reasons we do not yet understand. Within each domain, however, the coverage is boundary-to-boundary, although many enhancers in the BX-C are not active in embryos. Overall, H3K27ac is a mirror to H3K27me3, in extent, if not in density.<fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.02833.009</object-id><label>Figure 3.</label><caption><title>Additional features of the BX-C in PS7.</title><p>(<bold>A</bold>) The H3K27me3 distribution, repeated from <xref ref-type="fig" rid="fig2">Figure 2F</xref>, marks the border between active and repressed domains. (<bold>B</bold> and <bold>C</bold>) H3K27ac profiles are shown for mixed and PS7 nuclei; the acetylation mark in PS7 is largely restricted to the active domains. (<bold>D</bold> and <bold>E</bold>) Pol-II profiles show peaks at the promoters of <italic>Ubx</italic>, <italic>abd-A</italic>, and <italic>Abd-B</italic> both in whole embryo and in PS7 nuclei. In PS7, polymerase is distributed across the transcription units of <italic>Ubx</italic> and <italic>abd-A</italic>, but not <italic>Abd-B</italic>. The arrow in <bold>D</bold> marks the PS5/PS6 border; the arrow in <bold>E</bold> marks the major <italic>Abd-B</italic> promoter. (<bold>F</bold> and <bold>G</bold>) H3K4me3 profiles show prominent peaks over the <italic>Ubx</italic> and <italic>abd-A</italic> promoters, both of which drive strong transcription in PS7. (<bold>H</bold> and <bold>I</bold>) POLYCOMB profiles show sharp peaks over all known Polycomb Response Elements. In PS7, there is a reduction of the PC peak at the ‘bx PRE’ in the active PS5 domain. There is also a PC peak at the <italic>abd-A</italic> promoter, which is somewhat reduced in PS7, where <italic>Abd-A</italic> is transcribed. (<bold>J</bold> and <bold>K</bold>) POLYHOMEOTIC protein shows peaks at known PREs and the <italic>abd-A</italic> promoter in both the active and repressed domains. As with PC, the PH peak at the bx PRE is reduced in PS7. (<bold>L</bold> and <bold>M</bold>) SU(Z)12 profiles also mark PREs, but in PS7, the PRE peaks are reduced in all three active domains. The ranges for the vertical axes are indicated above the left edge of each trace.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02833.009">http://dx.doi.org/10.7554/eLife.02833.009</ext-link></p></caption><graphic xlink:href="elife02833f003"/></fig></p><p>ChIP of serine-5-phosphorylated RNA polymerase II (Pol-II) (transcriptionally engaged polymerase) in whole embryos shows the transcription units for <italic>Ubx</italic>, <italic>abd-A</italic>, and <italic>Abd-B</italic> (<xref ref-type="fig" rid="fig3">Figure 3D</xref>). Embryonic non-coding transcripts (<xref ref-type="bibr" rid="bib1">Bae et al., 2002</xref>; <xref ref-type="bibr" rid="bib36">Pease et al., 2013</xref>) are not apparent from the Pol-II density, either because they are limited to very early stages of embryogenesis, or because the levels of transcription are too low. There is an unexpected Pol-II peak ∼14.5 kb upstream of the <italic>Ubx</italic> promoter, coincident with the CTCF binding site at the border between the PS5 and PS6 domains (arrow in <xref ref-type="fig" rid="fig3">Figure 3D</xref>). There is no reported transcript initiating at this position (<xref ref-type="bibr" rid="bib36">Pease et al., 2013</xref>), although a short transcript could have been missed. Pol-II in PS7 (<xref ref-type="fig" rid="fig3">Figure 3E</xref>) marks the <italic>Ubx</italic> and <italic>abd-A</italic> transcription units, as expected from the UBX and ABD-A protein patterns (<xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2</xref>). There is also a Pol-II peak at the major <italic>Abd-B</italic> promoter (arrow in <xref ref-type="fig" rid="fig3">Figure 3E</xref>), although <italic>Abd-B</italic> RNA and protein are absent from PS7 (<xref ref-type="bibr" rid="bib10">Celniker et al., 1990</xref>; <xref ref-type="bibr" rid="bib5">Boulet et al., 1991</xref>) and this region is H3K27 trimethylated. Analogous localization of Pol-II at the <italic>Abd-B</italic> promoter in wing and haltere discs was reported by <xref ref-type="bibr" rid="bib11">Chopra et al. (2009)</xref>, who argued that these polymerase molecules were ‘paused’. The H3K4me3 mark aligns with the <italic>Ubx</italic> and <italic>abd-A</italic> promoters in PS7 nuclei (but not <italic>Abd-B</italic>; <xref ref-type="fig" rid="fig3">Figure 3G</xref>) and is reduced in mixed cells (<xref ref-type="fig" rid="fig3">Figure 3F</xref>); this histone mark has been seen at active promoters in other systems (<xref ref-type="bibr" rid="bib40">Ruthenburg et al., 2007</xref>).</p><p>The POLYCOMB subunit of PRC1 is known to bind to the H3K27me3 modification through its chromodomain (<xref ref-type="bibr" rid="bib15">Fischle et al., 2003</xref>)<italic>,</italic> and so PRC1 localization might be expected to correspond to the stair-step H3K27me3 pattern. Published ChIP–CHIP and ChIP-seq profiles for PC across the BX-C vary from broad coverage matching the H3K27me3 profile (<xref ref-type="bibr" rid="bib22">Kwong et al., 2008</xref>) to sharp peaks at the known PREs (<xref ref-type="bibr" rid="bib16">Gonzalez et al., 2014</xref>). The difference may be due to different procedures for chromatin fragmentation prior to immunoprecipitation (<xref ref-type="bibr" rid="bib47">Straub et al., 2013</xref>). Our PC profiles show sharp peaks at PREs in nuclei from whole embryos (<xref ref-type="fig" rid="fig3">Figure 3H</xref>), matching the profile for POLYHOMEOTIC, another PRC1 subunit (<xref ref-type="fig" rid="fig3">Figure 3J</xref>). In PS7 nuclei, we find both the POLYCOMB and POLYHOMEOTIC subunits of PRC1 are bound to the Polycomb Response Elements of the active PS6 and PS7 domains at levels comparable to those in the whole embryo profile (<xref ref-type="fig" rid="fig3">Figure 3H–K</xref>). In the active PS5 domain, the levels of PC and PHO at the PS5 PRE are reduced, relative to the whole embryo profile. <xref ref-type="bibr" rid="bib35">Papp and Müller (2006)</xref> have reported reduced retention of PRC1 and 2 components at the PRE in the PS5 domain in haltere and third leg imaginal discs, where the PS5 domain is in the active state and K27me3 is absent. <xref ref-type="bibr" rid="bib22">Kwong et al. (2008)</xref> have made analogous observations regarding PRC retention in imaginal discs of the third thoracic segment. PRC1 presence is apparently not a determinant of repressed domains, although its subunits are clearly needed for PcG repression (<xref ref-type="bibr" rid="bib43">Simon and Kingston, 2013</xref>). It will be important to map H2A ubiquitylation (a function of the dRING subunit of PRC1) across the domains, especially given recent findings that H2A ubiquitylation promotes PRC2 recruitment and H3K27 trimethylation (<xref ref-type="bibr" rid="bib4">Blackledge et al., 2014</xref>; <xref ref-type="bibr" rid="bib12">Cooper et al., 2014</xref>; <xref ref-type="bibr" rid="bib18">Kalb et al., 2014</xref>).</p><p>The profiles for SU(Z)12, a PRC2 component (<xref ref-type="fig" rid="fig3">Figure 3L,M</xref>), look similar to those of the PRC1 proteins, POLYCOMB and POLYHOMEOTIC. There are major peaks at the known PREs in both whole embryo and PS7 nuclei, although in PS7, the PREs in active domains have reduced levels of SU(Z)12. This result is again consistent with the prior work of <xref ref-type="bibr" rid="bib35">Papp and Müller (2006)</xref> on the PS5 PRE in imaginal discs. SU(Z)12 is also present at the transcription start sites of <italic>Ubx</italic>, <italic>abd-A</italic>, and <italic>Abd-B</italic>; binding at these promoters is more apparent than it is for PRC1 proteins.</p></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><p>The Polycomb Group repression system is often described as a cellular memory mechanism, which can impose lifelong silencing of a gene in response to a transitory signal. That view seems valid, but the concept of a PcG regulatory domain is much richer. In the PS6 domain of the BX-C, for example, there are many enhancers to drive <italic>Ubx</italic> expression in specific cells at specific developmental times, all of which are blocked in parasegments one through five, but active in parasegments 6 through 12 (<xref ref-type="bibr" rid="bib3">Bender and Lucas, 2013</xref>). Individual enhancers need not include a segmental address that is specified, for example, by gap and pair-rule DNA-binding factors; their function is segmentally restricted by the domain architecture. Indeed, these enhancers will drive expression in a different parasegment when inserted into a different domain (as in the <italic>Cbx</italic> transposition, <xref ref-type="bibr" rid="bib37">Peifer et al., 1987</xref>). Each domain has a distinctive collection of enhancers; the UBX pattern in PS5 is quite different from that in PS6. Thus, there are two developmental programs for <italic>Ubx</italic>, one in each of these parasegments, without the need for a duplication of the <italic>Ubx</italic> gene. Other loci with broad regions of H3K27 methylation may likewise be parsed into multiple domains, once we examine histone marks in specific cell types.</p><p>The all-or-nothing H3K27me3 coverage of the BX-C parasegmental domains validates and refines the domain model. In particular, K27me3 is uniformly removed across the PS5 and PS7 domains in PS5 and PS7, even though the activated genes in those parasegments (<italic>Ubx</italic> and <italic>abd-A</italic>, respectively) are only transcribed in a subset of cells. It is interesting that both PRC1 and PRC2 components have binding patterns that do not fully reflect function (repression and K27 methylation, respectively), indicating the possibility that function of these complexes is regulated separately from binding (<xref ref-type="bibr" rid="bib35">Papp and Müller, 2006</xref>). The challenges now are to understand how PcG regulated domains are established, differently in different parasegments, and to describe the molecular mechanisms, including changes in chromosome structure, that block gene activity in H3K27 trimethylated domains.</p></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Gal4 and Gal80 sources</title><p>A P element was built containing both Gal4 and Gal80 (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1A</xref>). The P promoter initially drives Gal4 transcription. The P-element used the Gal4 gene with an HSP70 terminator derived from pGawB (<xref ref-type="bibr" rid="bib7">Brand and Perrimon, 1993</xref>), fused in frame to the P transposase gene after the 130th amino acid. The Gal4 gene was followed by a synthetic FRT site plus a polylinker. The Gal80 coding sequence with an HSP70 terminator, derived from pBPG80Uw (a gift of B Pfeiffer and G Rubin) was inserted into this polylinker. Another synthetic FRT site was inserted near the start of the Gal4 coding region at a unique <italic>Sph</italic>I site. Thus, the Gal4 coding sequence is flanked by FRT sites, so that flipase-induced recombination can turn the P element into a Gal80 producer, with the same expression pattern. The P element was transformed into a random chromosomal position, and then used to swap into the position of an existing P enhancer trap in the BX-C (<xref ref-type="bibr" rid="bib2">Bender and Hudson, 2000</xref>) or at the <italic>Antennapedia</italic> promoter. P element swapping (<xref ref-type="bibr" rid="bib42">Sepp and Auld, 1999</xref>) is a low frequency event, but the lines were constructed with UAS/GFP as a marker for Gal4 activity, and thousands of first instar larvae could be quickly screened for one with the expected segmentally-restricted GFP pattern. A derivative of this vector was made by replacing the HSP70 terminator downstream of the Gal80 coding sequence with the poly(A) addition site from the <italic>Drosophila Alcohol Dehydrogenase</italic> (<italic>Adh</italic>) gene, recovered as a 317 bp PCR fragment from genomic DNA.</p><p>We have also taken known embryonic enhancers from the BX-C and inserted them into a second vector (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1B</xref>), which was derived from the initial vector with the <italic>Adh</italic> terminator. At a <italic>Bam</italic>H1 site immediately upstream of the 5′ FRT site, a fragment was inserted containing: (1) an 841 bp fragment from the 5′ end of the <italic>gypsy</italic> mobile element (bases 189-1029), (2) an <italic>Asc</italic>I site, and (3) a fragment from the <italic>engrailed</italic> gene (−595 bp to +243 bp relative to the major transcription start site). The <italic>gypsy</italic> fragment includes twelve tandem binding sites for SUPPRESSOR OF HAIRY WING (SU(HW)). The <italic>engrailed</italic> fragment includes the proximal PRE (‘PSE2’ [<xref ref-type="bibr" rid="bib14">DeVido et al., 2008</xref>]), and is designed to fuse the first nine amino acids of ENGRAILED to the amino terminal end of Gal4. Enhancer fragments from the BX-C were cloned by PCR with <italic>Asc</italic>I extensions on the primers, and inserted into the <italic>Asc</italic>I site between the <italic>gypsy</italic> and <italic>engrailed</italic> fragments. The engrailed PRE was included to maintain the segmentally-restricted pattern through most of embryonic development, and the SU(HW) binding sequences were intended to insulate the promoter from endogenous enhancers near the insertion site. Transformants were recognized by Gal4-driven GFP patterns in young larvae, and, again, any Gal4 producer could be converted to a Gal80 producer. Sequences of these P elements are available on request.</p></sec><sec id="s4-2"><title>Swap positions</title><p>P element swapping was used to replace a P element at the <italic>Antennapedia</italic> P1 promoter <bold>(</bold>P[XP] Antpd02480, target site duplication: 3R: 2,825,198-2,825,205 [<xref ref-type="bibr" rid="bib48">Thibault et al., 2004</xref>]), and a P element in the PS8 (iab-3) domain of the BX-C (HCJ192, target site duplication 3R: 12,673,279-12,673,286 [<xref ref-type="bibr" rid="bib2">Bender and Hudson, 2000</xref>]). Other swaps were made with P elements in the PS5 (bx) and PS6 (bxd) domains, but these did not give sufficiently strong or uniform expression patterns. The <italic>Antp</italic> swap was subjected to P transposase to induce ‘local hopping’ and thereby increase the Gal4 expression level.</p></sec><sec id="s4-3"><title>Enhancer fragments</title><p>Fragments used as enhancers inserted into the vector of <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1B</xref> included:<list list-type="simple"><list-item><p>abx enhancer (for PS5 anterior limit) 3R: 12,508,951-12,513,096</p></list-item><list-item><p>pbx enhancer (for PS6 anterior limit) 3R: 12,598,546-12,600,177</p></list-item><list-item><p>iab-2 enhancer (for PS7 anterior limit) 3R: 12,636,236-12,639,140</p></list-item><list-item><p>iab-3 enhancer (for PS8 anterior limit) 3R: 12,664,301-12,666,886</p></list-item></list></p><p>Other fragments tested covered the ‘bx’ and ‘bxd’ enhancers, but these did not give sufficiently strong or uniform expression patterns. One insertion of a P element with the iab-3 enhancer fortuitously inserted 1.4 kb upstream of the <italic>Ubx</italic> transcription start site (target site duplication, 3R: 12,561,577-12,561,584). This gave a strong pattern with a sharp PS6 anterior limit, and was used as the Gal4 source for PS6 nuclei. Insertions with the abx and pbx enhancers were subjected to P transposase to adjust the levels of Gal4 and Gal80. <italic>D. melanogaster</italic> genome coordinates are from release 5.57.</p></sec><sec id="s4-4"><title>INTACT marker</title><p>Flies with the INTACT reporter in a P element on the third chromosome (w<sup>1118</sup>; p[w<sup>+</sup>; UASRG]6) were generously provided by Paul Talbert and Steve Henikoff. This P element was crossed to a P transposase source (P{ry[+t7.2] = Delta2-3}99B), and offspring were screened initially for darker eye color, most likely due to multiple copies of the INTACT element. Such stocks with INTACT insertions on the second or third chromosome were then crossed to a Gal4 source, and those with brightest mCherry expression were used for subsequent efforts to mark single parasegments. The higher copy INTACT chromosomes are designated ‘INTACT<sub>n</sub> on II’ and ‘INTACT<sub>n</sub> on III’.</p></sec><sec id="s4-5"><title>Genotypes</title><p>To mark PS4, males homozygous for [w; abx en.>Gal80, pbx en.>Gal80; Antp swap>Gal4*, INTACT on III] were crossed to females homozygous for [w; INTACT<sub>n</sub> on II; INTACT<sub>n</sub> on III].</p><p>To mark PS5, males homozygous for [w; abx en.>Gal4*, pbx en.>Gal80*, UAS-GFP*] were crossed to females homozygous for [w; INTACT<sub>n</sub> on II; INTACT<sub>n</sub> on III].</p><p>To mark PS6, males homozygous for [w; INTACT<sub>n</sub> on II; iab-2 en.>Gal80, iab-3 swap>Gal80, INTACT on III] were crossed to females [w; bxd insert>Gal4, iab-2 en.>Gal80, iab-3 en.>Gal80, INTACT on III / TM6]. Only half of the resulting embryos had a Gal4-driven stripe.</p><p>To mark PS7, males homozygous for [w; iab-2 en.>Gal4, iab-3 swap>Gal80, INTACT on III] were crossed to females homozygous for [w; INTACT<sub>n</sub> on II; INTACT<sub>n</sub> on III].</p><p>Insertions treated with P transposase are marked above with an asterisk (*).</p></sec><sec id="s4-6"><title>Embryo collection and nuclear preps</title><p>Agar plates with yeast paste were put on the PS4, PS5 or PS7 laying cages for 8 hr at 25°C (or 16 hr at 18°C), and then the eggs were aged for another 5 hr at 25°C (or 10 hr at 18°C). For PS6, a 6 hr collection was aged for a further 4 hr. The eggs were dechorionated (2 min in 50% bleach) and fixed in fixation buffer (5% formaldehyde, 100 mM NaCl, 50 mM Hepes, 1 mM EDTA, 0.5 mM EGTA, pH 8.0) under heptane, on a rotator for 15 min at 25°C. The fixed embryos were washed with stop buffer (125 mM glycine, 130 mM NaCl, 7 mM Na<sub>2</sub>HPO<sub>4</sub>, 3 mM KH<sub>2</sub>PO<sub>4</sub>, 0.1% Triton X100, pH 8.0) for 2 min at 25°C, and then briefly with PBS (130 mM NaCl, 7 mM Na<sub>2</sub>HPO<sub>4</sub>, 3 mM KH<sub>2</sub>PO<sub>4</sub>) plus 0.1% Triton X100. The washed embryos were collected on a nytex filter, and examined under an epifluorescence stereoscope. Embryos that appeared too old, with fluorescent nuclei anterior to the desired parasegment, were manually removed. The remaining eggs were collected and weighed in a 1.5 ml microfuge tube, flash frozen in liquid nitrogen, and stored at −75°C.</p><p>For the isolation of nuclei, ∼100 mg of embryos were suspended in 5 ml of BBT buffer (55 mM NaCl, 40 mM KCl, 15 mM MgSO<sub>4</sub>, 5 mM CaCl<sub>2</sub>, 10 mM Tricine, 20 mM dextrose, 50 mM sucrose, 0.1% bovine serum albumin (Miles PENTEX), 0.01% Triton X-100, pH7.0) plus 100 µl protease inhibitor cocktail (Roche Complete, EDTA-free), and disrupted with 10 strokes in a Dounce homogenizer with a loose pestle, then 10 strokes with a tight pestle. The homogenate was spun at 275 × g for 1 min to remove large debris. The resulting supernatant was spun at 1000 × g for 10 min to pellet the nuclei. The pellet was resuspended in 2 ml BBT buffer plus 40 µl protease inhibitor, and homogenized again, 20 strokes with the tight pestle. The homogenate was passed through a cell strainer cap with a 40 µm nylon mesh and stored at 0°C. Hoechst 33,342 dye was added (2 µl of 10 mg/ml solution) before sorting.</p></sec><sec id="s4-7"><title>Sorting</title><p>Nuclei were sorted on a BD Biosciences (San Jose, CA) FACSAria IIu instrument. The fluorescence from Hoechst 33342 (405 nM excitation, 425–475 nm emission) was used to select for a 2N DNA content, thus avoiding clumps of nuclei. These single nuclei were secondarily selected for mCherry fluorescence (594 nM excitation, 619–640 nM emission) stronger than 99.9% of nuclei from control (<italic>Oregon R</italic>) embryos. Recovered nuclei constituted between 1 and 6% of total single nuclei (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1C</xref>), depending of the parasegment under selection. Recovered nuclei were kept at 0°C, and used for chromatin preparation within 5 hr.</p></sec><sec id="s4-8"><title>Chromatin preparation</title><p>The yield of sorted nuclei in sort buffer (PBS) was determined with a hemacytometer, and 50–200 K nuclei were used for each ChIP. Chromatin fragmentation was accomplished either with micrococcal nuclease (MNase) or bath sonication. For MNase fragmentation, nuclei in sort buffer were supplemented with PBS containing 0.1% Triton-X100 (PBS-Tx) to a volume of 400 µl, and CaCl2 to 1 mM. Nuclei were pre-warmed to 37°C and digested with 12U MNase (Worthington Biochemical) for 3 min. Digestion was stopped by moving the tubes to ice and adding 10 µl of 250 mM EDTA, 250 mM EGTA, and nuclei were briefly sonicated in a Diagenode Bioruptor (Denville, NJ) (3 min, high intensity, 30 s on, 30 s off). For bath sonication, the nuclear suspension in sort buffer was adjusted to ChIP buffer conditions (10 mM Tris pH8, 100 mM NaCl, 0.1% sodium deoxycholate, 0.5% sarkosyl, 1% Triton-X100) in a volume of 500 µl. Nuclei were sonicated for 30 min at high intensity (30 s on, 30 s off) in a Bioruptor. Fragmented chromatin was snap frozen and stored at −80°C prior to ChIP.</p></sec><sec id="s4-9"><title>ChIP-seq</title><p>MNase fragmented chromatin was adjusted to ChIP buffer conditions in a final volume of 500 µl, and incubated on ice for 5 min. Prior to ChIP, protease inhibitors (COMPLETE, Roche) were added to either MNase fragmented chromatin or sonicated chromatin. Chromatin was subjected to a high speed spin at 4°C for 10 min. Supernatant was moved to a new low retention tube, 1% input was removed and stored at 4°C, and and the remaining chromatin was incubated overnight with the appropriate antibody at 4°C. After a high speed spin at 4°C for 10 min, supernatant was moved to a low retention tube with 10 µl prewashed protein A dynabeads (Life Technologies), and incubated at 4°C for 1–2 hr with gentle rotation. After six rinses with ice cold ChIP buffer, immunoprecipitates were eluted from the beads with two successive additions of 125 µl freshly made elution buffer (0.2% SDS, 0.1 M NaHCO3, 5 mM DTT) incubated at 65°C for 10 min. Eluates were combined, and adjusted with Tris and EDTA to a final concentration of 10 mM Tris pH8 and 2 mM EDTA. 250 µl elution buffer was added to the reserved input and likewise adjusted. DNA cleanup began by incubating with RNase (DNase-free, Roche) at 37°C for 30 min, proteinase K (PCR-grade, Roche) at 55°C for 1 hr, and crosslinks were reversed by incubating at 65°C for 1 hr. After two phenol-chloroform extractions and one chloroform extraction, purified DNA was ethanol precipitated in the presence of sodium acetate and Glycoblue (Ambion). Sequencing library construction was performed according to (<xref ref-type="bibr" rid="bib6">Bowman et al., 2013</xref>). Libraries were sequenced on an Illumina (San Diego, CA) HiSeq2000, HiSeq2500, or MiSeq according to manufacturer's instructions.</p></sec><sec id="s4-10"><title>ChIP antisera</title><p>anti-K27me3 (Active Motif 39155), anti-K27ac (Active Motif 39,136), anti-K4me3 (07473; Millipore), anti-CTCF (<xref ref-type="bibr" rid="bib44">Smith et al., 2009</xref>), anti-Ph (residues 772–984 [<xref ref-type="bibr" rid="bib33">Oktaba et al., 2008</xref>]), anti-CP190 (<xref ref-type="bibr" rid="bib34">Pai et al., 2004</xref>), anti-Pol-II (ab5131; Abcam), anti-Pc (<xref ref-type="bibr" rid="bib41">Schuettengruber et al., 2009</xref>), anti-SU(Z)12 (<xref ref-type="bibr" rid="bib29">Müller et al., 2002</xref>).</p></sec><sec id="s4-11"><title>Data analysis</title><p>ChIP-seq reads were aligned to the dm3 genome using BWA and allowing up to one mismatch. Potential PCR duplicates, tags with multiple alignments, and paired-end reads with insert size greater than 1 kb were removed from further analysis. We calculated normalized positional coverage as previously described (<xref ref-type="bibr" rid="bib39">Pinter et al., 2012</xref>; <xref ref-type="bibr" rid="bib50">Yildirim et al., 2012</xref>). Briefly, experimental coverage was normalized by corresponding input coverage for each position: <italic>n</italic>norm = [(<italic>n</italic>+1)/(<italic>n</italic><sub>i</sub>+1)] * [<italic>N</italic><sub>i</sub>/<italic>N</italic>], where <italic>n</italic>, <italic>n</italic><sub>i</sub>, <italic>N</italic>, and <italic>N</italic><sub>i</sub> are positional coverages in experiment and input, and total genome coverages in experiment and input, respectively.</p><p>To determine regions of enrichment, we summed the coverage within sliding windows of size 1 kb and step 50 bp across the entire genome. The same was calculated for tags assigned a random location within the chromosome. The resulting distribution of random window coverages was then used to determine the significance of observed window coverages. Individual p-value cutoffs were selected based on manual inspection of each experiment. For samples with replicates, only regions significant in both replicates were used.</p><p>For genome-wide analysis of coverage difference between two parasegments for H3K27me3, we first merged the significant regions of enrichment from the different parasegments to create an aggregate set of regions of interest. Regions greater than 50 kb were split into equally sized sub-regions. For each region we calculated the density, or total normalized coverage divided by region length. Replicate density values were averaged. To find differences, region densities for two parasegments were scatter plotted against one another, and a line of best fit calculated. Points located furthest away from this fit line were identified as the candidate differing regions. This approach allows us to account for the variation in sequencing coverage and noise for each sample and parasegment.</p><p>SPP (<xref ref-type="bibr" rid="bib21">Kharchenko et al., 2008</xref>) was used to generate tag density profiles for visualization. For each chip ChIP experiment, we followed standard methods to estimate the binding peak separation distance, remove low quality or anomalous tags, and generate an input-subtracted, Gaussian-smoothed tag density profile.</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>The datasets supporting the results of this report are available in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE55257. We are grateful to Barret Pfeiffer for the pBPG80Uw plasmid containing a codon-optimized version of Gal80. Paul Talbert and Steve Henikoff supplied us with a UAS/INTACT P element. Stephanie Terrizzi and Jodi Moore assisted with nuclear sorting. PH and SU(Z)12 antiserum were a gift from Jürg Müller, and CTCF antiserum was a gift from Victor Lubanenkov. Matt Simon, Michele Markstein, and Jesse Cochrane gave helpful comments on the manuscript. SKB was supported by the Damon Runyon Cancer Research Foundation. This work was supported by grants from the NIH, GM43901 to REK and GM28630 to WB.</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>The authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>SKB, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting and revising the article</p></fn><fn fn-type="con" id="con2"><p>AMD, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting and revising the article</p></fn><fn fn-type="con" id="con3"><p>WB, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting and revising the article</p></fn><fn fn-type="con" id="con4"><p>HD, Conception and design, Acquisition of data</p></fn><fn fn-type="con" id="con5"><p>PIW, Analysis and interpretation of data, Drafting and revising the article</p></fn><fn fn-type="con" id="con6"><p>RIS, Analysis and interpretation of data, Drafting and revising the article</p></fn><fn fn-type="con" id="con7"><p>REK, Conception and design, Analysis and interpretation of data, Drafting and revising the article</p></fn></fn-group></sec><sec sec-type="supplementary-material"><title>Additional files</title><sec sec-type="datasets"><title>Major dataset</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>Bowman</surname><given-names>SK</given-names></name>, <name><surname>Deaton</surname><given-names>AM</given-names></name>, <name><surname>Domingues</surname><given-names>H</given-names></name>, <name><surname>Wang</surname><given-names>PI</given-names></name>, <name><surname>Sadreyev</surname><given-names>RI</given-names></name>, <name><surname>Kingston</surname><given-names>RE</given-names></name>, <name><surname>Bender</surname><given-names>W</given-names></name>, <year>2014</year><x>, </x><source>H3K27 modifications define segmental regulatory domains in the <italic>Drosophila</italic> bithorax complex</source><x>, </x><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE55257">http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE55257</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>Bae</surname><given-names>E</given-names></name><name><surname>Calhoun</surname><given-names>VC</given-names></name><name><surname>Levine</surname><given-names>M</given-names></name><name><surname>Lewis</surname><given-names>EB</given-names></name><name><surname>Drewell</surname><given-names>RA</given-names></name></person-group><year>2002</year><article-title>Characterization of the intergenic RNA profile at <italic>abdominal-A</italic> and <italic>Abdominal-B</italic> in the <italic>Drosophila</italic> bithorax complex</article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>99</volume><fpage>16847</fpage><lpage>16852</lpage><pub-id pub-id-type="doi">10.1073/pnas.222671299</pub-id></element-citation></ref><ref id="bib2"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bender</surname><given-names>W</given-names></name><name><surname>Hudson</surname><given-names>A</given-names></name></person-group><year>2000</year><article-title>P element homing to the Drosophila bithorax complex</article-title><source>Development</source><volume>127</volume><fpage>3981</fpage><lpage>3992</lpage></element-citation></ref><ref id="bib3"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bender</surname><given-names>W</given-names></name><name><surname>Lucas</surname><given-names>M</given-names></name></person-group><year>2013</year><article-title>The border between the <italic>Ultrabithorax</italic> and <italic>abdominal-A</italic> regulatory domains in the <italic>Drosophila</italic> bithorax complex</article-title><source>Genetics</source><volume>193</volume><fpage>1135</fpage><lpage>1147</lpage><pub-id pub-id-type="doi">10.1534/genetics.112.146340</pub-id></element-citation></ref><ref id="bib4"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Blackledge</surname><given-names>NP</given-names></name><name><surname>Farcas</surname><given-names>AM</given-names></name><name><surname>Kondo</surname><given-names>T</given-names></name><name><surname>King</surname><given-names>HW</given-names></name><name><surname>McGouran</surname><given-names>JF</given-names></name><name><surname>Hanssen</surname><given-names>LL</given-names></name><name><surname>Ito</surname><given-names>S</given-names></name><name><surname>Cooper</surname><given-names>S</given-names></name><name><surname>Kondo</surname><given-names>K</given-names></name><name><surname>Koseki</surname><given-names>Y</given-names></name><name><surname>Ishikura</surname><given-names>T</given-names></name><name><surname>Long</surname><given-names>HK</given-names></name><name><surname>Sheahan</surname><given-names>TW</given-names></name><name><surname>Brockdorff</surname><given-names>N</given-names></name><name><surname>Kessler</surname><given-names>BM</given-names></name><name><surname>Koseki</surname><given-names>H</given-names></name><name><surname>Klose</surname><given-names>RJ</given-names></name></person-group><year>2014</year><article-title>Variant PRC1 complex-dependent H2A ubiquitylation drives PRC2 recruitment and polycomb domain formation</article-title><source>Cell</source><volume>157</volume><fpage>1445</fpage><lpage>1459</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2014.05.004</pub-id></element-citation></ref><ref id="bib5"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Boulet</surname><given-names>AM</given-names></name><name><surname>Lloyd</surname><given-names>A</given-names></name><name><surname>Sakonju</surname><given-names>S</given-names></name></person-group><year>1991</year><article-title>Molecular definition of the morphogenetic and regulatory functions and the <italic>cis</italic>-regulatory elements of the <italic>Drosophila Abd-B</italic> homeotic gene</article-title><source>Development</source><volume>111</volume><fpage>393</fpage><lpage>405</lpage></element-citation></ref><ref id="bib6"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bowman</surname><given-names>SK</given-names></name><name><surname>Simon</surname><given-names>MD</given-names></name><name><surname>Deaton</surname><given-names>AM</given-names></name><name><surname>Tolstorukov</surname><given-names>M</given-names></name><name><surname>Borowsky</surname><given-names>ML</given-names></name><name><surname>Kingston</surname><given-names>RE</given-names></name></person-group><year>2013</year><article-title>Multiplexed Illumina sequencing libraries from picogram quantities of DNA</article-title><source>BMC Genomics</source><volume>14</volume><fpage>466</fpage><pub-id pub-id-type="doi">10.1186/1471-2164-14-466</pub-id></element-citation></ref><ref id="bib7"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Brand</surname><given-names>AH</given-names></name><name><surname>Perrimon</surname><given-names>N</given-names></name></person-group><year>1993</year><article-title>Targeted gene expression as a means of altering cell fates and generating dominant phenotypes</article-title><source>Development</source><volume>118</volume><fpage>401</fpage><lpage>415</lpage></element-citation></ref><ref id="bib8"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Carroll</surname><given-names>SB</given-names></name><name><surname>Laymon</surname><given-names>RA</given-names></name><name><surname>McCutcheon</surname><given-names>MA</given-names></name><name><surname>Riley</surname><given-names>PD</given-names></name><name><surname>Scott</surname><given-names>MP</given-names></name></person-group><year>1986</year><article-title>The localization and regulation of <italic>Antennapedia</italic> protein expression in Drosophila embryos</article-title><source>Cell</source><volume>47</volume><fpage>113</fpage><lpage>122</lpage><pub-id pub-id-type="doi">10.1016/0092-8674(86)90372-7</pub-id></element-citation></ref><ref id="bib9"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Casares</surname><given-names>F</given-names></name><name><surname>Bender</surname><given-names>W</given-names></name><name><surname>Merriam</surname><given-names>J</given-names></name><name><surname>Sanchez-Herrero</surname><given-names>E</given-names></name></person-group><year>1997</year><article-title>Interactions of Drosophila <italic>Ultrabithorax</italic> regulatory regions with native and foreign promoters</article-title><source>Genetics</source><volume>145</volume><fpage>123</fpage><lpage>137</lpage></element-citation></ref><ref id="bib10"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Celniker</surname><given-names>SE</given-names></name><name><surname>Sharma</surname><given-names>S</given-names></name><name><surname>Keelan</surname><given-names>DJ</given-names></name><name><surname>Lewis</surname><given-names>EB</given-names></name></person-group><year>1990</year><article-title>The molecular genetics of the bithorax complex of <italic>Drosophila: cis</italic>-regulation in the <italic>Abdominal-B</italic> domain</article-title><source>The EMBO Journal</source><volume>9</volume><fpage>4277</fpage><lpage>4286</lpage></element-citation></ref><ref id="bib11"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chopra</surname><given-names>VS</given-names></name><name><surname>Hong</surname><given-names>JW</given-names></name><name><surname>Levine</surname><given-names>M</given-names></name></person-group><year>2009</year><article-title>Regulation of hox gene activity by transcriptional elongation in <italic>Drosophila</italic></article-title><source>Current Biology</source><volume>19</volume><fpage>688</fpage><lpage>693</lpage><pub-id pub-id-type="doi">10.1016/j.cub.2009.02.055</pub-id></element-citation></ref><ref id="bib12"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cooper</surname><given-names>S</given-names></name><name><surname>Dienstbier</surname><given-names>M</given-names></name><name><surname>Hassan</surname><given-names>R</given-names></name><name><surname>Schermelleh</surname><given-names>L</given-names></name><name><surname>Sharif</surname><given-names>J</given-names></name><name><surname>Blackledge</surname><given-names>NP</given-names></name><name><surname>de Marco</surname><given-names>V</given-names></name><name><surname>Elderkin</surname><given-names>S</given-names></name><name><surname>Koseki</surname><given-names>H</given-names></name><name><surname>Klose</surname><given-names>R</given-names></name><name><surname>Heger</surname><given-names>A</given-names></name><name><surname>Brockdorff</surname><given-names>N</given-names></name></person-group><year>2014</year><article-title>Targeting polycomb to pericentric heterochromatin in embryonic stem cells reveals a role for H2AK119u1 in PRC2 recruitment</article-title><source>Cell Reports</source><volume>7</volume><fpage>1456</fpage><lpage>1470</lpage><pub-id pub-id-type="doi">10.1016/j.celrep.2014.04.012</pub-id></element-citation></ref><ref id="bib13"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Deal</surname><given-names>RB</given-names></name><name><surname>Henikoff</surname><given-names>S</given-names></name></person-group><year>2011</year><article-title>The INTACT method for cell type-specific gene expression and chromatin profiling in <italic>Arabidopsis thaliana</italic></article-title><source>Nature Protocols</source><volume>6</volume><fpage>56</fpage><lpage>68</lpage><pub-id pub-id-type="doi">10.1038/nprot.2010.175</pub-id></element-citation></ref><ref id="bib14"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>DeVido</surname><given-names>SK</given-names></name><name><surname>Kwon</surname><given-names>D</given-names></name><name><surname>Brown</surname><given-names>JL</given-names></name><name><surname>Kassis</surname><given-names>JA</given-names></name></person-group><year>2008</year><article-title>The role of Polycomb-group response elements in regulation of <italic>engrailed</italic> transcription in <italic>Drosophila</italic></article-title><source>Development</source><volume>135</volume><fpage>669</fpage><lpage>676</lpage><pub-id pub-id-type="doi">10.1242/dev.014779</pub-id></element-citation></ref><ref id="bib15"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Fischle</surname><given-names>W</given-names></name><name><surname>Wang</surname><given-names>Y</given-names></name><name><surname>Jacobs</surname><given-names>SA</given-names></name><name><surname>Kim</surname><given-names>Y</given-names></name><name><surname>Allis</surname><given-names>CD</given-names></name><name><surname>Khorasanizadeh</surname><given-names>S</given-names></name></person-group><year>2003</year><article-title>Molecular basis for the discrimination of repressive methyl-lysine marks in histone H3 by Polycomb and HP1 chromodomains</article-title><source>Genes & Development</source><volume>17</volume><fpage>1870</fpage><lpage>1881</lpage><pub-id pub-id-type="doi">10.1101/gad.1110503</pub-id></element-citation></ref><ref id="bib16"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gonzalez</surname><given-names>I</given-names></name><name><surname>Mateos-Langerak</surname><given-names>J</given-names></name><name><surname>Thomas</surname><given-names>A</given-names></name><name><surname>Cheutin</surname><given-names>T</given-names></name><name><surname>Cavalli</surname><given-names>G</given-names></name></person-group><year>2014</year><article-title>Identification of regulators of the three-dimensional polycomb organization by a microscopy-based genome-wide RNAi screen</article-title><source>Molecular Cell</source><volume>54</volume><fpage>485</fpage><lpage>499</lpage><pub-id pub-id-type="doi">10.1016/j.molcel.2014.03.004</pub-id></element-citation></ref><ref id="bib17"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Heintzman</surname><given-names>ND</given-names></name><name><surname>Hon</surname><given-names>GC</given-names></name><name><surname>Hawkins</surname><given-names>RD</given-names></name><name><surname>Kheradpour</surname><given-names>P</given-names></name><name><surname>Stark</surname><given-names>A</given-names></name><name><surname>Harp</surname><given-names>LF</given-names></name><name><surname>Ye</surname><given-names>Z</given-names></name><name><surname>Lee</surname><given-names>LK</given-names></name><name><surname>Stuart</surname><given-names>RK</given-names></name><name><surname>Ching</surname><given-names>CW</given-names></name><name><surname>Ching</surname><given-names>KA</given-names></name><name><surname>Antosiewicz-Bourget</surname><given-names>JE</given-names></name><name><surname>Liu</surname><given-names>H</given-names></name><name><surname>Zhang</surname><given-names>X</given-names></name><name><surname>Green</surname><given-names>RD</given-names></name><name><surname>Lobanenkov</surname><given-names>VV</given-names></name><name><surname>Stewart</surname><given-names>R</given-names></name><name><surname>Thomson</surname><given-names>JA</given-names></name><name><surname>Crawford</surname><given-names>GE</given-names></name><name><surname>Kellis</surname><given-names>M</given-names></name><name><surname>Ren</surname><given-names>B</given-names></name></person-group><year>2009</year><article-title>Histone modifications at human enhancers reflect global cell-type-specific gene expression</article-title><source>Nature</source><volume>459</volume><fpage>108</fpage><lpage>112</lpage><pub-id pub-id-type="doi">10.1038/nature07829</pub-id></element-citation></ref><ref id="bib18"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kalb</surname><given-names>R</given-names></name><name><surname>Latwiel</surname><given-names>S</given-names></name><name><surname>Baymaz</surname><given-names>HI</given-names></name><name><surname>Jansen</surname><given-names>PW</given-names></name><name><surname>Müller</surname><given-names>CW</given-names></name><name><surname>Vermeulen</surname><given-names>M</given-names></name><name><surname>Müller</surname><given-names>J</given-names></name></person-group><year>2014</year><article-title>Histone H2A monoubiquitination promotes histone H3 methylation in Polycomb repression</article-title><source>Nature Structural & Molecular Biology</source><volume>21</volume><fpage>569</fpage><lpage>571</lpage><pub-id pub-id-type="doi">10.1038/nsmb.2833</pub-id></element-citation></ref><ref id="bib19"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Karch</surname><given-names>F</given-names></name><name><surname>Bender</surname><given-names>W</given-names></name><name><surname>Weiffenbach</surname><given-names>B</given-names></name></person-group><year>1990</year><article-title><italic>abdA</italic> expression in <italic>Drosophila</italic> embryos</article-title><source>Genes & Development</source><volume>4</volume><fpage>1573</fpage><lpage>1587</lpage><pub-id pub-id-type="doi">10.1101/gad.4.9.1573</pub-id></element-citation></ref><ref id="bib20"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kharchenko</surname><given-names>PV</given-names></name><name><surname>Alekseyenko</surname><given-names>AA</given-names></name><name><surname>Schwartz</surname><given-names>YB</given-names></name><name><surname>Minoda</surname><given-names>A</given-names></name><name><surname>Riddle</surname><given-names>NC</given-names></name><name><surname>Ernst</surname><given-names>J</given-names></name><name><surname>Sabo</surname><given-names>PJ</given-names></name><name><surname>Larschan</surname><given-names>E</given-names></name><name><surname>Gorchakov</surname><given-names>AA</given-names></name><name><surname>Gu</surname><given-names>T</given-names></name><name><surname>Linder-Basso</surname><given-names>D</given-names></name><name><surname>Plachetka</surname><given-names>A</given-names></name><name><surname>Shanower</surname><given-names>G</given-names></name><name><surname>Tolstorukov</surname><given-names>MY</given-names></name><name><surname>Luquette</surname><given-names>LJ</given-names></name><name><surname>Xi</surname><given-names>R</given-names></name><name><surname>Jung</surname><given-names>YL</given-names></name><name><surname>Park</surname><given-names>RW</given-names></name><name><surname>Bishop</surname><given-names>EP</given-names></name><name><surname>Canfield</surname><given-names>TK</given-names></name><name><surname>Sandstrom</surname><given-names>R</given-names></name><name><surname>Thurman</surname><given-names>RE</given-names></name><name><surname>MacAlpine</surname><given-names>DM</given-names></name><name><surname>Stamatoyannopoulos</surname><given-names>JA</given-names></name><name><surname>Kellis</surname><given-names>M</given-names></name><name><surname>Elgin</surname><given-names>SC</given-names></name><name><surname>Kuroda</surname><given-names>MI</given-names></name><name><surname>Pirrotta</surname><given-names>V</given-names></name><name><surname>Karpen</surname><given-names>GH</given-names></name><name><surname>Park</surname><given-names>PJ</given-names></name></person-group><year>2011</year><article-title>Comprehensive analysis of the chromatin landscape in <italic>Drosophila melanogaster</italic></article-title><source>Nature</source><volume>471</volume><fpage>480</fpage><lpage>485</lpage><pub-id pub-id-type="doi">10.1038/nature09725</pub-id></element-citation></ref><ref id="bib21"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kharchenko</surname><given-names>PV</given-names></name><name><surname>Tolstorukov</surname><given-names>MY</given-names></name><name><surname>Park</surname><given-names>PJ</given-names></name></person-group><year>2008</year><article-title>Design and analysis of ChIP-seq experiments for DNA-binding proteins</article-title><source>Nature Biotechnology</source><volume>26</volume><fpage>1351</fpage><lpage>1359</lpage><pub-id pub-id-type="doi">10.1038/nbt.1508</pub-id></element-citation></ref><ref id="bib22"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kwong</surname><given-names>C</given-names></name><name><surname>Adryan</surname><given-names>B</given-names></name><name><surname>Bell</surname><given-names>I</given-names></name><name><surname>Meadows</surname><given-names>L</given-names></name><name><surname>Russell</surname><given-names>S</given-names></name><name><surname>Manak</surname><given-names>JR</given-names></name><name><surname>White</surname><given-names>R</given-names></name></person-group><year>2008</year><article-title>Stability and dynamics of polycomb target sites in <italic>Drosophila</italic> development</article-title><source>PLOS Genetics</source><volume>4</volume><fpage>e1000178</fpage><pub-id pub-id-type="doi">10.1371/journal.pgen.1000178</pub-id></element-citation></ref><ref id="bib23"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lewis</surname><given-names>EB</given-names></name></person-group><year>1978</year><article-title>A gene complex controlling segmentation in <italic>Drosophila</italic></article-title><source>Nature</source><volume>276</volume><fpage>565</fpage><lpage>570</lpage><pub-id pub-id-type="doi">10.1038/276565a0</pub-id></element-citation></ref><ref id="bib24"><element-citation publication-type="book"><person-group person-group-type="author"><name><surname>Lewis</surname><given-names>EB</given-names></name></person-group><year>1981</year><article-title>Developmental genetics of the bithorax complex in Drosophila</article-title><person-group person-group-type="editor"><name><surname>Brown</surname><given-names>DD</given-names></name><name><surname>Fox</surname><given-names>CF</given-names></name></person-group><source>Developmental Biology Using Purified Genes ICN-UCLA Symposia on Molecular and Cellular Biology XXIII</source><publisher-loc>New York</publisher-loc><publisher-name>Academic Press</publisher-name></element-citation></ref><ref id="bib25"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lewis</surname><given-names>PH</given-names></name></person-group><year>1947</year><article-title>Melanogaster-new mutants: report of Pamela H. Lewis</article-title><source>Drosophila Information Service</source><volume>21</volume><fpage>69</fpage></element-citation></ref><ref id="bib26"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Maeda</surname><given-names>RK</given-names></name><name><surname>Karch</surname><given-names>F</given-names></name></person-group><year>2009</year><article-title>The bithorax complex of <italic>Drosophila</italic>: an exceptional <italic>Hox</italic> cluster</article-title><source>Current Topics in Developmental Biology</source><volume>88</volume><fpage>1</fpage><lpage>33</lpage><pub-id pub-id-type="doi">10.1016/S0070-2153(09)88001-0</pub-id></element-citation></ref><ref id="bib27"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mito</surname><given-names>Y</given-names></name><name><surname>Henikoff</surname><given-names>JG</given-names></name><name><surname>Henikoff</surname><given-names>S</given-names></name></person-group><year>2005</year><article-title>Genome-scale profiling of histone H3.3 replacement patterns</article-title><source>Nature Genetics</source><volume>37</volume><fpage>1090</fpage><lpage>1097</lpage><pub-id pub-id-type="doi">10.1038/ng1637</pub-id></element-citation></ref><ref id="bib28"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mohan</surname><given-names>M</given-names></name><name><surname>Bartkuhn</surname><given-names>M</given-names></name><name><surname>Herold</surname><given-names>M</given-names></name><name><surname>Philippen</surname><given-names>A</given-names></name><name><surname>Heinl</surname><given-names>N</given-names></name><name><surname>Bardenhagen</surname><given-names>I</given-names></name><name><surname>Leers</surname><given-names>J</given-names></name><name><surname>White</surname><given-names>RA</given-names></name><name><surname>Renkawitz-Pohl</surname><given-names>R</given-names></name><name><surname>Saumweber</surname><given-names>H</given-names></name><name><surname>Renkawitz</surname><given-names>R</given-names></name></person-group><year>2007</year><article-title>The <italic>Drosophila</italic> insulator proteins CTCF and CP190 link enhancer blocking to body patterning</article-title><source>The EMBO Journal</source><volume>26</volume><fpage>4203</fpage><lpage>4214</lpage><pub-id pub-id-type="doi">10.1038/sj.emboj.7601851</pub-id></element-citation></ref><ref id="bib29"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Müller</surname><given-names>J</given-names></name><name><surname>Hart</surname><given-names>CM</given-names></name><name><surname>Francis</surname><given-names>NJ</given-names></name><name><surname>Vargas</surname><given-names>ML</given-names></name><name><surname>Sengupta</surname><given-names>A</given-names></name><name><surname>Wild</surname><given-names>B</given-names></name><name><surname>Miller</surname><given-names>EL</given-names></name><name><surname>O'Connor</surname><given-names>MB</given-names></name><name><surname>Kingston</surname><given-names>RE</given-names></name><name><surname>Simon</surname><given-names>JA</given-names></name></person-group><year>2002</year><article-title>Histone Methyltransferase activity of a <italic>Drosophila</italic> Polycomb group repressor complex</article-title><source>Cell</source><volume>111</volume><fpage>197</fpage><lpage>208</lpage><pub-id pub-id-type="doi">10.1016/S0092-8674(02)00976-5</pub-id></element-citation></ref><ref id="bib30"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Nègre</surname><given-names>N</given-names></name><name><surname>Brown</surname><given-names>CD</given-names></name><name><surname>Ma</surname><given-names>L</given-names></name><name><surname>Bristow</surname><given-names>CA</given-names></name><name><surname>Miller</surname><given-names>SW</given-names></name><name><surname>Wagner</surname><given-names>U</given-names></name><name><surname>Kheradpour</surname><given-names>P</given-names></name><name><surname>Eaton</surname><given-names>ML</given-names></name><name><surname>Loriaux</surname><given-names>P</given-names></name><name><surname>Sealfon</surname><given-names>R</given-names></name><name><surname>Li</surname><given-names>Z</given-names></name><name><surname>Ishii</surname><given-names>H</given-names></name><name><surname>Spokony</surname><given-names>RF</given-names></name><name><surname>Chen</surname><given-names>J</given-names></name><name><surname>Hwang</surname><given-names>L</given-names></name><name><surname>Cheng</surname><given-names>C</given-names></name><name><surname>Auburn</surname><given-names>RP</given-names></name><name><surname>Davis</surname><given-names>MB</given-names></name><name><surname>Domanus</surname><given-names>M</given-names></name><name><surname>Shah</surname><given-names>PK</given-names></name><name><surname>Morrison</surname><given-names>CA</given-names></name><name><surname>Zieba</surname><given-names>J</given-names></name><name><surname>Suchy</surname><given-names>S</given-names></name><name><surname>Senderowicz</surname><given-names>L</given-names></name><name><surname>Victorsen</surname><given-names>A</given-names></name><name><surname>Bild</surname><given-names>NA</given-names></name><name><surname>Grundstad</surname><given-names>AJ</given-names></name><name><surname>Hanley</surname><given-names>D</given-names></name><name><surname>MacAlpine</surname><given-names>DM</given-names></name><name><surname>Mannervik</surname><given-names>M</given-names></name><name><surname>Venken</surname><given-names>K</given-names></name><name><surname>Bellen</surname><given-names>H</given-names></name><name><surname>White</surname><given-names>R</given-names></name><name><surname>Gerstein</surname><given-names>M</given-names></name><name><surname>Russell</surname><given-names>S</given-names></name><name><surname>Grossman</surname><given-names>RL</given-names></name><name><surname>Ren</surname><given-names>B</given-names></name><name><surname>Posakony</surname><given-names>JW</given-names></name><name><surname>Kellis</surname><given-names>M</given-names></name><name><surname>White</surname><given-names>KP</given-names></name></person-group><year>2011</year><article-title>A cis-regulatory map of the <italic>Drosophila</italic> genome</article-title><source>Nature</source><volume>471</volume><fpage>527</fpage><lpage>531</lpage><pub-id pub-id-type="doi">10.1038/nature09990</pub-id></element-citation></ref><ref id="bib31"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Nègre</surname><given-names>N</given-names></name><name><surname>Brown</surname><given-names>CD</given-names></name><name><surname>Shah</surname><given-names>PK</given-names></name><name><surname>Kheradpour</surname><given-names>P</given-names></name><name><surname>Morrison</surname><given-names>CA</given-names></name><name><surname>Henikoff</surname><given-names>JG</given-names></name><name><surname>Feng</surname><given-names>X</given-names></name><name><surname>Ahmad</surname><given-names>K</given-names></name><name><surname>Russell</surname><given-names>S</given-names></name><name><surname>White</surname><given-names>RA</given-names></name><name><surname>Stein</surname><given-names>L</given-names></name><name><surname>Henikoff</surname><given-names>S</given-names></name><name><surname>Kellis</surname><given-names>M</given-names></name><name><surname>White</surname><given-names>KP</given-names></name></person-group><year>2010</year><article-title>A comprehensive map of insulator elements for the <italic>Drosophila</italic> genome</article-title><source>PLOS Genetics</source><volume>6</volume><fpage>e1000814</fpage><pub-id pub-id-type="doi">10.1371/journal.pgen.1000814</pub-id></element-citation></ref><ref id="bib32"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ohlsson</surname><given-names>R</given-names></name><name><surname>Bartkuhn</surname><given-names>M</given-names></name><name><surname>Renkawitz</surname><given-names>R</given-names></name></person-group><year>2010</year><article-title>CTCF shapes chromatin by multiple mechanisms: the impact of 20 years of CTCF research on understanding the workings of chromatin</article-title><source>Chromosoma</source><volume>119</volume><fpage>351</fpage><lpage>360</lpage><pub-id pub-id-type="doi">10.1007/s00412-010-0262-0</pub-id></element-citation></ref><ref id="bib33"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Oktaba</surname><given-names>K</given-names></name><name><surname>Gutierrez</surname><given-names>L</given-names></name><name><surname>Gagneur</surname><given-names>J</given-names></name><name><surname>Girardot</surname><given-names>C</given-names></name><name><surname>Sengupta</surname><given-names>AK</given-names></name><name><surname>Furlong</surname><given-names>EE</given-names></name><name><surname>Müller</surname><given-names>J</given-names></name></person-group><year>2008</year><article-title>Dynamic regulation by polycomb group protein complexes controls pattern formation and the cell cycle in <italic>Drosophila</italic></article-title><source>Developmental Cell</source><volume>15</volume><fpage>877</fpage><lpage>889</lpage><pub-id pub-id-type="doi">10.1016/j.devcel.2008.10.005</pub-id></element-citation></ref><ref id="bib34"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pai</surname><given-names>CY</given-names></name><name><surname>Lei</surname><given-names>EP</given-names></name><name><surname>Ghosh</surname><given-names>D</given-names></name><name><surname>Corces</surname><given-names>VG</given-names></name></person-group><year>2004</year><article-title>The centrosomal protein CP190 is a component of the gypsy chromatin insulator</article-title><source>Molecular Cell</source><volume>16</volume><fpage>737</fpage><lpage>748</lpage><pub-id pub-id-type="doi">10.1016/j.molcel.2004.11.004</pub-id></element-citation></ref><ref id="bib35"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Papp</surname><given-names>B</given-names></name><name><surname>Müller</surname><given-names>J</given-names></name></person-group><year>2006</year><article-title>Histone trimethylation and the maintenance of transcriptional ON and OFF states by trxG and PcG proteins</article-title><source>Genes & Development</source><volume>20</volume><fpage>2041</fpage><lpage>2054</lpage><pub-id pub-id-type="doi">10.1101/gad.388706</pub-id></element-citation></ref><ref id="bib36"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pease</surname><given-names>B</given-names></name><name><surname>Borges</surname><given-names>AC</given-names></name><name><surname>Bender</surname><given-names>W</given-names></name></person-group><year>2013</year><article-title>Noncoding RNAs of the <italic>Ultrabithorax</italic> domain of the <italic>Drosophila</italic> bithorax complex</article-title><source>Genetics</source><volume>195</volume><fpage>1253</fpage><lpage>1264</lpage><pub-id pub-id-type="doi">10.1534/genetics.113.155036</pub-id></element-citation></ref><ref id="bib37"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Peifer</surname><given-names>M</given-names></name><name><surname>Karch</surname><given-names>F</given-names></name><name><surname>Bender</surname><given-names>W</given-names></name></person-group><year>1987</year><article-title>The bithorax complex: control of segmental identity</article-title><source>Genes & Development</source><volume>1</volume><fpage>891</fpage><lpage>898</lpage><pub-id pub-id-type="doi">10.1101/gad.1.9.891</pub-id></element-citation></ref><ref id="bib38"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pengelly</surname><given-names>AR</given-names></name><name><surname>Copur</surname><given-names>O</given-names></name><name><surname>Jackle</surname><given-names>H</given-names></name><name><surname>Herzig</surname><given-names>A</given-names></name><name><surname>Müller</surname><given-names>J</given-names></name></person-group><year>2013</year><article-title>A histone mutant reproduces the phenotype caused by loss of histone-modifying factor Polycomb</article-title><source>Science</source><volume>339</volume><fpage>698</fpage><lpage>699</lpage><pub-id pub-id-type="doi">10.1126/science.1231382</pub-id></element-citation></ref><ref id="bib39"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pinter</surname><given-names>SF</given-names></name><name><surname>Sadreyev</surname><given-names>RI</given-names></name><name><surname>Yildirim</surname><given-names>E</given-names></name><name><surname>Jeon</surname><given-names>Y</given-names></name><name><surname>Ohsumi</surname><given-names>TK</given-names></name><name><surname>Borowsky</surname><given-names>M</given-names></name><name><surname>Lee</surname><given-names>JT</given-names></name></person-group><year>2012</year><article-title>Spreading of X chromosome inactivation via a hierarchy of defined Polycomb stations</article-title><source>Genome Research</source><volume>22</volume><fpage>1864</fpage><lpage>1876</lpage><pub-id pub-id-type="doi">10.1101/gr.133751.111</pub-id></element-citation></ref><ref id="bib40"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ruthenburg</surname><given-names>AJ</given-names></name><name><surname>Allis</surname><given-names>CD</given-names></name><name><surname>Wysocka</surname><given-names>J</given-names></name></person-group><year>2007</year><article-title>Methylation of lysine 4 on histone H3: intricacy of writing and reading a single epigenetic mark</article-title><source>Molecular Cell</source><volume>25</volume><fpage>15</fpage><lpage>30</lpage><pub-id pub-id-type="doi">10.1016/j.molcel.2006.12.014</pub-id></element-citation></ref><ref id="bib41"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Schuettengruber</surname><given-names>B</given-names></name><name><surname>Ganapathi</surname><given-names>M</given-names></name><name><surname>Leblanc</surname><given-names>B</given-names></name><name><surname>Portoso</surname><given-names>M</given-names></name><name><surname>Jaschek</surname><given-names>R</given-names></name><name><surname>Tolhuis</surname><given-names>B</given-names></name><name><surname>van Lohuizen</surname><given-names>M</given-names></name><name><surname>Tanay</surname><given-names>A</given-names></name><name><surname>Cavalli</surname><given-names>G</given-names></name></person-group><year>2009</year><article-title>Functional anatomy of polycomb and trithorax chromatin landscapes in Drosophila embryos</article-title><source>PLOS Biology</source><volume>7</volume><fpage>e13</fpage><pub-id pub-id-type="doi">10.1371/journal.pbio.1000013</pub-id></element-citation></ref><ref id="bib42"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sepp</surname><given-names>KJ</given-names></name><name><surname>Auld</surname><given-names>VJ</given-names></name></person-group><year>1999</year><article-title>Conversion of lacZ enhancer trap lines to GAL4 lines using targeted transposition in <italic>Drosophila melanogaster</italic></article-title><source>Genetics</source><volume>151</volume><fpage>1093</fpage><lpage>1101</lpage></element-citation></ref><ref id="bib43"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Simon</surname><given-names>JA</given-names></name><name><surname>Kingston</surname><given-names>RE</given-names></name></person-group><year>2013</year><article-title>Occupying chromatin: Polycomb mechanisms for getting to genomic targets, stopping transcriptional traffic, and staying put</article-title><source>Molecular Cell</source><volume>49</volume><fpage>808</fpage><lpage>824</lpage><pub-id pub-id-type="doi">10.1016/j.molcel.2013.02.013</pub-id></element-citation></ref><ref id="bib44"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Smith</surname><given-names>ST</given-names></name><name><surname>Wickramasinghe</surname><given-names>P</given-names></name><name><surname>Olson</surname><given-names>A</given-names></name><name><surname>Loukinov</surname><given-names>D</given-names></name><name><surname>Lin</surname><given-names>L</given-names></name><name><surname>Deng</surname><given-names>J</given-names></name><name><surname>Xiong</surname><given-names>Y</given-names></name><name><surname>Rux</surname><given-names>J</given-names></name><name><surname>Sachidanandam</surname><given-names>R</given-names></name><name><surname>Sun</surname><given-names>H</given-names></name><name><surname>Lobanenkov</surname><given-names>V</given-names></name><name><surname>Zhou</surname><given-names>J</given-names></name></person-group><year>2009</year><article-title>Genome wide ChIP-chip analyses reveal important roles for CTCF in <italic>Drosophila</italic> genome organization</article-title><source>Developmental Biology</source><volume>328</volume><fpage>518</fpage><lpage>528</lpage><pub-id pub-id-type="doi">10.1016/j.ydbio.2008.12.039</pub-id></element-citation></ref><ref id="bib45"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Soshnikova</surname><given-names>N</given-names></name><name><surname>Duboule</surname><given-names>D</given-names></name></person-group><year>2009</year><article-title>Epigenetic temporal control of mouse <italic>HOX</italic> genes in vivo</article-title><source>Science</source><volume>324</volume><fpage>1320</fpage><lpage>1323</lpage><pub-id pub-id-type="doi">10.1126/science.1171468</pub-id></element-citation></ref><ref id="bib46"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Steiner</surname><given-names>FA</given-names></name><name><surname>Talbert</surname><given-names>PB</given-names></name><name><surname>Kasinathan</surname><given-names>S</given-names></name><name><surname>Deal</surname><given-names>RB</given-names></name><name><surname>Henikoff</surname><given-names>S</given-names></name></person-group><year>2012</year><article-title>Cell-type-specific nuclei purification from whole animals for genome-wide expression and chromatin profiling</article-title><source>Genome Research</source><volume>22</volume><fpage>766</fpage><lpage>777</lpage><pub-id pub-id-type="doi">10.1101/gr.131748.111</pub-id></element-citation></ref><ref id="bib47"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Straub</surname><given-names>T</given-names></name><name><surname>Zabel</surname><given-names>A</given-names></name><name><surname>Gilfillan</surname><given-names>GD</given-names></name><name><surname>Feller</surname><given-names>C</given-names></name><name><surname>Becker</surname><given-names>PB</given-names></name></person-group><year>2013</year><article-title>Different chromatin interfaces of the <italic>Drosophila</italic> doasge compensation complex revealed by high-shear ChIP-seq</article-title><source>Genome Research</source><volume>23</volume><fpage>473</fpage><lpage>485</lpage><pub-id pub-id-type="doi">10.1101/gr.146407.112</pub-id></element-citation></ref><ref id="bib48"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Thibault</surname><given-names>ST</given-names></name><name><surname>Singer</surname><given-names>MA</given-names></name><name><surname>Miyazaki</surname><given-names>WY</given-names></name><name><surname>Milash</surname><given-names>B</given-names></name><name><surname>Dompe</surname><given-names>NA</given-names></name><name><surname>Singh</surname><given-names>CM</given-names></name><name><surname>Buchholz</surname><given-names>R</given-names></name><name><surname>Demsky</surname><given-names>M</given-names></name><name><surname>Fawcett</surname><given-names>R</given-names></name><name><surname>Francis-Lang</surname><given-names>HL</given-names></name><name><surname>Ryner</surname><given-names>L</given-names></name><name><surname>Cheung</surname><given-names>LM</given-names></name><name><surname>Chong</surname><given-names>A</given-names></name><name><surname>Erickson</surname><given-names>C</given-names></name><name><surname>Fisher</surname><given-names>WW</given-names></name><name><surname>Greer</surname><given-names>K</given-names></name><name><surname>Hartouni</surname><given-names>SR</given-names></name><name><surname>Howie</surname><given-names>E</given-names></name><name><surname>Jakkula</surname><given-names>L</given-names></name><name><surname>Joo</surname><given-names>D</given-names></name><name><surname>Killpack</surname><given-names>K</given-names></name><name><surname>Laufer</surname><given-names>A</given-names></name><name><surname>Mazzotta</surname><given-names>J</given-names></name><name><surname>Smith</surname><given-names>RD</given-names></name><name><surname>Stevens</surname><given-names>LM</given-names></name><name><surname>Stuber</surname><given-names>C</given-names></name><name><surname>Tan</surname><given-names>LR</given-names></name><name><surname>Ventura</surname><given-names>R</given-names></name><name><surname>Woo</surname><given-names>A</given-names></name><name><surname>Zakrajsek</surname><given-names>I</given-names></name><name><surname>Zhao</surname><given-names>L</given-names></name><name><surname>Chen</surname><given-names>F</given-names></name><name><surname>Swimmer</surname><given-names>C</given-names></name><name><surname>Kopczynski</surname><given-names>C</given-names></name><name><surname>Duyk</surname><given-names>G</given-names></name><name><surname>Winberg</surname><given-names>ML</given-names></name><name><surname>Margolis</surname><given-names>J</given-names></name></person-group><year>2004</year><article-title>A complementary transposon tool kit for <italic>Drosophila melanogaster</italic> using P and piggyBac</article-title><source>Nature Genetics</source><volume>36</volume><fpage>283</fpage><lpage>287</lpage><pub-id pub-id-type="doi">10.1038/ng1314</pub-id></element-citation></ref><ref id="bib49"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Tie</surname><given-names>F</given-names></name><name><surname>Banerjee</surname><given-names>R</given-names></name><name><surname>Stratton</surname><given-names>CA</given-names></name><name><surname>Prasad-Sinha</surname><given-names>J</given-names></name><name><surname>Stepanik</surname><given-names>V</given-names></name><name><surname>Zlobin</surname><given-names>A</given-names></name><name><surname>Diaz</surname><given-names>MO</given-names></name><name><surname>Scacheri</surname><given-names>PC</given-names></name><name><surname>Harte</surname><given-names>PJ</given-names></name></person-group><year>2009</year><article-title>CBP-mediated acetylation of histone H3 lysine 27 antagonizes <italic>Drosophila</italic> Polycomb silencing</article-title><source>Development</source><volume>136</volume><fpage>3131</fpage><lpage>3141</lpage><pub-id pub-id-type="doi">10.1242/dev.037127</pub-id></element-citation></ref><ref id="bib50"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yildirim</surname><given-names>E</given-names></name><name><surname>Sadreyev</surname><given-names>RI</given-names></name><name><surname>Pinter</surname><given-names>SF</given-names></name><name><surname>Lee</surname><given-names>JT</given-names></name></person-group><year>2012</year><article-title>X-chromosome hyperactivation in mammals via nonlinear relationships between chromatin states and transcription</article-title><source>Nature Structural & Molecular Biology</source><volume>19</volume><fpage>56</fpage><lpage>61</lpage><pub-id pub-id-type="doi">10.1038/nsmb.2195</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.02833.010</article-id><title-group><article-title>Decision letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Reinberg</surname><given-names>Danny</given-names></name><role>Reviewing editor</role><aff><institution>Howard Hughes Medical Institute, New York University School of Medicine</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>[Editors’ note: although it is not typical of the review process at <italic>eLife</italic>, in this case the editors decided to include the reviews in their entirety for the authors’ consideration as they prepared their revised submission.]</p><p>Thank you for sending your work entitled “H3K27 modifications define segmental regulatory domains in the Drosophila bithorax complex” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by K VijayRaghavan (Senior editor) and 3 reviewers, one of whom 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>We think that the study is solid and has its value. However, we collectively believe that the current data set (H3-K27me3/ PH/ CTCF) is too preliminary compared to earlier studies but that it might become suitable if the analysis is expanded as suggested below.</p><p>The authors should look minimally at PRC2 binding and RNA Pol II occupancy. These two profiles would help to clarify whether the 'constitutive' binding of PRC2, PRC1 etc. at the HOX gene <italic>Ubx</italic> (i.e. in <italic>Ubx</italic> OFF and <italic>Ubx</italic> ON cells) that was reported back in 2006 is a general principle. Similarly, it was shown that several components of the general transcription machinery are bound at <italic>Ubx</italic> in the OFF state, and so it would be good to know whether assembly of the RNA Pol II machinery even at permanently repressed Polycomb target genes is a general principle.</p><p>We also noticed that the citation of relevant scientific literature is missing.</p><p><italic>Reviewer #1</italic>:</p><p><xref ref-type="fig" rid="fig1">Figure 1</xref>:</p><p>Can the authors computationally overlay the different parasegments that are labeled at each time point? Currently, it is visually difficult to assess the level of overlap or lack thereof between each of the labeled parasegments.</p><p>Is it possible to label any upstream parasegments? The question of contamination of PS4 by upstream signal may thereby be resolved.</p><p>The authors should provide antibody stainings of genes within the BX-C cluster for us to align against the labeled parasegments.</p><p><xref ref-type="fig" rid="fig2">Figure 2</xref>:</p><p>What about the Antp-C locus? Does H3K27me3 profiling parallel what is seen in the BX-C locus? Does dCTCF localize to boundaries here as well?</p><p>As there are many characterized insulator proteins in the fruit fly, how does dCTCF co-localize with these factors? I.e. CP190, BEAF-32, Su(Hw)?</p><p>How is a given constitutive CTCF site chosen to be “active” in performing a boundary function? For example, in PS7, why are activation signals able to push through the most anterior dCTCF sites? Is specificity achieved through association with distinct insulator partners at different CTCF sites?</p><p>How does the H3K27me3 pattern compare to the distribution of E(Z) or other PRC2 components? Does PRC2 only sit at PREs, from which the modification spreads? Is PRC2 absent from upstream PREs present within an activated region of BX-C? If present, what inhibits PRC2 activity at these sites?</p><p>Since the authors are able to purify nuclei from distinct parasegments of the fly, they should be able to provide RNA-seq or Pol-II occupancy tracks to correlate with presence of repressive H3K27me3 and active H3K27ac.</p><p>The genetic system employed nicely allows for the isolation of parasegments during time points of development. What are the dynamics of the activation of the BX-C locus? Is the transition gradual or immediate? Is it possible to ChIP some of the transcription factors thought to activate these genes?</p><p>What is the 3D conformation of the parasegments within the BX-C cluster? How do they compare to each other? Are active and repressive domains spatially partitioned?</p><p>How is the expression and H3K27 distribution altered in mutants (null or hypomorphs) of Polycomb genes? Is there simply a decrease in H3K27me3, or is the H3K27me3 boundary shifted as well?</p><p><xref ref-type="fig" rid="fig3">Figure 3</xref>:</p><p>How does the distribution of H2AK119Ub correlate with Polyhomeotic? Does it form blocks within transcriptionally repressed chromatin, as is the case for PRC2?</p><p><italic>Reviewer #3</italic>:</p><p>1 )The authors should analyze the PRC2 binding profile (e.g. ChIP with antibody against E(z))in PS4, 5, 6 and 7, or at the minimum, just in PS7 as they did for Ph, CTCF etc.. This would address the conceptually important point whether the binding of PRC2 at PREs is constitutive (i.e. PRC2 bound not only in the inactive but also in the active state; see also below) and that the control takes place at the level of deposition of the modification. Given that the authors may already have chromatin from PS7 nuclei or can generate it as they did for several of their experiments in the manuscript, I do not think that it is asking too much to perform such a ChIP experiment.</p><p>2) This point is directly connected to the first point. The authors must discuss their findings in the context of the findings from earlier studies (i.e. <xref ref-type="bibr" rid="bib35">Papp and Müller, G&D 2006</xref>; Kwong et al., PLOS Genetics 2008; Soshnikova and Duboule, Science 2009). Directly relevant to the work by Bowman here, Papp and Muller had compared the chromatin of the HOX gene <italic>Ubx</italic> in wing imaginal disc cells where <italic>Ubx</italic> is inactive and in haltere and third leg discs where the gene is active. One key conclusion from that work was that PRC2 (as well as PRC1 and PhoRC) is bound at <italic>Ubx</italic> both in the inactive and in the active state (with subtle differences) but that the domains of H3-K27me3 are different in the two states, with H3-K27me3 covering the entire upstream, promoter and coding region in wing disc cells but being absent from the promoter and coding region in haltere and third leg disc cells. So the observation that H3-K27me3 domains correlate with inactivity of regulatory regions in the bithorax complex is not new but the nice data shown here in embryos (with modern genomic techniques) extend and refine these findings. From the above it also directly follows that it is key to find out about PRC2 binding at the <italic>bx</italic>, <italic>bxd</italic> and <italic>abd-A</italic> PREs in PS7 (Point 1).</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.02833.011</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p>Reviewer #1:</p><p><xref ref-type="fig" rid="fig1"><italic>Figure 1</italic></xref><italic>:</italic></p><p><italic>Can the authors computationally overlay the different parasegments that are labeled at each time point? Currently, it is visually difficult to assess the level of overlap or lack thereof between each of the labeled parasegments.</italic></p><p><italic>The authors should provide antibody stainings of genes within the BX-C cluster for us to align against the labeled parasegments.</italic></p><p><bold>Response:</bold> New figure with side-by-side embryos from each strain marking single parasegments, and a panel with UBX and ABD-A stainings.</p><p><bold>Location in revised manuscript:</bold> <xref ref-type="fig" rid="fig1s2">Figure 1–figure supplement 2</xref></p><p>This new figure should clarify the position of the labeled parasegments relative to each other and to UBX and ABD-A expression.</p><p><italic>Is it possible to label any upstream parasegments? The question of contamination of PS4 by upstream signal may thereby be resolved</italic>.</p><p>Construction of these strains takes many months. In any case, labeling more anterior parasegments would not resolve whether the PS4 (or PS5) nuclei are contaminated by anterior cells.</p><p><xref ref-type="fig" rid="fig2"><italic>Figure 2</italic></xref><italic>:</italic></p><p><italic>What about the Antp-C locus? Does H3K27me3 profiling parallel what is seen in the BX-C locus? Does dCTCF localize to boundaries here as well</italic>?</p><p><bold>Response:</bold> New figure depicting H3K27me3, CTCF, and CP190 in the Antp-C.</p><p><bold>Location in the revised manuscript:</bold> <xref ref-type="fig" rid="fig2s2">Figure 2–figure supplement 2</xref>.</p><p>H3K27me3 is depleted over the Antp transcription unit, as expected. CTCF is less prevalent in the Antp-C than in the BX-C. In the Antp-C, CP190 predominates.</p><p><italic>As there are many characterized insulator proteins in the fruit fly, how does dCTCF co-localize with these factors? I.e. CP190, BEAF-32, Su(Hw)</italic>?</p><p><bold>Experimental response:</bold> ChIP-seq for CP190 in mixed cells and parasegment 7.</p><p><bold>Location in revised manuscript:</bold> <xref ref-type="fig" rid="fig2">Figure 2</xref></p><p>Since CP190 is in a protein complex with CTCF and can alter local chromatin structure in a way that may promote boundary function, we thought it would be a worthwhile target of investigation. It co-occupies many sites with CTCF, but also binds to the BX-C and the ANTP-C at locations where we do not detect CTCF.</p><p><italic>How is a given constitutive CTCF site chosen to be “active” in performing a boundary function? For example, in PS7, why are activation signals able to push through the most anterior dCTCF sites? Is specificity achieved through association with distinct insulator partners at different CTCF sites</italic>?</p><p>Our results suggest that boundaries are constitutive. They may have nothing to do with whether an adjacent domain is active or repressed; they may only prevent the spread of activation or repression. The comparison of CTCF and CP190 profiles suggests that each boundary may be distinctive, which is borne out by sequence comparisons and swapping experiments from other labs, but this is no reason to suggest that the components of any boundary vary from one parasegment to another.</p><p><italic>How does the H3K27me3 pattern compare to the distribution of E(Z) or other PRC2 components? Does PRC2 only sit at PREs, from which the modification spreads? Is PRC2 absent from upstream PREs present within an activated region of BX-C? If present, what inhibits PRC2 activity at these sites</italic>?</p><p><bold>Experimental response:</bold> ChIP-seq for Su(z)12 in mixed cells and parasegment 7.</p><p><bold>Location in revised manuscript:</bold> <xref ref-type="fig" rid="fig3">Figure 3</xref></p><p>In mixed cells, Su(z)12 is present in sharp peaks at PREs and transcription start sites. In PS7, Su(z)12 peaks are somewhat diminished at the PREs in the active domains, while the peaks at the transcription start sites are strengthened. The reasons for these subtle changes will require further investigation.</p><p><italic>Since the authors are able to purify nuclei from distinct parasegments of the fly, they should be able to provide RNA-seq or Pol-II occupancy tracks to correlate with presence of repressive H3K27me3 and active H3K27ac.</italic></p><p><bold>Experimental response:</bold> ChIP-seq for Pol-II in mixed cells and parasegment 7.</p><p><bold>Location in revised manuscript:</bold> <xref ref-type="fig" rid="fig3">Figure 3</xref></p><p>Transcriptionally engaged Pol-II is present at the promoter and throughout gene bodies of <italic>Ubx</italic> and <italic>Abd-A</italic> in PS7, consistent with the expression of these genes. Pol-II is also enriched at the major promoter of <italic>Abd-B</italic>, even though this gene is transcriptionally silent in PS7 and covered by H3K27me3.</p><p><italic>The genetic system employed nicely allows for the isolation of parasegments during time points of development. What are the dynamics of the activation of the BX-C locus? Is the transition gradual or immediate? Is it possible to ChIP some of the transcription factors thought to activate these genes</italic>?</p><p>Unfortunately, the transgenes marking single parasegments do not turn on until at least 4 hours of development, well after repression via Pc-G has been established. These technical limitations make addressing this interesting developmental question in single parasegments quite challenging, and outside the scope of the current manuscript.</p><p><italic>What is the 3D conformation of the parasegments within the BX-C cluster? How do they compare to each other? Are active and repressive domains spatially partitioned</italic>?</p><p>Members of our lab have performed 4C with three different baits in PS7 (<italic>Abd-B</italic> promoter, the <italic>Ubx</italic> promoter, and the border between PS7 and PS8 regulatory domains). We have not observed contacts changing in an obvious manner that correlate with domain activation and repression. Given the significant amount of sorted material required for the experiments and the absence of robust differences between mixed cells and PS7, we have chosen to save a more thorough investigation of these data for another manuscript.</p><p><italic>How is the expression and H3K27 distribution altered in mutants (null or hypomorphs) of Polycomb genes? Is there simply a decrease in H3K27me3, or is the H3K27me3 boundary shifted as well</italic>?</p><p>We have developed the techniques to perform ChIP-seq from small numbers of precisely staged mutant embryos, and have begun to generate data that answer these questions. An analysis as thorough as this topic deserves is outside the scope of this work, and will be featured in a future manuscript.</p><p><xref ref-type="fig" rid="fig3"><italic>Figure 3</italic></xref>:</p><p><italic>How does the distribution of H2AK119Ub correlate with Polyhomeotic? Does it form blocks within transcriptionally repressed chromatin, as is the case for PRC2</italic>?</p><p>We are also interested to the answer to this question. Unfortunately, the two commercially available antisera for mammalian H2AK199Ub (monoclonal E65C, and cell signaling 8240) failed to enrich BX-C chromatin under our experimental conditions. This may be because the amino acid residues flanking the ubiquitinated lysine residue are different in mammalian and Drosophila H2A (as suggested in Scheuermann et al., Fly 2012), and a fly-specific antibody is necessary for ChIP.</p><p>Reviewer #3:</p><p><italic>1) The authors should analyze the PRC2 binding profile (e.g. ChIP with antibody against E(z))in PS4, 5, 6 and 7, or at the minimum, just in PS7 as they did for Ph, CTCF etc.</italic>.</p><p><bold>Experimental response:</bold> ChIP-seq for Su(z)12 in mixed cells and PS7.</p><p><bold>Location in revised manuscript:</bold> <xref ref-type="fig" rid="fig3">Figure 3</xref>.</p><p>We found that PRC1 components bind to PREs regardless of their activation state, and the PRC2 component Su(z)12 looks similar.</p><p><italic>This would address the conceptually important point whether the binding of PRC2 at PREs is constitutive (i.e. PRC2 bound not only in the inactive but also in the active state; see also below) and that the control takes place at the level of deposition of the modification. Given that the authors may already have chromatin from PS7 nuclei or can generate it as they did for several of their experiments in the manuscript, I do not think that it is asking too much to perform such a ChIP experiment</italic>.</p><p><italic>2) This point is directly connected to the first point. The authors must discuss their findings in the context of the findings from earlier studies (i.e. Papp and Müller, G&D 2006; Kwong et al, PLOS Genetics 2008; Soshnikova and Duboule, Science 2009)</italic>.</p><p>Thanks for pointing out this oversight. In the revised manuscript, we have included additional references to <xref ref-type="bibr" rid="bib35">Papp and Mueller (2006)</xref>, <xref ref-type="bibr" rid="bib22">Kwong et al. (2008)</xref>, and <xref ref-type="bibr" rid="bib45">Soshikova and Duboule (2009)</xref>.</p></body></sub-article></article> |