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.

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.

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.

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.

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 (Simon and Kingston, 2013). 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.

The prototypic targets of PcG action are the Drosophila homeotic genes that control segment identity. Indeed, a mutation in Polycomb, the founding member of the PcG, was discovered for its segmental transformations (Lewis, 1947). In Drosophila melanogaster, 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 (Lewis, 1978). 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 (Lewis, 1981). 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, Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B) (Maeda and Karch, 2009). The regulatory domains have been roughly defined by mutant lesions (Lewis, 1978), and by enhancer traps in the BX-C, which show different anterior limits of expression (Bender and Hudson, 2000). The molecular features that define these regions, as well as the precise locations of their boundaries, have not been defined.

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 (Schuettengruber et al., 2009; Nègre et al., 2011). This histone modification is necessary for maintaining repression of BX-C genes (Pengelly et al., 2013), 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, Papp and Müller (2006) observed loss of the K27me3 mark across the Ubx transcription unit in haltere and third leg imaginal discs, tissues where Ubx 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.

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 (Figure 1A). Gal4 and Gal80 expression domains were established either by enhancer trapping or by selecting an enhancer to achieve the desired expression pattern (Figure1—figure supplement 1). 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 (Figure 1B, Figure1—figure supplements 1 and 2; ‘Materials and methods’ for details).

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For isolation of marked nuclei, we used Gal4 to drive an mCherry-RanGAP fusion protein, from the INTACT system (Deal and Henikoff, 2011; Steiner et al., 2012). Staged embryos with single parasegment stripes of mCherry were formaldehyde-fixed and disrupted, and their nuclei were sorted on a FACS instrument (Figure1—figure supplement 1C). DNA enriched by chromatin immunoprecipitation (ChIP) was prepared for paired-end sequencing, using a picogram-scale library protocol (Figure 1—figure supplement 1D; Bowman et al., 2013).

The largest differences in H3K27me3 enrichment between parasegments occurred at the BX-C (Figure 2A, Figure 2—figure supplement 1). While the H3K27me3 profiles in our four adjacent parasegments were very similar elsewhere in the genome (Figure 2—figure supplement 1), 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 (Figure 2C–F), 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 (Soshnikova and Duboule, 2009). Our analysis of single parasegments locates distinct domain borders where K27me3 status changes abruptly. The contrast between the whole embryo profile (Figure 2B) and the single segment profiles (Figure 2C–F) shows how isolated cell types can identify chromatin changes linked to cell identity.

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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 (Figure 2C), with only a few narrow gaps at nucleosome-free regions (Mito et al., 2005). 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 bithorax (bx) mutant lesions, is defined by the low level of H3K27me3 modification across the leftmost (proximal) 92 kb of the complex (Figure 2D). There is a sharp transition to full H3K27me3 coverage at a position upstream of the Ubx 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 (Figure 1—figure supplement 2). In PS6, the leftmost 137 kb of the BX-C is clear of the K27me3 mark (Figure 2E); 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 bithoraxoid (bxd) phenotypes (Bender and Lucas, 2013). The transition point to full K27 methylation coincides with the genetically defined Fub border between the PS6 and the PS7 regulatory domains (Bender and Lucas, 2013). In PS7, an additional 36 kb was depleted of H3K27me3 (Figure 2F). This region includes the abd-A transcription unit, and is associated with the infraabdominal-2 (iab-2) class of genetic mutations. We observed a reproducible mound of H3K27me3 in PS7 nuclei close to the Ubx promoter (arrow in Figure 2F). This may reflect repression of Ubx by abd-A in this parasegment (Karch et al., 1990), mediated by the PcG.

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 (Bender and Hudson, 2000). 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 Ubx promoter. However, transgenic reporter elements inserted between the Ubx 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 Ubx 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 Ubx start site shows a PS4 anterior border (Casares et al., 1997), and a ncRNA with a PS4 anterior expression limit is transcribed from the region 5–15 kb downstream of the Ubx start (Pease et al., 2013). 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.

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 (Ohlsson et al., 2010). Figure 2G shows the CTCF ChIP pattern in unsorted nuclei, similar to that previously reported (Nègre et al., 2010). CTCF in PS7 nuclei gives a pattern virtually identical to the whole embryo pattern (Figure 2H). 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 (Nègre et al., 2010), notably at the border between regulatory regions for PS7 and PS8, and proximal to the 3′ end of the Ubx transcription unit.

CTCF is present in a protein complex with Centrosomal Protein 190 (CP190) (Mohan et al., 2007), and the binding sites of these proteins often overlap (Nègre et al., 2010). 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 (Figure 2I; Nègre et al., 2010) and in PS7 nuclei (Figure 2J). There is a noteworthy CP190 site immediately proximal to the 3′ end of the Ubx transcription unit, separating Ubx from the adjacent gene (modular serine protease). In the Antennapedia complex, the other homeotic gene complex in flies, CP190 peaks often appear without coincident CTCF peaks (Figure 2—figure supplement 2).

Acetylation of H3K27 (H3K27ac) is correlated with active enhancer regions (Heintzman et al., 2009; Kharchenko et al., 2011), and is anti-correlated with PcG function (Tie et al., 2009), 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 (Figure 3B,C). 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.

Figure 3

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ChIP of serine-5-phosphorylated RNA polymerase II (Pol-II) (transcriptionally engaged polymerase) in whole embryos shows the transcription units for Ubx, abd-A, and Abd-B (Figure 3D). Embryonic non-coding transcripts (Bae et al., 2002; Pease et al., 2013) 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 Ubx promoter, coincident with the CTCF binding site at the border between the PS5 and PS6 domains (arrow in Figure 3D). There is no reported transcript initiating at this position (Pease et al., 2013), although a short transcript could have been missed. Pol-II in PS7 (Figure 3E) marks the Ubx and abd-A transcription units, as expected from the UBX and ABD-A protein patterns (Figure 1—figure supplement 2). There is also a Pol-II peak at the major Abd-B promoter (arrow in Figure 3E), although Abd-B RNA and protein are absent from PS7 (Celniker et al., 1990; Boulet et al., 1991) and this region is H3K27 trimethylated. Analogous localization of Pol-II at the Abd-B promoter in wing and haltere discs was reported by Chopra et al. (2009), who argued that these polymerase molecules were ‘paused’. The H3K4me3 mark aligns with the Ubx and abd-A promoters in PS7 nuclei (but not Abd-B; Figure 3G) and is reduced in mixed cells (Figure 3F); this histone mark has been seen at active promoters in other systems (Ruthenburg et al., 2007).

The POLYCOMB subunit of PRC1 is known to bind to the H3K27me3 modification through its chromodomain (Fischle et al., 2003), 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 (Kwong et al., 2008) to sharp peaks at the known PREs (Gonzalez et al., 2014). The difference may be due to different procedures for chromatin fragmentation prior to immunoprecipitation (Straub et al., 2013). Our PC profiles show sharp peaks at PREs in nuclei from whole embryos (Figure 3H), matching the profile for POLYHOMEOTIC, another PRC1 subunit (Figure 3J). 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 (Figure 3H–K). In the active PS5 domain, the levels of PC and PHO at the PS5 PRE are reduced, relative to the whole embryo profile. Papp and Müller (2006) 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. Kwong et al. (2008) 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 (Simon and Kingston, 2013). 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 (Blackledge et al., 2014; Cooper et al., 2014; Kalb et al., 2014).

The profiles for SU(Z)12, a PRC2 component (Figure 3L,M), 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 Papp and Müller (2006) on the PS5 PRE in imaginal discs. SU(Z)12 is also present at the transcription start sites of Ubx, abd-A, and Abd-B; binding at these promoters is more apparent than it is for PRC1 proteins.

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 Ubx 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 (Bender and Lucas, 2013). 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 Cbx transposition, Peifer et al., 1987). 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 Ubx, one in each of these parasegments, without the need for a duplication of the Ubx 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.

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 (Ubx and abd-A, 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 (Papp and Müller, 2006). 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.

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Author details

1. Sarah K Bowman

   1. Department of Molecular Biology, Massachusetts General Hospital, Boston, United States
   2. Department of Genetics, Harvard Medical School, Boston, United States

   Contribution

   SKB, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting and revising the article

   Contributed equally with

   Aimee M Deaton

   Competing interests

   The authors declare that no competing interests exist.

2. Aimee M Deaton

   1. Department of Molecular Biology, Massachusetts General Hospital, Boston, United States
   2. Department of Genetics, Harvard Medical School, Boston, United States

   Contribution

   AMD, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting and revising the article

   Contributed equally with

   Sarah K Bowman

   Competing interests

   The authors declare that no competing interests exist.

3. Heber Domingues

   Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States

   Contribution

   HD, Conception and design, Acquisition of data

   Competing interests

   The authors declare that no competing interests exist.

4. Peggy I Wang

   1. Department of Molecular Biology, Massachusetts General Hospital, Boston, United States
   2. Department of Genetics, Harvard Medical School, Boston, United States

   Contribution

   PIW, Analysis and interpretation of data, Drafting and revising the article

   Competing interests

   The authors declare that no competing interests exist.

5. Ruslan I Sadreyev

   1. Department of Molecular Biology, Massachusetts General Hospital, Boston, United States
   2. Department of Pathology, Harvard Medical School, Boston, United States

   Contribution

   RIS, Analysis and interpretation of data, Drafting and revising the article

   Competing interests

   The authors declare that no competing interests exist.

6. Robert E Kingston

   1. Department of Molecular Biology, Massachusetts General Hospital, Boston, United States
   2. Department of Genetics, Harvard Medical School, Boston, United States

   Contribution

   REK, Conception and design, Analysis and interpretation of data, Drafting and revising the article

   For correspondence

   kingston@molbio.mgh.harvard.edu

   Competing interests

   The authors declare that no competing interests exist.

7. Welcome Bender

   Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States

   Contribution

   WB, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting and revising the article

   For correspondence

   +16174321906

   Competing interests

   The authors declare that no competing interests exist.

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