cis-Regulatory Sequences and Functions (part 2/3)

In Drosophila the Polycomb group genes are required for the long-term maintenance of the repressed state of many developmentally crucial regulatory genes. Their gene products are thought to function in a common multimeric complex that associates with Polycomb group response elements (PREs) in target genes and regulates higher-order chromatin structure. The chromodomain of Polycomb is necessary for protein-protein interactions within a Polycomb-Polyhomeotic complex. Posterior sexcombs protein coimmunoprecipitates Polycomb and Polyhomeotic, indicating that all three are members of a common multimeric protein complex. The B promoter of Abdominal-B is devoid of all three PcG proteins. Ph and Psc are not associated with the peak Pc binding element A (overlapping the gamma promoter). However, other fragments in the vicinity of gamma and C promoters are associated with Ph and/or Psc, and it may be that this regulatory region is unusually complex and contains several PREs that regulate the different Abd-B promoters. Both Ph and Psc are enriched for a restriction fragment in the 3' region of Abd-B, which is relatively poorly enriched by Pc. This element is strongly associated with GAGA factor. In the empty spiracles gene Psc is associated with an upstream fragment, covering a previously identified ems enhancer element. Pc and Ph are not found at this transcribed locus. These results suggest that there may be multiple different Polycomb group protein complexes which function at different target sites. Polyhomeotic and Posterior sexcombs are also associated with expressed genes. Polyhomeotic and Posterior sex combs may participate in a more general transcriptional mechanism that causes modulated gene repression, whereas the inclusion of Polycomb protein in the complex at PREs leads to stable silencing (Strutt, 1997b).

The bithorax complex contains three homeotic genes, and at least nine regulatory regions which control their expression in successive parasegments of the fly. A study of enhancer traps shows that these regulatory regions function to regulate gene expression to parasegmental domains and that these domains are mediated by Polycomb-mediated repression. In embryos mutant for genes of the Polycomb group, the lacZ expression from the enhancer traps spreads to all segments. Thus, the enhancer traps reveal parasegmental domains that are maintained by Polycomb-mediated repression. Such domains may be realized by parasegmental differences in chromatin structure (McCall, 1994).

Transcriptional silencing by the Polycomb Group of genes maintains the position-specific repression of homeotic genes throughout Drosophila development. The Polycomb Group of genes characterized to date encode chromatin-associated proteins that have been suggested to form heterochromatin-like structures. By studying the expression of reporter genes, a 725 bp fragment (called MCP725) in the homeotic gene Abdominal-B has been identified. It accurately maintains position-specific silencing during proliferation of imaginal cells. Complete repression of MCP725 directed gene expression is found in the wing disc (parasegments 4 and 5); repression in the anterior compartment (ps5) of the haltere disc and strong expression in the posterior compartment of the haltere disc (ps6). Therefore, the MCP725 element is a silencer that functions throughout proliferation of the imaginal discs. MCP725 contains sequences that have been proposed to act as a chromatin boundary influenceing Abd-B gene expression. Data presented here suggests that MCP725 does not act as a boundary element. Silencing by MCP725 requires the Polycomb and the Polycomblike genes, indicating that it contains a Polycomb response element. To investigate the mechanisms of transcriptional silencing by MCP725, its temporal requirements were studied by removing MCP725 from the transgene at various times during development. Excision of MCP725 during larval stages leads to loss of silencing. These findings indicate that the silencer is required for the maintenance of the repressed state throughout cell proliferation. They also suggest that propagation of the silenced state does not occur merely by templating of a heterochromatin structure by virtue of protein-protein interactions. Rather, they suggest that silencers play an active role in the maintenance of the position-specific repression throughout development. It is thought that silencers like MCP725 might serve as DNA binding sequences for proteins that functionally replace the position-specific repressor role of the gap genes at later stages in development. These proteins, if they exist, would likely have DNA binding specificity and be expressed in a spatially restricted pattern throughout development. Candidates for these include either the remaining uncharacterized members of the PcG genes or the homeotic proteins, which themselves might replace the role of the gap proteins as repressors. This latter suggestion is attractive because (1) homeotic proteins are transcriptional repressors as well as activators; (2) are known to bind the promoters of other homeotic gene family members; (3) are expressed and functionally reqired throughout development, and (4) they show highly complementary expression patterns with one another (Busturia, 1997).

The PcG proteins function through cis-regulatory elements called PcG response elements (PREs), which enable them to bind and to maintain the state of transcriptional silencing over many cell divisions. PcG proteins operate in two key evolutionarily conserved chromatin complexes, and reduced expression of these complexes, as found in PcG mutants, results in the derepression of PRE-controlled genes. To determine whether PcG silencing is modulated in regenerating tissue, the FLW-1 line, which contains a lacZ reporter gene under the control of the Fab7 PRE, was used. Prothoracic leg discs silent for lacZ expression were fragmented and transplanted into the abdomen of host flies. Flies were fed with 5-bromodeoxyuridine (BrdU) to mark the regenerated tissue (the blastema). In uncut discs, there was little proliferation and expression of lacZ was undetectable. On fragmentation, however, lacZ was expressed in the blastema. To confirm that this derepression was due to a reduction in PcG silencing and not simply to massive proliferation at the wound site, the line LW-1 was used; this line lacks the Fab7 PRE and is normally silent, but it can be activated by induction of GAL4. Neither uncut nor cut leg discs of the LW-1 line showed expression of lacZ after transplantation (Lee, 2005).

Different core promoters possess distinct regulatory activities in the Drosophila embryo

Binding the TFIID complex to a target promoter depends on at least three different core promoter elements located within a 50- to 60-base pair sequence flanking the transcription start site, the TATA box, the initiator element (Inr), and the downstream promoter element (Dpe). In general, promoters that lack a TATA sequence must possess conserved copies of the Inr and/or Dpe. Conversely, promoters containing optimal TATA sequences do not require Inr and Dpe elements for the binding of TFIID. The presences of these three elements define two common types of promoters: type I promoters contain a TATA box, whereas type II promoters contain Inr and Dpe sequences. There are numerous examples of shared enhancers interacting with just a subset of target promoters. These "shared enhancer" type of interactions are contrasted with a "competitive interaction" type. In some cases, specific enhancer-promoter interactions depend on promoter competition, whereby the activation of a preferred target promoter precludes expression of linked genes. A transgenic embryo assay was used to obtain evidence that promoter selection is influenced by the TATA element. Both the AE1 enhancer (located between Sex combs reduced and fushi tarazu) from the Drosophila Antennapedia gene complex (ANT-C) and the IAB5 enhancer (which selectively activates Abdominal-B, not abdominal-A) from the Bithorax complex (BX-C) preferentially activate the type I, TATA-containing, promoters when challenged with linked TATA-less promoters. The AE1 autoregulatory element in the ANT-C specifically interacts with the ftz promoter, but does not activate the equidistant Sex combs reduced gene. AE1 and IAB5 exhibit a competitive type of interaction. In contrast, the rho neuroectoderm enhancer (NEE) does not discriminate between type I and type II classes of promoters and exhibits a shared enhancer type of interaction. Thus, certain upstream activators, such as Ftz, prefer TATA-containing promoters, whereas other activators, including Dorsal, work equally well on both classes of promoters (Ohtsuki, 1998).

Related artifically constructed core promoter sequences were initially used for the analysis of AE1. ftz and eve contain optimal TATA sequences, but lack Inr (INIT) and Dpe (DPE) elements. AE1 also activates white and Tp promoters. white and Tp each contain conserved copies of the INIT and DPE sequences, but lack a TATA sequence (white) or contains a suboptimal TATA (Tp). AE1 can simultaneously activate linked TATA-containing promoters or linked INIT/DPE-containing promoters. In spite of AE1's ability to activate type I and type II promoters, promoter competition can be demonstrated. There is a substantial reduction in white expression when the Tp promoter is replaced with the core eve promoter sequence. This AE1-eve interaction appears to block the expression of the linked white gene. In the absence of eve, white is fully active. These observations are compatible with a promoter-competition mechanism whereby AE1-eve interactions inhibit white (Ohtsuki, 1998).

Similarly, IAB5 prefers the eve promoter. The 1-kb IAB5 enhancer exhibits a preference for TATA-containing promoters. IAB5 was placed downstream of an eve/lacZ fusion gene; the linked CAT reporter gene was placed under the control of the mini-white promoter. There is strong expression of the lacZ reporter gene in the presumptive abdomen, whereas CAT is not expressed above background levels. This result suggests that IAB5 prefers the eve promoter over white. An eve-white chimeric promoter was analyzed in an effort to assess the importance of the core elements, particularly the TATA sequence. An ~20-bp region of the eve sequence (the TATA region) was replaced with the corresponding region of white. This modified eve promoter (evewhite) is attenuated and mediates only weak expression of lacZ in the presumptive abdomen. In contrast, the linked white promoter directs strong expression of CAT. These results suggest that the removal of the eve TATA releases the IAB5 enhancer so that it can now interact with the white promoter (Ohtsuki, 1998).

The 300-bp rhomboid NEE is equally effective in activating the two classes of promoters. Additional experiments were done to determine whether the targeting of IAB5 to eve influences the activities of the nonspecific rho NEE. The latter enhancer is activated by the maternal gradient of Dorsal transcription factor in lateral stripes within the neurogenic ectoderm. A synthetic gene complex was prepared that contains both the NEE and IAB5 enhancers. white and CAT reporter genes were attached to the mini-white promoter, whereas lacZ is driven by eve. The rho NEE activates all three reporter genes, so that white, CAT, and lacZ are all expressed in lateral stripes. In contrast, IAB5 primarily activates the eve promoter, so that only lacZ exhibits strong expression within the presumptive abdomen. These results suggest that IAB5-eve interactions do not influence the nonspecific activities of the rho NEE (Ohtsuki, 1998).

It has been suggested that TATA-containing promoters are intrinsically stronger than TATA-less promoters, possibly because of higher affinity interactions with the TFIID complex. The divergent activities of the IAB5 and NEE enhancers, however, are most easily interpreted on the basis of qualitative, not quantitative, differences in type I and type II core promoter sequences. For example, the insertion of a TATA sequence in the white promoter allows it to compete with a linked eve promoter, whereas the removal of TATA from eve permits activation of white. These alterations in the white and eve promoters, the insertion and removal of TATA, dramatically alter the activities of IAB5, but have virtually no effect on the NEE enhancer. NEE is equally effective in activating the eve, white, evewhite, and whiteTATA promoters, and thereby serves as an internal control for normal promoter function (Ohtsuki, 1998).

These results suggest that the IAB5 and AE1 activators, particularly Ftz, prefer type I promoters. NEE activators, including Dorsal (dl) and bHLH proteins, appear to be promiscuous and work equally well on both classes of core promoters. The authors propose that the TFIID complex adopts different conformations on type I and type II promoters. Basal targets for the Ftz activator may be displayed in a more accessible conformation when TFIID binds TATA. In contrast, basal targets for the Dorsal and bHLH activators may be equally accessible whether TFIID binds TATA or Inr/Dpe elements (Ohtsuki, 1998).

Epigenetic inheritance of active chromatin after removal of the main transactivator

Fab-7 is a genetically identified element of the BX-C necessary to regulate spatial transcription of the Abdominal-B (Abd-B) gene. Polycomb group (PcG) and trithorax group (trxG) gene products are responsible for the maintenance of repressed and active expression patterns of many developmentally important regulatory genes, including Abd-B. In Drosophila embryos, Polycomb (Pc) protein and the trxG protein GAGA factor colocalize at the Fab-7 DNA element of the bithorax complex. There is a strong enrichment in GAGA factor and Pc at the Fab-7 site in 11-16 hr old embryos, as compared to mock immunoprecipitations. A major GAGA factor binding site was localized in a 422 bp fragment, which contains six putative GAGA binding sites. This fragment is located within the putative boundary element. A lower level of binding was detected in the flanking 1230 bp fragment, which contains three GAGA target consensus sequences and also the putative PRE. Little or no association with all other flanking sequences is observed. In contrast, Pc is found to be associated with the entire 3.6 kb region, with a rather broad peak centered at the boundary and the PRE regions. Peak binding for Pc and GAGA factor colocalize. Thus, the physical distribution of these two proteins at Fab-7 does not allow discrimination of the apparent insulator and PRE function identified by transgenic constructs. This might indicate that the chromosomal elements through which PcG and trxG proteins act (the PRE) are located in sequences partially overlapping the putative boundary region (Cavalli, 1998).

In transgenic lines, the Fab-7 element induces extensive silencing on a flanking GAL4-driven lacZ reporter and mini-white genes. The Fab-7 fragment acts as a silencer preventing trans-activators like GAL4 from binding to the UAS-site when Pc protein is bound at the PRE. However, a short single pulse of GAL4 during embryogenesis is sufficient to release PcG-dependent silencing from the transgene. Such an activated state of Fab-7 is mitotically inheritable through development and can be transmitted in a GAL4-independent manner to the subsequent generations through female meiosis. About 25% of the female progeny exhibit an activated state of Fab-7. Red-eyed flies, indicating an activated state, were again selected for two further crossings. In the F3 generation, strong beta-gal staining is still observed in 18% of the embryos, and about 27% of the adult females are red-eyed. Thus, inheritance of the active state can be propagated through multiple subsequent generations. Meiotic transmission of the activated Fab-7 state is a reversible process, however. Embryos from flies with an activated Fab-7 of the F1, F2, and F3 generation were allowed to develop at 28°C until the first instar larval stage and then transferred to 18°C. Silencing is reestablished, resulting in 100% yellow-eyed female progeny. Conversely, in those flies where repression is reestablished upon meiosis, this repressed state can be reactivated again. This complete reversibility strongly suggests that the observed partial efficiency of transmission of the active state does not depend on heterogeneity in the genetic background of the fly line, but on a stochastic process whereby some of the chromatin templates may lose the epigenetic information upon meiotic transmission. Crosses using GAL4-less females strongly suggest that meiotic transmission of the activated Fab-7 state is not dependent on a preondurance of GAL4 protein. It is concluded that Fab-7 is a switchable chromosomal element, which can convey memory of epigenetically determined active and repressed chromatin states (Cavalli, 1998).

Although in the test system described here only maternal inheritance of Fab-7-dependent epigenetic regulatory states was observed, paternal inheritance of heterochromatin states has also been documented in Drosophila. The molecular nature of this phenomenon is not understood, but the Y chromosome has been shown to be involved in this type of inheritance. Therefore, paternal inheritance of chromatin states is possible, and whether PcG/trxG-mediated meiotic inheritance is truly restricted to the female germline for all of their regulated sequences remains a fascinating question for future investigation. It is proposed that chromosomal elements such as Fab-7, where PcG and trxG proteins perform a coordinate maintenance function, be termed "cellular memory modules" (CMMs). CMMs may be thought of as switchable elements able to induce and heritably propagate both silenced and open chromatin conformations. The respective chromatin status determined by a regulatory cascade of transcription factors during early embryogenesis might be the primary switch. Activated transcription would drive a CMM into the trxG-dependent open chromatin mode, while inactive states would be maintained as silent chromatin (Cavalli, 1998).

The finding of meiotic inheritance in PcG/trxG-dependent regulation is surprising since these proteins control genes involved in developmental decisions. In the embryo, the zygotic genome has to develop different spatial patterns of homeotic gene expression. Therefore, the developing embryo must be able to erase the epigenetic information of its parental gametes in order to allow differentiation of a variety of cell lineages. It could be argued that PcG silencing at CMMs is the default state; that is, in the germ line, all CMMs remain "marked" by certain elements of the PcG silencing complex. As such, in the early zygote the occupation of all CMMs by PcG proteins would be retained, perpetuating silencing as the default state. Recent evidence speaks in favor of such a mechanism. In somatic cells, differential transcription induced by patterning factors would switch CMMs into the active mode, which would then be heritably maintained in subsequent cell generations. In the particular transgene combination used in this study, the strong GAL4 induction might have completely removed the PcG-silencing tag from this PRE, which subsequently remains in the active state through several rounds of mitotic and meiotic divisions until it becomes reinactivated by stochastic processes. The finding that a defined Drosophila chromosomal element can transmit an epigenetic state to the next generations in the absence of any apparent covalent modifications of the DNA suggests that chromatin proteins can faithfully maintain an epigenetic state, and will allow a detailed molecular analysis of this type of inheritance. Several questions can now be addressed: can mitotic and meiotic epigenetic inheritance of active and silenced chromatin states be driven by other known PRE-containing DNA elements, and which of the PcG and trxG proteins are involved in these processes? What are the molecular features of the chromosomal complexes involved in epigenetic inheritance through mitosis and meiosis? The answer to these questions should yield important insight into how developmental decisions are faithfully maintained, and has implications for a better understanding of other epigenetic phenomena like mammalian genomic imprinting and paramutation in plants. It could well be that cellular memory modules are used in a variety of mechanisms to maintain regulatory decisions about transcriptional states throughout mitotic and meiotic division (Cavalli, 1998).

The Mcp element from the Drosophila melanogaster Bithorax Complex mediates long-distance regulatory interactions

The Mcp element from the Drosophila melanogaster bithorax complex (BX-C) was initially identified because deletions of the element cause a dominant gain-of-function transformation of PS9 into PS10. This transformation in parasegmental identity is due to the inappropriate activation of the iab-5 cis-regulatory domain (which specifies PS10 identity) of the Abd-B gene in PS9 (a parasegment in which Abd-B is normally turned off). Two models have been proposed to explain the gain-of-function phenotypes associated with Mcp deletions. In the first, the Mcp deletions remove a PS10 silencer that functions to keep the iab-5 cis-regulatory domain off in PS9. When this silencer is removed, iab-5 is activated in PS9, turning on Abd-B. In the second model, Mcp corresponds to a boundary element that functions to preserve the functional autonomy of the iab-4 and iab-5 cis-regulatory domains. While the question of whether Mcp corresponds to a silencer, a boundary, or both (as is the case for the element deleted by another BX-C gain-of-function mutation, Fab-71, a novel activity has been uncovered. Sequences from the Mcp region of BX-C have properties characteristic of Polycomb response elements (PREs), and they silence adjacent reporters by means of a mechanism that requires trans-interactions between two copies of the transgene. However, Mcp trans-regulatory interactions have several novel features. In contrast to classical transvection, homolog pairing does not seem to be required. Thus, trans-regulatory interactions can be observed not only between Mcp transgenes inserted at the same site, but also between Mcp transgenes inserted at distant sites on the same chromosomal arm, or even on different arms. Trans-regulation can even be observed between transgenes inserted on different chromosomes. A small 800-bp Mcp sequence is sufficient to mediate these long-distance trans-regulatory interactions. This small fragment has little silencing activity on its own and must be combined with other Polycomb-Group-responsive elements to function as a 'pairing-sensitive' silencer. Finally, this pairing element can also mediate long-distance interactions between enhancers and promoters, activating mini-white expression (Muller, 1999).

The Mcp element in BX-C is defined by three overlapping deletions. Although these deletions differ in size and location, all three have indistinguishable, dominant gain-of-function phenotypes: they transform PS9 into PS10. This transformation in parasegmental identity is due to the ectopic activation of Abd-B in PS9, a parasegment in which Abd-B is normally off. The three deletions remove a common region of ~450 bp in length. This common region spans a major ~400-bp chromatin-specific, nuclease-hypersensitive site that is present throughout embryogenesis and in tissue culture cells. The smallest deletion, McpB116, is slightly larger than this common region, and it removes an additional ~350 bp proximal to the major nuclease-hypersensitive region. DNA fragments extending to either side of the small Mcp deletion have silencing activity when linked to either a mini-white or y reporter. Like other silencers in the PRE class, silencing activity depends on Pc-G proteins. Included in the Pc-G group is pleiohomeotic, a gene that encodes a DNA-binding protein that appears to be closely related to the mammalian YY1 transcription factor. As has been found for several other PREs in BX-C, the major Mcp nuclease-hypersensitive region has a consensus Pho/YY1-binding sequence. The presence of this sequence, together with the fact that silencing activity depends on the pho gene, argue that this DNA-binding protein may play a key role in the assembly of Pc-G-silencing complexes by the Mcp element (Muller, 1999).

The most unusual feature of the Mcp element is its ability to promote long-distance interactions. Regulatory interactions are observed between Mcp transgenes inserted at different sites on the same chromosomal arm, on different chromosomal arms, and even between transgenes inserted on different chromosomes. The long-distance regulatory activity of the Mcp element is unusual. Among the previously characterized PREs from BX-C and other genetic loci, pairing-sensitive silencing is generally observed only between interacting partners inserted at the same site. Only in a few instances have interactions been observed between partners inserted at different sites, and these usually involved PRE transgenes located in quite close proximity. How does Mcp promote regulatory interactions over long distances? One model suggests that Mcp might function by dragging paired DNA into a heterchromatic nuclear compartment. Contrary to the expectations of the compartmentalization model, the Mcp element can mediate not only long-distance silencing, but also long distance activation. An alternative, more plausible model suggests that the Mcp element facilitates long-distance regulatory interactions because it is able to locate and then pair with Mcp elements at other sites. After this locating process the formation and spread of a function silencing complex around each element would occur. Specificity is likely to be generated by a combination of proteins, some that are found in most PREs and some that are unique to the 800-bp MCP element (Muller, 1999).

Analysis of chromatin boundary activity in Drosophila cells.

Chromatin boundaries, also known as insulators, regulate gene activity by organizing active and repressive chromatin domains and modulate enhancer-promoter interactions. However, the mechanisms of boundary action are poorly understood, in part due to limited knowledge about insulator proteins, and a shortage of standard assays by which diverse boundaries could be compared. This paper reports the development of an enhancer-blocking assay for studying insulator activity in Drosophila cultured cells. The activities of diverse Drosophila insulators including suHw, SF1, SF1b, Fab7 and Fab8 are shown to be supported in these cells. It was further shown that double stranded RNA (dsRNA)-mediated knockdown of SuHw and dCTCF factors disrupts the enhancer-blocking function of suHw and Fab8, respectively, thereby establishing the effectiveness of using RNA interference in this cell-based assay for probing insulator function. It is concluded that the novel boundary assay provides a quantitative and efficient method for analyzing insulator mechanism and can be further exploited in genome-wide RNAi screens for insulator components. It provides a useful tool that complements the transgenic and genetic approaches for studying this important class of regulatory elements (Li, 2009).

Despite their diverse genomic origins and distinct cis- and trans- components, the Drosophila suHw, SF1, Fab7 and Fab8 elements function as potent enhancer-blockers in the Drosophila cells. This finding suggests that chromatin boundary represents a basic cell function that is shared by diverse tissues. The cell-based insulator assay was combined with RNAi-mediated gene knockdown to systematically test the requirement of SuHw and dCTCF in the function of several Drosophila insulators. RNAi-mediated knockdown of SuHw and dCTCF specifically disrupted the function of the suHw and Fab8 boundaries, respectively, thereby validating the functional specificity of the assay. The results suggest that multiple independent pathways in Drosophila mediate insulator function. This is in contrast with the pivotal role the CTCF protein plays in the enhancer-blocking activities in vertebrates (Li, 2009).

Cell culture assays have several important advantages that complement studies using in vivo system. The homogeneous cell populations in these assays can be used in biochemical and cell biological analyses. They allow more efficient and quantitative assessment of reporter readout from a large number of individual cells. Insulator activity has previously been demonstrated in Drosophila cells; this system has improved the assay with several novel features. First is the use of P-element-based transgene vector, which is known to mediate single to low copy number, non-tandem genomic integration of the assay transgenes. This would provide more native genomic and regulatory environment for studying chromatin boundary function. Large numbers of stably transfected cells with randomly integrated transgenes also provide a broader sampling of the genomic environment, a feature that can be exploited to examine boundary activity in blocking chromosomal position effect. The second improvement is the use of divergently transcribed dual reporters, which provides a linked readout to control for the 'off-targets' effects on the non-insulator components in the assay system, such as enhancers, promoters, reporters, the state of general transcription or other cellular functions that impact the reporter readout. It should also provide an important control for the chromosomal position effect near the transgene integration site in stably transfected cells. The use of fluorescent protein reporters further allows rapid and quantitative FACS assessment of the enhancer-blocking activity, a feature particular important in high-throughput applications. The activity of multiple Drosophila insulators has been established, along with the efficiency of RNAi-mediated gene knockdown; this should facilitate biochemical dissection of insulator function and genome-wide high throughput RNAi screens for novel boundary components (Li, 2009).

As most cell-based systems, the enhancer-blocking assay is limited in its application by potential tissue or developmental stage incompatibilities of the insulator and the cell. Studies have suggested that certain chromatin boundaries, such as Fab7 and SF1, are composed of distinct insulator activities that function in different tissues and/or developmental stages. Although this study has documented the functionality of several Drosophila insulators in S2 and Kc cells, both derived from embryonic cell lineages, other insulators may not function in these two cell lines. In addition, cultured cells may have, over the course of many passages, lost the physiological stoichiometry of relevant DNA or protein components, resulting in impaired function of certain insulators. Furthermore, the dynamic regulation of insulator activity in response to developmental and physiological cues would depend on the context of the whole animal. Therefore, the cell-based insulator assay presented in this study provides a useful tool that complements the transgenic and genetic approaches for studying this important class of regulatory elements (Li, 2009).

Chromatin insulator and the promoter targeting sequence modulate the timing of long-range enhancer-promoter interactions in the Drosophila embryo

The homeotic genes are essential to the patterning of the anterior-posterior axis along the developing Drosophila embryo. The expression timing and levels of these genes are crucial for the correct specification of segmental identity. Abdominal-B (Abd-B) is first detected in the most posterior abdominal segments at high levels and gradually appears in progressively anterior abdominal segments in lower amounts. Regulatory mutations affecting this expression pattern produce homeotic transformations in the abdomen. The promoter targeting sequences (PTS) from Abd-B locus overcome the enhancer blocking effect of insulators and facilitate long-range enhancer-promoter interactions in transgenic flies. This study found that transgene activation by the IAB5 enhancer can be delayed by inserting a 9.5 kb 3' Abd-B regulatory region containing the Frontabdominal-8 (Fab-8) insulator and the PTS element. The delay was found to be caused by the PTS and an insulator, and it is not specific to the enhancer or the promoter tested. Based on these findings, it is hypothesized that the delay of remote enhancers is responsible for the Abd-B expression pattern, which is at least in part due to the regulatory activities of the PTS elements and chromatin boundaries (Lin, 2010).

Homeotic selector genes control segmental identities along anterior-posterior axis during Drosophila development. Each of these genes contains complex regulatory regions necessary for its expression patterns. The Abdominal-B (Abd-B) locus from the Drosophila bithorax gene complex consists of four downstream located, Parasegment (PS) specific regulatory domains, called infraabdominal (iab)-5, iab-6, iab-7, and iab-8, each of these controls Abd-B in one of the corresponding Parasegments from PS10 to PS13 to generate defined temporal and spatial gradient of Abd-B. Abd-B is first expressed in the PS13 at stage 5 during embryogenesis and gradually appears in lower levels in more anterior segment during stages 9-11. In the CNS, Abd-B protein exhibits a gradient with higher levels in posterior segments and lower levels in PS10 and 11. Genetic mutations inactivating one regulatory region will result in the expression of Abd-B in the affected segment to duplicate that of an immediate anterior segment. For example, an iab-7 mutation would result in PS12 to PS11 transformation producing a duplication of PS11. To date, the exact mechanism of this transformation remains poorly understood (Lin, 2010).

Abd-B contains at least four classes of cis-regulatory elements responding to trans-regulators to orchestrate complex controls of Abd-B. At blastoderm stage, segmentation genes act on tissue specific enhancers such as IAB5, IAB7 and IAB8. In late embryogenesis, the Polycomb group (PcG) and trithorax group (TrxG) genes respond to the activating and silencing signals of these early enhancers through dedicated Polycomb or trithorax response elements (PREs or TREs) and maintain the pattern of their activities throughout late development and into adult. Individual iab domains function autonomously, and are protected by domain boundary elements such as miscadastral pigmentation (MCP), front abdominal (Fab)-7 and Fab-8, which may prevent the spread of active or repressed chromatin. Finally, a new class of cis-regulatory elements, the promoter targeting sequences (PTS) have been identified from the Abd-B locus. The PTS overcomes the enhancer blocking effect of an insulator, and facilitates enhancer-promoter communication. Thus, PTS elements may play important roles in enhancer-promoter communications over long distances and intervening boundary elements in Abd-B (Lin, 2010).

Currently, progressively lower levels of Abd-B in segments anterior to PS13 and 14 have been explained by the presence of insulators or silencers, which may attenuate distal enhancers. However, no systematic studies were reported to explain the temporal difference of Abd-B expression in these segments. A 'sequential opening' model hinted that different regulatory regions may become 'open' at different time during development. However, Abd-B enhancers such as IAB5 from iab-5, IAB7 from iab-7 and IAB8 from iab-8 become active at precisely the same time during cellular blastoderm stage, making it difficult to imagine how these enhancers could end up 'opening' the chromatin of each iab at a different time point during embryogenesis. The current study investigated whether the timing of enhancer-promoter interactions could generate the temporal and spatial gradient of Abd-B. For this purpose, transgenic promoter activation by enhancers and regulatory regions were tested from Abd-B. It was found that the reporter gene lacZ activation by IAB5 can be delayed by inserting a 9.5 kb Abd-B 3' regulatory region containing the Fab-8 insulator and the PTS. A similar delay of enhancers can be reproduced by simply inserting an insulator and the PTS. It is hypothesized that the process of overcoming insulators by the PTS delays enhancer-promoter interaction (Lin, 2010).

These results strongly suggest that the delayed Abd-B expression in more anterior abdominal segments is due to the delay in enhancer-promoter interaction. Consistent with this model, it was demonstrated that inserting a 9.5-kb tmr sequence from the 3' region of Abd-B containing an insulator and a PTS significantly delayed the timing of transgene activation by IAB5. It was further demonstrated that the delay is mainly caused by insulator and the PTS, and the delay is not specific to Abd-B enhancers, as a heterologous NEE enhancer can also be delayed. It was also found that enhancer delay depends on the presence of an insulator inserted between the enhancer and the promoter, and increasing the number of insulators prolongs the delay. This study strongly suggests that overcoming the enhancer-blocking effect of insulators in the endogenous locus contributes at least in part to the temporal delay of distal Abd-B enhancers. In addition, it is possible that overcoming multiple insulators may contribute a longer delay for the more remote enhancers such as IAB5 (Lin, 2010).

These analyses support an enhancer-timing model that accounts for, at least in part, the temporal and spatial gradient of Abd-B expression. It is proposed that domain boundaries and PTS elements from Abd-B regulate long-range enhancer-promoter interactions by establishing stable interactions between the Abd-B promoter and its regulatory domains (iabs). The stable interaction facilitates the activities of these distant enhancers, but in the process also delays promoter activation by these enhancers. Such interactions may require the assembly of complex structures linking the enhancer and its promoter, and/or involve complex movement of chromatin loops, processes that need significant longer time than that of a simple enhancer-promoter interaction. As a result, enhancers from iab-5 to iab-7 are significantly delayed. It is also proposed that a specific boundary and the nearby PTS mainly delay the enhancer they regulate. For example, Fab-8 and the linked PTS mainly delays IAB7 (Lin, 2010).

Enhancer-timing model could in part explain the spatial gradient of Abd-B. Since the delay of distal enhancers reduce the amount of time Abd-B protein accumulates in a specific segment, there would be high levels of Abd-B in PS13, but gradually lower levels in PS12, PS11 and PS10. However, this model does not exclude other possibilities such as distance and enhancer strength may play in generating the expression gradient. It is possible that long distance may both reduce enhancer strength and delay enhancer-promoter interactions, which explains the fact that IAB5 is further delayed compared to IAB7. The role of distance in delaying enhancer-promoter interaction was not tested because most embryonic enhancers are sensitive to distances and become undetectable when located at about 10 kb away from the promoter. When a shorter distance of 5 kb was tested, no obvious delay could be detected (data not shown) (Lin, 2010).

The enhancer delay model is compatible with the behavior of dominant gain of function mutations due to deletion of boundary elements from the 3' of Abd-B. For example, the Fab-7 deletions cause the expression of Abd-B in PS11 to elevate to that in PS12 and homeotic transformation of PS11 into a copy of PS12 . This could be due to the loss of an enhancer delay normally in place when Fab-7 is present. Its deletion allowed the two domains to fuse as one, thus, enhancer from iab-6 become regulated the same way as an iab-7 enhancer (IAB7). Consequently, both enhancers arrive at the promoter at about the same time, and activate the same amount of Abd-B transcription in both segments. Finally, the enhancer-timing model is compatible with the collinear arrangement of Abd-B regulatory domains. For example, to express high levels of Abd-B early in PS13, the IAB8 enhancer must be placed closer to the promoter. In contrast, to produce a lower level and delayed Abd-B expression in PS10, the enhancer IAB5 must be placed far away, and separated by multiple insulators and PTS elements to delay its interaction with the Abd-B promoter (Lin, 2010).

Drosophila chromosome condensation proteins Topoisomerase II and Barren colocalize with Polycomb and Maintain Fab-7 PRE silencing

Mechanisms of cellular memory control the maintenance of cellular identity at the level of chromatin structure. An investigation was carried out to see whether the converse is true; namely, if functions responsible for maintenance of chromosome structure play a role in epigenetic control of gene expression. Topoisomerase II (TopoII) and Barren (Barr) are shown to interact in vivo with Polycomb group (PcG) target sequences in the bithorax complex of Drosophila, including Polycomb response elements. In addition, the PcG protein Polyhomeotic (Ph) interacts physically with TopoII and Barr and Barr is required for Fab-7-regulated homeotic gene expression. Conversely, defects in chromosome segregation have been found associated with ph mutations. It is proposed that chromatin condensation proteins are involved in mechanisms acting in interphase that regulate chromosome domain topology and are essential for the maintenance of gene expression (Lupo, 2001).

PcG genes have been proposed to act as chromosomal components maintaining transcriptional repression by 'heterochromatinizing' their target sites. However, the molecular mechanisms underlying chromosomal silencing by the PcG, heterochromatin formation, and the transmission of the silenced state through mitosis are not known. It was reasoned that chromosome condensation machineries could provide an important functional link between the regulation of chromosome domain structure, gene silencing, and mitotic inheritance. Thus, the interaction of the PcG with the machinery involved in orchestrating chromosome dynamics has been investigated and in particular with those machines enabling mitotic chromosome condensation. The in vivo formaldehyde-fixed chromatin immunoprecipitation (X-ChIP) method was used to analyze the distribution in the BX-C locus of two proteins: TopoII, an enzyme involved in the regulation of DNA supercoiling, chromosome condensation, and segregation, and Barr. Barr is the homolog of the Xenopus XCAP-H and C. elegans DPY26 proteins, a TopoII-interacting protein associated with the SMC2/4 condensins complexes, known to be involved in mitotic chromosome condensation (Lupo, 2001).

A striking colocalization of TopoII and Barr with previously mapped PC binding sites was found, suggesting that the two groups of functions are at least acting on the same DNA regions. A clear colocalization was found at major PREs (Fab-7, Mcp, iab-3, bxd, and bx). In particular, the Fab-7 element appears to be a major TopoII/Barr binding site. Strong association of PC to Fab-7 is found. The expression of the major BX-C genes was examined by RT-PCR, and it was found that the AbdB gene is expressed, whereas Ubx and abdA are silent. No Barr/TopoII binding site was found at the Fab-8 PRE, which might define the border between the repressed and active BX-C domains in SL-2 cells (Lupo, 2001).

In iab-2 and iab-3, large fragments (11.0 and 11.5 kb, respectively) have PRE activity. Here specific Barr and TopoII sites are also found. These sites do not match the PC/GAGA peaks previously described. Yet, since these regions show considerable levels of PC, it is suggested that minor PC binding sites adjacent to the reported 'peaks' may also be functionally relevant. Another important aspect of PcG function is the interaction with promoters; major PC binding sites include core promoters, and it is known that PREs perform better when combined with their natural target promoters. Interestingly, a striking colocalization of TopoII and Barr is also found at promoters (AbdB gamma, abdA II, and Ubx) (Lupo, 2001).

Based on the mitotic phenotype and previous immunolocalization data, a direct association of TopoII and Barr with chromosomes mostly at mitosis is expected. In this context, the colocalization of TopoII and Barr in regulatory regions of the BX-C is striking. Although asyncronous tissue culture cells were used, it is believed that the association of Barr and TopoII with the regulatory regions of the BX-C occurs not only at mitosis but also in interphase. In particular, in X-ChIP experiments, the number of mitotic cells at the time of formaldehyde fixation is around 5%, thus, if only mitotic cells contributed to the overall precipitated DNA, this approach would have been below the detectable limit. Hence, it is proposed that TopoII and Barr are associated with their target sites throughout the cell cycle (Lupo, 2001).

The short proximal isoform of Ph (Ph 140p) can be copurified from nuclear extracts with TopoII and Barr. This isoform is not found coimmunoprecipitated with Pc and Psc, and neither Barr nor TopoII copurified with Pc and Psc. The three PcG members Pc, Psc, and the long proximal product Ph 170p have been shown to coimmunopurify from nuclear extracts with antibodies against one of the three. Due to the absence of Ph 140p signals in the Pc/Psc immunoprecipitations, these results might be taken to indicate that there is no functional connection between the presumptive TopoII/Barr/Ph 140p complex and the Pc/Psc/Ph 170p complex. For three reasons this is thought to be unlikely. (1) Both the 170p and the 140p isoforms of Ph are derived from the same transcript by posttranscriptional regulation and differ by a 244 N-terminal stretch of amino acids present only in the 170p isoform. Functional domains of Ph (zinc finger, coiled-coil region, GTP binding site, serine/threonine-rich region, and SAM/SPM domain) are all contained in both isoforms, suggesting that both proteins can fulfill related functions. (2) X-ChIP data, obtained with the same Ph antibodies used in this study, show an extended overlap of Pc and Ph binding regions in the BX-C. Together with the finding of a colocalization of TopoII and Barr with PcG binding sites in regulative regions of the BX-C, this suggests that these proteins act on the same DNA regions. (3) The data show that a reduction of the amount of Barren protein in barren heterozygotes parallels PcG-negative effects on the silencing function of the Fab-7 PRE (Lupo, 2001).

An additional finding supports the conclusion that Ph protein(s) are involved both in PcG function and mitotic chromosome condensation. ph null embryos show defects in chromosome segregation, the same phenotype observed for barren mutant embryos. Conversely, the results of Barren haplo-insufficiency on Fab-7 silencing are suggestive of a role for Barr in early embryogenesis. Since in early embryogenesis Ph 140p is the only Ph product made, these defects are diagnostic of a specific role of Ph 140p in mitosis. These results with regard to Barren protein and Fab-7 silencing are reminiscent of another previously documented role for SMCs in gene regulation. In C. elegans, the DPY27 protein, a homolog of the Xenopus XCAP-C (SMC4), has been shown to bind the X chromosome in females, whereas its absence results in lethality due to abnormally high gene expression levels from the X chromosome. Thus, it is concluded that Ph 140p shares an important role with the Barr/TopoII condensin complexes in mitosis and cell memory processes (Lupo, 2001).

In order to further study interactions between barren and the PcG the null alleles ph502 and ph602 were used for genetic analysis, and strains heterozygous for ph and barren mutations were crossed. Surprisingly, no effect was found. PcG genes, in contrast, show dosage effects, suggesting that the interaction between PcG and Barr/TopoII may imply a different, more dosage-insensitive regulation. However, it has been shown that barren mutations affect PRE silencing in the same way as mutations in PcG genes do. Taken together, these results may indicate a nonstoichiometric relationship between PcG and Barr/TopoII protein complexes. It is proposed that major PcG and condensin proteins belong to distinct protein complexes, but that they nevertheless cooperate at PREs and promoters to maintain the silenced state of homeotic genes. From the SMC standpoint, these results are intriguing because they show that proteins involved in chromosome condensation and segregation processes bind to regulatory elements in chromosomal domains responsible for the inheritance of transcription states. This would suggest that the 'structural maintenance of chromosome' function could also affect epigenetic control of gene expression (Lupo, 2001).

These data reveal novel molecular aspects of BX-C regulation. The distribution of PC and TopoII/Barr sites in the BX-C appears as a reiterated array suggestive of heterochromatic hallmarks, perhaps providing in cis information for higher-order organization of the BX-C chromosomal domain. In particular, TopoII oligomerizes in a DNA-dependent manner. Similar interactions in trans are proposed to occur between PcG proteins in vivo. According to this ability, spaced molecules at distant sites on the DNA could come into contact, giving rise to more condensed domains. A model has been proposed to explain how condensin proteins and Topoisomerases may act together in condensation. In this model, the size of the condensin complex (perhaps 1000 Å) could introduce (+) supercoils by affecting the global writhe of DNA, thus creating a more condensed state. In this study, Barr is found only at discrete sites, whereas PC and other PcG proteins are associated also with large chromosomal regions. Possibly, one aspect of PcG protein function and binding to chromatin in interphase is to stabilize and expand the condensed state by topological effects (Lupo, 2001 and references therein).

The positioning of TopoII at complex regulatory regions (e.g., abx/bx and iab-3-iab-8) may indicate the existence of minidomains providing tight control on the chromatin structure of intervening regulatory DNA sequences by localized changes of DNA superhelicity. The activity of TopoII could be locally regulated by the association with other proteins like Barr and perhaps some PcG and trxG members [e.g., Ph 140p, CCF, E(z), and Gaga]. Interestingly, Barr has been found to stimulate TopoII activity. It has to be pointed out that these data show, in a direct way, where in vivo TopoII binds to single-copy genes but they cannot tell if these sites correspond to TopoII cutting sites. However, it is likely that a tight association with DNA corresponds to enzymatic activity. Thus, it is proposed that in vivo TopoII activity may be enhanced at specific sites, whereas at others it could be reduced, resulting in local differences in chromatin condensation states controlled by DNA topology (Lupo, 2001).

The presence of multiple Barr and TopoII sites within the BX-C could thus provide a powerful way to fine-tune the structure of each of the parasegment-specific chromosomal subdomains. As a direct consequence of controlled condensation of specific parts of the BX-C, determined states could be fixed by enabling or not enabling specific interactions between cis elements. The mechanism by which Fab-7 regulates the AbdB promoters is, in fact, not known. It has been proposed that a combination of 'chromatin effects' and insulating activity may regulate enhancer-promoter interactions. It is proposed that the homeotic loss-of-function phenotypes observed in Fab-7 or Mcp deletions could be due to a change in local DNA topology altering the communication of segment-specific enhancers with the AbdB promoters. In this way, local differences in chromosome domain topology may contribute to stabilize or interfere with correct phasing between regulatory elements and promoters. If topological effects are at least part of Fab-7 function, this may also help to explain distance-dependent effects on enhancer-promoter interactions. Interestingly, in Drosophila, mutations in the Nipped-B gene facilitate enhancer-promoter interactions by overcoming the action of ectopic insulator elements in the Ubx domain. Nipped-B is the homolog of the yeast SMC-associated protein Scc2 (sister chromatid cohesion 2), suggesting that adherins may have a broader role in chromosomal domain organization and gene regulation. It is proposed that chromatin condensation proteins may be involved in a pathway acting also in interphase that regulates chromosome domain structure by DNA topology and is essential for maintenance of gene expression (Lupo, 2001).

The MCP silencer of the Drosophila Abd-B gene requires both Pleiohomeotic and GAGA factor for the maintenance of repression

Silencing of homeotic gene expression requires the function of cis-regulatory elements known as Polycomb Response Elements (PREs). The MCP silencer element of the Drosophila homeotic gene Abdominal-B has been shown to behave as a PRE and to be required for silencing throughout development. Using deletion analysis and reporter gene assays, a 138 bp sequence has been defined within the MCP silencer that is sufficient for silencing of a reporter gene in the imaginal discs. Within the MCP138 fragment, there are four binding sites for the Pleiohomeotic protein (Pho) and two binding sites for the GAGA factor, encoded by the Trithorax-like gene. PHO and the Trl proteins bind to these sites in vitro. Mutational analysis of Pho and Trl binding sequences indicate that these sites are necessary for silencing in vivo. Moreover, silencing by MCP138 depends on the function of Trl, and on the function of the PcG genes, including pleiohomeotic. Deletion and mutational analyses show that, individually, either Pho or Trl binding sites retain only weak silencing activity. However, when both Pho and Trl binding sites are present, they achieve strong silencing. A model is presented in which robust silencing is achieved by sequential and facilitated binding of Pho and Trl (Busturia, 2001).

How does Trl or perhaps another GAGA binding protein contribute to the silencing by MCP, and what is its relationship to the Pho protein function? Two models to explain their relationship which leads to strong silencing are suggested. These models are based on the following observations. (1) Pho binding sites by themselves show little silencing activity (MCP1 and MCP7* constructs). (2)Trl or some other protein that binds to MCP can weakly recruit silencing complexes in the absence of Pho binding (5MPho construct). (3) When present together, Trl and Pho binding sites exhibit robust silencing activity (MCP7 construct). In the first model, Trl and Pho bind to the MCP silencer in a sequential order. One version would be that Trl binding is absolutely required for binding or activity of Pho. Trl may open up chromatin at MCP, allowing binding of Pho. Upon binding, Pho may recruit PcG silencing complexes, although there is still little evidence that this happens. Trl has been shown to induce DNase I hypersensitive sites, or nucleosome-free regions, and this may create a prerequisite condition for Pho to bind to its recognition sites. There is indeed a DNase hypersensitive region associated with MCP that includes the location of the Trl binding site (Busturia, 2001).

In a second version of the model, Pho acts as a facilitator of Trl binding by creating some pre-condition, perhaps by bending DNA as YY1 does. Since Pho binding sites are not absolutely required for MCP silencing activity, Trl presumably can bind weakly to MCP in the absence of Pho. Enhanced binding of Trl leads to increased recruitment of silencing complexes. Trl bound to MCP may recruit PcG silencing complexes by directly interacting with PC or other members of PcG complexes. Alternatively, Trl could first recruit SIN3 histone deacetylation complexes through its interaction with SAP18, which then might generate a chromatin state favorable for PcG complex binding. Whichever version of the model is correct, the important feature of the model is the sequential recruitment of DNA binding proteins, Trl and Pho, to MCP. Binding of one protein creates a condition favorable to the binding of a second protein, eventually leading to the recruitment of PcG complexes. Note that the requirement of Trl and Pho proteins applies to MCP silencing, but not necessarily to all PREs. Other PREs may use other combinations of proteins. This model is analogous to Swi5 protein binding to the yeast HO promoter and recruiting the chromatin remodeling complex Swi/Snf. Swi/Snf in turn recruits the histone acetylase complex SAGA, eventually leading to the binding of the transcription factor SBF to the HO promoter. In such a sequential recruitment model, compromising one step in the sequence may become rate limitating so that combining two mutations that disable two different steps may not necessarily lead to synergistic effects. This may explain why no synergistic effects are observed when Trl and PcG mutations are combined. In the second model, Trl and Pho bind to MCP independently of one another. Each protein may induce a unique chromatin modification that, together, can have a positive synergistic effect on the recruitment of PcG silencing complexes (Busturia, 2001).

batman interacts with Polycomb and trithorax group genes to regulate homeotic genes

Polycomb and trithorax group genes maintain the appropriate repressed or activated state of homeotic gene expression throughout Drosophila development. lola like (lolal), also known as batman (ban), functions in both activation and repression of homeotic genes. The 127-amino acid Lolal protein consists almost exclusively of a BTB/POZ domain. This domain is involved in the interaction between Lolal and the DNA binding GAGA factor encoded by the Trithorax-like gene. The GAGA factor and Lolal codistribute on polytene chromosomes, coimmunoprecipitate from nuclear embryonic and larval extracts, and interact in the yeast two-hybrid assay. Lolal, together with the GAGA factor, binds to MHS-70, a 70-bp fragment of the bithoraxoid Polycomb response element. This binding, like that of the GAGA factor, requires the presence of d(GA)n sequences. lolal also interacts with polyhomeotic and, like Trl, both lolal and ph are needed for iab-7 polycomb response element mediated pairing dependent silencing of mini-white transgene. lolal was also identified as a strong interactor of GAGA factor in a yeast two-hybrid screen. lolal also interacts geneticially with polyhomeotic and, like Trl, both lolal and ph are needed for iab-7PRE mediated pairing dependent silencing of mini-white transgene. These observations suggest a possible mechanism for how Trl plays a role in maintaining the repressed state of target genes involving Lolal, which may function as a mediator to recruit PcG complexes (Faucheux, 2003; Mishra, 2003).

Several lines of evidence suggest a close association between Batman and Trl. In 0- to 18-h embryos, increasing the dose of Batman through the use of the Gal4/UAS system increases the formation of the Batman- and Trl-containing complexes on the MHS-70 Ubx PRE fragment, which are fully displaced by both anti-Trl and anti-Batman antibodies. This result suggests that Batman may be a Trl cofactor that modulates its binding to MHS-70. Consistent with this, lowering the dose of ban has the same effect as lowering the dose of Trl in at least two regulatory pathways: the repression of Scr, and pairing-sensitive silencing of a white reporter gene next to an AbdB PRE. In addition, ban function is necessary for the activity of Trl in the activation of Ubx. Finally, the increased lethality of Trl13c mutants when the dose of ban is reduced provides additional evidence for the functional significance of the interaction of ban with Trl (Faucheux, 2003).

Inheritance of Polycomb-dependent chromosomal interactions in Drosophila: Evidence using the Fab-7 cellular memory module

Maintenance of cell identity is a complex task that involves multiple layers of regulation, acting at all levels of chromatin packaging, from nucleosomes to folding of chromosomal domains in the cell nucleus. Polycomb-group (PcG) and trithorax-group (trxG) proteins maintain memory of chromatin states through binding at cis-regulatory elements named PcG response elements or cellular memory modules. Fab-7 is a well-defined cellular memory module involved in regulation of the homeotic gene Abdominal-B (Abd-B). In addition to its action in cis, it has been shown, by three-dimensional FISH, that the Fab-7 element leads to association of transgenes with each other or with the endogenous Fab-7, even when inserted in different chromosomes. These long-distance interactions enhance PcG-mediated silencing. They depend on PcG proteins, on DNA sequence homology, and on developmental progression. Once long-distance pairing is abolished by removal of the endogenous Fab-7, the derepressed chromatin state induced at the transgene locus can be transmitted through meiosis into a large fraction of the progeny, even after reintroduction of the endogenous Fab-7. Strikingly, meiotic inheritance of the derepressed state involves loss of pairing between endogenous and transgenic Fab-7. This suggests that transmission of nuclear architecture through cell division might contribute to inheritance of chromatin states in eukaryotes (Batignies, 2003).

In most experiments throughout this work, two constructs were used carrying a 3.6-kb Fab-7 fragment cloned in two different orientations (p5F24 and p5F3 transgenes) upstream to lacZ and mini-white reporter genes. These transgenes were inserted at different genomic locations and combined with deletions or with mutations in PcG or trxG genes. For simplicity, transgenic lines will be named after the CMM element present in the transgene and the chromosomal arm of insertion of the transgenes. For instance, one previously published line, 5F24 will be renamed here as Fab-X, to indicate insertion of Fab-7 in the X chromosome. As expected for CMM-mediated silencing, pairing-sensitive repression is observed and the eye color of homozygous females is strongly variegated, whereas in heterozygous females or in males (hemizygous) repression is weaker. Precise mapping of the transgene insertion indicates that the Fab-X line harbors two copies of the p5F24 transgene inserted in tandem 9.6 kb upstream of the scalloped (sd) gene. The sd gene product is required for wingblade development in Drosophila. Reduced expression of this gene leads to a characteristic wing phenotype (sd phenotype) that can have different degrees of severity ranging from small lesions in the wing margin to complete destruction of wing morphology. The insertion of the Fab-7 CMM at a distance of 18.4 kb from sd (8.8 kb DNA spanning the lacZ and mini-white regions plus 9.6 kb from the transgene insertion site to the sd promoter) induces a mutant phenotype, resulting in destruction of the wingblade. This phenotype is temperature sensitive and pairing dependent, since it is observed with a strong penetrance of up to 95% in homozygous females raised at 29°C, whereas it is almost absent in heterozygous females or hemizygous males. Both features are typical of PcG-mediated silencing and parallel effects on mini-white. The incomplete penetrance of the sd phenotype does not depend on genetic heterogeneity of the flies, preventing silencing in a fraction of the population. When flies were raised at 29°C and Fab-X females with wild-type wings were selected and remated with Fab-X males, the next generation females showed a sd phenotype with similar penetrance as nonselected Fab-X females. Finally, as observed for the white eye phenotype in the Fab-X line, the sd phenotype is strongly attenuated by mutations in PcG genes, whereas it is enhanced by a mutation in the trx gene (Batignies, 2003).

Surprisingly, repression of sd depends also on the presence of an intact copy of the endogenous Fab-7 element in the Abd-B locus, located in the right arm of the third chromosome (chromosome 3R). A genomic deletion of 4 kb encompassing the 3.6-kb Fab-7 transgenic element was introduced in the homozygous state into the Fab-X line to give the Fab-X; Fab-71 line. When raised at 29°C, Fab-X; Fab-71 females showed derepressed eye color and showed only 6%-12% of sd wing phenotype compared with 90%-95% of Fab-X females, suggesting that silencing of mini-white and sd is reduced. As a control, the presence of the Fab-7 deletion had no effect on the wing phenotype of a mutant line for the sd gene, indicating that endogenous Fab-7 does not play any role in sd regulation in the absence of the X-linked transgene. Moreover, a derepression both of mini-white and of sd was observed by introducing a homozygous Fab-712 deletion into Fab-X. This mutation deletes 1.5 Kb of DNA from the same region and has a similar effect on regulation of its endogenous target gene Abd-B, but it has an independent origin and genetic background from Fab-71. Therefore, derepression depends specifically on removal of Fab-7 (Batignies, 2003).

The results presented here show that a CMM element of 3.6 kb can mediate long-distance associations between distant chromosomal regions in embryonic nuclei. These interactions depend strongly on chromatin components of the PcG and on DNA sequence homology. Importantly, when disrupted in one generation, these pairing interactions are inefficiently re-established even upon reintroduction of sequence homology, and a large portion of the progeny maintains the loci unpaired in subsequent generations. This phenomenon is reversible, suggesting that PcG-mediated chromatin regulation is an equilibrium process that depends on the concentration of regulatory components and on the previous history of the cell. Perturbation of the balance between these regulatory cues might favor establishment as well as inheritance of active or repressed states at target genes. Several cases of inheritance of chromatin composition features have been reported in eukaryotes. The data described here suggest that inheritance of Fab-7 regulatory states depends not only on chromatin components, but also on nuclear compartmentalization of chromosomal domains (Batignies, 2003).

PcG proteins have been implicated previously in phenomena involving long-distance interactions among independent loci. Phenotypic interactions were documented both at transgenes containing CMM elements as well as in the phenomenon of cosuppression. In this last case, PcG proteins as well as mechanisms of RNA-dependent posttranscriptional gene silencing were shown to be involved. However, whether direct long-distance associations occur in these cases is presently unknown. In contrast, long-distance pairing has been observed in up to 30% of embryonic nuclei containing a euchromatic translocation of a region of ~900 kb, spanning the BX-C and flanking genes from chromosome 3R to chromosome X. Although the DNA sequence determinants and the proteins responsible for pairing in this large chromosomal region were not identified, several CMM, including Fab-7, are present in the BX-C and could contribute to this interaction. On the basis of these results, it is suggested that PcG proteins may mediate long-range pairing interactions in the case of Fab-7 as well as in transgenes containing other CMM and in some cases of cosuppression and silencing of repetitive DNA elements (Batignies, 2003).

Long-distance associations may not only involve transgenes, but also natural genes regulated by the Polycomb pathway. PcG proteins are distributed in specific nuclear compartments that have been termed PcG bodies. Although the significance of these bodies is presently unclear, it is speculated that endogenous PcG target genes may undergo physical associations at nuclear PcG bodies dedicated to their regulation. Compartmentalization of PcG target genes may not be required for primary recruitment of PcG complexes, but it may rather stabilize PcG and trxG-mediated gene regulation. This phenomenon may not be unique in eukaryotic nuclei, since evidence for gene clustering at specific intranuclear organelles has been found in vertebrates. These clustering phenomena were suggested to involve positioning of genes coregulated by the same set of proteins in the same nuclear compartments. PcG proteins may represent one class of factors acting in this manner (Batignies, 2003).

A hint for involvement of the Fab-7 element in heterologous associations at PcG bodies comes from experiments showing that in Fab-X; Fab-71 larvae, the Fab-7 transgene pairs with the BX-C locus to some extent, even in the absence of the endogenous Fab-7. This suggests that in the absence of sequence homology, Fab-7 may interact with other CMM present in the BX-C, albeit more weakly. An increase in Fab-7-dependent sd silencing is detected in the presence of a transgene containing the Mcp sequence from the BX-C, suggesting that these two elements may be able to interact (Batignies, 2003).

How is pairing achieved? First, homologous CMM must come in physical proximity. This may depend on constrained brownian motion of chromosomal territories. However, other processes may help this long-distance search. In particular, it may be postulated that genes containing CMM localize to PcG bodies. It is speculated that these bodies might not be immobile, but, as in the case of splicing speckles, they may rather undergo occasional movements, splitting, and mergers, although perhaps with different kinetics. Genes localized within these bodies may reside there for a certain time and then leave one PcG body to incorporate another one. Such a dynamic behavior may allow PcG target genes to explore part of the nucleus, but would, at the same time, allow them to stay in the vicinity of other PcG target genes and prevent them from diffusing away randomly in the nucleoplasm. This may increase the probability for a CMM to explore contacts with other CMM (Batignies, 2003).

Once proximity is established, strong association might be established by regulatory components of CMM chromatin. The identification of a 3.6-kb DNA sequence as a sufficient region of homology to induce long-range physical associations will allow, for the first time, to dissect DNA sequences and chromatin factors responsible for pairing at high resolution. The 3.6-kb Fab-7 element contains a chromatin boundary that can attenuate enhancer-promoter communications, and a PRE. GAGA factor binds to both the PRE and the boundary region of Fab-7. Moreover, this protein is able to bind cooperatively to DNA to form oligomers, bringing distant DNA sequences close together. Thus, GAGA factor-binding sites may be partly responsible for long-range interactions. Similarly, putative binding sites for Zeste, a protein that mediates trans-sensing phenomena, are also present in Fab-7, and they may contribute to pairing. Other chromatin components described previously to act at this element, such as proteins of the trxG, chromatin condensation proteins, and DNA topoisomerase II may also be involved in pairing of Fab-7 (Batignies, 2003).

However, all of these proteins associate also with other CMM in the genome. How do they distinguish between DNA sequence homologous and nonhomologous CMM? One possibility is that chromatin regulation and the DNA sequence determine a specific array of proteins and of histone modifications associated with it. For a given locus, this may result in the formation of a unique order of chromatin tags that can only be found at loci sharing strong sequence homology. Some of these components may undergo dimerization or oligomerization, leading to specific contacts that may maintain homologous chromatin stably associated. Similar contacts may also be involved in chromosome pairing in somatic cells or during meiosis (Batignies, 2003).

A remarkable finding involving long-distance pairing of the Fab-7 CMM is transmission through meiosis. What could be the role of this meiotic inheritance of chromatin states? This is particularly intriguing in the case of a CMM regulating a homeotic gene, as expression of homeotic genes must be reset at every generation in order to establish appropriate gene expression patterns along the anteroposterior embryonic axis. However, meiotic inheritance was reported previously to involve a phenotype associated with a chromosomal rearrangement at the BX-C locus, although no molecular determinant for this phenomenon could be found. In Caenorhabditis elegans, PcG proteins establish a germ-line-specific gene silencing that is heritable through meiosis. One possible way in which meiotic inheritance could be important in Drosophila homeotic gene regulation is to maintain a default silenced state during early embryogenesis. At the onset of homeotic gene transcription, spatial-specific transcriptional repressors maintain homeotic genes repressed outside of the appropriate expression domains. Maintenance of repression is crucial, as failure could cause homeotic transformations. Inheritance of chromatin silencing may stabilize this repression and contribute to developmental homeostasis (Batignies, 2003).

The fact that pairing interactions involving a chromosomal element regulated by PcG/trxG proteins are heritable raises the question of how transmission of chromatin architectural features is possible through cell division. Two different, but not mutually exclusive, mechanisms may contribute to explain this novel form of inheritance. Chromosomal contacts may depend on specific, heritable chromatin marks deposited in cis on the templates undergoing pairing. These marks may allow contacts to re-establish after they are broken during chromosome metabolism at mitosis and meiosis. Perturbation of these marks may change chromatin at Fab-7 and make it incapable of establishing pairing interactions with its homolog sequence in another chromosome. Chromatin marks, such as histone acetylation, histone methylation, and association of Swi6 protein to the mating type locus in Schizosaccharomyces pombe were shown previously to be heritable through meiosis. Initial chromatin characterization in the presence or absence of Fab-7-pairing interactions showed recruitment of PcG proteins to the transgene and to the region surrounding its site of insertion in both cases, and did not reveal significant changes in PcG protein binding or in histone modifications (Batignies, 2003).

A second mechanism for inheritance of long-range chromosomal interactions may depend on stable transmission of the relative chromosome positions and specific gene contacts through cell division. It was shown recently that global chromosome positioning can be transmitted in mammalian cells through the whole-cell cycle and mitosis, although the fidelity of mitotic transmission may depend on cell type. The data show that long-distance pairing is dynamic during development; it has a relatively weak frequency during embryonic stages, and it increases at larval stages. This dynamics may depend on the increased length of the cell cycle or on more robust PcG silencing in larvae, and it suggests that the actual physical contact between chromosomes may be lost, but regulation of nuclear compartmentalization may favor re-establishment of long-distance pairing at each cell generation (Batignies, 2003).

In summary, the present study suggests that features of the nuclear architecture of PcG target genes can be transmitted through cell division. It is proposed that this may represent a novel form of epigenetic inheritance that may be used to convey cellular memory of chromatin states in eukaryotic organisms (Batignies, 2003).

Developmental modulation of Fab-7 boundary function

The Fab-7 boundary functions to ensure the autonomous activity of the iab-6 and iab-7 cis-regulatory domains in the Drosophila Bithorax Complex from early embryogenesis through to the adult stage. Although Fab-7 is required only for the proper development of a single posterior parasegment, it is active in all tissues and stages of development that have been examined. In this respect, Fab-7 resembles conventional constitutive boundaries in flies and other eukaryotes that act through ubiquitous cis-elements and trans-acting factors. Surprisingly, however, the constitutive activity of Fab-7 is generated by combining sub-elements with developmentally restricted boundary function. In vivo evidence is provided that the Fab-7 boundary contains separable regions that function at different stages of development. These findings suggest that the units (domains) of genetic regulation that boundaries delimit can expand or contract by switching insulator function off or on in a temporally regulated fashion (Schweinsberg, 2004a).

The most thoroughly characterized of the BX-C boundary elements is Fab-7. It is located in between the iab-6 and iab-7 cis-regulatory domains and, like the other boundaries in BX-C, it functions to ensure the genetic autonomy of the two flanking cis-regulatory domains. Mutations that inactivate Fab-7 lead to the fusion of the iab-6 and iab-7 domains, and this disrupts the specification of PS11. In most Fab-7 mutant PS11 cells, positive regulatory elements in iab-6 inappropriately activate the iab-7 cis-regulatory domain. As a consequence, Abd-B expression in these cells is driven by iab-7 not iab-6, and they assume a PS12 identity. In the remaining mutant PS11 cells negative elements in iab-7 inappropriately silence iab-6 (and iab-7). When iab-6 is silenced Abd-B expression is driven by iab-5 and the cells assume a PS10 identity (Schweinsberg, 2004a and references therein).

Although the normal role of Fab-7 is to prevent crosstalk between the iab-6 and iab-7 cis-regulatory domains, it also has the ability to insulate promoters from the regulatory effects of nearby enhancers or silencers. Like other known boundaries, the insulating activity of the Fab-7 element does not appear to be restricted to specific enhancer-promoter combinations, nor is it stage or tissue specific. In the context of BX-C, the endogenous Fab-7 boundary insulates the Ubx and rosy promoters carried by 'bluetail' (blt), a transposon inserted into the iab-7 domain, from the regulatory effects of the iab-6 and iab-5 domains. This insulating activity is observed from early embryogenesis through the adult stage. In transgene assays in embryos, the Fab-7 boundary blocks the fushi tarzu (ftz) stripe (UPS) and neurogenic (NE) enhancers from activating the hsp70 promoter in the embryonic ectoderm and CNS, respectively. It also blocks eve, hairy, iab-5, rhomboid and twist enhancers from activating eve and white promoters in the embryo. In the adult, Fab-7 blocks the white eye and testes enhancers from activating the mini-white promoter (Schweinsberg, 2004a and references therein).

The minimal Fab-7 boundary defined in the ftz:hsp70-lacZ and wEN:mini-white enhancer blocking assays is 1.2 kb in length. It extends from the minor nuclease hypersensitive site on the proximal side to the iab-7 PRE (which corresponds HS3) on the distal side and includes two major chromatin-specific nuclease hypersensitive regions, HS1 and HS2. The largest hypersensitive region, HS1, contains six consensus GAGA factor binding sites arranged in three pairs, 1-2, 3-4 and 5-6. The ubiquitously expressed GAGA factor is encoded by the Trithorax-like (Trl) gene, and it is thought to function in the formation and/or maintenance of the nucleosome free regions of chromatin associated with a variety of cis-acting elements in flies, including enhancers, promoters, Polycomb Response Elements (PRES) and boundaries. Chromatin immunoprecipitation experiments demonstrate that GAGA is associated with the Fab-7 boundary in vivo. Moreover, the GAGA-binding sites in HS1 are important for boundary function. The enhancer blocking activity of the minimal 1.2 kb boundary is compromised in both the embryo and adult when GAGA sites 1-5 are mutated. Although this finding indicates that GAGA (or another protein that recognizes the GAGA consensus) is required for Fab-7 boundary activity throughout development, the GAGA sites are not functionally equivalent. When only the centromere proximal pair, 1-2, are mutated, blocking of the ftz UPS stripe enhancer in the ectoderm of early embryos by the minimal Fab-7 boundary is weakened, but there is no apparent effect on the blocking of either the ftz NE enhancer in the CNS of older embryos or the w enhancer in adults. By contrast, mutation of the central pair, 3-4, weakens blocking of the w enhancer in the eye, but has little effect on the blocking of the ftz enhancers in embryos (Schweinsberg, 2004a and references therein).

One interpretation of these results is that the constitutive boundary activity of the Fab-7 element is generated by sub-elements whose activities are developmentally restricted. In the studies reported here, this hypothesis was tested. Unlike other well characterized boundaries, the constitutive activity of Fab-7 is generated by combining a series of subelements that function at different stages of development. This unexpected finding indicates that chromatin domains are not always static units, but instead may be redefined by inactivating or activating a boundary element such that the chromatin domain can expand to include new genes or regulatory sequences, or alternatively contract eliminating genes or regulatory sequences (Schweinsberg, 2004a).

Thus, a fragment, pHS1, from the proximal half of the major Fab-7 nuclease hypersensitive region, HS1, can block the ftz UPS stripe enhancer in early embryos. However, this same fragment only has residual boundary activity in the CNS of older embryos and no detectable boundary activity in the adult eye. The opposite result is obtained with a fragment containing the remainder of the HS1, dHS1. Unlike pHS1, dHS1 can function as a boundary element in the adult eye. In fact, a multimerized version of dHS1 is more effective in blocking the white enhancer than the intact Fab-7 boundary. In embryos, dHS1 is nearly as effective in blocking the ftz NE enhancer in the CNS as is Fab-7. By contrast, dHS1 is comparatively ineffective in blocking the ftz UPS enhancer in the early embryo, functioning about as well as 5 su(Hw)-binding sites. Further subdivision of dHS1 localizes boundary function in the mini-white assay to dHS1A, a subfragment that is derived from the center of HS1. Like dHS1, the multimerized HS1A fragment is more effective in blocking the mini-white enhancer than the intact Fab-7 boundary. dHS1A also retains an ability to block the ftz NE enhancer in the CNS, though it is less effective than the larger dHS1 fragment. Finally, on the distal side of HS1, dHS1B can block, albeit weakly, both the UPS and NE enhancers in the ftz:hsp70-lacZ assay. However, like pHS1, it has little or no blocking activity in the wEN:mini-white assay. It should be noted that although the various sub-elements in HS1 seem to function most effectively at different stages of development, there is clearly some overlap in their activities (e.g. between pHS1 and dHS1B). It seems likely that this overlap is important in that it would allow the subelements (which in the endogenous locus are present in only single copies) to collaborate with each other to generate a functional Fab-7 boundary (Schweinsberg, 2004a).

The idea that the constitutive boundary function of Fab-7 depends upon combining subelements whose activity is developmentally restricted is supported by this analysis of three Fab-7 deletions generated by imprecise excisions of blt that retain an intact transposon. The largest of these, P14.1, removes a DNA segment closely corresponding to the minimal Fab-7 element defined in enhancer blocking transgene assays. This deletion has no discernable boundary activity at any stage of development and the blt Ubz-lacZ reporter is active in PS11 from early embryogenesis onwards. The two smaller deletions, P6.1 and P18.1, retain all of the sequences in pHS1 (plus sequences proximal to pHS1, which are important for the boundary function of the minimal 1.2 kb Fab-7 element). Because the pHS1 sequence (when multimerized) confers boundary activity in transgene assays during the early stages of embryogenesis, one might expect that these two smaller deletions will retain at least some boundary function in early embryos, and indeed they do. In both deletions, the anterior limit of Ubx-lacZ expression is initially PS12 just like wild-type Fab-7. However, because these two deletions lack sequences on the distal side of HS1 that confer enhancer blocking activity in the embryonic CNS and the adult eye in transgene assays, they might be expected to have little boundary function at later stages of development. Indeed, lacZ expression from the blt transposon in both deletion mutants spreads into PS11 in germband retracted embryos. In addition, the Fab-7 mutant phenotype of the two smaller deletions in adult flies is indistinguishable from that of the larger deletion. These findings indicate that although functionally autonomous iab-6 and iab-7 cis-regulatory domains can be established by the P6.1 and P18.1 mutants, the Fab-7 boundary sequences remaining in these mutants are unable to sustain autonomy as development proceeds. This would suggest that the process of establishing an autonomous domain is not irreversible and that boundary elements must remain continuously active in order to maintain independent units of genetic activity. Conversely, the properties of dHS1 or dHS1A would suggest that functionally independent domains can be established de novo by activating a previously inactive boundary element (Schweinsberg, 2004a).

A number of models could potentially account for the developmentally restricted activity of the different subelements from Fab-7. One idea is that the boundary function of each subelement is enhancer and/or promoter specific. Although this possibility cannot be excluded, it is noted that the Fab-7 boundary itself shows no evidence of enhancer or promoter specificity. In transgene assays and also in the context of BX-C itself, the boundary is able to block a wide range of enhancer-promoter combinations in many different tissues and cell types from early embryogenesis through to the adult. Another idea is that the subelements have target sequences for DNA-binding proteins and/or accessory factors whose expression or activity is developmentally restricted. In this model, the boundary function of the pHS1 multimer, the two deletions P6.1 and P18.1, and perhaps also dHS1B in early embryos would depend upon factors that are either deposited in the egg during oogenesis or expressed only in early stages of embryogenesis. In this case, one would expect that boundary activity would be lost when the complement of these factors is depleted as the embryo develops. Consistent with the idea that pHS1 function depends upon maternal factors, UPS blocking by the 4xpHS1 multimer is compromised in progeny of mothers heterozygous for several 3rd chromosome deficiencies. Conversely, because blocking by dHS1 (or dHS1A) is weak in early embryos, but then becomes stronger, it would be reasonable to think that its boundary activity depends more crucially upon factors that are zygotically expressed rather than of maternal origin. In this context, it is interesting to note that the interval in which the pHS1 subelement is active as a boundary corresponds roughly to the initiation phase of BX-C regulation, while it is not active once regulation switches to the maintenance mode. The converse seems to be true for the dHS1 subelement, which appears to become activated as BX-C regulation switches from initiation to maintenance (Schweinsberg, 2004a).

These overlapping patterns of activity suggest that one reason why Fab-7 might be composed of different subelements is that this would permit the use of boundary factors that are specialized with respect to their interactions with, in one case, initiation phase gap and pair-rule transcription factors, and, in the other case, with the maintenance phase trithorax and Polycomb group proteins. More generally, the fact that the boundary activity of the Fab-7 subelements is developmentally restricted suggests a hitherto unexpected plasticity in boundary function. This plasticity indicates that the activity of some boundary elements is likely to be subject to tissue or stage-specific regulation. If this is the case, the genes and regulatory elements included within a chromosomal domain, which is the unit of autonomous genetic activity, could change from one tissue or stage to the next by turning boundary function on or off. This would afford a novel mechanism of high order genetic regulation (Schweinsberg, 2004a).

The enhancer-blocking activity of the Fab-7 boundary from the Drosophila bithorax complex requires GAGA-factor-binding sites

The role of the GAGA factor [encoded by the Trithorax-like (Trl) gene] in the enhancer-blocking activity of Frontabdominal-7 (Fab-7), a domain boundary element from the Drosophila melanogaster bithorax complex (BX-C), was analyzed. One of the three nuclease hypersensitive sites in the Fab-7 boundary, HS1, contains multiple consensus-binding sequences for the GAGA factor, a protein known to be involved in the formation and/or maintenance of nucleosome-free regions of chromatin. GAGA protein has been shown to localize to the Fab-7 boundary in vivo, and it recognizes sequences from HS1 in vitro. Using two different transgene assays it has been demonstrated that GAGA-factor-binding sites are necessary but not sufficient for full Fab-7 enhancer-blocking activity. Distinct GAGA sites are required for different enhancer-blocking activities at different stages of development. The enhancer-blocking activity of the endogenous Fab-7 boundary is sensitive to mutations Trithorax-like (Schweinsberg, 2004b).

Assuming that GAGA factor interactions with the consensus-binding sites in HS1 are important to Fab-7 activity, one question of interest is whether GAGA plays a direct or indirect role in boundary function. Since GAGA-binding sites have been shown to be required for the enhancer-blocking activity of other fly elements in addition to Fab-7, one must consider the possibility that GAGA plays a direct role in boundary function analogous to that of, for example, the Su(Hw). However, this view is difficult to reconcile with the fact that GAGA is required for the functioning of other elements unrelated to boundaries such as promoters, enhancers, and PREs, not to mention its role in centromeric heterochromatin and chromosome segregation. Also arguing against a direct role in boundary function, it was found that enhancer-blocking activity cannot be reconstituted by multimerizing GAGA-factor-binding sites. In contrast, one known activity of the GAGA factor that would account for its ability to participate in the functioning of such a diverse array of regulatory elements is the formation and/or maintenance of nucleosome-free regions of chromatin. In this model, GAGA factor binding to sites in HS1 would ensure that this 400-bp sequence is nucleosome free and that target sequences within HS1 are readily accessible for the binding of other factors that actually confer boundary function. In this case, mutations in the HS1 GAGA sites would disrupt boundary function indirectly because of difficulties in generating a nucleosome-free region of chromatin, which is fully accessible for these other boundary proteins. While the idea is favored that a key function of the GAGA protein is to ensure DNA accessibility, it is reasonable to think that GAGA may also play a more central role in Fab-7 boundary function because of its ability to participate in protein:protein interactions. The N terminus of the GAGA protein has a BTB/POZ domain that is present in numerous other proteins. Moreover, depending upon the particular protein partners, the GAGA factor appears to have rather different activities. Thus, a plausible hypothesis is that GAGA can have boundary functions when it is combined with one set of proteins, transcriptional activation functions when it is combined with another, and Pc-G-silencing functions when combined with yet a third set of proteins. This would explain why no boundary function was observed with the multimermized GAGAG sites. Moreover, if this hypothesis is correct, then it would be reasonable to think that the partners for GAGA protein bound at sites 3-4 are likely to be different from the partners for GAGA protein bound at sites 1-2 or, presumably, 5-6. Further studies will be required to identify these putative partners and to understand how they function together with the GAGA factor to provide a scaffold for building a boundary element (Schweinsberg, 2004b).

dSAP18 and dHDAC1 contribute to the functional regulation of the Drosophila Fab-7 element

The Drosophila GAGA factor [Trithorax-like (Trl)] interacts with dSAP18, which, in mammals, is a component of the Sin3-HDAC co-repressor complex. GAGA-dSAP18 interaction has been proposed to contribute to the functional regulation of the bithorax complex (BX-C). Mutant alleles of Trl, dsap18 and drpd3/hdac1 enhance A6-to-A5 transformation indicating a contribution to the regulation of Abd-B expression at A6. In A6, expression of Abd-B is driven by the iab-6 enhancer, which is insulated from iab-7 by the Fab-7 element. GAGA, dSAP18 and dRPD3/HDAC1 co-localize to ectopic Fab-7 sites in polytene chromosomes, and mutant Trl, dsap18 and drpd3/hdac1 alleles affect Fab-7-dependent silencing. Consistent with these findings, chromatin immunoprecipitation analysis shows that, in Drosophila embryos, the endogenous Fab-7 element is hypoacetylated at histones H3 and H4. These results indicate a contribution of GAGA, dSAP18 and dRPD3/HDAC1 to the regulation of Fab-7 function (Canudas, 2005).

The conclusion that GAGA, dSAP18 and dRPD3/HDAC1 contribute to the function of the Fab-7 element of BX-C is based on the following observations:

  1. the localization of GAGA, dSAP18 and dRPD3/HDAC1 at ectopic Fab-7 elements (Canudas, 2005).
  2. the effects of Trl, dsap18 and drpd3/hdac1 mutations on Fab-7-dependent silencing. Ectopic Fab-7 constructs are known to mediate silencing of flanking reporter genes both in cis, as in heterozygous GCD6 flies, as well as in trans, as in 5F24 flies, where silencing is pairing-sensitive being observed only when the transgene is in a homozygous state. This study shows that Trl, dsap18 and drpd3/hdac1 mutations affect both cis- and trans-silencing mediated by Fab-7 (Canudas, 2005).
  3. the homeotic A6-to-A5 transformation observed in flies heterozygous for various Trl, dsap18 and drpd3/hdac1 mutant alleles and hemizygous for Df(3R)sbd45, which uncovers dsap18. This homeotic transformation results from the ectopic repression of the iab-6 enhancer at A6 that is insulated from the repressed iab-7 enhancer by the Fab-7 element. The fact that this homeotic transformation is very infrequent in hemizygous Df(3R)sbd45 flies, as well as in the heterozygous mutants, demonstrates that it is directly associated to the Trl, dsap18 and drpd3/hdac1 mutations. Moreover, a single copy of a transgene expressing dsap18 significantly rescues this phenotype. The results also indicate that an unidentified element(s) contained within Df(3R)sbd45 is also contributing to the establishment of the phenotype. In addition to sap18, Df(3R)sbd45 uncovers at least 11 other genes including the trithorax gene, taranis. However, the homeotic transformation described in this study does not appear to be associated to a loss of taranis function since no transformation is observed in flies trans-heterozygous for a null taranis allele and Trl, dsap18 or drpd3/hdac1 mutations (Canudas, 2005).

Together, these results indicate a contribution of GAGA, dSAP18 and dRPD3/HDAC1 to the structural and functional properties of Fab-7. What could this contribution be? Several models might account for these results. Fab-7 is known to contain two functional elements: a PRE, which is required for Pc-dependent silencing, and an adjacent boundary element that insulates iab-6 from iab-7. The finding that, in heterozygous GCD6 flies, mutant Trl, dsap18 and drpd3/hdac1 alleles enhance cis-silencing imposed by Fab-7 suggests that their functions might antagonize Pc-dependent silencing. Several observations, however, make this hypothesis unlikely: (1) at some PREs, GAGA helps recruitment of PcG complexes and contributes to silencing; (2) dRPD3/HDAC1 was shown to be a component of several PcG complexes, and genetic analysis indicates a contribution to homeotic silencing; (3) in mammals, SAP18 acts as a repressor when targeted to an active promoter (Canudas, 2005).

An alternative possibility is that GAGA, dSAP18 and dRPD3/HDAC1 contribute to the function of the Fab-7 boundary element. In fact, the Fab-7 boundary contains several GAGA-binding sites that are required for its enhancer blocking activity and, it is hypoacetylated at histones H3 and H4. In GCD-6 flies, the Fab-7 boundary element is located proximal to the reporter mini-white gene with respect to the PRE so that it might help to insulate the reporter gene from repression by the PRE. In this context, mutations that affect boundary function would result in a less efficient insulation and, therefore, would enhance silencing (Canudas, 2005).

In contrast to the enhancer effect observed in heterozygous GCD6 flies, mutations in Trl, dsap18 and drpd3/hdac1 suppress pairing-dependent trans-silencing in transgenic 5F24(25,2) flies. A contribution to boundary-functions might also account for this effect. Pairing-sensitive trans-silencing results from long-distance chromosomal interactions that involve the association of the transgenes with each other and with the endogenous Fab-7 element, even when located in different chromosomes. These long-distance interactions that require the contribution of PcG proteins might be facilitated by a functional boundary element as has been described for the gypsy insulator (Canudas, 2005).

The incomplete A6-to-A5 homeotic transformation observed in the presence of Trl, dsap18 and drpd3/hdac1 mutations might also reflect a contribution to the boundary function of Fab-7 as, in the mutant conditions, it might not properly insulate the iab-6 enhancer from the repressing activity of the Fab-7 PRE, thereby becoming partially inactivated. Interestingly, mutations that delete the Fab-7 boundary but not the PRE produce, in addition to strong A6-to-A7 transformation, incomplete A6-to-A5 transformation. Moreover, replacement of the Fab-7 boundary by the gypsy or the scs insulator (both of which are not functional in the context of BX-C) results in complete A6-to-A5 transformation (Canudas, 2005).

The results indicate that GAGA, dSAP18 and dRPD3/HDAC1 have similar effects on the functional properties of Fab-7 suggesting a functional link. A physical interaction between GAGA and dSAP18 has been reported. Moreover, in mammals, SAP18 is associated with the Sin3-HDAC co-repressor complex and, in Drosophila, dSAP18 modulates bicoid activity through the recruitment of dRPD3/HDAC1 and it is required to suppress bicoid activity in the anterior tip of the embryo. In this context, it is tempting to speculate that GAGA helps in the recruitment of dSAP18 and dRPD3/HDAC1 to Fab-7 resulting in a concerted contribution to its boundary function (Canudas, 2005).

In mammals, SAP18 is also associated with ASAP, a protein complex involved in RNA processing. In Drosophila, dSAP18 may also participate in RNA processing; in cultured S2 cells, a large proportion of dSAP18 co-immunoprecipitates with factors that participate in RNA processing. It is possible that, in response to cellular signals, the association of dSAP18 to different protein complexes would be regulated during development and/or cell cycle progression (Canudas, 2005).

The Mcp element mediates stable long-range chromosome-chromosome interactions in Drosophila

Chromosome organization inside the nucleus is not random but rather is determined by a variety of factors, including interactions between chromosomes and nuclear components such as the nuclear envelope or nuclear matrix. Such interactions may be critical for proper nuclear organization, chromosome partitioning during cell division, and gene regulation. An important, but poorly documented subset, includes interactions between specific chromosomal regions. Interactions of this type are thought to be involved in long-range promoter regulation by distant enhancers or locus control regions and may underlie phenomena such as transvection. Here, an in vivo microscopy assay was used, based on Lac Repressor/operator recognition to show that Mcp, a polycomb response element from the Drosophila bithorax complex, is able to mediate physical interaction between remote chromosomal regions. These interactions are tissue specific, can take place between multiple Mcp elements, and seem to be stable once established. It is speculated that this ability to interact may be part of the mechanism through which Mcp mediates its regulatory function in the bithorax complex (Vazquez, 2006).

Mcp is located between the iab-4 regulatory region that directs expression of abd-A in parasegment 9, and iab-5, that directs expression of Abd-B in parasegment 10. Deletions of Mcp cause ectopic activation of Abd-B in PS9, leading to the hypothesis that this element could function either as a silencer or as a boundary element located between and functionally separating iab-4 and iab-5. When present on a transgene, a 2.8-kb fragment containing Mcp was shown to mediate pairing-dependent silencing of a linked mini-white gene. The term pairing-dependent silencing describes a phenomenon in which the eye color of homozygous flies (containing two copies of the transgene) is lighter than the eye color observed in heterozygous flies. In general, the activity of the mini-white reporter gene is dosage dependent. Hence, eye pigmentation normally increases with the number of mini-white transgenes in the genome. Pairing-dependent silencing is position dependent and its strength is variable. Similar silencing effects could also be observed when the two P[Mcp, mini-white] copies were inserted at different locations in the genome or when they were present on rearranged chromosomes. These genetic interactions suggest that two copies of the Mcp element could physically interact, independently of homologous chromosome pairing (Vazquez, 2006 and references therein).

A variety of genetic phenomena are thought to rely on the physical interaction or communication between distant chromosomal elements. Examples include modulation of promoter activity by remote regulatory elements, homology search during DNA recombination and repair, and pairing-dependent phenomena such as transvection. For example, it has been proposed that developmentally regulated transcription at the human betaglobin locus relies on dynamic, short-lived interactions between promoter elements at the globin locus and a distant locus control region. More recently, long-range associations, both intra- and inter-chromosomal, were demonstrated in human T-helper cells (Vazquez, 2006).

Mcp is able to interact with other copies of the same element present at remote locations in the genome. After the direct demonstration of pairing of the Fab-7 PRE, this is the second example of a discrete chromosomal region able to mediate sequence-specific, long-range chromosomal interactions in the Drosophila nucleus (Vazquez, 2006).

The frequency of association of the Mpc construct in the eye disc was very high; it was observed in ~90% of nuclei. The frequency of association was substantially higher than that observed by in situ hybridization for Fab-7. It could be argued that the conditions used for in situ hybridization might disrupt potentially fragile interactions. However the fixation procedures generally used for in situ hybridization have now been shown not to significantly affect the frequency of paired sites in the eye disc, compared with the in vivo method. The differences, therefore, may reflect variable strengths of different pairing-sensitive elements, or stage or tissue-specific effects. Indeed, although pairing of the Mcp element was observed in other larval tissues such as brain and wing discs, the frequency of pairing in such tissues was often much lower (20%-60% of that observed in the eye disc). Other tissues, such as polytene nuclei, showed virtually no pairing. One possible explanation is that tissue-specific factors present in the eye disc might contribute to the pairing. Because the white gene present on the constructs is expressed in the eye, it is possible that white sequences might act in conjunction with Mcp to increase the level of association of the constructs in the eye disc. It is also possible that the embryonic stages analyzed in the Fab-7 studies may represent the early stages in the establishment of this type of long-range interactions. In agreement with the work on Fab-7, however, no evidence was found of pairing of the Mcp element in the male or female germ line (Vazquez, 2006).

The eye color assay for long-distance interactions showed that insertions located on the same chromosome are much more likely to show genetic interaction (as evidenced by the stronger silencing of white). The assay, however, revealed similar (and high) levels of association between sequences located on different chromosomes. These results are consistent with previous studies, where a substantial amount of residual pairing between alleles of the bithorax complex was still observed for translocations that abolished transvection. The assay also revealed that the long-range association may involve at least up to four elements located at three different chromosomal loci. Although not tested in this study, such interactions are also likely to involve the endogenous Mcp elements. This raises the possibility that Mcp and similar elements may be involved in the formation of higher order chromatin complexes comprising multiple genes or regulatory regions (Vazquez, 2006).

Previous studies have identified a mutation in grappa (gpp1A) that substantially suppresses the pairing-dependent silencing of white mediated by Mcp. grappa encodes the Drosophila homologue of the yeast Histone H3 methyltransferase Dot1p. The results clearly show that although gpp1A drastically reduces the level of pairing-dependent silencing mediated by Mcp, it has little or no effect on the observed pairing of Mcp elements in the eye imaginal disc. This suggests that pairing may be an initial necessary step in the regulatory process mediated by Mcp and that grappa acts subsequently to induce chromatin changes required for silencing. In the absence of additional data, however, other possibilities cannot be excluded. For example, the timing of pairing could be critical to allow developmentally regulated factors to associate to, and repress transcription around the Mcp element. In such a model, gpp1A could be delaying the onset of pairing, resulting in reduced levels of silencing. Additional studies will be necessary to establish the series of events that lead to pairing-dependent silencing of Mcp-associated genes (Vazquez, 2006).

The use of a live system has also allowed addressing of the dynamics of long-distance chromosome-chromosome interaction. Once established, the interactions seem to be stable, because no evidence of separation of initially paired loci was seen. Due to the finite resolution of the light microscope, this does not exclude local transient separation of short DNA regions. However, given that chromatin is naturally subject to diffusive motion, a complete separation of the paired regions, even for a brief moment, would be expected to lead to a drifting away of the tagged regions and the appearance of two separate GFP spots. Rare, unpaired loci were also never seen to associate. The presence of a small fraction of nuclei with unpaired loci at any given time therefore does not seem to be the result of an equilibrium state between a population of rapidly associating and dissociating loci. Therefore, Mcp elements, possibly by the action of specific chromosome-associated proteins, are able to lock remote chromosomal regions in the paired state for extended periods, even in the presence of substantial chromatin movement. The stable contacts described are in contrast to the short-lived dynamic interactions that have been postulated to occur between remote regulatory elements, such as between the human betaglobin LCR and promoter regions. Studies suggest that the rate-limiting step in the pairing process could be the establishment of the initial contact between remote Mcp elements early during development and possibly renewed early at the beginning of each new cell cycle. This situation is reminiscent of the rapid and stable pairing of homologous chromosomes observed in somatic cells and of meiotic pairing in Drosophila spermatocytes. This interpretation is consistent with the hypothesis that interactions between Polycomb-group response elements might be involved in the transmission of chromatin states during Drosophila development (Vazquez, 2006).

This study has presented a live system for the direct analysis of long-distance chromosome interactions in Drosophila. This system allowed identification of a discrete DNA sequence from the bithorax complex, Mcp, that is able to promote stable physical interactions between distant chromosomal regions. The presence of pairing elements at the bithorax complex had long been suspected, due to the susceptibility of this locus to transvection effects. Although the pairing properties of Mcp (and Fab-7) were originally inferred from the ability of this element to silence a linked white gene in a pairing-dependent manner, it is not clear at the moment what function pairing serves in the context of the bithorax complex. It has been proposed that association between these elements might play a role in the transmission of regulatory chromatin states. It is also possible that pairing elements might play a role in bringing together remote regulatory regions or stabilize regulatory interactions within the complex. The ability to track such associations both in live and fixed tissues should help clarify the relationship between chromosome organization and gene regulation (Vazquez, 2006).

RNAi components are required for nuclear clustering of Polycomb group response elements

Drosophila Polycomb group (PcG) proteins silence homeotic genes through binding to Polycomb group response elements (PREs). Fab-7 is a PRE-containing regulatory element from the homeotic gene Abdominal-B. When present in multiple copies in the genome, Fab-7 can induce long-distance gene contacts that enhance PcG-dependent silencing. Components of the RNA interference (RNAi) machinery are involved in PcG-mediated silencing at Fab-7 and in the production of small RNAs at transgenic Fab-7 copies. In general, these mutations do not affect the recruitment of PcG components, but they are specifically required for the maintenance of long-range contacts between Fab-7 copies. Dicer-2, PIWI, and Argonaute1, three RNAi components, frequently colocalize with PcG bodies, and their mutation significantly reduces the frequency of PcG-dependent chromosomal associations of endogenous homeotic genes. This suggests a novel role for the RNAi machinery in regulating the nuclear organization of PcG chromatin targets (Grimaud, 2006).

The RNAi machinery has been implicated in a wide variety of biological processes. One of these processes is the formation of heterochromatin. In S. pombe, this involves bidirectional transcription of RNA molecules from repetitive sequences and their cleavage into short interfering RNAs (siRNAs) of 21–23 nt by an RNase III enzyme called Dicer-1. siRNAs guide the RNA-induced initiation of transcriptional gene silencing (RITS) complex to homologous sequences in the nucleus (Noma, 2004; Verdel, 2004). Clr4, the homolog of the histone methyltransferase Su(Var) 3-9, is recruited along with the RITS complex to chromatin, where it methylates lysine 9 of histone H3 (H3K9). This epigenetic mark promotes the formation of heterochromatin by recruiting the heterochromatin protein Swi6, the homolog of HP1, via its chromodomain (Grewal, 2004). Consistent with these data, a redistribution of H3K9 methylation has been observed in Drosophila chromosomes in flies mutant for components of the RNAi machinery (Pal-Bhadra, 2004; Grimaud, 2006).

The RNAi machinery is also required for cosuppression, a phenomenon whereby the introduction of multiple transgenic copies of a gene phenocopies its loss of function instead of increasing its expression. In Drosophila, cosuppression can act at either the transcriptional or posttranscriptional level and involves PcG proteins as well as the RNAi machinery (Pal-Bhadra, 1997, Pal-Bhadra, 1999 and Pal-Bhadra, 2002; Grimaud, 2006 and references therein).

The Drosophila RNAi machinery includes two Dicer proteins encoded by the dicer-1 (dcr-1) and dicer-2 (dcr-2) genes. Dcr-2 is specifically required to process double-stranded RNAs into siRNAs and mediates the assembly of siRNAs into the RNA-induced silencing complex (RISC). Dcr-1 is involved in the metabolism of siRNAs as well as the processing of pre-microRNAs into microRNAs. RNA silencing also involves several highly conserved genes coding for PAZ-domain proteins. Argonaute1 (AGO1) and Argonaute2 (AGO2) are involved in microRNA biogenesis and RNA interference (RNAi). piwi is involved in cosuppression, silencing of retrotransposons, and heterochromatin formation. aubergine (aub) was first isolated based on its role in germline development but is also responsible for maintaining the silenced state of an X-linked male fertility gene locus (Stellate) via RNAi. The Aub protein is required for RNAi and RISC assembly in ovaries. In addition, homeless/spindle-E (hls) is involved in silencing of Stellate and in heterochromatin formation. This study tested whether RNAi components are involved in the PcG pathway. The results show that the RNAi machinery affects the PcG response via a novel regulatory function in nuclear organization (Grimaud, 2006).

The role of a variety of RNAi components in a specific transgenic line called Fab-X was tested. This line contains a construct carrying a 3.6 kb fragment from the Fab-7 region, cloned upstream of a mini-white reporter and inserted into the X chromosome. In the Fab-X line, the presence of the Fab-7 sequence is sufficient to induce PcG-dependent silencing, both of the mini-white eye-color reporter gene and of the endogenous scalloped (sd) gene, which is required for wing-blade morphogenesis and is located 18.4 kb downstream of Fab-7. These two repressed phenotypes are abolished in the presence of mutations in PcG genes and are not present in heterozygous females and hemizygous males, indicating that both mini-white and sd expression are subject to PSS (Grimaud, 2006).

The eye-color and wing phenotypes were used as a basis to analyze the effect of the RNAi machinery on PcG-dependent repression. Mutations in RNAi components were introduced into the Fab-X line and placed over a balancer chromosome containing a GFP marker. As the AGO1 mutant alleles involve P element insertions containing the mini-white reporter gene, they could not be tested using the eye phenotype. A null mutation in dcr-2 (dcr-2L811fsX) decreased silencing of the mini-white reporter gene relative to the Fab-X line when in the homozygous state. Likewise, two different mutant alleles of piwi (piwi1 and piwi2) decreased mini-white silencing, with the effect being more pronounced in piwi2 mutant flies. This effect was not restricted to the Fab-X line since it was also observed when piwi2 was recombined into another Fab-7-containing line. In contrast to the effects seen for dcr-2 and piwi alleles, Fab-X females homozygous mutant for hlsE1 or that carried the heteroallelic hlsE1/hlsE616 combination silenced mini-white like wt Fab-X females (Grimaud, 2006).

The sd phenotype was then analyzed in all mutant backgrounds at 28.5°C, a temperature inducing a strong wing phenotype in Fab-X. A preselection of non-GFP female larvae was carried out in order to selectively analyze homozygous or trans-heterozygous mutant adults. This analysis revealed that mutating any of the components of the RNAi machinery, except for hls and the heterozygous dcr-1 mutation, leads to a strong decrease in the sd phenotype. These data show that the RNAi machinery can affect PcG-mediated silencing. The fact that hls mutants had no effect suggests that this process might be mechanistically distinct from the role of RNAi components in heterochromatin formation (Grimaud, 2006).

While most RNAi components are not required for binding of PcG proteins to PREs, they are required to mediate long-range contacts between multiple copies of the Fab-7 element. Moreover, Dcr-2, PIWI, and AGO1 colocalize with PH in the cell nucleus, and their effect correlates with the presence of small RNAs homologous to Fab-7 sequences. Finally, in addition to their effects on transgenic Fab-7 copies, mutations in these genes also reduce the frequency of long-distance contacts between endogenous PcG target genes. Taken together, these results reveal a novel and unexpected role for the RNAi machinery in the regulation of euchromatic genes in the nuclear space (Grimaud, 2006).

The effects caused by mutations in different RNAi components suggest the existence of distinct molecular roles for these proteins in the regulation of PcG function. First, the hls gene does not seem to play a major role in silencing at the Fab-7 PRE or maintaining long-distance Fab-7 contacts. Since Hls has been shown to play a central role in heterochromatin formation (Pal-Bhadra, 2004), there may be different subtypes of nuclear RNAi machineries for heterochromatin formation and for regulating PcG function. No effect was found when a mutation in the dcr-1 gene was analyzed at the heterozygous state, but the elucidation of the function of dcr-1 in PcG-mediated silencing awaits further analysis in a homozygous mutant background. A second class of RNAi components that participate in PcG-mediated repression contains dcr-2, AGO1, and the aub gene. Loss of any of these RNAi gene products affects PcG-dependent silencing at Fab-7, although it does not impact the binding of PcG proteins to Fab-7. Only mutations in piwi affected the binding of PcG proteins to Fab-7, at least in polytene chromosomes, but even PIWI did not affect recruitment of PcG factors at endogenous genes. In S. pombe, both RNAi components as well as DNA binding proteins are involved in recruiting heterochromatin proteins to the mating-type region (Jia, 2004). At PREs, multiple DNA binding factors and chromatin-associated proteins are known to contribute to PcG protein recruitment, such as PHO, the GAGA factor, DSP1, and the CtBP proteins. Their combinatorial action might play a key role in the robust and specific chromatin tethering of PcG proteins, while, in contrast to the situation in S. pombe, the RNAi machinery might play a relatively minor role (Grimaud, 2006).

It is interesting to note that piwi mutations affected recruitment of E(Z) and PC to Fab-7 in polytene chromosomes but had no effects on PH, another PRC1 component. In the current model for recruitment of PcG proteins to PREs, histone H3 methylation by the E(Z) protein recruits PRC1 via the chromodomain of PC. The current results indicate that multiple mechanisms might be used to anchor different PRC1 components to PREs and that the loss of PC does not necessarily lead to the disintegration of the entire PRC1 complex at PREs (Grimaud, 2006).

To date, all transcriptional gene-silencing phenomena that depend on the RNAi machinery involve the production of small-RNA molecules. RNAi components were also shown to affect telomere clustering in S. pombe, although binding of Swi6 and H3K9me to individual telomeres is not affected. The production of siRNAs is believed to be essential for the nuclear clustering of telomeres since cells carrying a catalytically dead RNA-dependent RNA polymerase (which abolishes siRNA production) are defective in telomere clustering. Consistent with a role for small RNAs in mediating gene contacts, sense and antisense transcription of Fab-7 as well as small-RNA species were found in Fab-7 transgenic lines. Moreover, a mutant allele of dcr-2 producing a truncated polypeptide lacking the RNase III domain, which is required for dsRNA processing, is defective in long-range interactions of PcG target sequences as well as in accumulation of Fab-7 small RNAs. These data suggest that small-RNA species could be involved in these gene contacts. However, no small Fab-7 RNA species was detected in the wt situation, although RNAi mutants affect the contact of the endogenous Fab-7 locus with the Antp gene. This might indicate that other RNA species produced in the endogenous Hox genes could contribute to gene clustering. However, the possibility remains that RNA-independent functions of RNAi proteins contribute to the maintenance of gene contacts, in particular in the case of endogenous PcG target genes (Grimaud, 2006).

Interestingly, none of the RNAi mutants tested are defective in the establishment of long-distance chromosomal interactions. Fab-7 contacts are correctly established during embryogenesis but decay during later stages of development. This suggests that the RNAi machinery is not required to initiate contacts but rather to maintain them via the stabilization of gene clustering at specific nuclear bodies. This clustering could be important in cosuppression, where transgene silencing can occur at the transcriptional and posttranscriptional levels, both requiring the RNAi machinery (Pal-Bhadra, 2002). It is difficult, however, to understand how a relatively modest increase in transcript levels caused by an increase in the copy number of a gene could trigger a robust silencing of all copies. This is particularly puzzling considering that the transcript levels of endogenous single-copy genes can vary, e.g., during normal physiological gene regulatory processes, without triggering gene silencing. One explanation might be that cosuppressed genes are clustered in the cell nucleus. Indeed, clustering of multiple gene copies has been reported in plant cells (Grimaud, 2006).

It is proposed that the RNAi machinery, perhaps in conjunction with PcG proteins, might stabilize this gene-clustering phenomenon. Specifically, the colocalization of multiple gene copies with components of the RNAi machinery might increase the local concentration of RNA species. Once this concentration overcomes a critical threshold, double-stranded RNAs might assemble and be cleaved in situ by the enzymatic activity of the RNAi machinery. RNA molecules might contribute to hold together loci containing PcG proteins that produce noncoding transcripts encompassing PREs. This gene clustering might involve contacts with components of the RNAi machinery as well as PcG proteins assembled in the same nuclear compartments (Grimaud, 2006).

One important question is, what is the role of the RNAi machinery in the regulation of endogenous PcG target genes? The data indicate that RNAi components affect only a subset of these genes since the colocalization of PcG bodies with RNAi bodies is limited. Hox loci are characterized by extensive noncoding RNA transcription, and, recently, other PcG target genes have been shown to be associated to intergenic transcription. RNAi components might be targeted to this subset of PcG target genes, while other PcG target genes that are characterized by the absence of noncoding transcripts might be independent on RNAi factors (Grimaud, 2006).

The fact that no homeotic phenotypes are visible in RNAi mutant backgrounds suggests that the function of RNAi components can be rescued by other chromatin factors. Indeed, the decrease in the level of nuclear interaction between the homeotic complexes was incomplete in RNAi mutant backgrounds. The data suggest that, while the RNAi machinery does not act in the establishment of PcG-dependent gene silencing, RNAi factors might help stabilize silencing during development by clustering PcG target genes at RNAi nuclear bodies. Thus, in addition to its role in defending the genome against viruses, transposons, and gene duplications, the RNAi machinery might participate in fine tuning the expression of PcG target genes through the regulation of nuclear organization. Finally, it must be noted that the developmental expression profile of the components of the RNAi machinery is highly specific. The function of specific RNAi components is therefore likely to be highly variable in different cell types and as a function of time. It will be of great interest to explore this issue in the developmental context of the whole organism, in Drosophila as well as in other species (Grimaud, 2006).

The abdominal-B promoter tethering element mediates promoter-enhancer specificity at the Drosophila bithorax complex

At the Drosophila bithorax complex many distinct classes of cis-regulatory modules work collectively during development to control gene expression. Abdominal-B (Abd-B) is one of three homeotic genes in the BX-C and is expressed in specific presumptive abdominal segments in the embryo. The transcription of Abd-B is tightly controlled by an array of cis-regulatory modules that direct its expression over extended genomic distances. These regulatory modules include promoters, insulators, silencers, enhancers, promoter targeting sequences and the recently identified promoter tethering element (PTE). To activate gene expression at the endogenous complex, enhancers located >50 kb away must bypass intervening insulators to interact with the Abd-B promoter. The molecular mechanisms that allow enhancers to bypass insulators are not currently well understood. In this article, a novel mechanism is reported for insulator bypass involving the PTE. In addition, bioinformatic analysis across twelve Drosophila genomes was used to identify putative cis-regulatory sequences that may be capable of facilitating specific promoter-enhancer interactions at the bithorax complex, and a is proposed model for their molecular function during development (Akbari, 2007). It is possible that the conserved TGGT(T/C)(C/T) motif, detected in the PTE using cis-Decoder analysis, represents a protein factor-binding site. A model is proposed in which proteins bind the clustered motif in the PTE and a cluster in the Abd-B 3' regulatory region. Interactions between these factors may facilitate the formation of a chromatin loop, which mediates specific promoter-enhancer interactions. At the endogenous BX-C, there are several known enhancers capable of directing expression of Abd-B, but only one enhancer is active in a given spatial region of the developing embryo. The model is therefore that the tethering of a specific enhancer to the Abd-B promoter serves two critical functions: (1) it prevents promiscuous enhancer activity by physically restricting interactions at the PTE to a single enhancer and (2) it ensures that as the genomic DNA of the BX-C is subjected to rearrangement over evolutionary time, enhancers can still function properly even if they become distally located relative to their target promoter. This model is consistent with data from the Antennapedia complex (ANT-C), which contains the only other cluster of Hox genes in Drosophila melanogaster. In the ANT-C, the T1 enhancer preferentially activates the distal Scr gene over the proximal ftz gene. This activity is dependent on a 450bp tethering element located upstream of the Scr promoter, which recruits the T1 enhancer. Moreover, this tethering element contains a cluster of the hexamer motif TTCGAA, and a second cluster is also found near the T1 enhancer. This led to speculation that proteins binding to the clustered motif facilitate the formation of a chromatin loop. It is believed, therefore, that the molecular bridge mediated by promoter tethering elements represents a general transcription mechanism in the regulation of Hox genes. In order to more fully address this issue it will be critically important to investigate the functional activity of the PTE in the context of the endogenous BX-C (Akbari, 2007).

Functional interaction between the Fab-7 and Fab-8 boundaries and the upstream promoter region in the Drosophila Abd-B gene

Boundary elements have been found in the regulatory region of the Drosophila Abdominal-B gene, which is subdivided into a series of iab domains. The best-studied Fab-7 and Fab-8 boundaries flank the iab-7 enhancer and isolate it from the four promoters regulating Abd-B expression. Recently binding sites for the Drosophila homolog of the vertebrate insulator protein CTCF (dCTCF) were identified in the Fab-8 boundary and upstream of Abd-B promoter A, with no binding of CTCF to the Fab-7 boundary being detected either in vivo or in vitro. Taking into account the inability of the yeast GAL4 activator to stimulate the white promoter when its binding sites are separated by a 5-kb yellow gene, a study was performed of the functional interactions between the Fab-7 and Fab-8 boundaries and between these boundaries and the upstream promoter A region containing a dCTCF binding site. It was found that dCTCF binding sites are essential for pairing between two Fab-8 insulators. However, a strong functional interaction between the Fab-7 and Fab-8 boundaries suggests that additional, as yet unidentified proteins are involved in long-distance interactions between them. Fab-7 and Fab-8 boundaries effectively interact with the upstream region of the Abd-B promoter (Kyrchanova, 2008).

Previously it was found that the relative orientation of Mcp elements defines the mode of loop formation that either allows or blocks stimulation of the white promoter by the GAL4 activator. This study has demonstrated that two PTS/F8 boundaries or Fab-8 insulators alone are also capable of orientation-dependent interaction. When these elements are located in opposite orientations, the loop configuration is favorable for communication between regulatory elements located beyond the loop. The loop formed by two insulators located in the same orientation juxtaposes two elements located within and beyond the loop, which leads to partial isolation of the GAL4 binding sites and the white promoter placed on the opposite sides of the insulators (Kyrchanova, 2008).

The orientation-dependent interaction may be accounted for by at least two proteins bound to the insulator that are involved in specific protein-protein interactions. In the case of a Fab-8 insulator, dCTCF is likely to be directly involved in pairing between two insulators. Since mutated Fab-8 insulators devoid of dCTCF binding sites proved to be incapable of interacting with each other, it is hypothesized that dCTCF facilitates the binding of a certain as yet unidentified protein (or proteins) that, in combination with dCTCF, accounts for orientation-dependent interaction between the Fab-8 insulators. Functional interactions between the Fab-7 boundary devoid of dCTCF binding sites and PTS/F8 or the upstream Abd-B A promoter region are also evidence for the existence of unidentified proteins that support organization of distance interactions in the Abd-B locus (Kyrchanova, 2008).

Recently it was shown that in the repressed state of the bithorax complex, all of its major regulatory elements binding PcG proteins, including PREs with adjacent boundaries and core promoters, interact at a distance, giving rise to a topologically complex structure (Lanzuolo, 2007). The question arises as to what proteins are important for such interactions. All PREs tested (Lanzuolo, 2007) were flanked by boundaries, suggesting that all these regulatory elements may be involved in long-distance interactions. As shown previously, the Fab-7 or Mcp boundaries including PREs can support physical association between even transposons located on different chromosomes. One of relevant models proposes that PcG proteins are capable of supporting highly specific long-distance interactions between transposons (Lanzuolo, 2007). However, it is known that many PcG complexes with similar properties can bind to Drosophila chromosomes, which leaves open the question as to how such protein complexes can ensure a high specificity of interactions between distantly located transposons. Moreover, there is no experimental evidence that PREs without additional regulatory elements can support long-distance interactions. In contrast, there are many proven cases showing that insulator proteins are involved in physical association between distant chromosomal regions. For example, the interaction between gypsy insulators can support activation of the yellow promoter by enhancers separated by many megabases. The Mod(mdg4)-67.2 and Su(Hw) proteins bound to the gypsy insulator are essential for such long-distance interactions. In mammals, the interaction of the imprinting control region on chromosome 7 with the Wsb1/Nf1 locus on chromosome 11 depends on the presence of the CTCF protein. In vivo interaction between Fab-7 and the Abd-B promoter is absolutely dependent on the presence of the Fab-7 insulator. Finally, this study has demonstrated the functional interaction between the Fab-7 and Fab-8 boundaries and the Abd-B promoter. These results support the model that transcriptional factors bound to boundaries can facilitate enhancer-promoter interactions in the bithorax complex. Further studies are necessary for identifying new proteins involved in long-distance interactions and for elucidating the mechanisms that allow interactions either between proper active enhancers and promoters or between only silenced enhancers and promoters (Lanzuolo, 2008).

Stalled Hox promoters as chromosomal boundaries

Many developmental control genes contain stalled RNA Polymerase II (Pol II) in the early Drosophila embryo, including four of the eight Hox genes. Evidence is presented that the stalled Hox promoters possess an intrinsic insulator activity. The enhancer-blocking activities of these promoters are dependent on general transcription factors that inhibit Pol II elongation, including components of the DSIF (Spt4, and Spt5) and NELF complexes. The activities of conventional insulators are also impaired in embryos containing reduced levels of DSIF and NELF. Thus, promoter-proximal stalling factors might help promote insulator-promoter interactions. It is proposed that stalled promoters help organize gene complexes within chromosomal loop domains (Chopra, 2009b).

Hox genes are responsible for the anterior-posterior patterning of most metazoan embryos. They are typically organized in gene complexes containing a series of cis-regulatory DNAs, including enhancers, silencers, and insulator DNAs . In Drosophila, the eight Hox genes are contained within two gene complexes: the Antennapedia complex (ANT-C), which controls the patterning of anterior regions, and the Bithorax complex (BX-C), which controls posterior regions. The proper spatiotemporal transcription of Hox genes is achieved by the coordinated action of linked cis-regulatory DNAs that are organized in a colinear fashion across the ANT-C and BX-C complexes (Chopra, 2009b).

Chromosomal boundary elements, or insulators, are essential for the orderly regulation of Hox gene expression. They are thought to ensure proper cis-regulatory 'trafficking,' whereby the correct enhancers interact with the appropriate target promoters. Insulators might also help control the levels of transcription by attenuating enhancer-promoter interactions. Insulators are sometimes associated with promoter targeting sequences (PTS), which can facilitate enhancer-promoter interactions by modulating the activities of neighboring insulators (Chopra, 2009b).

Recently, long-range cis-regulatory interactions have been mapped in Drosophila Hox complexes using the DamID technique, chromosomal conformation capture (3C) assays, and transgenic approaches. These studies suggest that the Fab7 and Fab8 insulators are associated with the Abd-B promoter under repressed conditions, even though they map >30-50 kb downstream from the promoter. These long-range interactions depend on the CTCF boundary-binding protein, thereby raising the possibility that insulators interact with one another and organize Abd-B cis-regulatory DNAs within chromosomal loop domains. Similarly, the prototypic insulators flanking the heat-shock puff locus, scs and scs', have also been shown to interact with one another. Additional insulator-insulator loops have also been documented. These loops are thought to facilitate the interactions of remote enhancers and silencers with appropriate target promoters. This study presents evidence that Hox promoters with stalled RNA Polymerase II (Pol II) possess an intrinsic insulator activity, which might help foster the formation of insulator-promoter chromosomal loop domains (Chopra, 2009b).

Four of the eight Hox genes contained in the ANT-C and BX-C contain stalled Pol II. Interestingly, all four stalled genes map at the boundaries of the two Hox complexes. In contrast, internal Hox genes (pb, Dfd, and Scr within the ANT-C, and abd-A within the BX-C) lack stalled Pol II. This arrangement of stalled Hox genes raises the possibility that stalling contributes to the chromosomal organization of Hox complexes. All four stalled Hox genes (lab, Antp, Ubx, and Abd-B) were tested for enhancer-blocking activity in transgenic embryos, along with the promoter regions of two nonstalled genes (Scr and abd-A). Test promoters were placed 5' of lacZ and inserted between a divergent white reporter gene and 3' iab-5 enhancer (IAB5) (Chopra, 2009b).

IAB5 regulates Abd-B expression in posterior regions of the early embryo, corresponding to the primordia for parasegments 10-14. IAB5 is a robust enhancer, and can activate lacZ and white even when positioned far from the reporter genes. This assay was used to reveal an intrinsic enhancer-blocking activity of the eve promoter region. eve/lacZ fusion genes block the ability of IAB5 to activate a distal CAT reporter gene. However, mutagenized eve promoter sequences lacking a critical proximal GAGA element failed to block IAB5-white interactions. Similarly, the Abd-B proximal promoter (Abd-Bm) and Ubx promoter regions block activation of distal white expression, whereas the abd-A promoter does not interfere with the activation of white expression in the presumptive abdomen by the IAB5 enhancer (Chopra, 2009b).

These results suggest that the stalled Abd-B proximal promoter and Ubx promoters possess an enhancer-blocking activity, whereas abd-A does not. A similar trend was observed for Hox promoter sequences from the ANT-C. The Antp and lab promoters block IAB5-white interactions, whereas the Scr promoter (which lacks stalled Pol II) does not interfere with the activation of white expression in the presumptive abdomen. Stalled genes from the tinman complex (Tin-C), which encode NK homeobox proteins responsible for patterning mesodermal lineages, were also examined. All of the stalled promoters from the Tin-C contain insulator activities. In contrast, nonstalled promoters from lbl and C15 lack such activities when tested in similar transgenic assays. Even the Hsp70 promoter, the classic example of Pol II pausing, displayed insulator activity when tested in similar enhancer-blocking transgenic assays (Chopra, 2009b).

The preceding experiments suggest that stalled Hox gene promoters contain enhancer-blocking activities. However, an alternative possibility is that stalled promoters are 'stronger' than the white promoter, and are able to sequester the shared IAB5 enhancer. To distinguish between competition and insulator activities, the IAB5 enhancer was placed between the divergently transcribed white and lacZ reporter genes. When the white promoter sequence was placed 5' of the lacZ reporter gene, the shared IAB5 enhancer worked equally well to activate both white and lacZ expression. Similar results were obtained when the leftward lacZ reporter gene was placed under the control of either the stalled Abd-B or Ubx promoters. In all of these cases, both white and lacZ are expressed equally well in the presumptive abdomen. These results suggest that stalled promoters do not block enhancer-promoter interactions by a competition mechanism. Rather, they work like insulators and block such interactions only when positioned between the distal enhancer and target promoter (Chopra, 2009b).

To determine whether stalled Pol II is important for the enhancer-blocking activities of Ubx and Abd-B, mutant embryos were examined with reduced levels of critical Pol II elongation factors. Ubx and Abd-B were selected for further studies since optimal expression of both genes depends on the Pol II elongation factors Cdk9 (pTEFb) and Elo-A (Chopra, 2009a). It was reasoned that destabilization of stalled Pol II might reduce the enhancer-blocking activities of the Ubx and Abd-B promoter regions. However, reductions in Cdk9 and Elo-A are expected to stabilize, not destabilize, Pol II stalling since both are positive factors that promote elongation (Saunders, 2006). Indeed, reductions in Cdk9 or Elo-A activity do not alter the enhancer-blocking activities of the Ubx and Abd-B promoters (Chopra, 2009b).

To investigate the link between Pol II stalling and enhancer blocking, two negative elongation factors were examined: NELF (Lee, 2008) and DSIF (Wada, 1998; Yamaguchi, 1998; Kaplan, 2000). The NELF-E protein binds to the short nascent transcripts protruding from the active site of Pol II after transcription initiation and promoter clearance, and thereby inhibits Pol II elongation (Wu, 2005; Lee, 2008). Both NELF and DSIF are thought to help stabilize Pol II at the pause site, typically 20-50 base pairs (bp) downstream from the transcription start site (Saunders, 2006; Gilchrist, 2008; Lee et al. 2008). Since Pol II elongation factors are encoded by essential genes, it is not possible to examine the lacZ/white reporter genes in homozygous mutant embryos. Instead, the transgenes were expressed in embryos derived from heterozygous females, and thereby contain half the normal levels of NELF and DSIF (Spt) subunits. Reductions in Nelf-E, Nelf-A, Spt4, and Spt5 cause clear disruptions in the enhancer-blocking activities of both the Ubx and Abd-B promoters, as seen by the strong activation of the distal white reporter gene. In contrast, white expression is blocked when the same transgenes are expressed in a wild-type background. The simplest interpretation of these results is that reduced levels of the NELF and DSIF inhibitory complexes destabilize stalled Pol II at the pause site. Reduced pausing results in diminished enhancer-blocking activities. There is a similar loss in the enhancer-blocking activities of the eve promoter and Fab7 insulator when the transgenes are expressed in embryos containing reduced levels of the GAGA factor, Trl. It is conceivable that the GAGA factor also contributes to the enhancer-blocking activity of the Ubx promoter since Trl/+ embryos display augmented expression of white (Chopra, 2009b).

In principle, the augmented expression of the white reporter gene might not result from the impaired function of the stalled insulators, but might arise from enhanced activity of the white promoter. To investigate this issue, Pol II chromatin immunoprecipitation (ChIP) assays were performed, coupled with quantitative PCR (qPCR) assays. In DSIF and NELF mutant embryos, there is no increase in Pol II levels at either the white promoter or intronic regions as compared with wild-type embryos. These results suggest that augmented expression of white is due to diminished insulator activities of stalled promoters in embryos containing reduced levels of negative Pol II elongation factors (Chopra, 2009b).

It has been suggested that insulators might work, at least in part, via promoter mimicry. To explore this issue, the impact of reductions in NELF and DSIF on the activities of two known insulators, Fab7 and Fab8, from the BX-C, were examined. Previously published transgenic lines were used that contain Fab7 or Fab8 inserted between the IAB5 and 2XPE (twist) enhancers attached to a leftward lacZ reporter gene and rightward white reporter. In wild-type embryos, the reporter genes are activated only by the proximal enhancer. Thus, white is activated solely in the mesoderm by the 2XPE enhancer, while lacZ is activated in the presumptive abdomen by IAB5. The distal enhancers are blocked by the Fab7 or Fab8 insulators. Consequently, IAB5 fails to activate white and the 2XPE enhancer fails to activate lacZ (Chopra, 2009b).

Very different results are observed when the transgenes are crossed into mutant embryos containing reduced levels of NELF or DSIF (Spt) subunits. There is a loss in the enhancer-blocking activities of the Fab7 and Fab8 insulators and, as a result, white and lacZ display composite patterns of expression in the mesoderm and abdomen since they are now activated by both enhancers. These results suggest that negative Pol II elongation factors are required for the enhancer-blocking activities of the Fab7 and Fab8 insulators (Chopra, 2009b).

It is proposed that insulators interact with stalled promoters to form higher-order chromatin loop domains, similar to those created by insulator-insulator interactions. Perhaps proteins that bind insulators interact with components of the Pol II complex at stalled genes. Indeed, the recent documentation that the BEAF insulator protein binds to many of the same sites as NELF is consistent with a physical link between stalled Pol II and insulators (Jiang, 2009). The resulting chromatin loops can prevent the inappropriate activation of stalled genes by enhancers associated with neighboring loci. As discussed earlier, stalled Hox genes are located at the boundaries of the ANT-C and BX-C. This arrangement might help ensure that cis-regulatory sequences located outside the complexes do not fortuitously interact with genes contained inside the complex and vice versa. The demonstration that stalled Hox promoters possess an intrinsic insulator activity adds to the intricacy of the chromosomal landscapes that control Hox gene expression in both arthropods and vertebrates (Chopra, 2009b).

Stalled Hox promoters may help promote higher-order chromatin organization within the Hox loci (see illustration). These results suggest that the stalled promoters contain intrinsic insulator activity that requires NELF and DSIF proteins, and this may help define higher-order loops within gene complexes such as the Hox complex. The stalled Pol II along with the NELF and DSIF complex may interact with putative insulator sequences, as seen for the Abd-B promoter and the Fab7. These experiments also suggest that that putative insulator sequences also require NELF and DSIF proteins, and this could be due to sharing of these proteins via the formation of higher-order loops. Such loop domains may help in proper regulation of genes and prevent any aberrant activation from neighboring enhancers, thus favoring proper gene regulations at the higher-order level (Chopra, 2009b).

The transvection mediating region of the Abdominal-B locus

Continued Abdominal-B Promoter Structure part 3/3 | back to part 1/3

Abdominal-B: Biological Overview | Evolutionary Homologs | Transcriptional Regulation | Targets of activity | Protein Interactions | Developmental Biology | Effects of Mutation | References

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