Co-localization of Polycomb protein and GAGA factor on regulatory elements responsible for the maintenance of homeotic gene expression.

The Polycomb group and trithorax group genes of Drosophila are required for maintaining the differential expression state of developmental regulators, such as the homeotic genes, in a stable and heritable manner throughout development. The Polycomb group genes have been suggested to act by regulating higher order chromatin and packaging repressed chromosomal domains in a heterochromatin-like structure. We have mapped, at high resolution, the distribution of Polycomb protein on the bithorax complex of Drosophila tissue culture cells, using an improved formaldehyde cross-linking and immunoprecipitation technique. Polycomb protein is not distributed homogeneously on the regulatory regions of the repressed Ultrabithorax and abdominal-A genes, but is highly enriched at discrete sequence elements, many of which coincide with previously mapped Polycomb group response elements (PREs). Our results further suggest that Polycomb protein spreads locally over a few kilobases of DNA surrounding PREs, perhaps to stabilize silencing complexes. GAGA factor/Trithorax-like, a member of the trithorax group, is also bound at those PREs which contain GAGA consensus-binding sites. Two modes of binding can be distinguished: a high level binding to elements in the regulatory domain of the expressed Abdominal-B gene, and a low level of binding to Polycomb-bound PREs in the inactive domains of the bithorax complex. We propose that GAGA factor binds constitutively to regulatory elements in the bithorax complex, which function both as PREs and as trithorax group response elements.     

Functional reconstruction of trans regulation of the Ultrabithorax promoter by the products of two antagonistic genes, trithorax and Polycomb.

Maintenance of the "on-off" state of Drosophila homeotic genes in Antennapedia and bithorax complexes requires activities of the trithorax and Polycomb groups of genes. To identify cis-acting sequences for functional reconstruction of regulation by both trithorax and Polycomb, we examined the expression patterns of several Ubx-lacZ transgenes that carry upstream fragments corresponding to a region of approximately 50 kb. A 14.5-kb fragment from the postbithorax/bithoraxoid region of Ultrabithorax exhibited proper regulation by both trithorax and Polycomb in the embryonic central nervous system. Using a Drosophila haploid cell line for transient expression, we found that trithorax or Polycomb can function independently through this upstream fragment to activate or repress the Ultrabithorax promoter, respectively. Studies of deletion mutants of trithorax and Polycomb demonstrated that trithorax-dependent activation requires the central zinc-binding domain, while Polycomb-dependent repression requires the intact chromodomain. In addition, trithorax-dependent activity can be abrogated by increasing the amount of Polycomb, suggesting a competitive interaction between the products of trithorax and Polycomb. Deletion analysis of this fragment demonstrated that a 440-bp fragment contains response elements for both trithorax and Polycomb. Furthermore, we showed that the integrity of the proximal promoter region is essential for trithorax-dependent activation, implicating a long-range interaction for promoter activation.     

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, we defined a 138 bp sequence 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 (GAF), encoded by the Trithorax-like gene. PHO and the GAF proteins bind to these sites in vitro. Mutational analysis of PHO and GAF binding sequences indicate that these sites are necessary for silencing in vivo. Moreover, silencing by MCP138 depends on the function of the Trithorax-like gene, and on the function of the PcG genes, including pleiohomeotic. Deletion and mutational analyses show that, individually, either PHO or GAF binding sites retain only weak silencing activity. However, when both PHO and GAF binding sites are present, they achieve strong silencing. We present a model in which robust silencing is achieved by sequential and facilitated binding of PHO and GAF.     

Structure of a polycomb response element and in vitro binding of polycomb group complexes containing GAGA factor

Polycomb response elements (PREs) are regulatory sites that mediate the silencing of homeotic and other genes. The bxd PRE region from the Drosophila Ultrabithorax gene can be subdivided into subfragments of 100 to 200 bp that retain different degrees of PRE activity in vivo. In vitro, embryonic nuclear extracts form complexes containing Polycomb group (PcG) proteins with these fragments. PcG binding to some fragments is dependent on consensus sequences for the GAGA factor. Other fragments lack GAGA binding sites but can still bind PcG complexes in vitro. We show that the GAGA factor is a component of at least some types of PcG complexes and may participate in the assembly of PcG complexes at PREs.     

Site-Specific Recognition of a 70-Base-Pair Element Containing d(GA)n Repeats Mediates bithoraxoid Polycomb Group Response Element-Dependent Silencing

Polycomb group proteins act through Polycomb group response elements (PREs) to maintain silencing at homeotic loci. The minimal 1.5-kb bithoraxoid (bxd) PRE contains a region required for pairing-sensitive repression and flanking regions required for maintenance of embryonic silencing. Little is known about the identity of specific sequences necessary for function of the flanking regions. Using gel mobility shift analysis, we identify DNA binding activities that interact specifically with a multipartite 70-bp fragment (MHS-70) downstream of the pairing-sensitive sequence. Deletion of MHS-70 in the context of a 5.1-kb bxd Polycomb group response element derepresses maintenance of silencing in embryos. A partially purified binding activity requires multiple, nonoverlapping d(GA)(3) repeats for MHS-70 binding in vitro. Mutation of d(GA)(3) repeats within MHS-70 in the context of the 5.1-kb bxd PRE destabilizes maintenance of silencing in a subset of cells in vivo but gives weaker derepression than deletion of MHS-70. These results suggest that d(GA)(3) repeats are important for silencing but that other sequences within MHS-70 also contribute to silencing. Antibody supershift assays and Western analyses show that distinct isoforms of Polyhomeotic and two proteins that recognize d(GA)(3) repeats, the TRL/GAGA factor and Pipsqueak (Psq), are present in the MHS-70 binding activity. Mutations in Trl and psq enhance homeotic phenotypes of ph, indicating that TRL/GAGA factor and Psq are enhancers of Polycomb which have sequence-specific DNA binding activity. These studies demonstrate that site-specific recognition of the bxd PRE by d(GA)(n) repeat binding activities mediates PcG-dependent silencing.     

The Drosophila trithorax protein is a coactivator required to prevent re-establishment of polycomb silencing

Polycomb group (PcG) and Trithorax (TRX) complexes assemble at Polycomb response elements (PREs) and maintain respectively the repressed and active state of homeotic genes. Although PcG and TRX complexes are distinct, their binding to some PRE fragments in vitro depends on GAGA motifs. GAGA factor immunoprecipitates with both complexes. In presence of a PRE, TRX stimulates expression and prevents the return of repression at later stages. When TRX levels are reduced, repression is re-established in inappropriate regions of imaginal discs, suggesting that TRX insufficiency impairs the epigenetic memory of the active state. Targeting a GAL-TRX fusion shows that TRX is a coactivator that stimulates expression of an active gene but cannot initiate expression by itself. Targeting a histone acetylase to a PRE does not affect embryonic silencing but causes a loss of memory in imaginal discs, suggesting that deacetylation is required to establish the memory of the repressed state.     

Vertebrate Homologue of Drosophila GAGA Factor

Polycomb group (PcG) and trithorax group (trxG) proteins are chromatin-mediated regulators of a number of developmentally important genes including the homeotic genes. In Drosophila melanogaster, one of the trxG members, Trithorax like (Trl), encodes the essential multifunctional DNA binding protein called GAGA factor (GAF). While most of the PcG and trxG genes are conserved from flies to humans, a Trl-GAF homologue has been conspicuously missing in vertebrates. Here, we report the first identification of c-Krox/Th-POK as the vertebrate homologue of GAF on the basis of sequence similarity and comparative structural analysis. The in silico structural analysis of the zinc finger region showed preferential interaction of vertebrate GAF with GAGA sites similar to that of fly GAF. We also show by cross-immunoreactivity studies that both fly and vertebrate GAFs are highly conserved and share a high degree of structural similarity. Electrophoretic mobility shift assays show that vertebrate GAF binds to GAGA sites in vitro. Finally, in vivo studies by chromatin immunoprecipitation confirmed that vertebrate GAF binds to GAGA-rich DNA sequences present in hox clusters. Identification of vertebrate GAF and the presence of its target sites at various developmentally regulated loci, including hox complexes, highlight the evolutionarily conserved components involved in developmental mechanisms across the evolutionary lineage and answer a long-standing question of the presence of vertebrate GAF.