The trithorax (trx) gene functions in segment determination in Drosophika through interaction with genes of

The Polycomb (Pc) group (Pc-G) of repressors is essential for transcriptional silencing of homeotic genes that determine the axial development of metazoan animals. It is generally believed that the multimeric complexes formed by these proteins nucleate certain chromatin structures to silence promoter activity upon binding to Pc-G response elements (PRE). Little is known, however, about the molecular mechanism involved in sequence-specific binding of these complexes. Here, we show that an immunoaffinity-purified Pc protein complex contains a DNA binding activity specific to the (GA) n motif in a PRE from the bithoraxoid region. We found that this activity can be attributed primarily to the large protein isoform encoded by pipsqueak (psq) instead of to the well-characterized GAGA factor. The functional relevance of psq to the silencing mechanism is strongly supported by its synergistic interactions with a subset of Pc-G that cause misexpression of homeotic genes.

Deacetylation of the N-terminal tails of core histones plays a crucial role in gene silencing. Rpd3 and Hda1 represent two major types of genes encoding trichostatin A-sensitive histone deacetylases. Although they have been widely found, their cellular and developmental roles remain to be elucidated in metazoa. We show that Drosophila Hdac1, an Rpd3-type gene, interacts cooperatively with Polycomb group repressors in silencing the homeotic genes that are essential for axial patterning of body segments. The biochemical copurification and cytological colocalization of HDAC1 and Polycomb group repressors strongly suggest that HDAC1 is a component of the silencing complex for chromatin modification on specific regulatory regions of homeotic genes.Pc-G ͉ histone deacetylase ͉ Ubx ͉ PRE

Background:Genome-wide studies in higher eukaryotes have revealed the presence of paused RNA polymerase II (RNA-Pol) at about 30-50 bp downstream of the transcription start site of genes involved in developmental control, cell proliferation and intercellular signaling. Promoter-proximal pausing is believed to represent a critical step in transcriptional regulation. GAGA sequence motifs have frequently been found in the upstream region of paused genes in Drosophila, implicating a prevalent binding factor, GAF, in transcriptional pausing.Results: Using newly isolated mutants that retain only ~3 % normal GAF level, we analyzed its impacts on transcriptional regulation in whole animals. We first examined the abundance of three major isoforms of RNA-Pol on Hsp70 during heat shock. By cytogenetic analyses on polytene chromosomes and chromatin immunoprecipitation (ChIP), we show that paused RNA-Pol of Hsp70 is substantially reduced in mutants. Conversely, a global increase in paused RNA-Pol is observed when GAF is over-expressed. Coupled analyses of transcriptome and GAF genomic distribution show that 269 genes enriched for upstream GAF binding are down-regulated in mutants. Interestingly, ~15 % of them encode transcriptional factors, which might control ~2000 additional genes down-regulated in mutants. Further examination of RNA-Pol distribution in GAF targets reveals that a positive correlation exists between promoter-proximal RNA-Pol density and GAF occupancy in WT, but not in mutants. Comparison of nucleosome profiles indicates that nucleosome occupancy is preferentially attenuated by GAF in the upstream region that strongly favors nucleosome assembly. Using a dominant eye phenotype caused by GAF over-expression, we detect significant genetic interactions between GAF and the nucleosome remodeler NURF, the pausing factor NELF, and BAB1 whose binding sites are enriched specifically in genes displaying GAF-dependent pausing. Conclusion: Our results provide direct evidence to support a critical role of GAF in global gene expression, transcriptional pausing and upstream nucleosome organization of a group of genes. By cooperating with factors acting at different levels, GAF orchestrates a series of events from local nucleosome displacement to paused transcription. The use of whole animals containing broad tissue types attests the physiological relevance of this regulatory network.

Pluripotent stem cells often adopt a unique developmental program while retaining certain flexibility. The molecular basis of such properties remains unclear. Using differentiation of pluripotent Drosophila imaginal tissues as assays, we examined the contribution of epigenetic factors in ectopic activation of Hox genes. We found that over-expression of Trithorax H3K4 methyltransferase can induce ectopic adult appendages by selectively activating the Hox genes Ultrabithorax and Sex comb reduced in wing and leg discs, respectively. This tissue-specific inducibility correlates with the presence of paused RNA polymerase II in the promoter-proximal region of these genes. Although the Antennapedia promoter is paused in eye-antenna discs, it cannot be induced by Trx without a reduction in histone variants or their chaperones, suggesting additional control by the nucleosomal architecture. Lineage tracing and pulse-chase experiments revealed that the active state of Hox genes is maintained substantially longer in mutants deficient for HIRA, a chaperone for the H3.3 variant. In addition, both HIRA and H3.3 appeared to act cooperatively with the Polycomb group of epigenetic repressors. These results support the involvement of H3.3-mediated nucleosome turnover in restoring the repressed state. We propose a regulatory framework integrating transcriptional pausing, histone modification, nucleosome architecture and turnover for cell lineage maintenance.