The mechanisms of gene regulation by the trithorax-group (trxG) and Polycomb-group (PcG) of proteins remain 1 of the most actively pursued issues in the current developmental and molecular biology. This continuous interest is based on the realization that these protein families are employed in regulation of a large number of important genes during development of the multicellular organisms, including the key regulators of early development, the homeotic (HOX) genes. TrxG and PcG proteins exert their activities by altering the chromatin structure of their target genes. This is achieved in a variety of ways, by either changing the packaging of nucleosomes by the ATP-dependent nucleosome remodeling complexes, or by the enzymatic modifications of a number of residues in histone tails. Importantly, irrespective of the specific ways in which TrxG and PcG proteins exert their activities, they are capable of locking in a specific status of gene expression, which is then inherited in an epigenetic fashion in daughter cells. The studies of these 2 groups of proteins have an important health-related applications since some of these proteins, as for example ALL1/HRX/MLL, are believed to be involved in a number of cancers. Our recent data suggest that the TrxG protein complex TAC1 is recruited to the region downstream of the start site of the actively transcribed heat shock genes. This recruitment is accompanied by the specific TAG 1-dependent methylation and acetylation of histone residues and ultimately leads to an increase in heat shock gene transcription. The goal of this proposal is to test the hypothesis that this mode of functioning of TAG 1 is also true for the developmentally regulated genes, by using a target HOX gene Ultrabithorax (Ubx) as a model. Our preliminary results indicate that a key component of this complex, Trithorax (TRX), is found downstream of the start site of Ubx and at its promoter, suggesting that TRX is involved in 2 different steps of transcription, initiation and elongation of transcription by Poll. Based on these and previous studies, and on the functional similarities with the homologous proto-oncogene MLL, we propose the existence of 2 TRX complexes that may be involved in these different aspects of transcription. Two (2) TRX complexes may interact with other factors that are required for initiation and/or elongation of transcription. To test this hypothesis and to uncover the role of TRX complexes in the network of other transcription factors, we developed a sorting technique that allows to obtain nuclei in which Ubx is activated and silenced. Using this technique, we propose to address the following questions: (i) What is the difference in association of the TrxG and PcG proteins with the regulatory regions of the transcriptionally active and silenced Ubx gene? (ii) What are the features of the new MLL-like TRX complex? (iii) Which steps of Ubx transcription require different TRX protein complexes? (iv) What are the roles of TRX complexes in the network of other transcription factors? Answers to these questions will shed new light not only on the way TAC1 exerts its effects during transcriptional regulation, but will also reveal the roles of other TrxG and PcG proteins in epigenetic maintenance, as well as the relationship of these epigenetic regulators with factors that are involved in initiation and elongation of RNA polymerase. Given structural and functional similarities between TRX and MLL, these studies will also advance our knowledge of the basic mechanisms of transcriptional regulation in eukaryotes and their relevance to diseases like cancer.
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