| Abstract |
My studies were focused on different mechanistic aspects of tissue specific gene regulation by HNF-1 and HNF-4. HNF-4 and HNF-1 (Hepatic Nuclear Factor 4 and 1) are liver-enriched transcription factors which regulate genes that play important roles in various metabolic pathways in the adult liner. In humans , heterozygous mutations in the HNF-4 and HNF-1 genes are associated with an early onset form of type II diabetes called Maturity Onset Diabetes of the Young (MODY I and III, respectively), also proving the importance of these factors in pancreatic b-cell function. First, in an effort to explore the functional importance of the interaction between HNF-4 and the known coactivator CBP, which possesses histone acetyltransferase activity, we found that CBP acytylates HNF-4 DNA-binding activity and its affinity of interaction observation with CBP itself and is required for target gene activation. The most interesting observation of this work is that this modification is crucial for the proper nuclear retention of HNF-4, which binds referentially to non-acetylated HNF-4. The results of this study suggest that acetylation is a key posttranslational modification that affects several properties of HNF-4 critical for its biological functions. Secondly, while studying the molecular mechanism involved in HNF-1-dependent gene activation, we found that HNF-1 can physically interact with the histone acetyltransferases (HATs) CBP, P/CAF, SRC1 and RAC3 and that the synergistic action between these co-activators enhances HNF-1-dependent transcriptional activation. Our studies were mainly focused on the synergium observed between the more robust HATs CBP and P/CAF, which can independently interact with the N-terminal and the C-terminal domain of HNF-1, respectively. The transcriptional activation potential of HNF-1 on a genome integrated promoter was strictly dependent on the synergistic action of CBP and P/Caf and requirement for P/CAF HAT activity for HNF-1-dependent activation from a transiently transfected reporter. Employing in vitro assays, we found that the interaction of CBP with the N-terminal domain of HNF-1 greatly increased the binding affinity of P/CAF for the C-terminal activation domain, which provides a potential mechanism for the observed functional synergism. These results support a model which involves the combined action of multiple co-activator recruited by HNF-1 to activate transcription by coupling nucleosome modification and recruitment of the general transcription machinery. Thirdly, exploring the mechanism behind the impaired transactivation potential of two dominant-negative mutants of HNF-1 (P447L), occurring in maturity onset diabetes of the young (MODY3) patients, we found that they paradoxically exhibit stronger interactions with either CBP or P/CAF than the wild type protein both in vivo and in vitro. Further in vivo studies showed that the dominant-negative effect of MODY3 mutants is not due to a preferential recruitment of the corepressor NCOR or the deacetylase protein HDAC1, but not the MODY3 mutants, stimulated the HAT activity of CBP in vitro. Other transcription factors such as Sp! And HNF-4 also stimulated the HAT activity of CBP, demonstrating a more dynamic role for DNA-binding proteins in the transcription process. They are not only required for the recruitment of coactivators to the promoter, but they may also modulate their enzymatic activity. Finally, in order to explore the role/involvement of the HNF4 and HNF-1 preinitiation complex assembly and chromatin remodeling processes in a natural target gene, we analyzed the order of recruitment of factors to the alpha-1-antitrypin (a1-AT) promoter upon the initial activation of the gene during enterocyte differentiation. We found that a complete preinitiation complex, including the HNF-1 and HNF-4 transcription factors, componets of TFIID, TFIIH and the mediator complex, as well as, phosphorylated RNA pol-II, waw assembled at the promoter long before transcriptional activation. The histone acetyltransferases CBP and P/CAF were recruited subsequently, but local histone hyperacetylation was delayed. After transient recruitment of hBrm, remodeling of the neighboring nucleosome coincided with transcription initiation. The results suggest that at this promoter chromatin reconfiguration is a defining step of the initiation process, acting after the assembly of the Pol II machinery.
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