Antoine Dutilleul

Study of the enhancer/promoter function of the intragenic region of HIV-1: implications of the architectural proteins YY1 and CTCF

Abstract

The rate of replication of HIV-1, directly correlated to the progression of AIDS, is mainly regulated at the level of the transcription of viral genes. In addition to the cis-regulatory region located in the 5' LTR of the provirus, our laboratory has demonstrated an intragenic cis-regulatory region (IRR), comprised between nucleotides 4079 and 6026 of the HIV-1 provirus (where nt+1 equals the first nucleotide of the 5' LTR). The IRR exhibits dual promoter/enhancer transcriptional activity and is divided into three functional domains: fragments 5103 and 5105, responsible for PMA-inducible enhancer activity, flanking the DNase I (HSVII) hypersensitive site uniquely identified in a promonocyte lineage. Regarding the promoter activity of the IRR, our laboratory has previously identified four transcription initiation sites, including one major and three minor, at the D site of HSVII. In addition, the characterization of the promoter activity made it possible to identify the fragment 1012 (containing the D site) as being the basal minimal promoter of the IRR. Nevertheless, to date, the regulatory mechanisms of the dual promoter/enhancer activity of the IRR have not yet been characterized. Furthermore, the impact of chromatin structure, which is an important regulator of eukaryotic transcription, on the regulation of the different transcriptional activities of HIV-1 has not been studied. Interestingly, our laboratory recently identified five binding sites for the transcription factor YY1 within the IRR. This architectural protein is involved in the transcriptional regulation of many genes, as well as in the formation of chromatin loops. Therefore, the aim of our thesis is to study the involvement of YY1 in the regulation of the dual promoter/enhancer activity of the IRR. preferentially, we have characterized the enhancer activity of the IRR on the viral homologous promoter, as well as the promoter activity of the IRR in the context of an episomal plasmid by transient transfection experiments in a promonocyte line. These experiments confirmed that the IRR exhibits a weak enhancer activity on the 5' LTR but also exhibits a sense promoter activity, of which fragment 1012 is the basal minimal promoter. Second, we examined the functional effect of YY1 on IRR promoter/enhancer activity. Our transient co-transfection results indicate that YY1 is a repressor of the promoter activity of the 5' LTR, as well as of the dual transcriptional activity of the IRR. We also identified, in silico, seven novel binding sites for YY1 in fragment 1012, and demonstrated in vitro binding of YY1 at site D by gel migration retardation experiments. Finally, we have shown by chromatin immunoprecipitation experiments, the in vivo recruitment of YY1 at the level of the LTRs, as well as at the level of the IRR in the promonocyte lineage latently infected by HIV-1, and a decrease in this recruitment after treatment with ART. Our results contribute to improving the understanding of the different molecular mechanisms involved in the transcriptional regulation and latency control of HIV-1.