Role of CTIP2 and its post-translational modifications in transcriptional regulation of HTLV-1.

Abstract

- Introduction :Human T-lymphotropic Virus 1 (HTLV-1) infects 15-20 million people worldwide and is responsible for two major diseases :adult T-cell leukemia⁄lymphoma and HTLV-1-associated myelopathy⁄tropical spastic paraparesis. HTLV-1 infection is characterized by viral latency in the large majority of infected cells and by the absence of viremia. These features are thought to be due to the transcriptional repression of viral expression. This latency represents a viral strategy to escape from the host immune system allowing tumor development. Our laboratory has previously shown that the cellular transcriptional cofactor CTIP2 (COUP-TF interacting Factor 2)/Bcl11b (B-cell CLL/lymphoma 11b), involved in development and lymphomagenesis, is recruited to the HIV-1 and p21 promoters via its association with the transcription factor Sp1, thereby silencing gene transcription through interactions with histone deacetylases and histone methyltransferases. More recently, we have reported that CTIP2 interacts with and inhibits the positive transcription elongation factor b (P-TEFb) (Cherrier et al. PNAS, 2015), whose deregulations are associated with various types of human malignancies. Moreover, we showed that HMGA1 (High Mobility Group A1), a protein highly expressed during embryogenesis and all aggressive human cancers, is involved in the recruitment of the P-TEFb repressor CTIP2 and/or the CTIP2-repressed P-TEFb complex to target promoters (Eilebrecht et al. NAR, 2014).- Aims :The aim of this study is to further charaterize the epigenetic control of HTLV-1 gene expression in latently- and productively-infected cell lines with a special emphasis on the role of two cellular transcription factors, the oncogenic cofactor CTIP2 (COUP-TF Interacting Factor 2), also known as Bcl11b (B-cell CLL/lymphoma 11b), and the Specificity protein 1 (Sp1). This work should open new ways to better understand how chromatin modifications influence gene expression of the oncogenic retrovirus HTLV-1 and therefore the molecular basis underlying leukemogenesis.- Methods and results :Here, we used as a model the TAXHTLV-1-mediated transactivation of the HTLV-1 promoters in order to demonstrate that CTIP2 was also able to repress transcription of another complex retrovirus. It has been reported that CTIP2 interacts with the histone acetyltransferase p300 and is involved in transcriptional activation of the IL-2 promoter in T lymphocytes (Cismasiu et al. Blood, 2006). We postulate that the function of CTIP2 might be modulated by posttranslational modifications.To test this hypothesis, we evaluated posttranslational modifications of overexpressed CTIP2 protein and identified at least 5 acetylated residues. Interestingly, our recent results showed that the substitution of a single particular acetylable residue by an arginine, a non-acetylable residue, impeded the global acetylation of CTIP2. Moreover, this substitution also interfered with the ability of CTIP2 to inhibit TAXHTLV-1-mediated transactivation of the HTLV-1 promoters. To test whether this mutation alters CTIP2 recruitment to the HTLV-1 promoter, we will perform ChIP experiments using antibodies directed against CTIP2 and CTIP2-associated proteins, in the different HTLV-1-infected cell lines in which endogenous CTIP2 has been substituted by the lysine mutant using genome editing technology (CRISPR/Cas9). Moreover, we will identify by mass spectrometry and confirm by mutagenesis experiments the other potential post-translational modifications of CTIP2 in different lymphoblastic cell lines. Next, we will determine the functional role of these other modifications in CTIP2 activity by studying the effect of mutated CTIP2 proteins on HTLV-1 LTR activity in transient transfection and luciferase assays. Immunoprecipitation of genomicaly edited mutant versions of CTIP2 will be performed in parallel and the immunoprecipitated proteins will be analyzed by mass spectrometry to decipher their association with CTIP2 cofactors.- Conclusions :In this project, we will first elucidate the role of Sp1 in the epigenetic control of HTLV-1 latency. Next, we will characterize how CTIP2 is recruited to the HTLV-1 promoter and negatively regulates Tax-mediated HTLV-1 LTR transactivation, and finally we will study the functional role of post-translational modifications of CTIP2 and their implication in the formation of CTIP2-associated complexes. A better understanding of the epigenetic and non-epigenetic mechanisms responsible for HTLV-1 transcriptional repression, could serve as a model to further study oncogenic mechanisms associated with CTIP2 transcriptional regulation in humans.info:eu-repo/semantics/nonPublishe