Role of post-translational modifications of CTIP2 in transcriptional regulation of HTLV-1 (2017)

- Introduction :Human T-lymphotropic Virus 1 (HTLV-1) infects 15-20 million people worldwide and is responsible of two major diseases :adult T cell leukemia 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 in vivo. Latency, which results from transcriptional silencing in vivo, represents a viral strategy to escape from the host immune system and allow tumor development. Sp1 has been demonstrated as an important regulator of eukaryotic promoter transcriptional activity, we here analyzed the HTLV-1 Long Terminal Repeat (LTR) sequence for other potential Sp1 sites and studied the role of all the Sp1 sites in a nucleosomal context and in both HTLV-1 sense and antisense LTR promoter activity. In the context of HIV-1 latency, our laboratory have shown that the co-factor CTIP2 (COUP-TF interacting Factor 2)/Bcl11b (B-cell CLL/lymphoma 11b), a transcriptional factor involved in the development and lymphomagenesis, is recruited to the HIV-1 and p21 promoter via its association with the transcription factor Sp1 thereby silencing genes transcription through interactions with HDACs and the HMT SUV39H1. Recently, we have reported that CTIP2 interacts with and inhibits the positive transcription elongation factor b complex (P-TEFb, composed of CDK9 and human cyclin T1 or T2) for which deregulations are associated with various type of human malignancies and cardiomyocytes hypertrophy. Moreover, we showed that HMGA1 (High Mobility Group A1), a protein highly expressed during embryogenesis and in virtually all aggressive human cancers studied to date, is involved in the recruitment of the P-TEFb repressor CTIP2 and/or the CTIP2-repressed P-TEFb snRNP (small nuclear ribonucleo-protein complex) to target promoters. - Aims :The aim of this study is to further characterize the epigenetic control of the HTLV-1 gene expression in latently-infected cells with a special emphasis on the role of Sp1, CTIP-2 and their cofactors.- Methods and results :In silico analysis of the nucleotide sequence of the HTLV-1 LTR promoter revealed the presence of two additional potential Sp1 binding sites within the R region. We demonstrated that the Sp1 and Sp3 transcription factors bound in vitro to these sites by EMSAs and supershift experiments. By competition assays, we compared the binding affinity for Sp1 of all six different HTLV-1 Sp1 binding sites and demonstrated, by chromatin immunprecipitation experiments, Sp1 recruitment in vivo to the newly identified Sp1 sites. We demonstrated in the nucleosomal context of an episomal reporter vector that the Sp1 sites interfered with both the sense and antisense LTR transcription. Interestingly, we showed that the two Sp1 binding sites located in the R region of the LTR exhibited together a repressor effect on the LTR sense transcriptional activity but had no effect on the LTR antisense activity. Interestingly, our preliminary results demonstrated that CTIP2 is able to repress the TAXHTLV-1- mediated transactivation of the HTLV-1 promoters by luciferase reporter assays. CTIP2 interacts with the HAT p300 and is involved in transcriptional activation of the IL-2 promoter in T lymphocytes. We postulate that the function of CTIP2 might be modulated by posttranslational modifications of the protein. To test this hypothesis, we evaluated posttranslational modifications of overexpressed CTIP2 protein by western blot using antibodies directed against acetylated lysine and we identified by mass spectrometry at least 5 acetylated residues. Interestingly, our preliminary results showed that the substitution of a single acetylable residue by an arginine, a non-acetylable residue that mimics a constitutively hypoacetylated lysine, impede the global acetylation of CTIP2. Moreover, this substitution also interferes with the ability of CTIP2 to inhibit the Tax-mediated transactivation of the HTLV-1 promoters. Furthermore, we have demonstrated by ChIP assays the recruitment in vivo of CTIP2 to the HTLV-1 LTR in a latently-infected cell line (TL-Om1) but not in a HTLV-1 productive cell line (SLB1).- Conclusions :Taken together, our results demonstrate the presence of two new functional Sp1 binding sites located in the R region of the HTLV-1 LTR, which act as negative cis-regulatory elements of sense transcription. Furthermore, our recent data suggest that CTIP2 is a major regulator of the viral transcription and may be important for the viral latency potentially involving the multienzymatic complexes that interact with CTIP2.