Gwenaelle Robette

HMGA1 recruits CTIP2-repressed P-TEFb to the HIV-1 and cellular target promoters.

Active positive transcription elongation factor b (P-TEFb) is essential for cellular and human immunodeficiency virus type 1 (HIV-1) transcription elongation. CTIP2 represses P-TEFb activity in a complex containing 7SK RNA and HEXIM1. Recently, the inactive 7SK/P-TEFb small nuclear RNP (snRNP) has been detected at the HIV-1 core promoter as well as at the promoters of cellular genes, but a recruiting mechanism still remains unknown to date. Here we show global synergy between CTIP2 and the 7SK-binding chromatin master-regulator HMGA1 in terms of P-TEFb–dependent endogenous and HIV-1 gene expression regulation. While CTIP2 and HMGA1 concordingly repress the expression of cellular 7SK-dependent P-TEFb targets, the simultaneous knock-down of CTIP2 and HMGA1 also results in a boost in Tat-dependent and independent HIV-1 promoter activity. Chromatin immunoprecipitation experiments reveal a significant loss of CTIP2/7SK/P-TEFb snRNP recruitment to cellular gene promoters and the HIV-1 promoter on HMGA1 knock-down. Our findings not only provide insights into a recruiting mechanism for the inactive 7SK/P-TEFb snRNP, but may also contribute to a better understanding of viral latency.

Role of the cofactor CTIP2 (COUP-TF Interacting Protein 2) in the transcriptional repression of HTLV-1 (Human T-lymphotropic Virus 1)

Background Following entry and reverse transcription, HTLV-1 integrates into the host cell genome. The viral promoter activity is directly governed by its chromatin environment. Epigenetic modifications, such as DNA methylation and histone deacetylation, are crucial for transcriptional silencing of the virus. We have previously reported that the cofactor CTIP2 recruits a multienzymatic chromatin-modifying complex, including histone deacetylases and a methyltransferase to the HIV-1 promoter, thereby establishing a heterochromatic environment at the promoter. CTIP2 is recruited to the HIV-1 promoter via its association with the transcription factor Sp1. Here, we investigated the potential role of CTIP2 in transcriptional regulation of the HTLV-1 promoter. Methods Electrophoretic mobility shift assays (EMSAs), transient transfection and chromatin immunoprecipitation (ChIP) assays were performed. Results Analysis of the nucleotide sequence of the HTLV-1 promoter (LTRHTLV-1) revealed two Sp1 binding sites within the R region. We demonstrated that Sp1 and Sp3 bind in vitro to these sites by EMSAs. We showed, by cotransfection assays in epithelial HEK293T cells and T-lymphoid Jurkat cells, that the cofactor CTIP2 inhibited Tax-mediated transactivation of the HTLV-1 promoter. We demonstrated by ChIP assays the in vivo recruitment of CTIP2 to the LTRHTLV-1 in a latently HTLV-1 infected cell line (TLom1). Conversely, CTIP2 was absent from the proviral genome in a HTLV-1 productive cell line (SLB1). The identification of the multienzymatic complex recruited by CTIP2 to the viral promoter under latent conditions is currently under investigation.

Repression of Human T-lymphotropic virus type 1 Long Terminal Repeat sense transcription by Sp1 recruitment to novel Sp1 binding sites.

Human T-lymphotropic Virus type 1 (HTLV-1) infection is characterized by viral latency in the majority of infected cells and by the absence of viremia. These features are thought to be due to the repression of viral sense transcription in vivo. Here, our in silico analysis of the HTLV-1 Long Terminal Repeat (LTR) promoter nucleotide sequence revealed, in addition to the four Sp1 binding sites previously identified, the presence of two additional potential Sp1 sites within the R region. We demonstrated that the Sp1 and Sp3 transcription factors bound in vitro to these two sites and compared the binding affinity for Sp1 of all six different HTLV-1 Sp1 sites. By chromatin immunoprecipitation experiments, we showed 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 promoter activities. Interestingly, the Sp1 sites exhibited together a repressor effect on the LTR sense transcriptional activity but had no effect on the LTR antisense activity. Thus, our results demonstrate the presence of two new functional Sp1 binding sites in the HTLV-1 LTR, which act as negative cis-regulatory elements of sense viral transcription.

Identification and characterization of new Sp1 sites located in the R region of the Human T-lymphotropic virus 1 (HTLV-1) long terminal repeat

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. Specific protein 1 (Sp1) binds to more than 1000 different cellular promoters and regulates the expression of numerous genes involved in cell proliferation, apoptosis, and differentiation. In silico analysis of the nucleotide sequence of the HTLV-1 LTR revealed the presence of two new 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. We also performed competition assays with a probe corresponding to a Sp1 binding site consensus in order to compare Sp1 binding affinity for the four previously reported and the two newly identified Sp1-binding sites located in the HTLV-1 promoter. Point mutations of the known and newly identified Sp1 sites were introduced in the HTLV-I LTR cloned in either the sense or the antisense orientation in the context of an episomal reporter vector. We demonstrated that the Sp1 sites interfered with both the sense transcription from the 5’LTR and the antisense transcription from the 3’LTR (necessary for HBZ expression in vivo). ChIP experiments to evaluate in vivo Sp1 binding to the HTLV-I LTR U3 region are currently under investigation. Our results demonstrate the presence of two new functional Sp1 binding sites located in the R region of the HTLV-I LTR.