Marion Santangelo

Role of post-translational modifications of CTIP2 in the transcriptional regulation of the HTLV-1 virus

Abstract

The CTIP2 protein is a cellular co-factor associated with activation or repression of transcription depending on the cellular and genomic environment. Our laboratory has previously demonstrated that the acetylation of lysine K604 of the murine version of CTIP2 (MmCTIP2) is critical for its repressive function on the transactivation by Tax of the 5' LTR of the HTLV-1 retrovirus. At the level of the human version of the CTIP2 protein (HsCTIP2), our laboratory recently identified, by mass spectrometry, two lysines (K687 and K851) as being targets of acetylation. Moreover, literature has reported that other post-translational modifications of HsCTIP2 such as phosphorylation of serine S2 and SUMOylation of lysines K689 and K887 are involved in the activator or repressor role of HsCTIP2 on its target genes (such as genes encoding IL-2 and Id-2). During our thesis, we continued the biochemical and functional characterization of the acetylation of the cellular co-factor HsCTIP2 using as a model the transcriptional transactivation of the promoter (Long Terminal Repeat 5', LTR5') of the retrovirus HTLV-1 (Human T- cell leukemia virus type 1), by the viral protein Tax.
We performed immunoprecipitation and Western Blot experiments on wild-type or mutated HsCIP2 proteins at different residues. Our results demonstrate that only the acetylation of the K687 lysine of the HsCTIP2 protein is important for its acetylation state, and not the acetylation of the K851 lysine, the SUMOylation of the K689 and K887 lysines as well as the phosphorylation of the serine S2. Furthermore, we identified by mass spectrometry four new acetylated lysines (K434, K585, K597 and K802) in HsCTIP2 and new phosphorylation sites (in addition to confirming some phosphorylation sites previously reported in the literature). Finally, we performed transient co-transfection assays followed by luciferase assays with a reporter vector containing the HTLV-1 LTR5' and expression vectors for wild-type or mutated HsCIP2 in the absence or presence of Tax. Our functional results demonstrate the repression by wild-type HsCTIP2 of the Tax-mediated transactivation of the viral 5' LTR and the negative effect on this repression of the K687R and K687Q mutations and, to a lesser extent, of the S2D mutation. On the other hand, mutations K851R and K851Q as well as S2A, S2E, K689R, K689Q, K887R and K887A have no effect on this repression.
In conclusion, our results contribute to a better understanding of the post-translational modifications of the cellular factor HsCTIP2 and the role of these modifications in its transcriptional activity. These studies could be important in the context of all other cellular and viral genes regulated by CTIP2.