Activated signal transducer and activator of transcription STAT5 induces the expression of genes essential for cell differentiation, proliferation and inhibition of apoptosis. Previous work from our group demonstrated that the deacetylase inhibitor trichostatin A (TSA) attenuates transcriptional activation of STAT5 target genes at a step following STAT5 binding to its DNA binding sites by abrogating the recruitment of TBP and RNA polymerase II. The goal of this thesis was to better understand the mechanism of transcriptional regulation by STAT5 via the characterization of the mechanism underlying its inhibition by TSA. Specific aims were the identification of (i) the deacetylase (so-called HDAC) involved and of (ii) the acetylated substrate. The identification of the HDAC was performed using class-selective HDAC inhibitors and siRNA-mediated knock-down of HDAC expression. We found that, similarly to TSA, the deacetylase inhibitors valproic acid (VPA) and apicidin - but not MGCD0103 and MS-275 - inhibited expression of STAT5 target genes. However, siRNA-mediated knock-down experiments did not allow to identify the specific HDAC(s) involved in STAT5 target gene expression. To investigate whether STAT5 might be the acetylated substrate targeted by HDACs, selected lysine residues within STAT5 potentially targeted for acetylation were mutated and their effect on STAT5-mediated transcription was investigated. None of the mutations affected STAT5 transcriptional activity, arguing against STAT5 being the acetylated substrate targeted by the sought HDAC. Interestingly however, inhibition of STAT5-mediated transcription by TSA, VPA and apicidin correlated with an increase in global histone H3 and H4 acetylation. It also correlated with a redistribution of the acetylated-histone-binding protein BRD2, a member of the bromodomain and extra-terminal (BET) protein family described for its role in the recruitment of the transcriptional machinery and transcriptional activation. Notably, chromatin precipitation experiments revealed that BRD2 is associated with the actively-transcribed STAT5 target gene Cis in a STAT5-dependent manner, and that BRD2 binding to the Cis gene is lost upon TSA treatment. In agreement with a role of BRD2 in STAT5-mediated transcription, the BET inhibitor (+)-JQ1 inhibited STAT5-mediated transcription of the Cis gene. Together, our data support a model in which the HDAC inhibitors TSA, VPA and apicidin target histone acetylation, resulting in a global increase in chromatin acetylation. This change in chromatin acetylation would result in the redistribution of BRD2 to hyperacetylated chromatin and a departure of BRD2 from STAT5 target genes. BRD2 loss at STAT5 target genes would in turn prevent the proper recruitment and maintenance of the transcriptional machinery, resulting in transcriptional inhibition. In summary, this thesis identified BRD2 as an important co-factor of STAT5-mediated transcription and demonstrated that deacetylase inhibitors inhibit STAT5-mediated transcription by interfering with BRD2 function. This study thus identified BRD2 as a potential target for the development of novel therapies against STAT5-associated cancers
