Transcriptional regulation by the oncoprotein STAT5: role of acetylation and deacetylation processes

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

Signal transducer and activator of transcription STAT5 is recruited to c-Myc super-enhancer

Background: c-Myc has been proposed as a putative target gene of signal transducer and activator of transcription 5 (STAT5). No functional STAT5 binding site has been identified so far within the c-Myc gene locus, therefore a direct transcriptional regulation by STAT5 remains uncertain. c-Myc super-enhancer, located 1.7 Mb downstream of the c-Myc gene locus, was recently reported as essential for the regulation of c-Myc gene expression by hematopoietic transcription factors and bromodomain and extra-terminal (BET) proteins and for leukemia maintenance. c-Myc super-enhancer is composed of five regulatory regions (E1-E5) which recruit transcription and chromatin-associated factors, mediating chromatin looping and interaction with the c-Myc promoter. Results: We now show that STAT5 strongly binds to c-Myc super-enhancer regions E3 and E4, both in normal and transformed Ba/F3 cells. We also found that the BET protein bromodomain-containing protein 2 (BRD2), a co-factor of STAT5, co-localizes with STAT5 at E3/E4 in Ba/F3 cells transformed by the constitutively active STAT5-1*6 mutant, but not in non-transformed Ba/F3 cells. BRD2 binding at E3/E4 coincides with c-Myc transcriptional activation and is lost upon treatment with deacetylase and BET inhibitors, both of which inhibit STAT5 transcriptional activity and c-Myc gene expression. Conclusions: Our data suggest that constitutive STAT5 binding to c-Myc super-enhancer might contribute to BRD2 maintenance and thus allow sustained expression of c-Myc in Ba/F3 cells transformed by STAT5-1*6

SFN treatment does not affect STAT5 phosphorylation.

<p>Ba/F3 (<b>A</b>), Ba/F3-1*6 (<b>B</b>) and K562 (<b>C</b>) cells were treated 60 minutes with DMSO (vehicle) or the indicated concentrations of TSA, SFN or Imatinib. Ba/F3 cells (<b>A</b>) were stimulated with 5 ng/mL IL-3 for 30 minutes following 30 minutes of drug pre-treatment. Whole-cell Brij protein lysates were analyzed by Western blot using antibodies specific for phospho-STAT5 (pSTAT5), STAT5A, STAT5B, STAT5A and B, and α-tubulin (loading control).</p

SFN treatment does not affect histone acetylation at the promoters of STAT5 target (<i>Cis, Osm</i>) and control (<i>p21</i>) genes.

<p>Ba/F3 cells were pre-treated 30 minutes with DMSO (vehicle), 0.2 µM TSA or 10 µM SFN and further stimulated 30 minutes with 5 ng/mL IL-3. Chromatin immunoprecipitation (ChIP) was performed using antibodies directed against acetylated histone H3 (Ac-H3) and H4 (Ac-H4) and against histone H3 proteins (total H3). Co-precipitated genomic DNA was analyzed by quantitative PCR using primers specific for the transcription start sites of the mouse <i>Cis</i> (<b>A</b>) and <i>Osm</i> (<b>B</b>) genes (amplicons B and J respectively in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099391#pone.0099391.s002" target="_blank">Figure S2</a>), as well as for the proximal promoter region of the mouse <i>p21</i> gene (amplicon K in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099391#pone.0099391.s002" target="_blank">Figure S2</a>) as a control (<b>C</b>). Ac-H3 and Ac-H4 ChIP data were normalized to total Histone H3, to more accurately estimate histone acetylation levels at the investigated gene loci. Corresponding raw ChIP data for Ac-H3, Ac-H4 and H3 immunoprecipitations (expressed as % of input DNA) are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099391#pone.0099391.s006" target="_blank">Figure S6</a>. While histone acetylation levels were dramatically affected by TSA at all three gene loci, no major change in histone H3 and H4 acetylation was monitored in SFN-treated cells.</p

SFN treatment does not affect STAT5 phosphorylation.

<p>Ba/F3 (<b>A</b>), Ba/F3-1*6 (<b>B</b>) and K562 (<b>C</b>) cells were treated 60 minutes with DMSO (vehicle) or the indicated concentrations of TSA, SFN or Imatinib. Ba/F3 cells (<b>A</b>) were stimulated with 5 ng/mL IL-3 for 30 minutes following 30 minutes of drug pre-treatment. Whole-cell Brij protein lysates were analyzed by Western blot using antibodies specific for phospho-STAT5 (pSTAT5), STAT5A, STAT5B, STAT5A and B, and α-tubulin (loading control).</p

SFN treatment does not affect global histone acetylation level in Ba/F3 cells.

<p>Ba/F3 cells were treated for the indicated times with either 0.2 µM TSA or 10 µM SFN. Whole-cell Freeze-Thaw protein lysates were analyzed by Western blot using antibodies specific for acetylated histone H3 (Ac-H3) and H4 (Ac-H4) and for total histone H3 proteins as a reference. While global histone acetylation was markedly increased in cells treated with TSA, no apparent effect was detected upon SFN treatment.</p

Effect of SFN treatment on cytotoxicity and viability of normal (Ba/F3) and transformed (Ba/F3-1*6, K562) cells.

<p>(<b>A</b>) The WST-1 reagent was added to cells following 30 minutes of pre-treatment with 0.001, 0.01, 0.1 and 1 µM TSA or with 0.1, 1, 10 and 100 µM SFN. IL-3 (5 ng/mL) was supplemented to rested Ba/F3 cells at the same time as the WST-1 reagent to mimic the IL-3 stimulation conditions used in other assays. OD measurement was performed after 90 minutes incubation with the WST-1 reagent, and the percentage of cytotoxicity was normalized to the vehicle control. (<b>B</b>) Growing Ba/F3, Ba/F3-1*6 and K562 cells were incubated for 24 and 48 hours in the presence of the indicated concentrations of TSA and SFN. Cell viability was measured by Trypan Blue exclusion assay.</p

MOESM1 of Signal transducer and activator of transcription STAT5 is recruited to c-Myc super-enhancer

Additional file 1: Fig. S1. Protein interaction between STAT5A-1*6 and BRD2 cannot be evidenced in co-immunoprecipitation assays. Nuclear lysates from formaldehyde-crosslinked (A-C) or non-crosslinked (D, E) STAT5A-1*6-expressing cells were prepared as described in the Methods section. Nuclear protein enrichment was verified by Western blot using antibodies specific for the nuclear and cytosolic proteins HDAC1 and α-tubulin respectively, and STAT5A-1*6 expression was monitored using the FLAG antibody (A, D). Immunoprecipitations (IP) were performed as described in the “Methods” section using the indicated antibodies. Input (In), immunoprecipitation supernatants (SN) and eluted bead fractions (B) were analysed by immunoblot (IB) using the indicated antibodies (B, E). In panel B, arrow points to BRD2 and (*) indicates a non-specific signal associated with the bead fractions. Bead samples from the IP experiment shown in panel B (crosslinked cells) were further processed for ChIP analysis by qPCR, using the Cis-specific primers depicted in Fig. 2a (C). In panel C, background cut-off (dotted line) was defined as in legend to Fig. 4 (mean IgG background + 2x SD). One-way ANOVA with Dunnett’s multiple comparison test was used to evaluate BRD2 and STAT5 enrichment at the STAT5 binding site (STAT5) and transcription start site (TSS) of the Cis gene, in comparison to the “ORF” region, used as a reference and background control; ***P < 0.001; a P value < 0.05 was considered statistically significant

SFN treatment inhibits STAT5 constitutive activity in the transformed cell lines Ba/F3-1*6 and K562.

<p>Ba/F3 (<b>A</b>), its transformed counterpart Ba/F3-1*6 (<b>B</b>) and human leukemic K562 (<b>C</b>) cells were treated 90 minutes with DMSO (vehicle), 0.2 µM TSA, 10 µM SFN or 1 µM Imatinib. Ba/F3 cells (<b>A</b>) were stimulated with 5 ng/mL IL-3 for 60 minutes following 30 minutes of drug pre-treatment. Expression of STAT5-dependent (<i>Cis</i>, <i>c-Myc, Pim-1, Socs-1, Osm</i>,) and -independent (<i>JunB</i>, <i>c-Fos</i>, <i>36b4</i>) genes was analyzed by quantitative RT-PCR. Gene expression data were normalized to cDNA levels derived from mouse ribosomal <i>S9</i> (<b>A, B</b>) or human Lamin A/C (<i>LMNA</i>) (<b>C</b>) mRNAs. (<b>A, B</b>) The Y-axis scales were adjusted to allow a direct comparison of relative expression levels in Ba/F3 and Ba/F3-1*6 cells.</p

STAT5 binding and RNA polymerase II recruitment to the promoter of STAT5 target genes are marginally affected by SFN treatment.

<p>Ba/F3 cells were pre-treated 30 minutes with DMSO (vehicle), 10 µM or 20 µM SFN and further stimulated with 5 ng/mL IL-3 for 30 minutes. Cells were harvested for both gene expression analysis of the <i>Cis</i> and <i>Osm</i> genes by quantitative RT-PCR (A) and for chromatin immunoprecipitation (ChIP) (B–D). ChIP was performed using antibodies directed against STAT5 (B) or RNA polymerase II (RNA Pol II; C, D) proteins. Co-precipitated genomic DNA was analyzed by quantitative PCR using primers specific for the STAT5 binding sites (amplicons A and I in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099391#pone.0099391.s002" target="_blank">Figure S2</a>) (STAT5 ChIP; B) or the transcription start site (amplicons B and J in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099391#pone.0099391.s002" target="_blank">Figure S2</a>) (RNA Pol II ChIP; C) of the mouse <i>Cis</i> and <i>Osm</i> genes, as well as with primers spanning the open reading frame of the <i>Cis</i> gene (RNA Pol II ChIP; D). Schematic representation of the <i>Cis</i> gene with its transcribed region (dark grey arrow), the coding sequence (white arrow with exons in light grey), the four STAT5 binding sites within its proximal promoter region, and the quantitative PCR amplicons investigated (A to H-labeled black boxes) is shown in (D). The RNA polymerase II occupancy along the transcribed region of the <i>Cis</i> gene is slightly but consistently reduced in SFN-treated cells. Two-tailed paired Student's t-test, SFN-treated compared to vehicle control (IL-3-stimulated); *<i>P</i><0.05, **<i>P</i><0.005, ***<i>P</i><0.001, ****<i>P</i><0.0001; ns, not significant.</p