Deacetylase activity is required for recruitment of the basal transcription machinery and transactivation by STAT5

Abstract

The signal transducer and activator of transcription STAT5 plays a major role in the cellular response to cytokines, but the mechanism by which it activates transcription remains poorly understood. We show here that deacetylase inhibitors (trichostatin A, suberoylanilide hydroxamic acid, and sodium butyrate) prevent induction of endogenous STAT5 target genes, implying that a deacetylase activity is required for that process. Microarray analyses revealed that this requirement is common to all STAT5 target genes. Using chromatin immunoprecipitation, we show that, following STAT5 DNA binding, deacetylase inhibitors block transcription initiation by preventing recruitment of the basal transcription machinery. This inhibition is not due to effects on histone H3 and H4 acetylation or chromatin remodeling within the promoter region. This novel mechanism of transactivation by STAT5 provides a rationale for the use of deacetylase inhibitors for therapeutic intervention in STAT5-associated cancers.

FIG. 1.

Cytokine induction of Cis and c-Myc proteins is abolished in TSA-treated murine lymphocytes. (A) Effects of TSA on protein expression in cytokine-stimulated murine B and T lymphocytes. Ba/F3-β and CTLL-2 were stimulated with IL-3 and IL-2, respectively, for the indicated times, as described in Materials and Methods. Cells were pretreated with 200 nM TSA (+) or DMSO (−) for 30 min prior to cytokine stimulation. Protein lysates were subjected to a Western blot analysis with the indicated antibodies. (B) c-Myc constitutive expression in a STAT5Aca cell line is repressed by TSA. Ba/F3-β and Ba/F3-1*6 were stimulated with IL-2 or IL-3 for 2 h in the presence of 200 nM TSA or DMSO, as described for panel A. Protein lysates were subjected to a Western blot analysis with a polyclonal antibody specific for c-Myc.

FIG. 2.

TSA inhibits induction of STAT5 target genes at the RNA level, through a primary effect. TSA specifically inhibits transcriptional induction of STAT5 target genes. (A and B) Ba/F3-β cells were stimulated with IL-3 in the absence or presence of TSA, as described in the legend to Fig. 1. mRNA levels were analyzed by real-time PCR, as described in Materials and Methods, with primers specific for STAT5 target genes (A) or control genes (B). (C) Inhibition of transcription by TSA does not require de novo protein synthesis. CTLL-2 cells were stimulated with IL-2 in the absence or presence of TSA and/or CHX, as described in Materials and Methods. mRNA levels were analyzed by real-time PCR as described above. (D) p21 expression is differentially affected by TSA in Ba/F3-β and CTLL-2 cells. Cells were treated, and mRNA levels were analyzed with primers specific for mouse p21, as described above.

FIG. 3.

A deacetylase activity is required for cytokine induction of STAT5 target genes. (A) Three unrelated inhibitors of deacetylases block induction of STAT5 target genes. Ba/F3-β cells were stimulated with IL-3 in the presence of 200 nM TSA, 10 μM SAHA, 10 mM NaB, or DMSO (−), and mRNA levels were analyzed by real-time PCR, as described for Fig. 2. (B) Titration of TSA and SAHA. Ba/F3-β cells were stimulated with IL-3 for 30 min in the presence of increasing concentrations of TSA or SAHA, and mRNA levels were analyzed by real-time PCR, as described above. Data are expressed as the fold induction relative to untreated unstimulated cells.

FIG. 4.

TSA blocks transcription initiation by preventing recruitment of the basal transcription machinery to the Cis promoter. (A) TSA does not interfere with STAT5 phosphorylation or its nuclear translocation upon cytokine stimulation. CTLL-2 cells were stimulated with IL-2 for 1 h in the absence or presence of TSA, as described for Fig. 2. Nuclear and cytosolic lysates were subjected to Western blot analysis with antibodies specific for phospho-STAT5 and STAT5b, as described in Materials and Methods. Identical results were obtained with a STAT5a-specific antibody (data not shown). (B) Structure of the murine Cis promoter. Gray boxes represent STAT5 binding sites, together with their positions relative to the transcription start site or CAP site (+1). Positions and sizes of the amplicons analyzed by ChIP in panels C to F are shown. (C to F) TSA does not interfere with STAT5 DNA binding but prevents histone acetylation and recruitment of TBP and RNA polymerase II at the CAP site of the Cis promoter. Ba/F3-β cells were stimulated with IL-3 in the absence or presence of TSA, as described for Fig. 1. At the times indicated, cells were harvested and analyzed by ChIP, with antibodies specific for STAT5a and -b (STAT5) (C), RNA polymerase II (RNA Pol II) and TBP (E), acetylated histone H3 (Ac-H3) and histone H4 (Ac-H4) (F), or no antibody as a control (D), as described in Materials and Methods. DNA samples were analyzed by real-time PCR, with primers amplifying the −184/−102 region (STAT5) or the −17/+55 CAP site region (RNA Pol II, TBP, Ac-H3, Ac-H4, and No Antibody). Identical results were obtained for STAT5 binding when the −256/−195 region was amplified (data not shown). (G and H) Effect of TSA on histone acetylation at the Osm and c-Fos genes. Ba/F3-β cells were treated, and histone acetylation was analyzed by ChIP, as described above, with primers specific for the CAP site (−21/+40, Osm) or the open reading frame (+1273/+1325, c-Fos). Histone acetylation in panels F to H is represented at identical scales for better comparison.

FIG. 5.

TSA inhibits transcription reinitiation by preventing reloading of the transcription machinery. (A) TSA rapidly inhibits Cis transcription in cytokine-stimulated cells. Ba/F3-β cells were stimulated with IL-3 in the absence of TSA for 30 min before either DMSO (−TSA) or 200 nM TSA (+TSA) was added to the cells (indicated by an arrowhead). Cells were harvested at the indicated times, and Cis mRNA levels were analyzed by real-time PCR. (B) TBP and RNA polymerase II are rapidly released from the Cis promoter upon TSA treatment of cytokine-stimulated cells. Ba/F3-β cells were treated as described for panel A, and cells were harvested at the indicated times and analyzed by ChIP, as described for Fig. 4C to F.

FIG. 6.

TSA does not affect chromatin remodeling on the Cis promoter. (A) Structure of the Cis promoter, indicating the positions of the restriction sites analyzed for panel B. Gray boxes represent STAT5 binding sites. (B) IL-3-mediated chromatin remodeling on the Cis promoter is not affected by TSA. Ba/F3-β cells were stimulated with IL-3 for 30 min in the absence or presence of 200 nM TSA, as described for Fig. 1. Unstimulated and stimulated cells were harvested, nuclei were prepared, and chromatin accessibility was analyzed by CHART-PCR, as described in Materials and Methods. Purified genomic (naked) DNA was included in a parallel reaction as a control for the cutting efficiency of the restriction enzyme. Data are expressed as the percentages of nondigested DNA (% Protection).