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Deacetylase inhibitors repress STAT5-mediated transcription by interfering with bromodomain and extra-terminal (BET) protein function.

Pinz S, Unser S, Buob D, Fischer P, Jobst B, Rascle A - Nucleic Acids Res. (2015)

Bottom Line: We showed previously that the deacetylase inhibitor trichostatin A (TSA) inhibits STAT5-mediated transcription by preventing recruitment of the transcriptional machinery at a step following STAT5 binding to DNA.The mechanism and factors involved in this inhibition remain unknown.Instead, they induce a rapid increase in global histone acetylation apparently resulting in the delocalization of the bromodomain and extra-terminal (BET) protein Brd2 and of the Brd2-associated factor TBP to hyperacetylated chromatin.

View Article: PubMed Central - PubMed

Affiliation: Stat5 Signaling Research Group, Institute of Immunology, University of Regensburg, 93053 Regensburg, Germany.

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The nuclear BET protein Brd2 is rapidly relocalized to the chromatin fraction together with acetylated histones upon treatment with deacetylase inhibitors that inhibit STAT5 activity. (A) Ba/F3 cells were treated with 200 nM TSA, 10 μM MG132, both (T+MG) or vehicle (0.12% DMSO final in all conditions). Cells were treated for a total duration of ∼2 h, with a 45 min MG132 pre-treatment followed by a 90 min TSA treatment. Soluble and insoluble nuclear fractions were analysed by western blot using antibodies against Brd2 and the nuclear marker HDAC1 as loading control. Similar results were obtained upon 3 h pre-treatment with 10 μM MG132 followed by 90 min incubation with 200 nM TSA (4.5 h treatment in total; not shown). (B) Ba/F3 cells were rested for 6 h and stimulated with IL-3 for 60 min. Prior to IL-3 stimulation, cells were pre-treated 3.5 h with 10 μM MG132, 30 min with 200 nM TSA, both (3h MG132 followed by 30 min TSA) or vehicle (0.12% DMSO final in all conditions). Hence cells were incubated 4.5 h in total with 10 μM MG132 and 90 min with 200 nM TSA. Expression of the STAT5 target gene Cis, of the MG132-regulated gene hsp70 and of Brd2 and 36b4 genes as controls was monitored by quantitative RT-PCR. (C) Ba/F3 cells were treated for the indicated times with 200 nM TSA, 3 mM valproic acid (VPA), 500 nM apicidin (Api.), 1 μM MGCD0103 (MGC), 5 μM MS-275 (MS), or vehicle (Veh.; 0.02% DMSO final in all conditions). Cytosolic as well as soluble and insoluble nuclear fractions were analysed by western blot using the indicated antibodies. As before, α-tubulin and HDAC1 served as cytosolic and nuclear markers, respectively, to control cell fractionation.
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Figure 10: The nuclear BET protein Brd2 is rapidly relocalized to the chromatin fraction together with acetylated histones upon treatment with deacetylase inhibitors that inhibit STAT5 activity. (A) Ba/F3 cells were treated with 200 nM TSA, 10 μM MG132, both (T+MG) or vehicle (0.12% DMSO final in all conditions). Cells were treated for a total duration of ∼2 h, with a 45 min MG132 pre-treatment followed by a 90 min TSA treatment. Soluble and insoluble nuclear fractions were analysed by western blot using antibodies against Brd2 and the nuclear marker HDAC1 as loading control. Similar results were obtained upon 3 h pre-treatment with 10 μM MG132 followed by 90 min incubation with 200 nM TSA (4.5 h treatment in total; not shown). (B) Ba/F3 cells were rested for 6 h and stimulated with IL-3 for 60 min. Prior to IL-3 stimulation, cells were pre-treated 3.5 h with 10 μM MG132, 30 min with 200 nM TSA, both (3h MG132 followed by 30 min TSA) or vehicle (0.12% DMSO final in all conditions). Hence cells were incubated 4.5 h in total with 10 μM MG132 and 90 min with 200 nM TSA. Expression of the STAT5 target gene Cis, of the MG132-regulated gene hsp70 and of Brd2 and 36b4 genes as controls was monitored by quantitative RT-PCR. (C) Ba/F3 cells were treated for the indicated times with 200 nM TSA, 3 mM valproic acid (VPA), 500 nM apicidin (Api.), 1 μM MGCD0103 (MGC), 5 μM MS-275 (MS), or vehicle (Veh.; 0.02% DMSO final in all conditions). Cytosolic as well as soluble and insoluble nuclear fractions were analysed by western blot using the indicated antibodies. As before, α-tubulin and HDAC1 served as cytosolic and nuclear markers, respectively, to control cell fractionation.

Mentions: Since the above results suggested a possible implication of Brd2 in STAT5-mediated transcription in Ba/F3 cells, we investigated whether and how TSA could interfere with Brd2 function. We first assessed the subcellular localization of Brd2 in TSA-treated and untreated Ba/F3 cells by cell fractionation and western blot analysis (Figure 10). Brd2 was exclusively detected in nuclear extracts of Ba/F3 cells (Figure 10C), within both the soluble and insoluble (i.e. mainly chromatin-associated) nuclear fractions (Figure 10A and C). Remarkably, upon TSA treatment, Brd2 protein was depleted from the soluble nuclear fraction while it was still detectable in the insoluble nuclear fraction (Figure 10A and C). The depletion of Brd2 from the soluble nuclear fraction was not due to proteasome-dependent protein degradation since it was not prevented by pre-treatment with the proteasome inhibitor MG132 (Figure 10A). MG132 activity was verified by measuring the mRNA levels of hsp70 (Figure 10B), an acknowledged MG132-induced gene (87). The drop in the level of soluble nuclear Brd2 protein in TSA-treated cells was also not the consequence of a reduced Brd2 gene expression, since Brd2 mRNA levels were not affected by TSA (Figure 10B). These observations suggest that delocalization of nuclear Brd2 from the soluble to the insoluble chromatin fraction following TSA treatment could limit its availability to support STAT5-mediated transcription.


Deacetylase inhibitors repress STAT5-mediated transcription by interfering with bromodomain and extra-terminal (BET) protein function.

Pinz S, Unser S, Buob D, Fischer P, Jobst B, Rascle A - Nucleic Acids Res. (2015)

The nuclear BET protein Brd2 is rapidly relocalized to the chromatin fraction together with acetylated histones upon treatment with deacetylase inhibitors that inhibit STAT5 activity. (A) Ba/F3 cells were treated with 200 nM TSA, 10 μM MG132, both (T+MG) or vehicle (0.12% DMSO final in all conditions). Cells were treated for a total duration of ∼2 h, with a 45 min MG132 pre-treatment followed by a 90 min TSA treatment. Soluble and insoluble nuclear fractions were analysed by western blot using antibodies against Brd2 and the nuclear marker HDAC1 as loading control. Similar results were obtained upon 3 h pre-treatment with 10 μM MG132 followed by 90 min incubation with 200 nM TSA (4.5 h treatment in total; not shown). (B) Ba/F3 cells were rested for 6 h and stimulated with IL-3 for 60 min. Prior to IL-3 stimulation, cells were pre-treated 3.5 h with 10 μM MG132, 30 min with 200 nM TSA, both (3h MG132 followed by 30 min TSA) or vehicle (0.12% DMSO final in all conditions). Hence cells were incubated 4.5 h in total with 10 μM MG132 and 90 min with 200 nM TSA. Expression of the STAT5 target gene Cis, of the MG132-regulated gene hsp70 and of Brd2 and 36b4 genes as controls was monitored by quantitative RT-PCR. (C) Ba/F3 cells were treated for the indicated times with 200 nM TSA, 3 mM valproic acid (VPA), 500 nM apicidin (Api.), 1 μM MGCD0103 (MGC), 5 μM MS-275 (MS), or vehicle (Veh.; 0.02% DMSO final in all conditions). Cytosolic as well as soluble and insoluble nuclear fractions were analysed by western blot using the indicated antibodies. As before, α-tubulin and HDAC1 served as cytosolic and nuclear markers, respectively, to control cell fractionation.
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Figure 10: The nuclear BET protein Brd2 is rapidly relocalized to the chromatin fraction together with acetylated histones upon treatment with deacetylase inhibitors that inhibit STAT5 activity. (A) Ba/F3 cells were treated with 200 nM TSA, 10 μM MG132, both (T+MG) or vehicle (0.12% DMSO final in all conditions). Cells were treated for a total duration of ∼2 h, with a 45 min MG132 pre-treatment followed by a 90 min TSA treatment. Soluble and insoluble nuclear fractions were analysed by western blot using antibodies against Brd2 and the nuclear marker HDAC1 as loading control. Similar results were obtained upon 3 h pre-treatment with 10 μM MG132 followed by 90 min incubation with 200 nM TSA (4.5 h treatment in total; not shown). (B) Ba/F3 cells were rested for 6 h and stimulated with IL-3 for 60 min. Prior to IL-3 stimulation, cells were pre-treated 3.5 h with 10 μM MG132, 30 min with 200 nM TSA, both (3h MG132 followed by 30 min TSA) or vehicle (0.12% DMSO final in all conditions). Hence cells were incubated 4.5 h in total with 10 μM MG132 and 90 min with 200 nM TSA. Expression of the STAT5 target gene Cis, of the MG132-regulated gene hsp70 and of Brd2 and 36b4 genes as controls was monitored by quantitative RT-PCR. (C) Ba/F3 cells were treated for the indicated times with 200 nM TSA, 3 mM valproic acid (VPA), 500 nM apicidin (Api.), 1 μM MGCD0103 (MGC), 5 μM MS-275 (MS), or vehicle (Veh.; 0.02% DMSO final in all conditions). Cytosolic as well as soluble and insoluble nuclear fractions were analysed by western blot using the indicated antibodies. As before, α-tubulin and HDAC1 served as cytosolic and nuclear markers, respectively, to control cell fractionation.
Mentions: Since the above results suggested a possible implication of Brd2 in STAT5-mediated transcription in Ba/F3 cells, we investigated whether and how TSA could interfere with Brd2 function. We first assessed the subcellular localization of Brd2 in TSA-treated and untreated Ba/F3 cells by cell fractionation and western blot analysis (Figure 10). Brd2 was exclusively detected in nuclear extracts of Ba/F3 cells (Figure 10C), within both the soluble and insoluble (i.e. mainly chromatin-associated) nuclear fractions (Figure 10A and C). Remarkably, upon TSA treatment, Brd2 protein was depleted from the soluble nuclear fraction while it was still detectable in the insoluble nuclear fraction (Figure 10A and C). The depletion of Brd2 from the soluble nuclear fraction was not due to proteasome-dependent protein degradation since it was not prevented by pre-treatment with the proteasome inhibitor MG132 (Figure 10A). MG132 activity was verified by measuring the mRNA levels of hsp70 (Figure 10B), an acknowledged MG132-induced gene (87). The drop in the level of soluble nuclear Brd2 protein in TSA-treated cells was also not the consequence of a reduced Brd2 gene expression, since Brd2 mRNA levels were not affected by TSA (Figure 10B). These observations suggest that delocalization of nuclear Brd2 from the soluble to the insoluble chromatin fraction following TSA treatment could limit its availability to support STAT5-mediated transcription.

Bottom Line: We showed previously that the deacetylase inhibitor trichostatin A (TSA) inhibits STAT5-mediated transcription by preventing recruitment of the transcriptional machinery at a step following STAT5 binding to DNA.The mechanism and factors involved in this inhibition remain unknown.Instead, they induce a rapid increase in global histone acetylation apparently resulting in the delocalization of the bromodomain and extra-terminal (BET) protein Brd2 and of the Brd2-associated factor TBP to hyperacetylated chromatin.

View Article: PubMed Central - PubMed

Affiliation: Stat5 Signaling Research Group, Institute of Immunology, University of Regensburg, 93053 Regensburg, Germany.

Show MeSH
Related in: MedlinePlus