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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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Lysine acetylation is not required for STAT5A-1*6 transcriptional activity in Ba/F3 cells. (A–C) Ba/F3 cells were transfected with empty pcDNA3 (-) or pcDNA3-based plasmids expressing wild-type mSTAT5A, constitutively active mSTAT5A-1*6 or mSTAT5A-1*6 mutants (K>Q, K>R and Y694F), as indicated. Transfected cells were maintained for 10 h in IL-3-free medium to prevent activation of endogenous STAT5. In (C), transfected cells were treated with 200 nM TSA or 0.02% DMSO (vehicle) for the last 90 min before harvest. Transgene expression and phosphorylation of STAT5 proteins were verified by western blot analysis of Brij whole-cell lysates using FLAG- and pSTAT5-specific antibodies, respectively. α-Tubulin was used as a loading control. Expression of the STAT5 target gene Cis was investigated by quantitative RT-PCR, as before. Of note, data not shown suggest that the weaker pSTAT5 signal consistently detected for the 3xQ mutant is likely due to epitope masking, probably as a result of multiple adjacent K>Q mutations surrounding the phospho-tyrosine residue. (D) Ba/F3-tet-on-1*6 cells were grown for 22 h in the absence or presence of 1 μg/ml doxycycline (Dox) to induce mSTAT5A-1*6 expression. Cells were kept in IL-3-containing medium for the first 12 h, washed twice in RPMI 1640 and further cultivated in IL-3-deprived medium for 10 h (±Dox, -IL-3) until harvest to inactivate endogenous STAT5 activity. As a positive control for endogenous STAT5 activity, non-induced cells were maintained in IL-3-containing medium (-Dox, +IL-3) for the duration of the experiment (22 h). Cytosolic and nuclear protein lysates were analysed by western blot to monitor endogenous nuclear STAT5B protein level (STAT5B) before and after induction of STAT5A-1*6 (FLAG). α-Tubulin and HDAC1 served as cytosolic and nuclear markers, respectively, to verify the quality of cell fractionation.
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Figure 3: Lysine acetylation is not required for STAT5A-1*6 transcriptional activity in Ba/F3 cells. (A–C) Ba/F3 cells were transfected with empty pcDNA3 (-) or pcDNA3-based plasmids expressing wild-type mSTAT5A, constitutively active mSTAT5A-1*6 or mSTAT5A-1*6 mutants (K>Q, K>R and Y694F), as indicated. Transfected cells were maintained for 10 h in IL-3-free medium to prevent activation of endogenous STAT5. In (C), transfected cells were treated with 200 nM TSA or 0.02% DMSO (vehicle) for the last 90 min before harvest. Transgene expression and phosphorylation of STAT5 proteins were verified by western blot analysis of Brij whole-cell lysates using FLAG- and pSTAT5-specific antibodies, respectively. α-Tubulin was used as a loading control. Expression of the STAT5 target gene Cis was investigated by quantitative RT-PCR, as before. Of note, data not shown suggest that the weaker pSTAT5 signal consistently detected for the 3xQ mutant is likely due to epitope masking, probably as a result of multiple adjacent K>Q mutations surrounding the phospho-tyrosine residue. (D) Ba/F3-tet-on-1*6 cells were grown for 22 h in the absence or presence of 1 μg/ml doxycycline (Dox) to induce mSTAT5A-1*6 expression. Cells were kept in IL-3-containing medium for the first 12 h, washed twice in RPMI 1640 and further cultivated in IL-3-deprived medium for 10 h (±Dox, -IL-3) until harvest to inactivate endogenous STAT5 activity. As a positive control for endogenous STAT5 activity, non-induced cells were maintained in IL-3-containing medium (-Dox, +IL-3) for the duration of the experiment (22 h). Cytosolic and nuclear protein lysates were analysed by western blot to monitor endogenous nuclear STAT5B protein level (STAT5B) before and after induction of STAT5A-1*6 (FLAG). α-Tubulin and HDAC1 served as cytosolic and nuclear markers, respectively, to verify the quality of cell fractionation.

Mentions: Lysine to glutamine (K>Q) and lysine to arginine (K>R) substitutions that mimic the acetylated or unmodified lysine respectively (70) were then introduced into STAT5A-1*6. Lysine residues that were proposed to be critical for the regulation of STAT5 but also of STAT3 at equivalent positions (8–10,53–56,60)—namely K84, K359, K384, K675, K681, K689, K696 and K700—were mutated either individually or in combination (Figure 2). Transcriptional activity of the STAT5A-1*6 lysine mutants was assessed upon transfection in Ba/F3 cells maintained in IL-3-free medium, hence in conditions where endogenous wild-type STAT5 is inactive. Expression (protein level) and activation (phosphorylation) of the respective STAT5A-1*6 mutants were verified by western blot, and their transcriptional activity was evaluated by measuring expression of the STAT5 target gene Cis by quantitative RT-PCR. Neither single nor multiple lysine mutations impaired STAT5A-1*6 transcriptional activity (Figure 3A and B). Of note, STAT5A-1*6 proteins mutated at lysine 675 (K675Q, K675R as well as 5xQ and 5xR) were poorly detectable in western blot, likely due to protein instability, and thus their activity could not be readily assessed. Nevertheless, these results indicate that acetylation at the investigated lysines, notably at K359, K689 or K696 previously reported to be acetylated and important for STAT5 activity (8,10,56), is not required for STAT5A-1*6 activity in Ba/F3 cells. These experiments therefore indicate that the impaired STAT5 transcriptional activity induced by deacetylase inhibitors is unlikely to be due to a change in STAT5 acetylation, at least at the investigated lysines. This proposition is further supported by the important finding that the transcriptional activity of STAT5A-1*6 3xQ/R proteins, mutated simultaneously at K689, K696 and K700, remained sensitive to TSA (Figure 3C). Of note, the observation that the STAT5A-1*6 K696Q/R mutants are as active as the non-mutated STAT5A-1*6 protein indicates that not only acetylation but also SUMOylation at K696 is not required for STAT5A-1*6 transcriptional activity in Ba/F3 cells.


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)

Lysine acetylation is not required for STAT5A-1*6 transcriptional activity in Ba/F3 cells. (A–C) Ba/F3 cells were transfected with empty pcDNA3 (-) or pcDNA3-based plasmids expressing wild-type mSTAT5A, constitutively active mSTAT5A-1*6 or mSTAT5A-1*6 mutants (K>Q, K>R and Y694F), as indicated. Transfected cells were maintained for 10 h in IL-3-free medium to prevent activation of endogenous STAT5. In (C), transfected cells were treated with 200 nM TSA or 0.02% DMSO (vehicle) for the last 90 min before harvest. Transgene expression and phosphorylation of STAT5 proteins were verified by western blot analysis of Brij whole-cell lysates using FLAG- and pSTAT5-specific antibodies, respectively. α-Tubulin was used as a loading control. Expression of the STAT5 target gene Cis was investigated by quantitative RT-PCR, as before. Of note, data not shown suggest that the weaker pSTAT5 signal consistently detected for the 3xQ mutant is likely due to epitope masking, probably as a result of multiple adjacent K>Q mutations surrounding the phospho-tyrosine residue. (D) Ba/F3-tet-on-1*6 cells were grown for 22 h in the absence or presence of 1 μg/ml doxycycline (Dox) to induce mSTAT5A-1*6 expression. Cells were kept in IL-3-containing medium for the first 12 h, washed twice in RPMI 1640 and further cultivated in IL-3-deprived medium for 10 h (±Dox, -IL-3) until harvest to inactivate endogenous STAT5 activity. As a positive control for endogenous STAT5 activity, non-induced cells were maintained in IL-3-containing medium (-Dox, +IL-3) for the duration of the experiment (22 h). Cytosolic and nuclear protein lysates were analysed by western blot to monitor endogenous nuclear STAT5B protein level (STAT5B) before and after induction of STAT5A-1*6 (FLAG). α-Tubulin and HDAC1 served as cytosolic and nuclear markers, respectively, to verify the quality of cell fractionation.
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Figure 3: Lysine acetylation is not required for STAT5A-1*6 transcriptional activity in Ba/F3 cells. (A–C) Ba/F3 cells were transfected with empty pcDNA3 (-) or pcDNA3-based plasmids expressing wild-type mSTAT5A, constitutively active mSTAT5A-1*6 or mSTAT5A-1*6 mutants (K>Q, K>R and Y694F), as indicated. Transfected cells were maintained for 10 h in IL-3-free medium to prevent activation of endogenous STAT5. In (C), transfected cells were treated with 200 nM TSA or 0.02% DMSO (vehicle) for the last 90 min before harvest. Transgene expression and phosphorylation of STAT5 proteins were verified by western blot analysis of Brij whole-cell lysates using FLAG- and pSTAT5-specific antibodies, respectively. α-Tubulin was used as a loading control. Expression of the STAT5 target gene Cis was investigated by quantitative RT-PCR, as before. Of note, data not shown suggest that the weaker pSTAT5 signal consistently detected for the 3xQ mutant is likely due to epitope masking, probably as a result of multiple adjacent K>Q mutations surrounding the phospho-tyrosine residue. (D) Ba/F3-tet-on-1*6 cells were grown for 22 h in the absence or presence of 1 μg/ml doxycycline (Dox) to induce mSTAT5A-1*6 expression. Cells were kept in IL-3-containing medium for the first 12 h, washed twice in RPMI 1640 and further cultivated in IL-3-deprived medium for 10 h (±Dox, -IL-3) until harvest to inactivate endogenous STAT5 activity. As a positive control for endogenous STAT5 activity, non-induced cells were maintained in IL-3-containing medium (-Dox, +IL-3) for the duration of the experiment (22 h). Cytosolic and nuclear protein lysates were analysed by western blot to monitor endogenous nuclear STAT5B protein level (STAT5B) before and after induction of STAT5A-1*6 (FLAG). α-Tubulin and HDAC1 served as cytosolic and nuclear markers, respectively, to verify the quality of cell fractionation.
Mentions: Lysine to glutamine (K>Q) and lysine to arginine (K>R) substitutions that mimic the acetylated or unmodified lysine respectively (70) were then introduced into STAT5A-1*6. Lysine residues that were proposed to be critical for the regulation of STAT5 but also of STAT3 at equivalent positions (8–10,53–56,60)—namely K84, K359, K384, K675, K681, K689, K696 and K700—were mutated either individually or in combination (Figure 2). Transcriptional activity of the STAT5A-1*6 lysine mutants was assessed upon transfection in Ba/F3 cells maintained in IL-3-free medium, hence in conditions where endogenous wild-type STAT5 is inactive. Expression (protein level) and activation (phosphorylation) of the respective STAT5A-1*6 mutants were verified by western blot, and their transcriptional activity was evaluated by measuring expression of the STAT5 target gene Cis by quantitative RT-PCR. Neither single nor multiple lysine mutations impaired STAT5A-1*6 transcriptional activity (Figure 3A and B). Of note, STAT5A-1*6 proteins mutated at lysine 675 (K675Q, K675R as well as 5xQ and 5xR) were poorly detectable in western blot, likely due to protein instability, and thus their activity could not be readily assessed. Nevertheless, these results indicate that acetylation at the investigated lysines, notably at K359, K689 or K696 previously reported to be acetylated and important for STAT5 activity (8,10,56), is not required for STAT5A-1*6 activity in Ba/F3 cells. These experiments therefore indicate that the impaired STAT5 transcriptional activity induced by deacetylase inhibitors is unlikely to be due to a change in STAT5 acetylation, at least at the investigated lysines. This proposition is further supported by the important finding that the transcriptional activity of STAT5A-1*6 3xQ/R proteins, mutated simultaneously at K689, K696 and K700, remained sensitive to TSA (Figure 3C). Of note, the observation that the STAT5A-1*6 K696Q/R mutants are as active as the non-mutated STAT5A-1*6 protein indicates that not only acetylation but also SUMOylation at K696 is not required for STAT5A-1*6 transcriptional activity in Ba/F3 cells.

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