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Histone Deacetylase 3 and 4 Complex Stimulates the Transcriptional Activity of the Mineralocorticoid Receptor.

Lee HA, Song MJ, Seok YM, Kang SH, Kim SY, Kim I - PLoS ONE (2015)

Bottom Line: The transcriptional activity of MR was significantly decreased by inhibitors of PKA (H89), PP1/2 (calyculin A), class I HDACs (MS-275), but not class II HDACs (MC1568).Interaction between MR and HDAC3 was significantly decreased by H89, calyculin A, and HDAC4 siRNA.A non-genomic effect of MR via PKA and PP1/2 induced nuclear translocation of HDAC4 to facilitate the interaction between MR and HDAC3.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu, 41944, Republic of Korea; Cardiovascular Research Institute, School of Medicine, Kyungpook National University, Daegu, 41944, Republic of Korea; Cell and Matrix Research Institute, School of Medicine, Kyungpook National University, Daegu, 41944, Republic of Korea.

ABSTRACT
Histone deacetylases (HDACs) act as corepressors in gene transcription by altering the acetylation of histones, resulting in epigenetic gene silencing. We previously reported that HDAC3 acts as a coactivator of the mineralocorticoid receptor (MR). Although HDAC3 forms complexes with class II HDACs, their potential role in the transcriptional activity of MR is unclear. We hypothesized that HDAC4 of the class II family stimulates the transcriptional activity of MR. The expression of MR target genes was measured by quantitative real-time PCR. MR and RNA polymerase II recruitment to promoters of MR target genes was analyzed by chromatin immunoprecipitation. The association of MR with HDACs was investigated by co-immunoprecipitation. MR acetylation was determined with an anti-acetyl-lysine antibody after immunoprecipitation with an anti-MR antibody. Among the class II HDACs, HDAC4 interacted with both MR and HDAC3 after aldosterone stimulation. The nuclear translocation of HDAC4 was mediated by protein kinase A (PKA) and protein phosphatases (PP). The transcriptional activity of MR was significantly decreased by inhibitors of PKA (H89), PP1/2 (calyculin A), class I HDACs (MS-275), but not class II HDACs (MC1568). MR acetylation was increased by H89, calyculin A, and MS-275, but not by MC1568. Interaction between MR and HDAC3 was significantly decreased by H89, calyculin A, and HDAC4 siRNA. A non-genomic effect of MR via PKA and PP1/2 induced nuclear translocation of HDAC4 to facilitate the interaction between MR and HDAC3. Thus, we have uncovered a crucial role for a class II HDAC in the activation of MR-dependent transcription.

No MeSH data available.


HDAC4 acts as a scaffold between the MR and HDAC3.Treatment with Aldo resulted in increased expression of GILZ (A) and SGK-1 (B), which was decreased by knockdown of HDAC4. Graph shows the means ± SE of three independent experiments (*p < 0.05 vs. vehicle, #p<0.05 Scramble vs. siRNA). HDAC4 knockdown decreased the Aldo-induced recruitment of MR and Pol II to GILZ (C) and SGK-1 (D) promoters. Schematic diagrams show the locations of HRE and PCR amplification after chromatin immunoprecipitation (ChIP) in GILZ (C) and SGK-1 (D) promoters. Graph shows the means ± SE of three independent experiments (*p < 0.05 vs. vehicle, #p<0.05 Scramble vs. siRNA). E, HDAC4 protein was significantly decreased after 48 h of siRNA transfection. F, The interaction between MR and HDAC3 induced by Aldo was inhibited by knockdown of HDAC4. G, Acetylation of MR was increased by HDAC4 knockdown when HEK293 cells were stimulated by Aldo.
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pone.0136801.g003: HDAC4 acts as a scaffold between the MR and HDAC3.Treatment with Aldo resulted in increased expression of GILZ (A) and SGK-1 (B), which was decreased by knockdown of HDAC4. Graph shows the means ± SE of three independent experiments (*p < 0.05 vs. vehicle, #p<0.05 Scramble vs. siRNA). HDAC4 knockdown decreased the Aldo-induced recruitment of MR and Pol II to GILZ (C) and SGK-1 (D) promoters. Schematic diagrams show the locations of HRE and PCR amplification after chromatin immunoprecipitation (ChIP) in GILZ (C) and SGK-1 (D) promoters. Graph shows the means ± SE of three independent experiments (*p < 0.05 vs. vehicle, #p<0.05 Scramble vs. siRNA). E, HDAC4 protein was significantly decreased after 48 h of siRNA transfection. F, The interaction between MR and HDAC3 induced by Aldo was inhibited by knockdown of HDAC4. G, Acetylation of MR was increased by HDAC4 knockdown when HEK293 cells were stimulated by Aldo.

Mentions: The effect of MS-275, a class I HDAC inhibitor (HDAC1, 2, and 3), and MC1568, a class II HDAC inhibitor (HDAC4 and 6), on MR target gene expression was investigated by qRT-PCR. Aldo treatment increased the expression of Glucocorticoid-induced leucine zipper (GILZ) by approximately 209% which was about 65% attenuated by MS-275 but not by MC1568 (Fig 2A). Expression of serum/glucocorticoid-induced protein kinase-1 (SGK-1) showed a similar pattern to that of GILZ in the presence of MS-275 and MC1568 (Fig 2B). Recruitment of MR and Pol II to the GILZ and SGK-1 promoters was analyzed by chromatin immunoprecipitation (ChIP). The results of the ChIP assays showed that treatment with Aldo enriched MR (~306%) and Pol II (~232%) on the HRE sequence of Gilz, which was decreased by MS-275 (MR: ~70%; PolII: ~83%) but not by MC1568 (Fig 2C). Similarly, the enrichment of MR and Pol II on the SGK-1 promoter was significantly induced by Aldo (MR: ~294%; PolII: ~206%), which was inhibited by MS-275 (MR: ~71%; PolII: ~74%) but not by MC1568 (Fig 2D). The interaction between MR and HDAC3 in the nucleus increased when the cells were treated with Aldo, which was unaffected by MS-275 or MC1568 (Fig 2E). To check if the enrichments were specific to the promoter regions examined, we analyzed the enrichment of MR and PolII at a 2 kb fragment upstream of each HRE. As expected, Aldo treatment did not enrich the proteins at the upstream genomic region (S1 Fig). We also analyzed MR acetylation, which reduces the DNA binding affinity of MR. HDAC3 inhibition by MS-275 significantly increased MR acetylation, whereas HDAC4 inhibition by MC1568 showed little effect on the acetylation status of MR (Fig 2F). Therefore, we speculated that HDAC4 has a role other than MR deacetylation in regulating MR activity. To address this hypothesis, we knocked down HDAC4 with siRNA and then analyzed the transcriptional activity of MR. Depletion of HDAC4 resulted in an approximate 77% and 73% decreased Aldo-induced expression of GILZ (Fig 3A) and SGK-1 (Fig 3B), respectively. Recruitment of MR and Pol II to the GILZ (Fig 3C) and SGK-1 (Fig 3D) promoters was significantly attenuated by knockdown of HDAC4 (GILZ (MR: 42%; PolII: 60%)) (SGK-1 (MR: 83%; PolII: 67%). As expected, HDAC4 protein levels significantly decreased in HEK293 cells transfected with siRNA targeting HDAC4 but HDAC3 protein levels remained unchanged (Fig 3E). Interestingly, HDAC4 knockdown decreased the interaction between MR and HDAC3 in the nucleus of Aldo-treated HEK293 cells (Fig 3F). HDAC4 knockdown showed little effect on MR translocation to the nucleus (Fig 3F), whereas it increased Aldo-stimulated MR acetylation in the nucleus (Fig 3G). Together, these data reveal that HDAC4 has a critical role in MR transcriptional activation and increases the interaction between MR and HDAC3.


Histone Deacetylase 3 and 4 Complex Stimulates the Transcriptional Activity of the Mineralocorticoid Receptor.

Lee HA, Song MJ, Seok YM, Kang SH, Kim SY, Kim I - PLoS ONE (2015)

HDAC4 acts as a scaffold between the MR and HDAC3.Treatment with Aldo resulted in increased expression of GILZ (A) and SGK-1 (B), which was decreased by knockdown of HDAC4. Graph shows the means ± SE of three independent experiments (*p < 0.05 vs. vehicle, #p<0.05 Scramble vs. siRNA). HDAC4 knockdown decreased the Aldo-induced recruitment of MR and Pol II to GILZ (C) and SGK-1 (D) promoters. Schematic diagrams show the locations of HRE and PCR amplification after chromatin immunoprecipitation (ChIP) in GILZ (C) and SGK-1 (D) promoters. Graph shows the means ± SE of three independent experiments (*p < 0.05 vs. vehicle, #p<0.05 Scramble vs. siRNA). E, HDAC4 protein was significantly decreased after 48 h of siRNA transfection. F, The interaction between MR and HDAC3 induced by Aldo was inhibited by knockdown of HDAC4. G, Acetylation of MR was increased by HDAC4 knockdown when HEK293 cells were stimulated by Aldo.
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pone.0136801.g003: HDAC4 acts as a scaffold between the MR and HDAC3.Treatment with Aldo resulted in increased expression of GILZ (A) and SGK-1 (B), which was decreased by knockdown of HDAC4. Graph shows the means ± SE of three independent experiments (*p < 0.05 vs. vehicle, #p<0.05 Scramble vs. siRNA). HDAC4 knockdown decreased the Aldo-induced recruitment of MR and Pol II to GILZ (C) and SGK-1 (D) promoters. Schematic diagrams show the locations of HRE and PCR amplification after chromatin immunoprecipitation (ChIP) in GILZ (C) and SGK-1 (D) promoters. Graph shows the means ± SE of three independent experiments (*p < 0.05 vs. vehicle, #p<0.05 Scramble vs. siRNA). E, HDAC4 protein was significantly decreased after 48 h of siRNA transfection. F, The interaction between MR and HDAC3 induced by Aldo was inhibited by knockdown of HDAC4. G, Acetylation of MR was increased by HDAC4 knockdown when HEK293 cells were stimulated by Aldo.
Mentions: The effect of MS-275, a class I HDAC inhibitor (HDAC1, 2, and 3), and MC1568, a class II HDAC inhibitor (HDAC4 and 6), on MR target gene expression was investigated by qRT-PCR. Aldo treatment increased the expression of Glucocorticoid-induced leucine zipper (GILZ) by approximately 209% which was about 65% attenuated by MS-275 but not by MC1568 (Fig 2A). Expression of serum/glucocorticoid-induced protein kinase-1 (SGK-1) showed a similar pattern to that of GILZ in the presence of MS-275 and MC1568 (Fig 2B). Recruitment of MR and Pol II to the GILZ and SGK-1 promoters was analyzed by chromatin immunoprecipitation (ChIP). The results of the ChIP assays showed that treatment with Aldo enriched MR (~306%) and Pol II (~232%) on the HRE sequence of Gilz, which was decreased by MS-275 (MR: ~70%; PolII: ~83%) but not by MC1568 (Fig 2C). Similarly, the enrichment of MR and Pol II on the SGK-1 promoter was significantly induced by Aldo (MR: ~294%; PolII: ~206%), which was inhibited by MS-275 (MR: ~71%; PolII: ~74%) but not by MC1568 (Fig 2D). The interaction between MR and HDAC3 in the nucleus increased when the cells were treated with Aldo, which was unaffected by MS-275 or MC1568 (Fig 2E). To check if the enrichments were specific to the promoter regions examined, we analyzed the enrichment of MR and PolII at a 2 kb fragment upstream of each HRE. As expected, Aldo treatment did not enrich the proteins at the upstream genomic region (S1 Fig). We also analyzed MR acetylation, which reduces the DNA binding affinity of MR. HDAC3 inhibition by MS-275 significantly increased MR acetylation, whereas HDAC4 inhibition by MC1568 showed little effect on the acetylation status of MR (Fig 2F). Therefore, we speculated that HDAC4 has a role other than MR deacetylation in regulating MR activity. To address this hypothesis, we knocked down HDAC4 with siRNA and then analyzed the transcriptional activity of MR. Depletion of HDAC4 resulted in an approximate 77% and 73% decreased Aldo-induced expression of GILZ (Fig 3A) and SGK-1 (Fig 3B), respectively. Recruitment of MR and Pol II to the GILZ (Fig 3C) and SGK-1 (Fig 3D) promoters was significantly attenuated by knockdown of HDAC4 (GILZ (MR: 42%; PolII: 60%)) (SGK-1 (MR: 83%; PolII: 67%). As expected, HDAC4 protein levels significantly decreased in HEK293 cells transfected with siRNA targeting HDAC4 but HDAC3 protein levels remained unchanged (Fig 3E). Interestingly, HDAC4 knockdown decreased the interaction between MR and HDAC3 in the nucleus of Aldo-treated HEK293 cells (Fig 3F). HDAC4 knockdown showed little effect on MR translocation to the nucleus (Fig 3F), whereas it increased Aldo-stimulated MR acetylation in the nucleus (Fig 3G). Together, these data reveal that HDAC4 has a critical role in MR transcriptional activation and increases the interaction between MR and HDAC3.

Bottom Line: The transcriptional activity of MR was significantly decreased by inhibitors of PKA (H89), PP1/2 (calyculin A), class I HDACs (MS-275), but not class II HDACs (MC1568).Interaction between MR and HDAC3 was significantly decreased by H89, calyculin A, and HDAC4 siRNA.A non-genomic effect of MR via PKA and PP1/2 induced nuclear translocation of HDAC4 to facilitate the interaction between MR and HDAC3.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu, 41944, Republic of Korea; Cardiovascular Research Institute, School of Medicine, Kyungpook National University, Daegu, 41944, Republic of Korea; Cell and Matrix Research Institute, School of Medicine, Kyungpook National University, Daegu, 41944, Republic of Korea.

ABSTRACT
Histone deacetylases (HDACs) act as corepressors in gene transcription by altering the acetylation of histones, resulting in epigenetic gene silencing. We previously reported that HDAC3 acts as a coactivator of the mineralocorticoid receptor (MR). Although HDAC3 forms complexes with class II HDACs, their potential role in the transcriptional activity of MR is unclear. We hypothesized that HDAC4 of the class II family stimulates the transcriptional activity of MR. The expression of MR target genes was measured by quantitative real-time PCR. MR and RNA polymerase II recruitment to promoters of MR target genes was analyzed by chromatin immunoprecipitation. The association of MR with HDACs was investigated by co-immunoprecipitation. MR acetylation was determined with an anti-acetyl-lysine antibody after immunoprecipitation with an anti-MR antibody. Among the class II HDACs, HDAC4 interacted with both MR and HDAC3 after aldosterone stimulation. The nuclear translocation of HDAC4 was mediated by protein kinase A (PKA) and protein phosphatases (PP). The transcriptional activity of MR was significantly decreased by inhibitors of PKA (H89), PP1/2 (calyculin A), class I HDACs (MS-275), but not class II HDACs (MC1568). MR acetylation was increased by H89, calyculin A, and MS-275, but not by MC1568. Interaction between MR and HDAC3 was significantly decreased by H89, calyculin A, and HDAC4 siRNA. A non-genomic effect of MR via PKA and PP1/2 induced nuclear translocation of HDAC4 to facilitate the interaction between MR and HDAC3. Thus, we have uncovered a crucial role for a class II HDAC in the activation of MR-dependent transcription.

No MeSH data available.