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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.


Location of mutant HDAC4 is not affected by H89 or calyculin A.A, HEK293 cells were transfected with GFP-tagged-mutant HDAC4 plasmid. After 48 h of transfection, cells were stimulated with Aldo or FSK for 30 min with or without pretreatment with H89 or calyculin A for 6 h, and analyzed by fluorescence microscopy. HEK293 cells were co-transfected with HA-tagged MR and Flag-tagged wild-type HDAC4 (B) or mutant HDAC4 (C). The cells were stimulated with Aldo for 30 min with or without pretreatment with H89 or calyculin A, and then fractionated. The interaction between MR and HDAC4 in each fraction was analyzed by a co-immunoprecipitation assay.
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pone.0136801.g007: Location of mutant HDAC4 is not affected by H89 or calyculin A.A, HEK293 cells were transfected with GFP-tagged-mutant HDAC4 plasmid. After 48 h of transfection, cells were stimulated with Aldo or FSK for 30 min with or without pretreatment with H89 or calyculin A for 6 h, and analyzed by fluorescence microscopy. HEK293 cells were co-transfected with HA-tagged MR and Flag-tagged wild-type HDAC4 (B) or mutant HDAC4 (C). The cells were stimulated with Aldo for 30 min with or without pretreatment with H89 or calyculin A, and then fractionated. The interaction between MR and HDAC4 in each fraction was analyzed by a co-immunoprecipitation assay.

Mentions: We investigated the effect of H89 and calyculin A on the transcriptional activity of MR, because H89 and calyculin A showed inhibitory effects on HDAC4 translocation (Fig 5). In addition, HDAC4 knockdown decreased the transcriptional activity of MR (Fig 3). Expression of GILZ (Fig 6A) and SGK-1 (Fig 6B) induced by Aldo was significantly inhibited by pretreatment of cells with H89 (GILZ: ~74%; SGK-1: ~48%) or calyculin A (GILZ: ~68%; SGK-1: ~39%). Enrichment of MR and Pol II on the GILZ (Fig 6C) and SGK-1 (Fig 6D) promoters induced by Aldo were also significantly reduced by H89 and calyculin A. The interaction between MR and HDAC3 induced by Aldo was also inhibited by H89 and calyculin A treatment (Fig 6E). However, MR translocation into the nucleus was not affected by H89 or calyculin A (Fig 6E). Since H89 and calyculin A treatments decreased the interaction between MR and HDAC3, we investigated the effect of H89 and calyculin A on MR acetylation. H89 and calyculin A resulted in increased acetylation of the MR when HEK293 cells were stimulated with Aldo (Fig 6F). Previous studies showed that dephosphorylation by PP2A induced the translocation of HDAC4 from the cytosol into the nucleus [24, 25]. The effect of HDAC4 phosphorylation on its localization was investigated using phosphomutant HDAC4 (S246A, S467A, S632A). Strikingly, phosphomutant HDAC4 was nuclear even without Aldo stimulation, and H89 and calyculin A had no effect on the nuclear localization of phosphomutant HDAC4 (Fig 7A). Aldo induced an interaction between MR and wild-type HDAC4 in the nucleus of HEK293 cells, which was inhibited by H89 and calyculin A (Fig 7B). In contrast, although phosphomutant HDAC4 interacted with MR in the nucleus of cells treated with Aldo, this association was not affected by H89 and calyculin A (Fig 7C).


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)

Location of mutant HDAC4 is not affected by H89 or calyculin A.A, HEK293 cells were transfected with GFP-tagged-mutant HDAC4 plasmid. After 48 h of transfection, cells were stimulated with Aldo or FSK for 30 min with or without pretreatment with H89 or calyculin A for 6 h, and analyzed by fluorescence microscopy. HEK293 cells were co-transfected with HA-tagged MR and Flag-tagged wild-type HDAC4 (B) or mutant HDAC4 (C). The cells were stimulated with Aldo for 30 min with or without pretreatment with H89 or calyculin A, and then fractionated. The interaction between MR and HDAC4 in each fraction was analyzed by a co-immunoprecipitation assay.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4549324&req=5

pone.0136801.g007: Location of mutant HDAC4 is not affected by H89 or calyculin A.A, HEK293 cells were transfected with GFP-tagged-mutant HDAC4 plasmid. After 48 h of transfection, cells were stimulated with Aldo or FSK for 30 min with or without pretreatment with H89 or calyculin A for 6 h, and analyzed by fluorescence microscopy. HEK293 cells were co-transfected with HA-tagged MR and Flag-tagged wild-type HDAC4 (B) or mutant HDAC4 (C). The cells were stimulated with Aldo for 30 min with or without pretreatment with H89 or calyculin A, and then fractionated. The interaction between MR and HDAC4 in each fraction was analyzed by a co-immunoprecipitation assay.
Mentions: We investigated the effect of H89 and calyculin A on the transcriptional activity of MR, because H89 and calyculin A showed inhibitory effects on HDAC4 translocation (Fig 5). In addition, HDAC4 knockdown decreased the transcriptional activity of MR (Fig 3). Expression of GILZ (Fig 6A) and SGK-1 (Fig 6B) induced by Aldo was significantly inhibited by pretreatment of cells with H89 (GILZ: ~74%; SGK-1: ~48%) or calyculin A (GILZ: ~68%; SGK-1: ~39%). Enrichment of MR and Pol II on the GILZ (Fig 6C) and SGK-1 (Fig 6D) promoters induced by Aldo were also significantly reduced by H89 and calyculin A. The interaction between MR and HDAC3 induced by Aldo was also inhibited by H89 and calyculin A treatment (Fig 6E). However, MR translocation into the nucleus was not affected by H89 or calyculin A (Fig 6E). Since H89 and calyculin A treatments decreased the interaction between MR and HDAC3, we investigated the effect of H89 and calyculin A on MR acetylation. H89 and calyculin A resulted in increased acetylation of the MR when HEK293 cells were stimulated with Aldo (Fig 6F). Previous studies showed that dephosphorylation by PP2A induced the translocation of HDAC4 from the cytosol into the nucleus [24, 25]. The effect of HDAC4 phosphorylation on its localization was investigated using phosphomutant HDAC4 (S246A, S467A, S632A). Strikingly, phosphomutant HDAC4 was nuclear even without Aldo stimulation, and H89 and calyculin A had no effect on the nuclear localization of phosphomutant HDAC4 (Fig 7A). Aldo induced an interaction between MR and wild-type HDAC4 in the nucleus of HEK293 cells, which was inhibited by H89 and calyculin A (Fig 7B). In contrast, although phosphomutant HDAC4 interacted with MR in the nucleus of cells treated with Aldo, this association was not affected by H89 and calyculin A (Fig 7C).

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.