Deacetylase-independent function of HDAC3 in transcription and metabolism requires nuclear receptor corepressor - PubMed (original) (raw)
. 2013 Dec 26;52(6):769-82.
doi: 10.1016/j.molcel.2013.10.022. Epub 2013 Nov 21.
Dan Feng 1, Bin Fang 1, Shannon E Mullican 1, Seo-Hee You 1, Hee-Woong Lim 1, Logan J Everett 1, Christopher S Nabel 1, Yun Li 1, Vignesh Selvakumaran 1, Kyoung-Jae Won 1, Mitchell A Lazar 2
Affiliations
- PMID: 24268577
- PMCID: PMC3877208
- DOI: 10.1016/j.molcel.2013.10.022
Deacetylase-independent function of HDAC3 in transcription and metabolism requires nuclear receptor corepressor
Zheng Sun et al. Mol Cell. 2013.
Abstract
Histone deacetylases (HDACs) are believed to regulate gene transcription by catalyzing deacetylation reactions. HDAC3 depletion in mouse liver upregulates lipogenic genes and results in severe hepatosteatosis. Here we show that pharmacologic HDAC inhibition in primary hepatocytes causes histone hyperacetylation but does not upregulate expression of HDAC3 target genes. Meanwhile, deacetylase-dead HDAC3 mutants can rescue hepatosteatosis and repress lipogenic genes expression in HDAC3-depleted mouse liver, demonstrating that histone acetylation is insufficient to activate gene transcription. Mutations abolishing interactions with the nuclear receptor corepressor (NCOR or SMRT) render HDAC3 nonfunctional in vivo. Additionally, liver-specific knockout of NCOR, but not SMRT, causes metabolic and transcriptomal alterations resembling those of mice without hepatic HDAC3, demonstrating that interaction with NCOR is essential for deacetylase-independent function of HDAC3. These findings highlight nonenzymatic roles of a major HDAC in transcriptional regulation in vivo and warrant reconsideration of the mechanism of action of HDAC inhibitors.
Copyright © 2013 Elsevier Inc. All rights reserved.
Figures
Figure 1. HDI-dependent histone hyperacetylation does not upregulate gene expression as seen in HDAC3-depletion
(A) Primary hepatocytes from HDAC3f/f mice were treated with adenovirus (Ad) expressing GFP or Cre, or increasing concentrations of HDAC inhibitors (HDIs) including Trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA), or sodium butyrate (NaB). Cells were directly lysed in SDS sampling buffer after 48 h followed by immunoblot analysis. (B) Primary hepatocytes were treated with adenovirus or the highest concentrations of HDIs. Histones were acid extracted after the indicated time and analyzed by immunoblot. (C) Quantitative RT-PCR (RT-qPCR) analysis of primary hepatocytes after 48 h since HDAC3 depletion. * P < 0.05 compared with GFP. (D) RT-qPCR analysis of primary hepatocytes treated for 48 h with the highest concentrations of HDIs. p57, cyclin-dependent kinase inhibitor 1C; Bmal1, aryl hydrocarbon receptor nuclear translocator-like; Plin2, perilipin 2; Acacb, acetyl-CoA carboxylase beta; Gpam, glycerol-3-phosphate acyltransferase, mitochondrial; Fasn, fatty acid synthase; Scd1, stearoyl-Coenzyme A desaturase 1; Elovl6, fatty acid elongase 6; G6pdx, glucose-6-phosphate dehydrogenase X-linked; Me1, malic enzyme 1; Cidec, fat specific protein 27; Fitm1, fat storage-inducing transmembrane protein 1; Cd36, fatty acid translocase. * P < 0.05 compared with the mock treatment group. All error bars, s.e.m. from triplicate plates. See also Figure S1.
Figure 2. Mutations Y298F (YF) and K25A (KA) abolish HDAC3 enzymatic activity by distinct mechanisms
(A) Key residues within the HDAC3 catalytic site. (B) In vitro-translated (IVT) HDAC3 wild-type (WT) or YF mutant were mixed with IVT GAL-tagged SMRT-DAD (amino acid 1–763), followed by HDAC enzyme assay. Values are fluorescence signal intensities. (C) Immunoblot analysis of the above IVT proteins mixtures. (D) Flag-tagged HDAC3 and GAL-tagged SMRT (1–763) were co-expressed in HEK 293T cells. Cell lysates were immunoprecipitated with Flag antibodies followed by HDAC assay. (E) Immunoblot analysis of the above immunoprecipitates (IP). (F) Potential residues involved in binding of HDAC3 with SMRT-DAD and inositol phosphates Ins(1,4,5,6)P4 (IP4). (G) Flag-HDAC3 mutants and SMRT (1–763) were co-expressed in HEK 293T cells. Cell lysates were immunoprecipitated with Flag antibodies followed by Immunoblot analysis. (H) HDAC assay of the above immunoprecipitates. All error bars, s.e.m. from triplicate assays. See also Figure S2.
Figure 3. Deacetylase-dead HDAC3 mutants rescue derangement in both gene transcription and lipid metabolism in HDAC3-depleted liver
(A) HDAC3f/f mice were injected with AAV-Tbg-GFP or AAV-Tbg-Cre along with AAV vectors expressing Flag-tagged HDAC3 mutants. Total liver lysates (upper panel), or anti-Flag immunoprecipitates (IP) after washing with buffer containing 1% NP-40 (lower panel), were analyzed by immunoblot (IB). (B) Liver protein lysates were immunoprecipitated with either HDAC3 antibodies or normal IgG, followed by HDAC assay, n = 4. (C) Hepatic triglyceride (TG) measurement, n = 4. * P < 0.05. ns, not significant. (D) Oil red O (ORO) staining of livers. (E) Immunoprecipitation from the liver lysates were washed with more stringent buffer containing either 1% NP-40 plus 1% sodium deoxycholate (condition #1) or 1% NP-40 plus 1% sodium deoxycholate plus 0.1% SDS (condition #2), followed by immunoblot analysis with different exposure time (exp). * indicates non-specific signals. (F) RT-qPCR analysis of livers from mice described above, n = 4. * P < 0.05 between Cre and YF. All error bars, s.e.m. See also Figures S3 and S4.
Figure 4. Deacetylase-dead HDAC3 rescues HDAC3-dependent transcriptional repression despite failing to repress genome-wide histone acetylation
(A) GAL-tagged SMRT (1–763) and Flag-HDAC3 were co-expressed in HEK 293T cells. Cell lysates were immunoprecipitated with Flag antibodies followed by immunoblot analysis. (B) HDAC assay of the above immunoprecipitates in triplicates. (C) HDAC3f/f mice were injected with AAV-Tbg-GFP or AAV-Tbg-Cre along with AAV vectors expressing Flag-tagged HDAC3 mutants. Total liver lysates were analyzed by immunoblot analysis. (D) Liver lysates were immunoprecipitated with anti-HDAC3 antibodies and assayed for enzyme activities, n = 4. (E) ORO staining of livers. (F) RT-qPCR analysis of livers, n = = 4. * P < 0.05 between Cre and HAHA. (G) Livers were subjected to chromatin immunoprecipitation (ChIP) with HDAC3 antibodies followed by qPCR analysis using primers for HDAC3 sites near the indicated genes, n = 4. * P < 0.05 between WT and HAHA. (H) ChIP-qPCR analysis with antibodies against acetylated lysine 9 of histone 3 (H3K9ac), n = 4. * P < 0.05 between WT and HAHA. (I) Heat map of H3K9ac ChIP-seq signals from −1 kb to +1 kb surrounding the center of HDAC3 sites within 50 kb of genes that are upregulated upon HDAC3 depletion. rpm, reads per million. (J) Average H3K9ac signals, represented by reads per bp in 10 million total reads, from −2 kb to +2 kb surrounding the center of the same HDAC3 sites as shown above. G0S2, G0/G1 switch 2; Arbp, ribosomal protein, large, P0, also known as 36B4. All error bars, s.e.m. See also Figure S5.
Figure 5. A mutant HDAC3 loses interactions with not only DAD but also the full-length NCOR/SMRT
(A) Sequence alignment of HDAC3 and HDAC1. Identical and similar residues are highlighted in yellow and green respectively. Clusters of residues chosen for mutagenesis are boxed and labeled “A”– “I”. (B) GAL-SMRT (1–763) containing DAD and Flag-tagged HDAC3 mutants were co-expressed in HEK 293T cells. Cell lysates were immunoprecipitated with Flag antibodies and analyzed by immunoblot. (C) HDAC assay of the above immunoprecipitates in triplicates. (D) Protein lysates from HEK 293T cells expressing either a GAL-tagged SMRT truncation or a Flag-tagged HDAC3 mutant were mixed together, immunoprecipitated with Flag antibodies, and analyzed by immunoblot. DAD, the N-terminal 1–763 region of SMRT; M, the middle 1531–1961 region of SMRT containing the second interaction domain with HDAC3; C, the C-terminal 1961–2473 region of SMRT that does not interact with HDAC3 serving as a negative control. * Non-specific signals. (E) HEBI mutations on the crystal structure of HDAC3. (F) HDAC3f/f mice were injected with AAV-Tbg-GFP or AAV-Tbg-Cre along with AAV vectors expressing Flag-tagged HDAC3 mutants. Total liver lysates were analyzed by immunoblot analysis. (G) The liver lysates were immunoprecipitated with either anti-HDAC3 antibodies or normal IgG, followed by HDAC enzyme assay, n = 3–4. (H) The liver lysates were immunoprecipitated with Flag antibodies and washed with buffer containing 1% NP-40, followed by immunoblot analysis. * indicates non-specific signals. All error bars, s.e.m. See also Figure S5.
Figure 6. Loss of interaction with NCOR renders hepatic HDAC3 completely nonfunctional in vivo
(A) ORO stain of livers from HDAC3f/f mice injected with the indicated AAV vectors. (B) Hepatic triglyceride (TG) measurement, n = 4. (C) RT-qPCR analysis of livers from mice described above, n = 4. (D) The livers were subjected to ChIP with HDAC3 antibodies followed by qPCR analysis using primers specific for HDAC3 sites near the indicated genes, n = 4. * P < 0.05 between WT and YF. (E) ChIP-qPCR analysis of the livers with H3K9ac antibodies, n = 4. * P < 0.05 between WT and any of the other three groups. (F) Heat map of H3K9ac ChIP-seq signals from −1 kb to +1 kb surrounding the center of HDAC3 sites within 50 kb of genes that are upregulated upon HDAC3 depletion. (G) Average H3K9ac signals, represented by reads per bp in 10 million total reads, from −2 kb to +2 kb surrounding the center of HDAC3 sites. All error bars, s.e.m.
Figure 7. Liver-specific knockout of NCOR, but not SMRT, causes metabolic and transcriptomal alterations resembling those of mice without hepatic HDAC3
(A) RT-qPCR analysis of livers from SMRTf/f mice injected with AAV-Tbg-GFP or AAV-Tbg-Cre, n = 2–3, * P < 0.05 compared to the GFP group. **(B)** ORO staining of livers from SMRTf/f mice injected with AAV. **(C)** Immunoblot analysis of livers from NCORf/f mice injected with AAV-Tbg-GFP or AAV-Tbg-Cre. **(D)** RT-qPCR analysis of livers from NCORf/f mice injected with AAV, n = 4. * P < 0.05 compared to GFP. **(E)** ORO staining of livers from NCORf/f mice injected with AAV. **(F)** Hepatic TG and glycogen measurement in NCORf/f; AAV mice, n = 4–5, * P < 0.05 compared to GFP. **(G)** Heat map of microarray results from NCOR or HDAC3 depleted livers. Genes that were upregulated (294 genes) or downregulated (272 genes) in HDAC3-depleted livers versus their wild-type controls were selected (fold-change > 1.4, q < 0.05), and were sorted by fold-changes in NCOR-depleted livers versus their controls. (H) Gene ontology analysis using top 200 upregulated genes (by fold-change) in either the HDAC3 KO versus WT array or the NCOR KO versus WT array, with p < 0.05. All error bars, s.e.m. See also Figure S7.
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References
- Adrain C, Freeman M. New lives for old: evolution of pseudoenzyme function illustrated by iRhoms. Nat Rev Mol Cell Biol. 2012;13:489–498. - PubMed
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