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. 2018 Dec 3;215(12):3194-3212.
doi: 10.1084/jem.20180520. Epub 2018 Nov 21.

HDAC Stimulates Gene Expression Through BRD4 Availability in Response to IFN and in Interferonopathies

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Free PMC article

HDAC Stimulates Gene Expression Through BRD4 Availability in Response to IFN and in Interferonopathies

Isabelle J Marié et al. J Exp Med. .
Free PMC article

Abstract

In contrast to the common role of histone deacetylases (HDACs) for gene repression, HDAC activity provides a required positive function for IFN-stimulated gene (ISG) expression. Here, we show that HDAC1/2 as components of the Sin3A complex are required for ISG transcriptional elongation but not for recruitment of RNA polymerase or transcriptional initiation. Transcriptional arrest by HDAC inhibition coincides with failure to recruit the epigenetic reader Brd4 and elongation factor P-TEFb due to sequestration of Brd4 on hyperacetylated chromatin. Brd4 availability is regulated by an equilibrium cycle between opposed acetyltransferase and deacetylase activities that maintains a steady-state pool of free Brd4 available for recruitment to inducible promoters. An ISG expression signature is a hallmark of interferonopathies and other autoimmune diseases. Combined inhibition of HDAC1/2 and Brd4 resolved the aberrant ISG expression detected in cells derived from patients with two inherited interferonopathies, ISG15 and USP18 deficiencies, defining a novel therapeutic approach to ISG-associated autoimmune diseases.

Figures

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Figure 1.
Figure 1.
Class I HDAC mediates ISG expression. (A and B) HeLa cells were treated with IFN-α for 6 h in the absence or presence of increasing concentrations of Romidepsin or TSA, as indicated. RNA was quantified by real-time RT-PCR for ISG54 and p21WAF1/Cip1 expression, normalized to GAPDH, and represented as fold induction over untreated cells. (C) HeLa cells were treated with IFN-α for 6 h in the absence or presence of increasing concentrations of romidepsin, RGFP966, or RGFP233, as indicated. RNA was quantified by real-time RT-PCR for IRF9 expression, normalized to GAPDH, and represented as fold induction over untreated cells. (D and E) HEK293 cells were transfected with siRNA against HDAC1, HDAC2, or E2F4 separately (D) or with a combination of HDAC1 and HDAC2 targeting oligonucleotides (E), and cells were stimulated with IFN-α for 6 h (α) in the presence (αΤ) or absence of TSA, as indicated. Whole-cell extracts were analyzed for expression of the indicated proteins by Western blotting (left). ISG54 expression was quantified by real-time RT-PCR and represented in arbitrary units (right). MW, molecular weight. A.U., arbitrary units. (F) HEK293T cells were transfected with pcDNA3 (Ctl), HDAC1, or HDAC2 expression constructs, and ISG54 expression was quantified after stimulation with IFN-α for 10 h. Representative data from two experiments are shown. (G) Sin3A F/– and Sin3A F/– Sin3B F/– immortalized mouse embryonic fibroblasts expressing Cre-ERT2 were treated for 3 d with 4OH-tamoxifen (4OH-T) or left untreated. Nuclear extracts were analyzed for expression of Sin3B by Western blotting (middle). Expression of Sin3A, represented as percent expression relative to levels before tamoxifen treatment (left) and expression of IRF9 mRNA before and after tamoxifen (right), were quantified by real-time RT-PCR and normalized to GAPDH mRNA expression. Weak expression of Sin3B protein in Sin3A F/– Sin3B F/– fibroblasts before 4OH-T treatment is likely due to some leakiness of Cre recombinase expression. Quantitative data are representative examples of three (A and F) or two (B, C, E, and G) independent experiments, each performed in duplicate, and error bars represent +SD. *, P < 0.05; **, P < 0.005; ***, P < 0.001.
Figure 2.
Figure 2.
HDAC inhibition does not prevent transcription in vitro or IFN-stimulated chromatin remodeling in vivo. (A) Nuclear extracts from HEK293T cells expressing activated recombinant ISGF3 (STAT1, STAT2, IRF9, and JAK1) or transfected with vector (Ctl) were programmed with G6TI-CAT (G6TI) or p107 (ISG54) templates. Radiolabeled RNA transcripts were resolved by polyacrylamide-urea gel electrophoresis. Arrow and arrowhead indicate position of the transcripts from G6TI-CAT and p107, respectively. Molecular sizes indicated in nucleotides. (B) HeLa cells were either left untreated or treated with IFN-α in the absence or presence of TSA (TSA in vivo) before nuclei were isolated for run-on transcription. Where indicated, TSA was added to the elongation reaction in vitro (TSA in vitro). Specific signals from γ-actin, ISG15, ISG56, 6-16, 9-27, IRF9, and GBP transcription were quantified following filter hybridization and normalized to the signal for GAPDH, arbitrarily set to 100. Representative data from three independent experiments are shown. A.U., arbitrary units. (C and D) FS2 human diploid fibroblasts were starved for 72 h before being treated with IFN-α for 7.5 h in the absence or presence of TSA. Mononucleosomal DNA fraction was purified from MNase-digested nuclei and analyzed for nucleosome positions by PCR quantitation of protected fragments. All samples were quantified by real-time PCR, except the −53 to +1 ISG54 fragment, which was quantified by gel electrophoresis (C). The same mononucleosomal fractions were assayed for two fragments of the IFN-β promoter as control (D). Normalized protection factor for each sample was expressed proportionally to the signal obtained for fragment +40/+116 protected by the fixed nucleosome N2 at IFN-β promoter-proximal region, which was arbitrarily set at 100. Putative nucleosome positions are diagramed. **, P < 0.002 from representative experiment (of two) performed in duplicate, and error bars represent +SD.
Figure 3.
Figure 3.
RNAPII recruitment and activation do not require HDAC activity. (A) 2fTGH cells were either left untreated or treated with IFN-α in the absence or presence of TSA for the indicated time (minutes). ChIP assays were performed with antibodies against Pol II, and recovered ISG56 promoter sequences were quantified by real-time PCR relative to input and represented as fold induction relative to untreated cells. (B) As in A, except that antibodies against phosphorylated Pol II at serine-5 in the CTD were used for immunoprecipitation, following 60-min IFN-α treatments. Recovered ISG54, ISG56, and ISG15 promoter sequences were quantified by real-time PCR relative to input and represented as fold induction relative to untreated cells, which were arbitrarily set to 1. (C) As in A, except that cells were treated with IFN-α for 60 min and proximal, middle, and distal fragments along the ISG54 gene (left) and ISG56 gene (right) were assayed. Regions of ISG54 analyzed were centered around +2380 (proximal), +4540 (middle), and +7590 (distal); regions of ISG56 were +2940 (proximal), 7120 (middle), and +11040 (distal), relative to the TSS. (D) As in A, except that antibodies against trimethylated lysine-4 on histone H3 were used. Promoter regions of ISG54, ISG56, and 6-16 genes were quantified by real-time PCR and reported as fold over the signal detected with a nonspecific antibody. **, P < 0.05; ***, P < 0.001 from representative experiment (of two) performed in duplicate, and error bars represent +SD.
Figure 4.
Figure 4.
HDAC activity is required for P-TEFb recruitment. (A) HeLa cells were either left untreated or treated with IFN-α for 1 h in the presence or absence of 100 or 200 nM of the CDK9 inhibitor flavopiridol (Flav). Using primers spanning intron-exon junctions, nuclear pre-mRNA abundance for ISG54 and RPS11 was quantified using real-time RT-PCR and normalized to GAPDH mRNA. (B) 2fTGH cells were either left untreated or treated with IFN-α in the absence or presence of TSA for 60 min. ChIP assays were performed with antibodies against CDK9, and recovered ISG56 and ISG15 promoter sequences were quantified by real-time PCR relative to input and represented as percentage of the input signal. (C) BT474 cells were either left untreated or treated with IFN-α in the absence or presence of TSA after exposure to geldanamycin (Gelda) for 24 h, as indicated. mRNAs for ISG56, OAS2, and c-Myc were quantified by real-time RT-PCR and normalized to GAPDH. (D) Control and NELF-E KD cells were either left untreated or treated with IFN-α in the absence or presence of TSA. ISG56 mRNA was quantified using real-time RT-PCR and normalized to GAPDH mRNA abundance (left). Knockdown was verified by Western blotting using anti–NELF-E antibodies (right). (E) Control and Spt5 KD cells were either left untreated or treated with IFN-α in the absence or presence of TSA. ISG56, Spt5 mRNA, and ISG54 pre-mRNA were quantified using real-time RT-PCR and normalized to GAPDH mRNA abundance. **, P < 0.03; ***, P < 0.005 from representative experiment (of two) performed in duplicate, and error bars represent +SD. A.U., arbitrary units.
Figure 5.
Figure 5.
Brd4 is required for ISG transcription. (A) HeLa cells were either left untreated or treated with IFN-α for 1 h in the presence or absence of JQ-1 or HMBA. Using primers spanning intron-exon junctions, nuclear pre-mRNA for ISG54 and γ-actin was quantified by real-time RT-PCR and normalized to GAPDH mRNA. (B) Control and Brd4 KD cells were either left untreated or treated with IFN-α. ISG56 and Brd4 mRNA were quantified using real-time RT-PCR and normalized to GAPDH mRNA abundance. A.U., arbitrary units. (C) HEK293T cells were transfected with pcDNA3 (Ctl) or Brd4 expression constructs along with a luciferase reporter driven by the ISG54 promoter. 24 h after transfection, cells were either left untreated or treated with IFN-α in the absence or presence of TSA before being assayed for luciferase activity (left). HEK293T cells were transfected with pcDNA3 (Ctl) or Brd4 expression constructs along with a luciferase reporter driven by the constitutive Rous sarcoma virus promoter and assayed for luciferase activity after 36 h (right). R.L.U., relative light units. (D) HeLa cells were either left untreated or treated with IFN-α for 2 h in the presence or absence of TSA. Nuclear proteins were extracted as a soluble fraction and a chromatin-bound fraction and analyzed by Western blotting using anti-Brd4, H4-AcK5, and actin antibodies, as indicated. (E) Soluble nuclear proteins from HeLa cells stimulated with IFN-α in the absence or presence of TSA or anacardic acid (AA) for 60 min were analyzed by Western blotting. (F) HeLa cells were treated for 60 min as shown, and ChIP assays were performed with antibodies against Brd4 and analyzed for recovered ISG56, Mx1, and IRF9 TSS spanning sequences by real-time PCR, normalized to input, and represented as fold enrichment over an untreated sample. (G) Profile and heat map of the enrichment of Brd4 peaks ±2.5 kb relative to TSS for genes showing IFN-dependent enrichment of Brd4. Each row represents a unique gene segment. *, P < 0.05; ***, P < 0.001 for representative experiment (of two) performed in duplicate, and error bars represent +SD.
Figure 6.
Figure 6.
Combined HDAC and Brd4 inhibition is a potential therapy for type I interferonopathies. (A) HEK293T cells were either left untreated or treated with IFN-α for 6 h in the presence or absence of JQ-1, TSA, or a combination of the two inhibitors, as indicated (nM). ISG56 and Viperin mRNA were quantified using real-time RT-PCR and normalized to GAPDH mRNA abundance. (B) hTert-immortalized human fibroblasts from an ISG15-deficient patient and a healthy donor (C21) were treated with IFN-α for 8 h, washed with PBS, and incubated in the absence of IFN for 3 d. Where indicated, cells were treated with JQ-1, romidepsin, or a combination of the two drugs (nM) for the final 24 h before RNA extraction. Mx1 and Viperin mRNA were quantified using real-time RT-PCR and normalized to GAPDH mRNA abundance. (C) As in B, except that hTert-immortalized fibroblasts from a USP18-deficient patient were used. (D) As in B, except that nuclear pre-mRNA for ISG54 from ISG15-deficient cells was scored. (E) As in D, except that pre-mRNA for ISG54 from hTert-immortalized USP18-deficient fibroblasts was quantified. A.U., arbitrary units. (F) Proposed model of Brd4 sequestration in the absence of constitutive HDAC function, as described in the text. *, P < 0.02; **, P < 0.01; ***, P < 0.005; ****, P < 0.002 for representative experiments (of three) performed in duplicate, and error bars represent +SD.

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