Role of Brg1 and HDAC2 in GR trans-repression of the pituitary POMC gene and misexpression in Cushing disease

Abstract

Negative feedback regulation of the proopiomelanocortin (POMC) gene by the glucocorticoid (Gc) receptor (GR) is a critical feature of the hypothalamo-pituitary-adrenal axis, and it is in part exerted by trans-repression between GR and the orphan nuclear receptors related to NGFI-B. We now show that Brg1, the ATPase subunit of the Swi/Snf complex, is essential for this trans-repression and that Brg1 is required in vivo to stabilize interactions between GR and NGFI-B as well as between GR and HDAC2. Whereas Brg1 is constitutively present at the POMC promoter, recruitment of GR and HDAC2 is ligand-dependent and results in histone H4 deacetylation of the POMC locus. In addition, GR-dependent repression inhibits promoter clearance by RNA polymerase II. Thus, corecruitment of repressor and activator at the promoter and chromatin modification jointly contribute to trans-repression initiated by direct interactions between GR and NGFI-B. Loss of Brg1 or HDAC2 should therefore produce Gc resistance, and we show that approximately 50% of Gc-resistant human and dog corticotroph adenomas, which are the hallmark of Cushing disease, are deficient in nuclear expression of either protein. In addition to providing a molecular basis for Gc resistance, these deficiencies may also contribute to the tumorigenic process.

Figure 1.

Cell lines can/cannot support trans-repression between GR and NGFI-B. Assessment of trans-repression between GR and NGFI-B using either NurRE (A–D) or GRE (E–H) reporters and expression vectors for NGFI-B (25 ng of expression vector in A–D, resulting in eightfold to 10-fold activation) and GR (100 ng of expression plasmid in E–H). CV-1 (A,E) and MEFs from Rb^−/−, p107^−/−, and p130^−/− mouse (MEF TKO) cells (D,H) support trans-repression, whereas the Brg1/Brm-deficient C33A (B,F) and SW13 (C,G) cells do not. (H) GR is expressed at significant levels in MEF TKO cells and was not overexpressed in these experiments. (I) Western blot analysis of cellular proteins showing that the ability for trans-repression correlates with expression of Brg1 or Brm. Whereas GR and NGFI-B are expressed at various levels in all six cell lines, only the trans-repression-proficient CV-1, AtT-20, and MEF TKO cells express Brg1 and/or Brm. (J) Brg1/Brm-deficient A-427 cells do not support trans-repression, which can be reconstituted by expression of Brg1 (K) or Brm (L) as assessed using NurRE reporter and expression vectors for GR and NGFI-B (25 ng of expression vector resulting in eightfold activation). Total DNA is kept constant in all transfections, and data represent the means ± SEM of three experiments, each performed in duplicate.

Figure 2.

Brg1 is required for POMC promoter activity and trans-repression by GR. (A) Colocalization of Brg1 (red) with the corticotroph marker ACTH (green) in mouse pituitary section. (B) Relative mRNA expression (RT–QPCR) of Brg1 and Brm in corticotroph AtT-20 cells showing a preponderance of Brg1. (C) Western blot analysis of Brg1 expression in AtT20 cells transfected with expression plasmids for Brg1 or control shRNA; GAPDH was measured as loading control. (D,E) Effects of Brg1 (D) or control (E) shRNA knockdown on the activity (ctrl) and Gc (Dex) repression of a POMC promoter luciferase reporter assessed in transfected AtT-20 cells. (F,G) Effects of the same shRNAs on endogenous POMC (F) and Brg1 (G) mRNAs in transfected and FACS-sorted AtT-20 cells. Endogenous mRNAs were quantitated by RT–QPCR. Data of three experiments, each performed in duplicate, are shown as means ± SEM.

Figure 3.

Brg1 and Brm ATPase activity is required for repression but not for interaction with NGFI-B or GR. (A) Standard model of trans-repression by GR. On the POMC promoter, dimers of NGFI-B or heterodimers between NGFI-B and the related orphan NRs Nurr1 and NOR1 bind the NurRE; their activity is enhanced by CRH signaling. Gc repression is thought to be initiated through protein:protein interactions between GR and promoter-bound NGFI-B. (B) Coimmunoprecipitation of Brg1 or its ATPase-deficient K798R mutant with Flag-tagged NGFI-B. GR- and Brg1-deficient C33A cells were transfected with expression vectors as indicated, and relevant proteins were revealed by immunoblotting (IB) after immunoprecipitation (IP) of Flag-NGFI-B. (C) Coimmunoprecipitation of Brm or its indicated mutants following immunoprecipitation of Flag-NGFI-B. (D) Coimmunoprecipitation of Brm or its indicated mutants following immunoprecipitation of GR. (E) In vitro pulldown of [³⁵S]-labeled Brg1 or Brm with MBP–NGFI-B, MBP-Pitx1, MBP-Tpit, or MBP-βGal as control. The Swi/Snf protein BAF155 was used as control. (F,G) Reconstitution of trans-repression using a NurRE reporter in A-427 cells with hBrm and mutants of this protein (F) as well as with Brg1 and the ATPase deficiency Brg1 mutant K798R (G). Band reporter activity was similar for all Brg1 and Brm proteins. (Insets) The relative expression levels of each Brm or Brg1 protein were assessed by Western blot. Data represent the means ± SEM. of three experiments, each performed in duplicate.

Figure 4.

Brg1 is required for complex formation between NGFI-B and GR. (A) Schematic representation of the mouse POMC gene indicating the position of PCR-amplified regions in ChIP experiments. (B) Abundance of POMC mRNA measured by RT–QPCR in control and AtT-20 cells treated as indicated. Both the synthetic Gc dexamethasone (Dex) and the hypothalamic hormone CRH were used at 10⁻⁷ M. (C) ChIP analysis of Brg1 recruitment at the POMC promoter and exon 3 of the POMC gene in AtT-20 cells. The dashed line indicates background ChIP signal observed with control IgG. (D) ChIP analysis of Pitx1 recruitment to the POMC promoter and exon 3. (E) ChIP analysis of GR recruitment to the POMC promoter and exon 3. (F) ChIP analysis of NGFI-B recruitment to the POMC promoter and exon 3. (G) Coimmunoprecipitation of GR requires Brg1 or Brm with Flag-NGFI-B in cotransfected C33A cells.

Figure 5.

Brg1-dependent recruitment of HDAC2 to the trans-repression complex. AtT-20 cells were treated with valproate (VPA) (A) or nicotinamide (B) at the indicated concentrations and assessed for Dex repression of POMC-luciferase activity. Class I and II (A), but not class III (B), HDAC inhibitors show a complete loss of Dex repression. (C) ChIP analysis of the POMC promoter showing Dex-dependent recruitment of HDAC2 to the promoter, but not HDAC1 or HDAC3. (D) ChIP analysis of HDAC2 recruitment to the POMC promoter, but not to POMC exon 3, following treatment of AtT-20 cells with Dex. (E) Coimmunoprecipitation of GR with Flag-HDAC2 requires the presence of Brg1 or Brm in transfected C33A cells. (F) Coimmunoprecipitation of Brg1, the Nur-related factor Nurr1, and HDAC2 with endogenous GR in AtT-20 cell nuclear extracts (lane 3) is impaired following siRNA knockdown (lane 2) of Brg1 (lane 4). (G) Coimmunoprecipitation of endogenous Brg1, GR, and Nurr1 in AtT-20 cells stimulated (S) or not (NS) with CRH and Dex following HDAC2 immunoprecipitation. Neither immunoprecipitation with antibody against HDAC1 nor control IgG brought down these proteins.

Figure 6.

POMC promoter function in repressed (Dex) and activated (CRH) conditions. (A) Initiation of POMC transcription as measured in nuclear run-on assays in pituitary primary culture treated with Dex and/or CRH. (B) ChIP analyses of RNA Pol II recruitment to the POMC promoter and exon 3 in control and treated AtT-20 cells. (C) ChIP analysis of POMC gene occupancy by Pol II with phospho-Ser2 CTD. (D) ChIP analysis of POMC gene occupancy by Pol II with phospho-Ser5 CTD. (E) ChIP analysis of acetylated histone H4 over the POMC promoter and exon 3. Dashed lines indicate background ChIP signals.

Figure 7.

Deficient Brg1 or HDAC2 expression in Cushing disease corticotroph adenomas. Immunohistochemical analysis of the corticotroph marker TPIT (A,D,G,J), Brg1 (B,E,H,K), and HDAC2 (C,F,I,L) in representative corticotroph adenomas. (A–C) Tumor revealed by nuclear TPIT staining (A) with normal nuclear Brg1 (B) and nuclear HDAC2 (C). (D–F) Tumor with cytoplasmic Brg1 (E) and normal nuclear HDAC2 (F). Note nuclear Brg1 in contiguous normal tissue. (G–I) Tumor with no nuclear Brg1 (H) and normal nuclear HDAC2 (I). (J–L) Tumor with normal nuclear Brg1 (K) and no nuclear HDAC2 (L). Each row presents data from one patient who is representative of the others in the same group as summarized in M. Human patients were subdivided into moderate or complete Gc resistance groups depending on their response to low or high doses of dexamethasone. In 19 cases, expression of Brg1 and HDAC2 were similar in tumor and normal pituitary tissues. Overall, 17 of 36 patients (47%) present with abnormal expression of Brg1 or HDAC2. The panel of 12 dog corticotroph adenomas (Supplementary Fig. S2) had four with no or low Brg1 expression and two with no tumor Brg1 and low HDAC2.

Figure 8.

Model of Brg1-dependent trans-repression. (A) The pituitary POMC gene is constitutively expressed in corticotroph cells. This basal transcription relies on many ubiquitous and cell-restricted factors such as Pitx1, Tpit, and NeuroD1 (not shown), and on signal-regulated factors such as NGFI-B. This activity is partly dependent on Brg1 and is reflected by the presence of RNA Pol II at the promoter and by the presence of acetylated histone H4 over the promoter and gene. For the sake of clarity, nucleosomes are not shown in these diagrams and histones H4, which are always within nucleosomes, are portrayed as isolated entities in the promoter and gene regions. (B) CRH activation through the PKA and MAPK pathways enhances NGFI-B activity through phosphorylation of its AF-1 domain and its recruitment to the promoter. This leads to increased Pol II at the promoter and increased transcription initiation without changes in H4 acetylation. (C) Ligand (Dex) activation of GR initiates formation of a Brg1-dependent protein complex that also contains NGFI-B and HDAC2. Both GR and HDAC2 are recruited to the promoter, with neither contacting DNA directly. This results in deacetylation of H4 and inhibition of transcription initiation without changing promoter occupancy of Pol II; promoter clearance thus appears to be reduced. (D) Trans-repression of transcription initiation by GR is dominant over CRH activation, as GR and HDAC2 are still recruited in the presence of both regulators and H4 are deacetylated. The presence of the trans-repression complex containing Brg1, NGFI-B, GR, and HDAC2 may prevent promoter recruitment of Pol II by steric hindrance or through protein interactions.