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

Important aspects of glucocorticoid (Gc) action are exerted through repression of transcription (Helmberg et al. 1995; Reichardt et al. 1998), such as their anti-inflammatory action (Wintermantel et al. 2004). Contrary to activation of transcription resulting from DNA binding of the glucocorticoid receptor (GR), repression by GR is often achieved through protein:protein interactions and mutual antagonism with other transcription factors. The first proposal of a mechanism for this type of transcriptional repression, now known as trans-repression, involved GR antagonism of AP-1-dependent transcription on the collagenase 1 gene (Jonat et al. 1990; Schule et al. 1990; Yang-Yen et al. 1990). Many features of this mechanism of repression were revealed in this early work. Mainly, Gc-dependent repression is mediated by GR, but without direct GR:DNA interactions as for GR- activated transcription (McKenna and O'Malley 2002). Rather, GR represses transcription through protein:protein interactions with DNA-bound AP-1. In contrast to GR activation of transcription, Gc-dependent trans-repression by GR is exerted by monomers (Heck et al. 1994) and, consequently, is independent of dimerization (Reichardt et al. 1998; Martens et al. 2005). Direct protein interactions between GR and AP-1 (jun/fos) were observed and initially led to the suggestion that complex formation between these factors titrated AP-1 away from transcription targets, thus resulting in apparent repression. It was later shown by in vivo footprinting that pro-moter occupancy is not altered in the repressed state, and thus a model of repressors interacting with promoter-bound activators was proposed (Konig et al. 1992). This mechanism was shown to be reciprocal, such that activators (such as GR, AP-1, NFκB, or NGFI-B) can behave as either activator or repressor, with the DNA- bound factor acting as activator. Trans-repression was also shown to occur between AP-1 and different nuclear receptors (NR) in addition to GR (for review, see De Bosscher et al. 2003). The anti-inflammatory action of Gc is largely exerted by its repressor activity (Hayashi et al. 2004). Indeed, repression by GR of NFκB action on genes encoding pro- inflammatory cytokines such as interleukin 8 (IL-8) (Wintermantel et al. 2004) was suggested to use similar mechanisms of trans-repression as those between GR and AP-1 (De Bosscher et al. 2003). Recent insights into the molecular mechanism of GR trans-repression of the NFκB-activated IL-8 gene followed the introduction of the chromatin immunoprecipitation (ChIP) technique, which confirmed and extended the trans-repression model: In this case, GR does not inhibit formation of a preinitiation complex, but rather interferes with phosphorylation of the RNA polymerase II (Pol II) C-terminal repeat (CTD) at Ser2 (Nissen and Yamamoto 2000) and with recruitment of p-TEFb, the Ser2 CTD kinase. In this system, failure to recruit p-TEFb appeared to decrease gene expression at a post-initiation step (Luecke and Yamamoto 2005). More recently, trans-repression initiated by another NR, PPARγ, was shown to depend on ligand-induced SUMOylation of the PPARγ ligand- binding domain and the resulting stabilization of its interaction with the corepressor NCor (Pascual et al. 2005). We have investigated the mechanism of Gc repression of the pituitary proopiomelanocortin (POMC) gene. Pituitary POMC is at the center of the hypothalamo–pituitary–adrenal (HPA) axis that ultimately controls Gc synthesis and modulates the stress response, energy metabolism, and immune response. Central activation of the HPA axis is mediated through secretion of hypothalamic CRH into the pituitary portal system, where CRH stimulates secretion of presynthesized POMC-derived ACTH and transcription of the POMC gene (Philips et al. 1997b). Blood-borne ACTH is the major stimulus of adrenal Gc synthesis, and maintenance of physiological levels of cortisol requires adequate negative feedback regulation by Gc of pituitary ACTH secretion and POMC gene transcription. Disregulation of this negative feedback loop has severe metabolic consequences that characterize Cushing disease. Cushing syndrome, or hypercortisolism, is characterized by upper body obesity (moon face and buffalo hump), muscle weakness, high blood pressure, and glucose intolerance. This condition is caused by elevated blood Gc that may result from high-dose Gc treatment, excessive cortisol production by adrenal tumors, and, in most noniatrogenic cases, overproduction of ACTH by ectopic or pituitary tumors (Arnaldi et al. 2003). Typically, ACTH-producing pituitary tumors are micro- adenomas that are not malignant, produce excessive amounts of ACTH, and are resistant to Gc negative feedback. These tumors define Cushing disease (as opposed to syndrome), and they appear to be due to tumorigenic transformation of anterior pituitary corticotroph cells (Vallette-Kasic et al. 2003). The corticotrophs are one of two proopiomelanocortin (POMC)-expressing pituitary lineages (Pulichino et al. 2003), and they are at the center of the HPA axis. The development of Gc resistance in corticotroph adenomas may be a critical (and possibly primary) step in tumorigenesis. Although various human pathological conditions, such as Cushing disease and depressive illness, have been associated with deficient Gc feedback, little mechanistic insight exists into the molecular defects causing Gc resistance, except for a few rare mutations in GR itself (Lamberts 2002). We have used Gc/GR repression of POMC gene transcription to gain insight into mechanisms of Gc resistance. Repression of POMC gene transcription by Gc results, at least in part, from trans-repression exerted by GR on the activity of orphan NRs related to NGFI-B (Philips et al. 1997b). Indeed, transcription elicited by NGFI-B (Nur77) and by the closely related orphan NRs, Nurr1 and NOR1, is subject to GR repression by a mechanism that is very similar to trans-repression between GR and AP-1 and between GR and NFκB (Philips et al. 1997b; Martens et al. 2005). In pituitary corticotroph cells that produce ACTH, NGFI-B, and its related NRs are mediators of the stimulatory signals elicited by CRH (Philips et al. 1997a; Maira et al. 1999). Acting through its membrane receptors, CRH leads to activation of protein kinase A (PKA) and mitogen- activated protein kinase (MAPK) pathways that quickly result in: (1) dephosphorylation of the NGFI-B DNA- binding domain (DBD), which is required for NGFI-B interaction with DNA; (2) formation of Nur factor dimers that recognize the POMC promoter NurRE sequence; and (3) recruitment of the NR coactivator SRC-2 (TIF2) to the AF-1 domain of NGFI-B (Maira et al. 2003b). CRH signals also act on the POMC promoter through SRC-2 coactivation of Tpit (Maira et al. 2003a), a highly cell-restricted T-box transcription factor (Lamolet et al. 2001). Although both NGFI-B and Tpit activities are enhanced in response to SRC2 and CRH signals, only NurRE-dependent activity is subject to Gc repression (Martens et al. 2005). We now report on the molecular mechanism of trans-repression between GR and NGFI-B, and in particular on the requirement for the Swi/Snf chromatin remodeling protein Brg1 and its ATPase activity in this mechanism. Brg1 is critical for formation of stable in vivo complexes between GR and NGFI-B, and between GR and HDAC2. Promoter recruitment of both GR and HDAC2 are Gc- dependent and are associated with decreased acetylated histone H4 at the promoter and throughout the gene. Assembly of a trans-repression machinery at the POMC promoter also appears to hamper initiation of transcription by blocking Pol II at the promoter. Protein:protein interactions at the promoter between activator (NGFI-B) and repressor (GR) together with histone deacetylation result in inhibition of transcription initiation. Further, we found that ∼50% of pituitary adenomas from Cushing disease patients or dogs have misexpression of either Brg1 or HDAC2 in tumor tissue but not in adjacent normal pituitary tissue. The high frequency of misexpression of these proteins in corticotroph adenomas clearly supports their importance in Gc negative feedback regulation and in Gc resistance syndromes.