Coordinated Recruitment of Histone Methyltransferase G9a and Other Chromatin-Modifying Enzymes in SHP-Mediated Regulation of Hepatic Bile Acid Metabolism

Abstract

SHP has been implicated as a pleiotropic regulator of diverse biological functions by its ability to inhibit numerous nuclear receptors. Recently, we reported that SHP inhibits transcription of CYP7A1, a key gene in bile acid biosynthesis, by recruiting histone deacetylases (HDACs) and a Swi/Snf-Brm complex. To further delineate the mechanism of this inhibition, we have examined whether methylation of histones is also involved and whether a functional interplay between chromatin-modifying enzymes occurs. The histone methyltransferase G9a, but not SUV39, was colocalized with SHP in the nucleus and directly interacted with SHP in vitro. G9a, which was coimmunoprecipitated with hepatic SHP, methylated Lys-9 of histone 3 (H3K9) in vitro. Expression of G9a enhanced inhibition of CYP7A1 transcription by SHP, while a catalytically inactive G9a dominant negative (DN) mutant reversed the SHP inhibition. G9a was recruited to and H3K9 was methylated at the CYP7A1 promoter in a SHP-dependent manner in bile acid-treated HepG2 cells. Expression of the G9a-DN mutant inhibited H3K9 methylation, blocked the recruitment of the Brm complex, and partially reversed CYP7A1 inhibition by bile acids. Inhibition of HDAC activity with trichostatin A blocked deacetylation and methylation of H3K9 at the promoter, and, conversely, inhibition of H3K9 methylation by G9a-DN partially blocked deacetylation. Hepatic expression of G9a-DN in mice fed cholic acid disrupted bile acid homeostasis, resulting in increased bile acid pools and partial de-repression of Cyp7a1 and Cyp8b1. Our studies establish a critical role for G9a methyltransferase, histone deacetylases, and the Swi/Snf-Brm complex in the SHP-mediated inhibition of hepatic bile acid synthesis via coordinated chromatin modification at target genes.The small heterodimer partner (SHP, or NR0B2), an unusual orphan nuclear receptor that lacks a DNA binding domain but contains a putative ligand binding domain, interacts with a number of nuclear receptors, including liver receptor homologue 1, hepatic nuclear factor 4 (HNF-4), estrogen-related receptor, constitutive androstane receptor, liver X receptor, glucocorticoid receptor, and estrogen receptor, and inhibits their transcriptional activities (1,4,7,30,39,40). Thus, SHP acts as a pleiotropic transcriptional repressor affecting diverse biological functions, including cholesterol and glucose metabolic pathways, energy homeostasis, and reproductive biology (2). SHP has been shown to be a key regulator in the negative feedback regulation of bile acid biosynthesis from cholesterol in the liver (15,25,31,48). Bile acid-activated farnesoid X receptor (FXR), upon heterodimerization with retinoid X receptor, binds to the promoter of the SHP gene and increases transcription (15,31). The bile acid-induced SHP then suppresses transcription of CYP7A1 and CYP8B1, key hepatic genes involved in the neutral pathway of bile acid biosynthesis, by interacting with liver receptor homologue 1 and/or HNF-4, which are bound to the promoters of the genes. In addit...

Loss of orphan receptor small heterodimer partner sensitizes mice to liver injury from obstructive cholestasis

“…The primary basis for this effect is the induction of SHP by FXR in the presence of elevated bile acid levels, inhibiting transactivation by the nuclear receptors liver receptor homolog-1 (LRH-1) and possibly hepatocyte nuclear factor 4 alpha (HNF4␣) and repressing expression of Cyp7A1, the rate-limiting enzyme of bile acid biosynthesis, as well as a number of other bile acid biosynthetic enzymes. 7,8,[23][24][25] This function of SHP has been strongly supported by results with Shp Ϫ/Ϫ mice, which reveal defective repression in response to synthetic FXR agonists 10,11 or very short term dietary bile acid treatment. 26 However, such studies have also demonstrated the importance of SHP-independent pathways that can efficiently repress Cyp7A1 and other negative targets in response to longer term treatments with dietary bile acids.…”

“…20 It is a transcriptional repressor and exerts its regulatory functions through protein-protein interactions with other nuclear hormone receptors and possibly other transcription factors, which can inhibit or even reverse their transactivation. [20][21][22][23][24][25] Regulation of bile acid metabolism is the most prominent of a number of physiological roles proposed for SHP. The primary basis for this effect is the induction of SHP by FXR in the presence of elevated bile acid levels, inhibiting transactivation by the nuclear receptors liver receptor homolog-1 (LRH-1) and possibly hepatocyte nuclear factor 4 alpha (HNF4␣) and repressing expression of Cyp7A1, the rate-limiting enzyme of bile acid biosynthesis, as well as a number of other bile acid biosynthetic enzymes.…”

Recruitment of Histone Methyltransferase G9a Mediates Transcriptional Repression of Fgf21 Gene by E4BP4 Protein*

“…G9a-associated transcription repression might be also involved in the postdevelopmental stage and participate in a short term gene regulation. Indeed, G9a-dependent methyltransferase activity has been found to be involved in the expression of Th2-associated cytokines (27), terminal differentiation of B-lymphocytes (28), bile acid metabolism (29), cocaine-induced neuronal plasticity (30), tumor growth and metastasis (31)(32)(33), and the interferon response (34). No previous studies have reported the role of G9a in lipid metabolism in the liver.…”

Structural insights into gene repression by the orphan nuclear receptor SHP

“…A likely explanation for this difference is that SHP recruits other corepressors through the helix H12 site that act independently of EID1. Indeed, previous studies have shown SHP helix H12 is crucial for its repressor activity (24), and EID1 is known to form a multiple-component complex with other corepressors to regulate SHP's inhibitory activity (20,22,23). For this reason, mutation of the SHP-EID1 interface residues F72, L76, and F178 might be expected to compromise both EID1 binding and repression activity, whereas mutations of other SHP-EID1 interface residues may still retain SHP repressor activity because they permit the assembly of other non-EID1-dependent corepressors through the H12 helix.…”

“…In the second step, SHP actively recruits corepressors to inhibit gene transcription. Identification of SHP-interacting corepressors has been a subject of intense study in recent years (19)(20)(21)(22)(23)(24)(25). As a result, a number of proteins have been isolated that bind to SHP in in vitro or in vivo assays, one of which is E1A-like inhibitor of differentiation (EID1) (24).…”