Post-translational modification of chromatin has profound effects on many biological processes including transcriptional regulation, heterochromatin organization, and X-chromosome inactivation. Recent studies indicate that methylation on specific histone lysine (K) residues participates in many of these processes. Lysine methylation occurs in three distinct states, having either one (me1), two (me2) or three (me3) methyl groups attached to the amine group of the lysine side chain. These differences in modification state have an important role in defining how methylated chromatin is recognized and interpreted. Until recently, histone lysine methylation was considered a stable modification, but the identification of histone demethylase enzymes has demonstrated the reversibility of this epigenetic mark. So far, all characterized histone demethylases show enzymatic activity towards lysine residues modified in the me1 or me2 state, leaving open the possibility that me3 constitutes an irreversible modification. Here we demonstrate that JHDM3A (jumonji C (JmjC)-domain-containing histone demethylase 3A; also known as JMJD2A) is capable of removing the me3 group from modified H3 lysine 9 (H3K9) and H3 lysine 36 (H3K36). Overexpression of JHDM3A abrogates recruitment of HP1 (heterochromatin protein 1) to heterochromatin, indicating a role for JHDM3A in antagonizing methylated H3K9 nucleated events. siRNA-mediated knockdown of JHDM3A leads to increased levels of H3K9 methylation and upregulation of a JHDM3A target gene, ASCL2, indicating that JHDM3A may function in euchromatin to remove histone methylation marks that are associated with active transcription.

During bone homeostasis, osteoblast and osteoclast differentiation is coupled and regulated by multiple signaling pathways and their downstream transcription factors. Here, we show that microRNA 34 (miR-34) is significantly induced by BMP2 during osteoblast differentiation. In vivo, osteoblast-specific gain of miR-34c in mice leads to an age-dependent osteoporosis due to the defective mineralization and proliferation of osteoblasts and increased osteoclastogenesis. In osteoblasts, miR-34c targets multiple components of the Notch signaling pathway, including Notch1, Notch2 and Jag1 in a direct manner, and influences osteoclast differentiation in a non-cell-autonomous fashion. Taken together, our results demonstrate that miR-34c is critical during osteoblastogenesis in part by regulating Notch signaling in bone homeostasis. Furthermore, miR-34c-mediated post-transcriptional regulation of Notch signaling in osteoblasts is one possible mechanism to modulate the proliferative effect of Notch in the committed osteoblast progenitors which may be important in the pathogenesis of osteosarcomas. Therefore, understanding the functional interaction of miR-34 and Notch signaling in normal bone development and in bone cancer could potentially lead to therapies modulating miR-34 signaling.

The role of the constitutive androstane receptor (CAR) in xenobiotic metabolism by inducing expression of cytochromes P450 is well known, but CAR has also been implicated in the down-regulation of key genes involved in bile acid synthesis, gluconeogenesis, and fatty acid ␤-oxidation by largely unknown mechanisms. Because a key hepatic factor, hepatic nuclear factor-4 (HNF-4), is crucial for the expression of many of these genes, we examined whether CAR could suppress HNF-4 transactivation. Expression of CAR inhibited HNF-4 transactivation of CYP7A1, a key gene in bile acid synthesis, in HepG2 cells, and mutation of the DNA binding domain of CAR impaired this inhibition. Gel shift assays revealed that CAR competes with HNF-4 for binding to the DR1 motif in the CYP7A1 promoter. TCPOBOP, a CAR agonist that increases the interaction of CAR with coactivators, potentiated CAR inhibition of HNF-4 transactivation. Furthermore, inhibition by CAR was reversed by expression of increasing amounts of GRIP-1 or PGC-1␣, indicating that CAR competes with HNF-4 for these coactivators. Treatment of mice with phenobarbital or TCPOBOP resulted in decreased hepatic mRNA levels of the reported genes down-regulated by CAR, including Cyp7a1 and Pepck. In vivo recruitment of endogenous CAR to the promoters of Cyp7a1 and Pepck was detected in mouse liver after phenobarbital treatment, whereas association of HNF-4 and coactivators, GRIP-1, p300, and PGC-1␣, with these promoters was significantly decreased. Our data suggest that CAR inhibits HNF-4 activity by competing with HNF-4 for binding to the DR1 motif and to the common coactivators, GRIP-1 and PGC-1␣, which may be a general mechanism by which CAR down-regulates key genes in hepatic lipid and glucose metabolism.

The orphan receptor SHP interacts with many nuclear receptors and inhibits their transcriptional activities. SHP is central to feedback repression of cholesterol 7␣ hydroxylase gene (CYP7A1) expression by bile acids, which is critical for maintaining cholesterol homeostasis. Using CYP7A1 as a model system, we studied the molecular mechanisms of SHP repression at the level of native chromatin. Chromatin immunoprecipitation studies showed that mSin3A and a Swi/Snf complex containing Brm as a central ATPase were recruited to the promoter. This recruitment was associated with chromatin remodeling after bile acid treatment that was blunted by inhibition of the endogenous Swi/Snf function by dominant-negative ATPase mutants. Biochemical studies indicated that SHP was associated with the mSin3A-Swi/Snf complex by direct interaction with Brm and mSin3A through its repression domain. Expression of Brm, but not an ATPase mutant, inhibited CYP7A1 promoter activity and further enhanced SHP-mediated repression. Bile acid-induced recruitment of mSin3A/ Brm, chromatin remodeling, and concomitant repression of endogenous CYP7A1 expression were impaired when SHP expression was inhibited by SHP small interfering RNA. Our results suggest that SHP mediates recruitment of mSin3A-Swi/Snf to the CYP7A1 promoter, resulting in chromatin remodeling and gene repression, which may also be a mechanism for the repression by SHP of genes activated by many nuclear receptors. Our study establishes the first link between a Swi/Snf complex and regulation of cholesterol metabolism.The orphan nuclear receptor small heterodimer partner (SHP) protein is an atypical nuclear receptor that lacks a conventional DNA binding domain but contains a putative ligand binding domain (45). SHP has been reported to interact with a number of nuclear receptors, including both ligandregulated receptors, such as ER (estrogen receptor), GR, TR, AR, RAR, and RXR (retinoid X receptor), and orphan receptors, such as LRH-1 (liver receptor homologue 1), HNF-4 (hepatic nuclear factor 4), ERR, CAR, LXR, and PPAR, and to inhibit their transcriptional activities (1-3, 19, 29, 30, 43, 45). SHP, therefore, has been implicated in diverse biological activities, including cholesterol/bile acid and glucose/energy metabolic pathways.SHP has been reported to play a key role in the negative feedback regulation of cholesterol 7␣ hydroxylase gene (CYP7A1) expression in the liver (15,32). This hepatic enzyme catalyzes the first and rate-limiting step of the neutral pathway for the conversion of cholesterol into bile acids and thus plays a crucial role in enterohepatic cholesterol-bile acid homeostasis (42). Previous studies showed that the bile acid-activated farnesoid X receptor (FXR) binds as a heterodimer with RXR to the promoter of the SHP gene and increases transcription. The bile acid-induced SHP then interacts with LRH-1 and/or HNF-4, bound to the bile acid response element (BARE) in the CYP7A1 promoter, which results in transcriptional repression (7,15,29,32). Although the key regu...

SUMMARY Bone metastasis is a major health threat to breast cancer patients. Tumor-derived Jagged1 represents a central node in mediating tumor-stromal interactions that promote osteolytic bone metastasis. Here, we report the development of a highly effective fully human monoclonal antibody against Jagged1 (clone 15D11). In addition to its inhibitory effect on bone metastasis of Jagged1-expressing tumor cells, 15D11 dramatically sensitizes bone metastasis to chemotherapy, which induces Jagged1 expression in osteoblasts to provide a survival niche for cancer cells. We further confirm the bone metastasis-promoting function of osteoblast-derived Jagged1 using osteoblast-specific Jagged1 transgenic mouse model. These findings establish 15D11 as a potential therapeutic agent for the prevention or treatment of bone metastasis.