Yangjin Bae

The transcriptional repressor JHDM3A demethylates trimethyl histone H3 lysine 9 and lysine 36.

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.

Functional Inhibitory Cross-talk between Constitutive Androstane Receptor and Hepatic Nuclear Factor-4 in Hepatic Lipid/Glucose Metabolism Is Mediated by Competition for Binding to the DR1 Motif and to the Common Coactivators, GRIP-1 and PGC-1α

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 β-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-1α, 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-1α, 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-1α, which may be a general mechanism by which CAR down-regulates key genes in hepatic lipid and glucose metabolism.

miRNA-34c regulates Notch signaling during bone development

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.

Role of an mSin3A-Swi/Snf Chromatin Remodeling Complex in the Feedback Repression of Bile Acid Biosynthesis by SHP

ABSTRACT The orphan receptor SHP interacts with many nuclear receptors and inhibits their transcriptional activities. SHP is central to feedback repression of cholesterol 7α 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.

Yunis-Varón Syndrome Is Caused by Mutations in FIG4, Encoding a Phosphoinositide Phosphatase

Yunis-Varón syndrome (YVS) is an autosomal-recessive disorder with cleidocranial dysplasia, digital anomalies, and severe neurological involvement. Enlarged vacuoles are found in neurons, muscle, and cartilage. By whole-exome sequencing, we identified frameshift and missense mutations of FIG4 in affected individuals from three unrelated families. FIG4 encodes a phosphoinositide phosphatase required for regulation of PI(3,5)P(2) levels, and thus endosomal trafficking and autophagy. In a functional assay, both missense substitutions failed to correct the vacuolar phenotype of Fig4-null mouse fibroblasts. Homozygous Fig4-null mice exhibit features of YVS, including neurodegeneration and enlarged vacuoles in neurons. We demonstrate that Fig4-null mice also have small skeletons with reduced trabecular bone volume and cortical thickness and that cultured osteoblasts accumulate large vacuoles. Our findings demonstrate that homozygosity or compound heterozygosity for null mutations of FIG4 is responsible for YVS, the most severe known human phenotype caused by defective phosphoinositide metabolism. In contrast, in Charcot-Marie-Tooth disease type 4J (also caused by FIG4 mutations), one of the FIG4 alleles is hypomorphic and disease is limited to the peripheral nervous system. This genotype-phenotype correlation demonstrates that absence of FIG4 activity leads to central nervous system dysfunction and extensive skeletal anomalies. Our results describe a role for PI(3,5)P(2) signaling in skeletal development and maintenance.

Repression of CAR-Mediated Transactivation of CYP2B Genes by the Orphan Nuclear Receptor, Short Heterodimer Partner (SHP)

The induction of CYP2B gene expression by phenobarbital (PB) is mediated by the translocation of the constitutive androstane receptor (CAR) from the cytoplasm to the nucleus. The CAR/RXR heterodimer binds to two DR-4 sites in a complex phenobarbital responsive unit (PBRU) in the CYP2B gene. The short heterodimer partner (SHP), an orphan nuclear receptor that lacks a conventional DNA binding domain, was initially identified by its interaction with CAR. We have examined the role of SHP in CAR-mediated transactivation of the CYP2B gene. Coexpression of SHP inhibited the transactivation of the CYP2B gene by CAR in cultured hepatoma cells and the p160 coactivator GRIP1 reversed the inhibition. The interaction of CAR with SHP was confirmed by GST pulldown experiments. SHP did not block the binding of either CAR/RXR to the PBRU or binding of GRIP1 to the CAR/RXR complex in gel mobility shift assays, but slightly increased CAR/RXR binding and slightly altered the mobility of the CAR/RXR/GRIP1 complex, suggesting an interaction of SHP with these complexes. The presence of SHP in the complexes, however, could not be detected in an antibody supershift assay. Recombinant corepressors mSin3A, SMRT, and HDAC1, but not NCoR1, interacted with GST-SHP but each of these corepressors in liver nuclear extracts bound to GST-SHP. SMRT and NCoR1 inhibited CAR-mediated activation independent of SHP, but mSin3A and HDAC1 had little effect alone, and were additive with SHP. These studies demonstrate that SHP does not inhibit CAR-mediated trans-activation by interfering with DNA binding or by competition with GRIP1. Instead, SHP may either inhibit recruitment of other coactivators by GRIP1 or actively recruit corepressors directly to the CAR/RXR/PBRU complex.

Whole-exome sequencing identifies mutations in the nucleoside transporter gene SLC29A3 in dysosteosclerosis, a form of osteopetrosis

Dysosteosclerosis (DSS) is the form of osteopetrosis distinguished by the presence of skin findings such as red-violet macular atrophy, platyspondyly and metaphyseal osteosclerosis with relative radiolucency of widened diaphyses. At the histopathological level, there is a paucity of osteoclasts when the disease presents. In two patients with DSS, we identified homozygous or compound heterozygous missense mutations in SLC29A3 by whole-exome sequencing. This gene encodes a nucleoside transporter, mutations in which cause histiocytosis-lymphadenopathy plus syndrome, a group of conditions with little or no skeletal involvement. This transporter is essential for lysosomal function in mice. We demonstrate the expression of Slc29a3 in mouse osteoclasts in vivo. In monocytes from patients with DSS, we observed reduced osteoclast differentiation and function (demineralization of calcium surface). Our report highlights the pleomorphic consequences of dysfunction of this nucleoside transporter, and importantly suggests a new mechanism for the control of osteoclast differentiation and function.

Notch gain of function inhibits chondrocyte differentiation via Rbpj-dependent suppression of Sox9

Notch signaling plays a critical role during development by directing the binary cell fate decision between progenitors and differentiated cells. Previous studies have shown sustained Notch activation in cartilage leads to chondrodysplasia. Genetic evidence indicates that Notch regulates limb bud mesenchymal stem cell differentiation into chondrocytes via an Rbpj‐dependent Notch pathway. However, it is still unknown how Notch governs chondrogenesis in the axial skeleton where Notch serves a primary patterning function. We hypothesized that both Rbpj‐dependent and Rbpj‐independent Notch signaling mechanisms might be involved. Cartilage‐specific Notch gain‐of‐function (GOF) mutant mice display chondrodysplasia accompanied by loss of Sox9 expression in vertebrae. To evaluate the contribution of an Rbpj‐dependent Notch signaling to this phenotype, we deleted Rbpj on the Notch GOF background. These mice showed persistent spine abnormalities characterized by “butterfly” vertebrae suggesting that removal of Rbpj does not fully rescue the axial skeleton deformities caused by Notch GOF. However, Sox9 protein level was restored in Rbpj‐deficient Notch GOF mice compared with Notch GOF mutants, demonstrating that regulation of Sox9 expression is canonical or Rbpj‐dependent. To further understand the molecular basis of this regulation, we performed chromatin immunoprecipitation (ChIP) assays and detected the recruitment of the Rbpj/NICD transcription complex to Rbpj‐binding sites upstream of the Sox9 promoter. The association of the Rbpj/NICD complex with the Sox9 promoter is associated with transcriptional repression of Sox9 in a cellular model of chondrocyte differentiation. Hence, Notch negatively regulates chondrocyte differentiation in the axial skeleton by suppressing Sox9 transcription, and Rbpj‐independent Notch signaling mechanisms may also contribute to axial skeletogenesis.

Notch Signaling in Skeletal Stem Cells

The skeleton originates from stem cells residing in the sclerotome and neural crest that undergo proliferation, migration, and commitment. The development of the skeletal stem cells is influenced by many signaling pathways that govern cell fate determination, proliferation, differentiation, and apoptosis. This review will focus on Notch signaling functions in regulating the different cell types that form the skeletal system as well as the interplay between them to maintain homeostasis. Osteochondroprogenitors require Notch signaling to maintain multipotency and to prevent premature differentiation into osteoblasts. Subsequently, overactivation of Notch signaling suppresses osteoblast maturation. Moreover, Notch signaling in osteochondroprogenitors is required for chondrocyte proliferation and hypertrophy and suppresses terminal differentiation. Translational studies demonstrated a crucial role of Notch signaling in osteosarcoma and osteoarthritis, where concepts derived from developmental pathways are often recapitulated. This brings hope of taking advantage of the molecular mechanisms learned from development to approach the pathological processes underlying abnormal bone/cartilage metabolism or tumorigenesis. Pharmacological agents that target Notch receptors or ligands in a tissue-specific fashion would offer new opportunities for treating bone/cartilage diseases caused by dysregulation of Notch signaling.

E-selectin ligand 1 regulates bone remodeling by limiting bioactive TGF-β in the bone microenvironment

TGF-β is abundantly produced in the skeletal system and plays a crucial role in skeletal homeostasis. E-selectin ligand-1 (ESL-1), a Golgi apparatus-localized protein, acts as a negative regulator of TGF-β bioavailability by attenuating maturation of pro–TGF-β during cartilage homeostasis. However, whether regulation of intracellular TGF-β maturation by ESL-1 is also crucial during bone homeostasis has not been well defined. Here, we show that Esl-1−/− mice exhibit a severe osteopenia with elevated bone resorption and decreased bone mineralization. In primary culture, Esl-1−/− osteoclast progenitors show no difference in osteoclastogenesis. However, Esl-1−/− osteoblasts show delayed differentiation and mineralization and stimulate osteoclastogenesis more potently in the osteoblast–osteoclast coculture, suggesting that ESL-1 primarily acts in osteoblasts to regulate bone homeostasis. In addition, Esl-1−/− calvaria exhibit an elevated mature TGF-β/pro–TGF-β ratio, with increased expression of TGF-β downstream targets (plasminogen activator inhibitor-1, parathyroid hormone-related peptide, connective tissue growth factor, and matrix metallopeptidase 13, etc.) and a key regulator of osteoclastogenesis (receptor activator of nuclear factor κB ligand). Moreover, in vivo treatment with 1D11, a pan–TGF-β antibody, significantly improved the low bone mass of Esl-1−/− mice, suggesting that elevated TGF-β signaling is the major cause of osteopenia in Esl-1−/− mice. In summary, our study identifies ESL-1 as an important regulator of bone remodeling and demonstrates that the modulation of TGF-β maturation is pivotal in the maintenance of a homeostatic bone microenvironment and for proper osteoblast–osteoclast coupling.