Min-g Youn

A histone lysine methyltransferase activated by non-canonical Wnt signalling suppresses PPAR-gamma transactivation.

Histone modifications induced by activated signalling cascades are crucial to cell-lineage decisions. Osteoblast and adipocyte differentiation from common mesenchymal stem cells is under transcriptional control by numerous factors. Although PPAR-γ (peroxisome proliferator activated receptor-γ) has been established as a prime inducer of adipogenesis, cellular signalling factors that determine cell lineage in bone marrow remain generally unknown. Here, we show that the non-canonical Wnt pathway through CaMKII–TAK1–TAB2–NLK transcriptionally represses PPAR-γ transactivation and induces Runx2 expression, promoting osteoblastogenesis in preference to adipogenesis in bone marrow mesenchymal progenitors. Wnt-5a activates NLK (Nemo-like kinase), which in turn phosphorylates a histone methyltransferase, SETDB1 (SET domain bifurcated 1), leading to the formation of a co-repressor complex that inactivates PPAR-γ function through histone H3-K9 methylation. These findings suggest that the non-canonical Wnt signalling pathway suppresses PPAR-γ function through chromatin inactivation triggered by recruitment of a repressing histone methyltransferase, thus leading to an osteoblastic cell lineage from mesenchymal stem cells.

DNA demethylation in hormone-induced transcriptional derepression

Epigenetic modifications at the histone level affect gene regulation in response to extracellular signals. However, regulated epigenetic modifications at the DNA level, especially active DNA demethylation, in gene activation are not well understood. Here we report that DNA methylation/demethylation is hormonally switched to control transcription of the cytochrome p450 27B1 (CYP27B1) gene. Reflecting vitamin-D-mediated transrepression of the CYP27B1 gene by the negative vitamin D response element (nVDRE), methylation of CpG sites (5mCpG) is induced by vitamin D in this gene promoter. Conversely, treatment with parathyroid hormone, a hormone known to activate the CYP27B1 gene, induces active demethylation of the 5mCpG sites in this promoter. Biochemical purification of a complex associated with the nVDRE-binding protein (VDIR, also known as TCF3) identified two DNA methyltransferases, DNMT1 and DNMT3B, for methylation of CpG sites, as well as a DNA glycosylase, MBD4 (ref. 10). Protein-kinase-C-phosphorylated MBD4 by parathyroid hormone stimulation promotes incision of methylated DNA through glycosylase activity, and a base-excision repair process seems to complete DNA demethylation in the MBD4-bound promoter. Such parathyroid-hormone-induced DNA demethylation and subsequent transcriptional derepression are impaired in Mbd4-/- mice. Thus, the present findings suggest that methylation switching at the DNA level contributes to the hormonal control of transcription.

Nuclear Receptors in Bone Physiology and Diseases

During the last decade, our view on the skeleton as a mere solid physical support structure has been transformed, as bone emerged as a dynamic, constantly remodeling tissue with systemic regulatory functions including those of an endocrine organ. Reflecting this remarkable functional complexity, distinct classes of humoral and intracellular regulatory factors have been shown to control vital processes in the bone. Among these regulators, nuclear receptors (NRs) play fundamental roles in bone development, growth, and maintenance. NRs are DNA-binding transcription factors that act as intracellular transducers of the respective ligand signaling pathways through modulation of expression of specific sets of cognate target genes. Aberrant NR signaling caused by receptor or ligand deficiency may profoundly affect bone health and compromise skeletal functions. Ligand dependency of NR action underlies a major strategy of therapeutic intervention to correct aberrant NR signaling, and significant efforts have been made to design novel synthetic NR ligands with enhanced beneficial properties and reduced potential negative side effects. As an example, estrogen deficiency causes bone loss and leads to development of osteoporosis, the most prevalent skeletal disorder in postmenopausal women. Since administration of natural estrogens for the treatment of osteoporosis often associates with undesirable side effects, several synthetic estrogen receptor ligands have been developed with higher therapeutic efficacy and specificity. This review presents current progress in our understanding of the roles of various nuclear receptor-mediated signaling pathways in bone physiology and disease, and in development of advanced NR ligands for treatment of common skeletal disorders.

JMJD5, a Jumonji C (JmjC) domain-containing protein, negatively regulates osteoclastogenesis by facilitating NFATc1 protein degradation.

Background: NFATc1 is a necessary and sufficient transcription factor for osteoclastogenesis. Results: JMJD5 negatively regulates NFATc1 protein level through its hydroxylase activity. Conclusion: JMJD5 is a novel osteoclastogenic repressor that induces the degradation of NFATc1 protein. Significance: This study revealed a novel mechanism that regulates NFATc1 activity during osteoclastogenesis. Osteoclastogenesis is a highly regulated process governed by diverse classes of regulators. Among them, nuclear factor of activated T-cells calcineurin-dependent 1 (NFATc1) is the primary osteoclastogenic transcription factor, and its expression is transcriptionally induced during early osteoclastogenesis by receptor activation of nuclear factor κB ligand (RANKL), an osteoclastogenic cytokine. Here, we report the novel enzymatic function of JMJD5, which regulates NFATc1 protein stability. Among the tested Jumonji C (JmjC) domain-containing proteins, decreased mRNA expression levels during osteoclastogenesis were found for JMJD5 in RAW264 cells stimulated by RANKL. To examine the functional role of JMJD5 in osteoclast differentiation, we established stable JMJD5 knockdown cells, and osteoclast formation was assessed. Down-regulated expression of JMJD5 led to accelerated osteoclast formation together with induction of several osteoclast-specific genes such as Ctsk and DC-STAMP, suggesting that JMJD5 is a negative regulator in osteoclast differentiation. Although JMJD5 was recently reported as a histone demethylase for histone H3K36me2, no histone demethylase activity was detected in JMJD5 in vitro or in living cells, even for other methylated histone residues. Instead, JMJD5 co-repressed transcriptional activity by destabilizing NFATc1 protein. Protein hydroxylase activity mediated by the JmjC domain in JMJD5 was required for the observed functions of JMJD5. JMJD5 induced the association of hydroxylated NFATc1 with the E3 ubiquitin ligase Von Hippel-Lindau tumor suppressor (VHL), thereby presumably facilitating proteasomal degradation of NFATc1 via ubiquitination. Taken together, the present study demonstrated that JMJD5 is a post-translational co-repressor for NFATc1 that attenuates osteoclastogenesis.

Phosphorylation of Williams Syndrome Transcription Factor by MAPK Induces a Switching between Two Distinct Chromatin Remodeling Complexes

Changes in the environment of a cell precipitate extracellular signals and sequential cascades of protein modification and elicit nuclear transcriptional responses. However, the functional links between intracellular signaling-dependent gene regulation and epigenetic regulation by chromatin-modifying proteins within the nucleus are largely unknown. Here, we describe novel epigenetic regulation by MAPK cascades that modulate formation of an ATP-dependent chromatin remodeling complex, WINAC (WSTF Including Nucleosome Assembly Complex), an SWI/SNF-type complex containing Williams syndrome transcription factor (WSTF). WSTF, a specific component of two chromatin remodeling complexes (SWI/SNF-type WINAC and ISWI-type WICH), was phosphorylated by the stimulation of MAPK cascades in vitro and in vivo. Ser-158 residue in the WAC (WSTF/Acf1/cbpq46) domain, located close to the N terminus of WSTF, was identified as a major phosphorylation target. Using biochemical analysis of a WSTF mutant (WSTF-S158A) stably expressing cell line, the phosphorylation of this residue (Ser-158) was found to be essential for maintaining the association between WSTF and core BAF complex components, thereby maintaining the ATPase activity of WINAC. WINAC-dependent transcriptional regulation of vitamin D receptor was consequently impaired by this WSTF mutation, but the recovery from DNA damage mediated by WICH was not impaired. Our results suggest that WSTF serves as a nuclear sensor of the extracellular signals to fine-tune the chromatin remodeling activity of WINAC. WINAC mediates a previously unknown MAPK-dependent step in epigenetic regulation, and this MAPK-dependent switching mechanism between the two functionally distinct WSTF-containing complexes might underlie the diverse functions of WSTF in various nuclear events.

Transcriptionally active nuclei are selective in mature multinucleated osteoclasts.

Multinucleation is indispensable for the bone‐resorbing activity of mature osteoclasts. Although multinucleation is evident in mature osteoclasts and certain other cell types, putative regulatory networks among nuclei remain poorly characterized. To address this issue, transcriptional activity of each nucleus in a multinucleated osteoclast was assessed by detecting the distributions of nuclear proteins by immunocytochemistry and primary transcripts by RNA FISH. Patterns of epigenetic histone markers governing transcription as well as localization of tested nuclear receptor proteins appeared indistinguishable among nuclei in differentiated Raw264 cells and mouse mature osteoclasts. However, RNAPII‐Ser5P/2P and NFATc1 proteins were selectively distributed in certain nuclei in the same cell. Similarly, the distributions of primary transcripts for osteoclast‐specific genes (Nfatc1, Ctsk and Acp5) as well as a housekeeping gene (beta‐tubulin) were limited in certain nuclei within individual cells. By fusing two Raw264 cell lines that stably expressed ZsGreen‐NLS and DsRed‐NLS proteins, transmission of nuclear proteins across all of the nuclei in a cell could be observed, presumably through the shared cytoplasm. Taken together, we conclude that although nuclear proteins are diffusible among nuclei, only certain nuclei within a multinucleated osteoclast are transcriptionally active.

Retraction: DNA demethylation in hormone-induced transcriptional derepression

This corrects the article DOI: 10.1038/nature08456

Emergence of the osteo-epigenome in bone biology

Bone development and bone remodeling are accompanied by bone cell-specific gene regulation. Recent progress in gene expression has revealed that chromatin remodeling and histone modifications are indispensable for transcriptional activation as well as repression. Previously known transcriptional coregulators have consistently been found to regulate chromatin reorganization, constituting an epigenetic platform on chromatin. As in other tested cells, bone cell type-specific chromatin reorganization is thought to mediate the function of prime transcriptional factors, which are responsible for bone cell fate decision and cellular differentiation. In this Perspective, we review chromatin reorganization and its regulators in bone cells, along with recent pioneering work describing osteo-epigenetic regulators. IBMS BoneKEy. 2010 September;7(9):314-324. 2010 International Bone & Mineral Society The Transcriptional Network in Bone Development and Remodeling From its first appearance in embryos, bones formation and maintenance is a dynamic and complex process. Complicated but highly regulated differentiation steps guide the generation of bone tissue, processes that continue until full growth is achieved. In adulthood, even though net increases in bone have ceased, bone remodeling remains active to keep serum mineral levels physiologically normal in response to demands, and to maintain a physically solid bone structure against mechanical stress. In this respect, bone cells are like other cell types as their proliferation and differentiation are highly regulated through the entire life of the animal (1). Osteoblasts, osteocytes and chondrocytes originate from mesenchymal stem cells, whereas osteoclast precursors arise from hematopoietic stem cells (2-5). Autonomous cell proliferation and differentiation of these bone cells are regulated in a bone cell type-specific manner but “cross-talk” among different types of bone cells is significant during bone tissue development and bone remodeling (6;7). Reflecting the complexity of the proliferation and differentiation of bone cells, a number of regulators have been identified and their physiological impact in intact bone has been confirmed by a variety of experimental approaches (1;8). Among such osteoregulators, transcriptional regulators appear pivotal in cell lineage decisions (9-11). Transcriptional controls at a given type II gene locus require in general three classes of transcription factors. Basic transcription factors are essential for a transcriptional reaction by RNA polymerase II. Assembly of a set of basic transcription factors assures the minimum conditions for initiation of transcription (12). The enhancement or suppression of transcription depends on the participation of DNA-binding transcription factors. Since DNA-binding transcription factors direct transcriptional regulation, the expression and function of this class of transcription factors are prime determinants in tissue-specific gene cascades. These two classes of transcription factors are emphasized in the current literature from a classical point of view. However, a third class of factors, transcriptional coregulators, has emerged and is now understood to be essential for transcriptional control (13) (Fig. 1). The role of these coIBMS BoneKEy. 2010 September;7(9):314-324 http://www.bonekey-ibms.org/cgi/content/full/ibmske;7/9/314 doi: 10.1138/20100464

Purification and identification of estrogen receptor alpha co‐regulators in osteoclasts

Mature osteoclasts are multinuclear, macrophage‐like cells derived from hematopoietic stem cells in the bone marrow. Several transcription factors regulating osteoclast differentiation have been identified. However, the molecular basis of transcriptional regulation in osteoclasts at epigenetic levels is largely unknown. In fact, no osteoclast‐specific transcriptional co‐regulators have been characterized. Recently, selective ablation of estrogen receptor alpha (ERα) in mature osteoclasts derived from female mice (ERαΔoc/Δoc) exhibited trabecular bone loss due to induced apoptosis via upregulated expression of Fas ligand mRNA. In general, the component composition of the ERα‐associated co‐activator complex and its expression levels are distinct among tissues. However, ERα transcriptional co‐regulators in mature osteoclasts remain unclear. In the present study, we achieved large‐scale cultivation of mature, multinucleated osteoclasts and established a purification system for ERα‐associated proteins. In addition to co‐regulators previously found in other ERα target cells, several unexpected factors were found such as CAP‐H. The mRNA expression level of CAP‐H was high during osteoclast differentiation. These results demonstrate the existence of osteoclast‐specific transcriptional co‐regulators supporting ERα function.

DNA demethylation for hormone-induced transcriptional derepression

This corrects the article DOI: 10.1038/nature08456