Regulation of the osteoblast-specific transcription factor Osterix by NO66, a Jumonji family histone demethylase.
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ABSTRACT:
SUBMITTER: Sinha KM
PROVIDER: S-EPMC2780536 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Osterix (Osx) is an osteoblast-specific transcriptional factor and is required for osteoblast differentiation and bone formation. A JmjC domain-containing protein NO66 was previously found to participate in regulation of Osx transcriptional activity and plays an important role in osteoblast differentiation through interaction with Osx. Here, we report the crystal structure of NO66 forming in a functional tetramer. A hinge domain links the N-terminal JmjC domain and C-terminal winged helix-turn-helix domain of NO66, and both domains are essential for tetrameric assembly. The oligomerization interface of NO66 interacts with a conserved fragment of Osx. We show that the hinge domain-dependent oligomerization of NO66 is essential for inhibition of Osx-dependent gene activation. Our findings suggest that homo-oligomerization of JmjC domain containing proteins might play a physiological role through interactions with other regulatory factors during gene expression.
| S-EPMC3675579 | biostudies-literature
Project description:Commitment of Runx2-expressing precursor osteoblasts to functional osteoblasts and then to osteocytes is triggered by Osterix (Osx), which activates its target genes in those cells during bone formation. It is not yet known whether Osx has a role in remodeling the chromatin architecture of its target genes during the transition from preosteoblast to osteoblast. In testing the hypothesis that Osx is indispensable for active chromatin architecture, we first showed that in Osx-null calvarial cells occupancy of the transcriptional activators, including lysine 4 methyl transferase (Wdr5), c-Myc, and H2A.Z, at the Osx target gene Bsp was very markedly decreased. The levels of methylation of lysines 4 and 36 and acetylation of histone H3, markers for active chromatin, were also reduced at the Bsp gene in these cells. In contrast, occupancy of the transcriptional repressors HP1 and the nucleolar protein 66 (NO66), a histone demethylase previously identified as an Osx-interacting protein, was increased at the Bsp gene in Osx-null calvarial cells. Furthermore, the Bsp promoter was hypermethylated in embryonic stem (ES) cells and in embryonic day 9.5 (E9.5) embryos but was markedly hypomethylated in the calvaria of E18.5 embryos, coinciding with robust Bsp expression. In contrast, CpG methylation in the Bsp promoter remained high in Osx-null calvaria compared to Osx-wild-type calvaria. Our data also revealed that NO66 interacted with DNA Methyltransferase 1A (DNMT1A), histone deacetylase 1A (HDAC1A), and HP1, which are known to control histone and DNA methylation. In addition, HP1 stimulated the demethylase activity of NO66 for its substrates "trimethylation of histone H3 at lysine 4" (H3K4me3) and "trimethylation of histone H3 at lysine 36" (H3K36me3). Our findings strongly suggest that in the absence of Osx, the chromatin of Osx target genes is transcriptionally inactive. We propose that Osx is a molecular switch for the formation of an active chromatin state during osteoblast differentiation, whereas NO66 helps gene repression through histone demethylation and/or formation of a repressor complex, resulting in multilayered control of the chromatin architecture of specific osteoblast genes.
| S-EPMC3961497 | biostudies-literature
Project description:The full length human histone 3 lysine 4 demethylase KDM5B (PLU-1/Jarid1B) has been studied using Hydrogen/Deuterium exchange mass spectrometry, homology modelling, sequence analysis, small angle X-ray scattering and electron microscopy. This first structure on an intact multi-domain Jumonji histone demethylase reveal that the so-called PLU region, in the central region of KDM5B, has a curved α-helical three-dimensional structure, that acts as a rigid linker between the catalytic core and a region comprising four α-helices, a loop comprising the PHD2 domain, two large intrinsically disordered loops and the PHD3 domain in close proximity. The dumbbell shaped and curved KDM5B architecture observed by electron microscopy is complementary to the nucleosome surface and has a striking overall similarity to that of the functionally related KDM1A/CoREST complex. This could suggest that there are similarities between the demethylation mechanisms employed by the two histone 3 lysine 4 demethylases at the molecular level.
| S-EPMC6411775 | biostudies-literature
Project description:Osterix (Osx) is an osteoblast-specific transcription factor required for osteoblast differentiation and bone formation. Osx knock-out mice lack bone completely. Recent findings that Osx inhibits Wnt signaling provide a feedback control mechanism involved in bone formation. Mechanisms of Osx inhibition on Wnt signaling are not fully understood. Our results in this study revealed that the expression of a Wnt antagonist Sclerostin (Sost) was downregulated in Osx-null calvaria. Overexpression of Osx in stable C2C12 mesenchymal cell line resulted in Sost upregulation. Transient transfection assay showed that Osx activated 1kb Sost promoter reporter activity in a dose-dependent manner. To define Sost promoter activated by Osx, we made a series of deletion mutants of Sost constructs, and narrowed down the minimal region to the proximal 260bp. Gel shift assay indicated that Osx bound to GC-rich site within this minimal region, and that point mutations of this binding site disrupted Osx binding. Moreover, the same point mutations in 260bp Sost promoter reporter disrupted the promoter activation by Osx, suggesting that the GC-rich binding site was responsible for Sost promoter activation by Osx. To further examine physical association of Osx with Sost promoter in vivo, Chromatin immunoprecipitation (ChIP) assays were performed using primary osteoblasts from mouse calvaria. Osx was found to associate with endogenous Sost promoter. Taken together, these findings support our hypothesis that Sost is a direct target of Osx. This provides a new additional mechanism through which Osx inhibits Wnt signaling during bone formation.
| S-EPMC4041335 | biostudies-literature
Project description:The transcription factor osterix (Sp7) is essential for osteoblastogenesis and bone formation in mice. Genome wide association studies have demonstrated that osterix is associated with bone mineral density in humans; however, the molecular significance of osterix in human osteoblast differentiation is poorly described. In this study we have characterized the role of osterix in human mesenchymal progenitor cell (hMSC) differentiation. We first analyzed temporal microarray data of primary hMSC treated with bone morphogenetic protein-6 (BMP6) using clustering to identify genes that are associated with osterix expression. Osterix clusters with a set of osteoblast-associated extracellular matrix (ECM) genes, including bone sialoprotein (BSP) and a novel set of proteoglycans, osteomodulin (OMD), osteoglycin, and asporin. Maximum expression of these genes is dependent upon both the concentration and duration of BMP6 exposure. Next we overexpressed and repressed osterix in primary hMSC using retrovirus. The enforced expression of osterix had relatively minor effects on osteoblastic gene expression independent of exogenous BMP6. However, in the presence of BMP6, osterix overexpression enhanced expression of the aforementioned ECM genes. Additionally, osterix overexpression enhanced BMP6 induced osteoblast mineralization, while inhibiting hMSC proliferation. Conversely, osterix knockdown maintained hMSC in an immature state by decreasing expression of these ECM genes and decreasing mineralization and hMSC proliferation. Overexpression of the osterix regulated gene OMD with retrovirus promoted mineralization of hMSC. These results suggest that osterix is necessary, but not sufficient for hMSC osteoblast differentiation. Osterix regulates the expression of a set of ECM proteins which are involved in terminal osteoblast differentiation.
| S-EPMC3241898 | biostudies-literature
Project description:The recent identification of the genes responsible for several human genetic diseases affecting bone homeostasis and the characterization of mouse models for these diseases indicated that canonical Wnt signaling plays a critical role in the control of bone mass. Here, we report that the osteoblast-specific transcription factor Osterix (Osx), which is required for osteoblast differentiation, inhibits Wnt pathway activity. First, in calvarial cells of embryonic day (E)18.5 Osx-null embryos, expression of the Wnt antagonist Dkk1 was abolished, and that of Wnt target genes c-Myc and cyclin D1 was increased. Moreover, our studies demonstrated that Osx bound to and activated the Dkk1 promoter. In addition, Osx inhibited beta-catenin-induced Topflash reporter activity and beta-catenin-induced secondary axis formation in Xenopus embryos. Importantly, in calvaria of E18.5 Osx-null embryos harboring the TOPGAL reporter transgene, beta-galactosidase activity was increased, suggesting that Osx inhibited the Wnt pathway in osteoblasts in vivo. Our data further showed that Osx disrupted binding of Tcf to DNA, providing a likely mechanism for the inhibition by Osx of beta-catenin transcriptional activity. We also showed that Osx decreased osteoblast proliferation. Indeed, E18.5 Osx-null calvaria showed greater BrdU incorporation than wild-type calvaria and that Osx overexpression in C2C12 mesenchymal cells inhibited cell growth. Because Wnt signaling has a major role in stimulating osteoblast proliferation, we speculate that Osx-mediated inhibition of osteoblast proliferation is a consequence of the Osx-mediated control of Wnt/beta-catenin activity. Our results add a layer of control to Wnt/beta-catenin signaling in bone.
| S-EPMC2383965 | biostudies-literature
Project description:In the present study, we evaluated JIB-04, a small-molecule epigenetic inhibitor initially discovered to inhibit cancer growth, to determine its ability to affect deep intrinsic resistance in a breast cancer model. The model was based on a function-based approach to the selection of cancer cells in a cell culture that can survive a variety of challenges in prolonged, but reversible, quiescence. These resistant cancer cells possessed a variety of mechanisms, including modifications of the epigenome and transcriptome, for generating a high degree of cellular heterogeneity. We found that long pretreatment with JIB-04 sensitized resistant triple-negative inflammatory breast cancer cells and their parental cell line SUM149 to the chemotherapeutic drugs doxorubicin and paclitaxel. Resistant cancer cells derived from another inflammatory breast cancer cell line, FC-IBC02, were considerably more sensitive to JIB-04 than the parental cell line. Investigating a mechanism of sensitization, we found that JIB-04 exposure increased the expression of PD-L1 in resistant cells, suggesting that JIB-04 may also sensitize resistant breast cancer cells to anti-PD-L1 immune therapy. Finally, these results support the usefulness of a cell culture-based experimental strategy for evaluating anticancer agents, such as JIB-04, that may halt cancer evolution and prevent the development of cancer resistance to currently used therapies.
| S-EPMC9179491 | biostudies-literature
Project description:Our previous studies indicated that the Jumonji C (JmjC)-domain-containing NO66 is a histone demethylase with specificity for methylated histone H3K4 and H3K36. NO66 binds to the transcription factor Osterix (Osx) and inhibits its transcriptional activity in promoter assays. However, the physiological role of NO66 in formation of mammalian bones is unknown. Here, using a genetically engineered mouse model, we show that during early skeletal development, Prx1-Cre-dependent mesenchymal deletion of NO66 promotes osteogenesis and formation of both endochondral as well as intramembranous skeletal elements, leading to a larger skeleton and a high bone mass phenotype in adult mice. The excess bone formation in mice where NO66 was deleted in cells of mesenchymal origin is associated with an increase in the number of preosteoblasts and osteoblasts. Further analysis revealed that in the embryonic limbs and adult calvaria of mice with deletion of NO66 in cells of mesenchymal origin, expression of several genes including bone morphogenetic protein 2 (Bmp2), insulin-like growth factor 1 (Igf1), and osteoclast inhibitor osteoprotegerin was increased, concurrent with an increase in expression of bone formation markers such as osterix (Osx), type I collagen, and bone sialoprotein (Bsp). Taken together, our results provide the first in vivo evidence that NO66 histone demethylase plays an important role in mammalian osteogenesis during early development as well as in adult bone homeostasis. We postulate that NO66 regulates bone formation, at least in part, via regulating the number of bone-forming cells and expression of multiple genes that are critical for these processes.
| S-EPMC4780322 | biostudies-literature
Project description:The histone demethylase Jumonji domain-containing 1A (JMJD1A) is overexpressed in multiple cancers and promotes cancer progression. However, the role and mechanism of JMJD1A in gastric cancer (GC) remains poorly understood. Here, we found that JMJD1A could suppress GC cell proliferation and xenograft tumor growth. Using RNA sequencing, we identified runt-related transcription factor 3 (RUNX3) as a novel target gene of JMJD1A. Mechanistically, we identified that JMJD1A upregulated RUNX3 through co-activating Ets-1 and reducing the H3K9me1/2 levels at the RUNX3 promoter in GC cells. Functionally, JMJD1A inhibits the growth of GC cells in vivo, which is partially dependent on RUNX3. Moreover, JMJD1A expression was decreased in GC and low expression of JMJD1A was correlated with an aggressive phenotype and a poor prognosis in patients with GC. Importantly, JMJD1A expression was positively associated with RUNX3 expression in GC samples. These studies indicated that JMJD1A upregulates RUNX3 expression via co-activation of transcription factor Ets-1 to inhibit proliferation of GC cells. Our findings provide new insight into the mechanism by which JMJD1A regulates RUNX3 transcription and suggest that JMJD1A and/or RUNX3 may be used as a therapeutic intervention for GC.
| S-EPMC7541000 | biostudies-literature
Project description:Osterix (Osx) is an osteoblast-specific transcription factor which is essential for bone formation. MicroRNAs (miRNAs) have been previously shown to be involved in osteogenesis. However, it is unclear whether Osx is involved in the regulation of miRNA expression. In this study, we have identified groups of miRNAs that are differentially expressed in calvaria of the E18.5 Osx(-/-) embryos compared to wild type embryos. The correlation between the levels of miRNAs and Osx expression was further verified in cultured M-Osx cells in which over-expression of Osx is inducible. Our results suggest that Osx down-regulates expression of a group of miRNAs including mir-133a and -204/211, but up-regulates expression of another group of miRNAs such as mir-141/200a. Mir-133a and -204/211 are known to target the master osteogenic transcription factor Runx2. Further assays suggest that Sost, which encodes the Wnt signaling antagonist Sclerostin, and alkaline phosphatase (ALP) are two additional targets of mir-204/211. Mir-141/200a has been known to target the transcription factor Dlx5. Thus, we postulate that during the process of Osx-controlled osteogenesis, Osx has the ability to coordinately modulate Runx2, Sclerostin, ALP and Dlx5 proteins at levels appropriate for optimal osteoblast differentiation and function, at least in part, through regulation of specific miRNAs. Our study shows a tight correlation between Osx and the miRNAs involved in bone formation, and provides new information about molecular mechanisms of Osx-controlled osteogenesis.
| S-EPMC3589352 | biostudies-literature