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Transcription factor Runx2 controls bone development and osteoblast differentiation by regulating expression of a significant number of bone-related target genes. Here, we report that transcriptional activation and repression of the Runx2 gene via its osteoblast-specific P1 promoter (encoding mRNA for the Runx2/p57 isoform) is accompanied by selective deposition and elimination of histone marks during differentiation of mesenchymal cells to the osteogenic and myoblastic lineages. These epigenetic profiles are mediated by key components of the Trithorax/COMPASS-like and Polycomb group complexes together with histone arginine methylases like PRMT5 and lysine demethylases like JARID1B/KDM5B. Importantly, knockdown of the H3K4me2/3 demethylase JARID1B, but not of the demethylases UTX and NO66, prevents repression of the Runx2 P1 promoter during myogenic differentiation of mesenchymal cells. The epigenetically forced expression of Runx2/p57 and osteocalcin, a classical bone-related target gene, under myoblastic-differentiation is accompanied by enrichment of the H3K4me3 and H3K27ac marks at the Runx2 P1 promoter region. Our results identify JARID1B as a key component of a potent epigenetic switch that controls mesenchymal cell fate into myogenic and osteogenic lineages., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Mitotic inheritance of gene function is obligatory to sustain biological control. Emerging evidence suggests that epigenetic mechanisms are linked to transmission of cell fate, lineage commitment, and maintenance of cellular phenotype in progeny cells. Mechanisms of epigenetic memory include gene silencing by DNA methylation, transcriptional regulation by histone modifications, regulation of gene expression by noncoding small RNA molecules, and retention of regulatory machinery on target gene loci for activation and repression. We will focus on the regulatory implications of epigenetic memory for physiological control and for the onset and progression of disease.

Transcriptional control of Runx2 gene expression through two alternative promoters (P1 and P2) is critical for the execution of its function as an osteogenic cell fate determining factor. In all vertebrates examined to date, the bone related P1 promoter contains a purine-rich region (-303 to -128 bp in the rat) that separates two regulatory domains. The length of this region differs dramatically between species even within the same order. Using deletion analysis, we show that part of this purine-rich region (-200 to -128) containing a duplicated element (Y-repeat) positively regulates Runx2 P1 transcription. Electrophoretic mobility assays and chromatin immunoprecipitations reveal that Y-repeat binds at least two different classes of transcription factors related to GC box binding proteins (e.g. SP1 and SP7/Osterix) and ETS-like factors (e.g. ETS1 and ELK1). Forced expression of SP1 increases Runx2 P1 promoter activity through the Y-repeats, and small interfering RNA depletion of SP1 decreases Runx2 expression. Similarly, exogenous expression of wild type ELK1, but not a defective mutant that cannot be phosphorylated, enhances Runx2 gene expression. SP1 is most abundant in proliferating cells, and ELK1 is most abundant in postconfluent cells; during MC3T3-E1 osteoblast differentiation, both proteins are transiently co-expressed when Runx2 expression is enhanced. Taken together, our data suggest that basal Runx2 gene transcription is regulated by dynamic interactions between SP1 and ETS-like factors during progression of osteogenesis.

Changes in local chromatin structure accompany transcriptional activation of eukaryotic genes. In vivo these changes in chromatin organization can be catalyzed by ATP-dependent chromatin-remodeling complexes, such as SWI/SNF. These complexes alter the tight wrapping of DNA in the nucleosomes and can facilitate the mobilization of the histone octamer to adjacent DNA segments, leaving promoter regulatory elements exposed for transcription factor binding. To gain understanding of how the activity of SWI/SNF complexes may be modulated by the different DNA sequences within a natural promoter, we have reconstituted nucleosomes containing promoter segments of the transcriptionally active cell type-specific osteocalcin (OC) gene and determined how they affect the directional movements of the nucleosomes. Our results indicate that SWI/SNF complexes induce octamer sliding to preferential positions in the OC promoter, leading to a nucleosomal organization that resembles that described in intact cells expressing the OC gene. Our studies demonstrate that the position of the histone octamer is primarily determined by sequences within the OC promoter that include or exclude nucleosomes. We propose that these sequences are critical components of the regulatory mechanisms that mediate expression of this tissue-specific gene.

Tissue-specific activation of the osteocalcin (OC) gene is associated with changes in chromatin structure at the promoter region. Two nuclease-hypersensitive sites span the key regulatory elements that control basal tissue-specific and vitamin D3-enhanced OC gene transcription. To gain understanding of the molecular mechanisms involved in chromatin remodeling of the OC gene, we have examined the requirement for SWI/SNF activity. We inducibly expressed an ATPase-defective BRG1 catalytic subunit that forms inactive SWI/SNF complexes that bind to the OC promoter. This interaction results in inhibition of both basal and vitamin D3-enhanced OC gene transcription and a marked decrease in nuclease hypersensitivity. We find that SWI/SNF is recruited to the OC promoter via the transcription factor CCAAT/enhancer-binding protein beta, which together with Runx2 forms a stable complex to facilitate RNA polymerase II binding and activation of OC gene transcription. Together, our results indicate that the SWI/SNF complex is a key regulator of the chromatin-remodeling events that promote tissue-specific transcription in osteoblasts.

The molecular logistics of nuclear regulatory processes necessitate temporal and spatial regulation of protein-protein and protein-DNA interactions in response to physiological cues. Biochemical, in situ, and in vivo genetic evidence demonstrates the requirement for intranuclear localization of regulatory complexes that functionally couple cellular responses to signals that mediate combinatorial control of gene expression. We have summarized evidence that subnuclear targeting of transcription factors mechanistically links gene expression with architectural organization and assembly of nuclear regulatory machinery for biological control. The compromised intranuclear targeting of regulatory proteins under pathological conditions provides options for the diagnosis and treatment of disease.

Vitamin D receptor (VDR) and Runx2 are key regulators of tissue-specific gene transcription. Using the bone-related osteocalcin (OC) gene, we have previously shown that Runx2 is required for the extensive chromatin remodeling that accompanies gene activation. Here, we have addressed the direct contribution of the VDR to chromatin remodeling events necessary for regulation of OC transcription using mutational analysis. Our studies demonstrate that both the distal and proximal DNase I-hypersensitive sites characteristic of the transcriptionally active OC promoter are not enhanced in the absence of a functional vitamin D response element (VDRE). Furthermore, restriction enzyme accessibility studies reveal that nucleosomal reorganization of the proximal promoter occurs in response to vitamin D and this reorganization is abrogated by mutation of the VDRE. These findings indicate that binding of liganded VDR in the distal promoter directly impacts the chromatin structure of the proximal promoter. We find that, in the absence of functional Runx sites, the VDR cannot be recruited to the OC promoter and, therefore, the VDRE is not competent to mediate vitamin D responsiveness. On the other hand, chromatin immunoprecipitation assays show that Runx2 association with the OC promoter is not significantly impaired when the VDRE is mutated. Chromatin immunoprecipitation assays also demonstrate that basal levels of histone acetylation occur in the absence of Runx2 binding but that the VDRE and vitamin D are required for enhanced acetylation of histones H3 and H4 downstream of the VDRE. Together our results support a stepwise model for chromatin remodeling of the OC promoter and show that binding of the liganded VDR.retinoid X receptor directly impacts both the distal and proximal regulatory domains.

The accessibility of regulatory elements in chromatin represents a principal rate-limiting parameter of gene transcription and is modulated by enzymatic transcriptional co-factors that alter the topology of chromatin or covalently modify histones (e.g. by acetylation). The bone-specific activation and 1,25-dihydroxyvitamin D(3) enhancement of osteocalcin (OC) gene transcription are both functionally linked to modifications in nucleosomal organization. The initiation of tissue-specific basal transcription is accompanied by the induction of two DNase I hypersensitive sites, and this chromatin remodeling event requires binding of the key osteogenic factor RUNX2/CBFA1 to the OC promoter. Here, we analyzed the acetylation status of histones H3 and H4 when the OC gene is active (in osteoblastic ROS17/2.8 cells) or inactive (in fibroblastic ROS24/1 cells) using chromatin immunoprecipitation assays. We find that acetylated histone H3 and H4 proteins are associated with the OC promoter only when the gene is transcriptionally active and that the acetylation status is relatively uniform across the OC locus under basal conditions. Acetylation of H4 at the OC gene is selectively increased following vitamin D(3) enhancement of OC transcription, with the most prominent changes occurring in the region between the vitamin D(3) enhancer and basal promoter. Thus, our results suggest functional linkage of H3 and H4 acetylation in specific regions of the OC promoter to chromatin remodeling that accompanies tissue-specific transcriptional activation and vitamin D enhancement of OC gene expression. These findings provide mechanistic insights into bone-specific gene activation within a native genomic context in response to steroid hormone-related regulatory cues.

CCAAT/enhancer-binding proteins (C/EBP) are critical determinants for cellular differentiation and cell type-specific gene expression. Their functional roles in osteoblast development have not been determined. We addressed a key component of the mechanisms by which C/EBP factors regulate transcription of a tissue-specific gene during osteoblast differentiation. Expression of both C/EBPbeta and C/EBPdelta increases from the growth to maturation developmental stages and, like the bone-specific osteocalcin (OC) gene, is also stimulated 3-6-fold by vitamin D(3), a regulator of osteoblast differentiation. We characterized a C/EBP enhancer element in the proximal promoter of the rat osteocalcin gene, which resides in close proximity to a Runx2 (Cbfa1) element, essential for tissue-specific activation. We find that C/EBP and Runx2 factors interact together in a synergistic manner to enhance OC transcription (35-40-fold) in cell culture systems. We show by mutational analysis that this synergism is mediated through the C/EBP-responsive element in the OC promoter and by a direct interaction between Runx2 and C/EBPbeta. Furthermore, we have mapped a domain in Runx2 necessary for this interaction by immunoprecipitation. A Runx2 mutant lacking this interaction domain does not exhibit functional synergism. We conclude that, in addition to Runx2 DNA binding functions, Runx2 can also form a protein complex at C/EBP sites to regulate transcription. Taken together, our findings indicate that C/EBP is a principal transactivator of the OC gene and the synergism with Runx2 suggests that a combinatorial interaction of these factors is a principal mechanism for regulating tissue-specific expression during osteoblast differentiation.