p300 is a multifunctional transcriptional coactivator that serves as an adapter for several transcription factors including nuclear steroid hormone receptors. p300 possesses an intrinsic histone acetyltransferase (HAT) activity that may be critical for promoting steroid-dependent transcriptional activation. In osteoblastic cells, transcription of the bone-specific osteocalcin (OC) gene is principally regulated by the Runx2/Cbfa1 transcription factor and is stimulated in response to vitamin D 3 via the vitamin D 3 receptor complex. Therefore, we addressed p300 control of basal and vitamin D 3 -enhanced activity of the OC promoter. We find that transient overexpression of p300 results in a significant dose-dependent increase of both basal and vitamin D 3 -stimulated OC gene activity. This stimulatory effect requires intact Runx2/Cbfa1 binding sites and the vitamin D-responsive element. In addition, by coimmunoprecipitation, we show that the endogenous Runx2/ Cbfa1 and p300 proteins are components of the same complexes within osteoblastic cells under physiological concentrations. We also demonstrate by chromatin immunoprecipitation assays that p300, Runx2/Cbfa1, and 1␣,25-dihydroxyvitamin D 3 receptor interact with the OC promoter in intact osteoblastic cells expressing this gene. The effect of p300 on the OC promoter is independent of its intrinsic HAT activity, as a HAT-deficient p300 mutant protein up-regulates expression and cooperates with P/CAF to the same extent as the wild-type p300. On the basis of these results, we propose that p300 interacts with key transcriptional regulators of the OC gene and bridges distal and proximal OC promoter sequences to facilitate responsiveness to vitamin D 3 .The rat osteocalcin (OC) gene encodes a 10-kDa bonespecific protein that is induced in osteoblasts with the onset of mineralization at late stages of differentiation (26). Transcription of the OC gene is controlled by modularly distributed basal and hormone-responsive elements, located within two DNase I-hypersensitive sites (distal site, positions Ϫ600 to Ϫ400; proximal site, positions Ϫ170 to Ϫ70) that are present only in bone-derived cells expressing this gene (23,24). Thus, chromatin remodeling of the OC gene promoter accompanies the onset in OC gene expression during osteoblast differentiation (Fig. 1). A key regulatory element that controls OC gene expression is recognized by the 1␣,25-dihydroxyvitamin D 3 receptor (VDR) complex upon ligand activation. This vitamin D 3 -responsive element (VDRE) is located in the distal region (Fig. 1) of the OC promoter (positions Ϫ465 to Ϫ437) and functions as an enhancer to increase OC gene transcription by three-to fivefold (20). Binding of the ligand 1␣,25-dihydroxyvitamin D 3 (vitamin D 3 ) induces conformational changes in the receptor that enable it to interact with several coactivators, such as NCoA-1/SRC-1 (nuclear receptor coactivator 1/steroid receptor coactivator 1), NCoA-2/GRIP/TIF2 (nuclear receptor coactivator 2/glucocorticoid receptor-interacting protein/transcrip...

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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 D 3 -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 D 3 -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 ␤, 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.Within the eukaryotic nucleus, the packaging of DNA into nucleosomes and higher order chromatin structures have been implicated in the regulation of key cellular events, such as replication and transcription. During the last decade, a large family of protein complexes that promote transcription by altering chromatin structure have been described (1-3). Among them is the SWI/SNF complex subfamily that remodels chromatin in an ATP-dependent manner (1-3). SWI/SNF complexes are composed of several subunits, which have been implicated in a wide range of cellular events, including gene regulation, cell cycle control, development, and differentiation (1, 3). The mammalian SWI/SNF complexes contain a catalytic subunit that can be either BRG1 or BRM, which includes ATPase activity. Mutations in the ATPase domain of BRG1 or BRM that abrogate the ability of these proteins to bind ATP result in the formation of inactive SWI/SNF complexes (4 -6). Furthermore, expression of mutant BRG1 or BRM proteins in NIH3T3 cells impairs the ability of these cells to activate endogenous stress response genes in the presence of arsenite (5) and to differentiate into muscle or adipocytic cells (4, 5, 7). In addition, we have recently shown that the presence of the mutant BRG1 protein in these NIH3T3 cell lines inhibits BMP2-induced differentiation into the osteoblast lineage (8).The rat osteocalcin (OC) 3 gene encodes a 10-kDa bone-specific protein that is expressed at late stages of osteoblast differentiation, concomitant with the mineralization of the extracellular matrix (9). Osteoblast-specific transcription of the OC gene is controlled by modularly organized basal and hormoneresponsive elements located within two DNase I-hypersensitive sites (distal site Ϫ605 to Ϫ400 and proximal site Ϫ170 to Ϫ70; see Fig. 1) that are present only in osteoblastic cells expressing this gene (10). Thus, chromatin remodeling of the OC gene p...

BackgroundIncreased expression of the cyclooxygenase-2 enzyme (COX2) is one of the main characteristics of gastric cancer (GC), which is a leading cause of death in the world, particularly in Asia and South America. Although the Wnt/β-catenin signaling pathway has been involved in the transcriptional activation of the COX2 gene, the precise mechanism modulating this response is still unknown.Methodology/Principal FindingsHere we studied the transcriptional regulation of the COX2 gene in GC cell lines and assessed whether this phenomenon is modulated by Wnt/β-catenin signaling. We first examined the expression of COX2 mRNA in GC cells and found that there is a differential expression pattern consistent with high levels of nuclear-localized β-catenin. Pharmacological treatment with either lithium or valproic acid and molecular induction with purified canonical Wnt3a significantly enhanced COX2 mRNA expression in a dose- and time-dependent manner. Serial deletion of a 1.6 Kbp COX2 promoter fragment and gain- or loss-of-function experiments allowed us to identify a minimal Wnt/β-catenin responsive region consisting of 0.8 Kbp of the COX2 promoter (pCOX2-0.8), which showed maximal response in gene-reporter assays. The activity of this pCOX2-0.8 promoter region was further confirmed by site-directed mutagenesis and DNA-protein binding assays.Conclusions/SignificanceWe conclude that the pCOX2-0.8 minimal promoter contains a novel functional T-cell factor/lymphoid enhancer factor (TCF/LEF)-response element (TBE Site II; -689/-684) that responds directly to enhanced Wnt/β-catenin signaling and which may be important for the onset/progression of GC.

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The Runx2 transcription factor is essential for skeletal development as it regulates expression of several key bone-related genes. Multiple lines of evidence indicate that expression of the Runx2/p57 isoform in osteoblasts is controlled by the distal P1 promoter. Alterations of chromatin structure are often associated with transcription and can be mediated by members of the SWI/SNF family of chromatin remodeling complexes, or by transcriptional co-activators that possess enzymatic activities that covalently modify structural components of the chromatin. Here, we report that a specific chromatin remodeling process at the proximal region (−400 to +35) of the Runx2 gene P1 promoter accompanies transcriptional activity in osteoblasts. This altered chromatin organization is reflected by the presence of two DNase I hypersensitive sites that span key regulatory elements for Runx2/p57 transcription. Chromatin remodeling and transcription of the Runx2 gene are associated with elevated levels of histone acetylation at the P1 promoter region and binding of active RNA polymerase II, and are independent of the activity of the SWI/SNF chromatin remodeling complex. Changes in chromatin organization at the P1 promoter are stimulated during differentiation of C2C12 mesenchymal cells to the osteoblastic lineage by treatment with BMP2. Together, our results support a model in which changes in chromatin organization occur at very early stages of mesenchymal differentiation to facilitate subsequent expression of the Runx2/p57 isoform in osteoblastic cells.The Runx2 transcription factor is essential for skeletal formation as it regulates the expression of numerous key bone-related genes (1,2). Elimination of the Runx2 gene causes developmental defects in osteogenesis (3), and hereditary mutations in this gene in humans are linked to specific ossification defects, as observed in Cleidocranial Dysplasia (4). The Runx2 proteins are expressed in early mesenchyme of developing skeletal tissues (embryonic age E9.5) (5,6). Expression of the bone-related Runx2/p57 protein is controlled by the P1 upstream promoter, which contains regulatory elements that are recognized by several transcription factors to either activate or repress expression (see Figure 1). Among these factors are the homeodomain factors Msx2, CDP/cut, Dlx3 and Dlx5 (7,8), β-catenin/TCF (9), Hoxa10 (10), AP-1 (11), Nkx3.2 (5), and Runx2 (12).*To whom correspondence should be addressed: Departamento de Bioquimica y Biologia Molecular, Facultad de Ciencias Biologicas, Universidad de Concepcion, Casilla 160-C, Concepcion, Chile. Phone: 56-41-2203815; Fax: 56-41-2239687; mmonteci@udec.cl. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 August 4. Published in final edited form as:Biochemistry. Also essential for commitment and differentiation of mesenchymal cells to the osteoblast lineage during bone formation are the BMP2/4/7 members of the TGFβ superfamily of signaling factors (13,14). BMP2 can block differentiation of mesenc...

Calcium/calmodulin-dependent protein kinase IV (CaMKIV) plays a key role in the regulation of calcium-dependent gene expression. The expression of CaMKIV and the activation of CREB regulated genes are involved in memory and neuronal survival. We report here that: (a) a bioinformatic analysis of 15,476 promoters of the human genome predicted several Wnt target genes, being CaMKIV a very interesting candidate; (b) CaMKIV promoter contains TCF/LEF transcription motifs similar to those present in Wnt target genes; (c) biochemical studies indicate that lithium and the canonical ligand Wnt-3a induce CaMKIV mRNA and protein expression levels in rat hippocampal neurons as well as CaMKIV promoter activity; (d) treatment of hippocampal neurons with Wnt-3a increases the binding of beta-catenin to the CaMKIV promoter: (e) In vivo activation of the Wnt signaling improve spatial memory impairment and restores the expression of CaMKIV in a mice double transgenic model for Alzheimer's disease which shows decreased levels of the kinase. We conclude that CaMKIV is regulated by the Wnt signaling pathway and that its expression could play a role in the neuroprotective function of the Wnt signaling against the Alzheimer's amyloid peptide.

c Self-renewal of human pluripotent embryonic stem cells proceeds via an abbreviated cell cycle with a shortened G 1 phase. We examined which genes are modulated in this abbreviated period and the epigenetic mechanisms that control their expression. Accelerated upregulation of genes encoding histone proteins that support DNA replication is the most prominent gene regulatory program at the G 1 /S-phase transition in pluripotent cells. Expedited expression of histone genes is mediated by a unique chromatin architecture reflected by major nuclease hypersensitive sites, atypical distribution of epigenetic histone marks, and a region devoid of histone octamers. We observed remarkable differences in chromatin structure-hypersensitivity and histone protein modifications-between human embryonic stem (hES) and normal diploid cells. Cell cycle-dependent transcription factor binding permits dynamic three-dimensional interactions between transcript initiating and processing factors at 5= and 3= regions of the gene. Thus, progression through the abbreviated G 1 phase involves cell cycle stage-specific chromatin-remodeling events and rapid assembly of subnuclear microenvironments that activate histone gene transcription to promote nucleosomal packaging of newly replicated DNA during stem cell renewal. Human embryonic stem (hES) and induced pluripotent stem (iPS) cells maintain an undifferentiated state, are competent to proliferate indefinitely, and possess the ability to differentiate to all three germ layers (25,33,42,45,51,52,54,60). The unique ability to self-renew and to give rise to any cell type of an organism reflects the therapeutic potential of pluripotent stem cells in regenerative medicine. Human ES and iPS cells have an abbreviated G 1 phase and lack a classical restriction (R) point that normally controls commitment for progression into S phase (3,4,23,24). In contrast, proliferation of somatic cells is linked to growth factor-dependent passage through the R point in G 1 phase (43,44). The precise mechanisms by which cell cycle kinetics are modulated as cells switch between pluripotent and phenotype-committed states are complex and remain to be established. Key cell cyclerelated gene-activating events that occur between mitosis and S phase must be accelerated in the pluripotent state relative to those in phenotype-committed cells. More importantly, the absence of an R point in pluripotent cells necessitates reliance on other G 1 /Sphase-related gene-regulatory mechanisms to control entry into S phase. To understand molecular events at the G 1 /S-phase transition in pluripotent embryonic stem cells, it is necessary to identify genes that can be mechanistically examined for chromatin remodeling that accompanies gene activation.There are fundamental architectural modifications in genome configurations during the abbreviated self-renewal cell cycle of pluripotent hES cells to establish competency for DNA replication. As hES cells exit mitosis during self-renewal, chromosome decondensation and immediate assembly of c...