Biomedical subjects

Martin Montecino

Publications and source records attributed to Martin Montecino.

At least 19 recordsLinked to original sources

PubMed2007

Crystallization and preliminary X-ray analysis of a domain in the Runx2 transcription factor that interacts with the 1alpha,25 dihydroxy vitamin D3 receptor.

The Runx2 transcription factor is a key regulator of osteoblast differentiation. In response to 1alpha,25 dihydroxy vitamin D3, Runx2 may interact with the 1alpha,25 dihydroxy vitamin D3 receptor (VDR) in the promoter of target genes, producing a synergic activation of their transcription. Previous studies have suggested that the motifs responsible for the VDR-Runx2 interaction are contained within the 230-361 domain of Runx2. In this work, we confirmed by GST-pull down that Runx2(I(209-361)) is sufficient to interact with the VDR. To obtain structural information, GST-Runx2(I(209-361)) protein was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapor-diffusion method and polyethyleneglycol as a precipitant. The crystals were found to diffract to a maximum resolution of 2.7 A and a complete data set to a 3.3 A resolution was collected and analyzed. The crystals belong to the tetragonal system, with a space group P4 and unit-cell parameters of a = b = 90.8, and c = 57.2 A. The presence of a monomer of the recombinant GST-Runx2(I(209-361)) in the asymmetric unit gives a V(M) of 2.7 A(3) Da(-1) and a solvent content of 54.8%.

Absorptiometry, Photon↗

PubMed2007

The 1alpha,25-dihydroxy Vitamin D3 receptor preferentially recruits the coactivator SRC-1 during up-regulation of the osteocalcin gene.

Binding of 1alpha,25-dihydroxy Vitamin D3 to the C-terminal domain (LBD) of its receptor (VDR), induces a conformational change that enables interaction of VDR with transcriptional coactivators such as the members of the p160/SRC family or the DRIP (Vitamin D interacting complex)/Mediator complex. These interactions are critical for VDR-mediated transcriptional enhancement of target genes. Recent reports indicate that nuclear receptors, including VDR, interact with p160/SRC members and the DRIP/Mediator complex in a sequential, cyclical, and mutually exclusive manner when bound to a target promoter, exhibiting also a high exchange rate. Here, we present an overview of how these coactivators are recruited to the bone-specific osteocalcin (OC) gene in response to short and long exposures to 1alpha,25-dihydroxy Vitamin D3. We find that in intact osteoblastic cells VDR and SRC-1 rapidly bind to the OC promoter in response to the ligand. This recruitment correlates with transcriptional enhancement of the OC gene and with increased histone acetylation at the OC promoter. In contrast, binding of the DRIP205 subunit, which anchors the DRIP/Mediator complex to the VDR, is detected at the OC promoter after several hours of incubation with 1alpha,25-dihydroxy Vitamin D3. Together, our results indicate that VDR preferentially recruits SRC-1 to enhance basal bone-specific OC gene transcription. We propose a model where specific protein-DNA and protein-protein interactions that occur within the context of the OC gene promoter in osteoblastic cells stabilize the preferential association of the VDR-SRC-1 complex.

Animals↗

PubMed2006

The classic receptor for 1alpha,25-dihydroxy vitamin D3 is required for non-genomic actions of 1alpha,25-dihydroxy vitamin D3 in osteosarcoma cells.

1alpha,25-dihydroxy vitamin D3 has a major role in the regulation of the bone metabolism as it promotes the expression of key bone-related proteins in osteoblastic cells. In recent years it has become increasingly evident that in addition to its well-established genomic actions, 1alpha,25-dihydroxy vitamin D3 induces non-genomic responses by acting through a specific plasma membrane-associated receptor. Results from several groups suggest that the classical nuclear 1alpha,25-dihydroxy vitamin D3 receptor (VDR) is also responsible for these non-genomic actions of 1alpha,25-dihydroxy vitamin D3. Here, we have used siRNA to suppress the expression of VDR in osteoblastic cells and assessed the role of VDR in the non-genomic response to 1alpha,25-dihydroxy vitamin D3. We report that expression of the classic VDR in osteoblasts is required to generate a rapid 1alpha,25-dihydroxy vitamin D3-mediated increase in the intracellular Ca(2+) concentration, a hallmark of the non-genomic actions of 1alpha,25-dihydroxy vitamin D3 in these cells.

Animals↗

PubMed2006

Chromatin remodeling and transcriptional activity of the bone-specific osteocalcin gene require CCAAT/enhancer-binding protein beta-dependent recruitment of SWI/SNF activity.

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.

Animals↗

PubMed2006

Brg1, the ATPase subunit of the SWI/SNF chromatin remodeling complex, is required for myeloid differentiation to granulocytes.

Many mammalian SWI/SNF complexes use Brahma-related gene 1 (Brg1) as a catalytic subunit to remodel nucleosomes for transcription regulation. In several mesenchymal cells and tissues, expression of a defective Brg1 protein negates the normal activity of the SWI/SNF complex and delays or blocks differentiation. To investigate the role of SWI/SNF complexes during myelopoiesis, we stably expressed a dominant negative (dn) Brg1 mutant in the myeloid lineage. Forced expression of dnBrg1 in IL-3-dependent murine 32Dcl3 myeloid progenitor cells results in a profound delay in the granulocyte-colony stimulating factor (G-CSF) induced granulocytic maturation. These cells also exhibit a significant decrease in the expression of both CD11b and Gr-1 surface receptors, which are normally upregulated during granulopoiesis, and show sustained expression of myeloperoxidase, which is synthesized primarily during the promyelocytic (blast) stage of myeloid development. Thus, dnBrg1 expression causes a developmental block at the promyelocytic/metamyelocytic stage of myeloid differentiation. Our findings indicate that the normal chromatin remodeling function of Brg1 is necessary for the G-CSF dependent differentiation of myeloid cells towards the granulocytic lineage. This dependency on Brg1 may reflect a stringent requirement for chromatin remodeling at a critical stage of hematopoietic cell maturation.

Adenosine Triphosphatases↗

PubMed2006

An architectural perspective of cell-cycle control at the G1/S phase cell-cycle transition.

A prominent role for the execution of cell cycle and growth regulatory mechanisms within the three-dimensional context of nuclear architecture is becoming increasingly evident. Signaling pathways and regulatory networks that govern activation and suppression of genes controlling proliferation are functionally integrated for the organization and assembly of transcriptional machinery in nuclear microenvironments. The transcriptional activation of histone genes at the G1/S phase transition (S-point) is temporarily, functionally, and spatially distinct from transcriptional mechanisms at the restriction point (R-point). The spatial distinction in R-point versus S-point control is the localization of clustered histone gene loci at cajal bodies, which is modulated during the cell cycle. Histone nuclear factor P (HiNF-P), the principal factor mediating H4 histone gene transcription, is the final link in the signaling cascade that is initiated with growth factor dependent induction of cyclin E/CDK2 kinase activity at the R-point and culminates in the NPAT-mediated activation of histone H4 genes through HiNF-P at the G1/S phase cell-cycle transition.

Cell Cycle↗

PubMed2006

Networks and hubs for the transcriptional control of osteoblastogenesis.

We present an overview of the concepts of tissue-specific transcriptional control mechanisms essential for development of the bone cell phenotype. BMP2 induced transcription factors constitute a network of activities and molecular switches for bone development and osteoblast differentiation. Among these regulators are Runx2 (Cbfa1/AML3), the principal osteogenic master gene for bone formation, as well as homeodomain proteins and osterix. Runx2 has multiple regulatory activities, including activation or repression of gene expression, and integration of biological signals from developmental cues, such as BMP/TGFbeta, Wnt and Src signaling pathways. Runx2 provides a new paradigm for transcriptional control by functioning as a principal scaffolding protein in nuclear microenvironments to control gene expression in response to physiologic signals (growth factors, cytokines and hormones). The protein serves as a hub for the coordination of activities essential for the expansion and differentiation of osteogenic lineage cells through the formation of co-regulatory protein complexes organized in subnuclear domains. Mechanisms by which Runx2 supports commitment to osteogenesis and determines cell fate involve its retention on mitotic chromosomes. Disruption of a unique protein module, the subnuclear targeting signal of Runx2, has profound effects on osteoblast differentiation and metastasis of cancer cells in the bone microenvironment. Runx2 target genes include regulators of cell growth control, components of the bone extracellular matrix, angiogenesis, and signaling proteins for development of the osteoblast phenotype and bone turnover. The specificity of Runx2 regulatory activities provides a basis for novel therapeutic strategies to correct bone disorders.

Animals↗

PubMed2005

Combinatorial organization of the transcriptional regulatory machinery in biological control and cancer.

The architecturally associated subnuclear organization of nucleic acids and cognate regulatory factors suggests functional interrelationships between nuclear structure and gene expression. Mechanisms that contribute to the spatial distribution of transcription factors within the three dimensional context of nuclear architecture control the sorting and integration of regulatory information as well as the combinatorial assembly, organization and activities of transcriptional machinery at scaffold-associated subnuclear sites that support gene expression. During the past several years our laboratory has been addressing intranuclear trafficking mechanisms that direct transcription factors to transcriptionally active nuclear microenvironments. We are pursuing these studies using the AML/Runx/Cbfa transcription factors that govern hematopoietic and bone-specific transcription as a paradigm. Our objective is to gain insight into linkage of intranuclear organization of genes, transcripts, and regulatory proteins with fidelity of biological control and contributions of aberrant nuclear structure/function relationships to the onset and progression of tumorigenesis.

Animals↗

PubMed2005

SWI/SNF chromatin remodeling complex is obligatory for BMP2-induced, Runx2-dependent skeletal gene expression that controls osteoblast differentiation.

Development of bone tissue requires maturation of osteoblasts from mesenchymal precursors. BMP2, a member of the TGFbeta superfamily, and the Runx2 (AML3/Cbfa1) transcription factor, a downstream BMP2 effector, are regulatory signals required for osteoblast differentiation. While Runx2 responsive osteogenic gene expression has been functionally linked to alterations in chromatin structure, the factors that govern this chromatin remodeling remain to be identified. Here, we address the role of the SWI/SNF chromatin remodeling enzymes in BMP2-induced, Runx2-dependent development of the osteoblast phenotype. For these studies, we have examined calvarial cells from wild-type (WT) mice and mice that are homozygous for the Runx2 null allele, as well as the C2C12 model of BMP2-induced osteogenesis. By the analysis of microarray data, we find that several components of the SWI/SNF complex are regulated during BMP2-mediated osteoblast differentiation. Brg1 is an essential DNA dependent ATPase subunit of the SWI/SNF complex. Thus, functional studies were carried out using a fibroblast cell line that conditionally expresses a mutant Brg1 protein, which exerts a dominant negative effect on SWI/SNF function. Our findings demonstrate that SWI/SNF is required for BMP2-induced expression of alkaline phosphatase (APase), an early marker reflecting Runx2 control of osteoblast differentiation. In addition, Brg1 is expressed in cells within the developing skeleton of the mouse embryo as well as in osteoblasts ex vivo. Taken together these results support the concept that BMP2-mediated osteogenesis requires Runx2, and demonstrates that initiation of BMP2-induced, Runx2-dependent skeletal gene expression requires SWI/SNF chromatin remodeling complexes.

Alkaline Phosphatase↗

PubMed2005

The dynamic organization of gene-regulatory machinery in nuclear microenvironments.

Nuclear components are functionally linked with the dynamic temporal and spatial compartmentalization, sorting and integration of regulatory information to facilitate its selective use. For example, the subnuclear targeting of transcription factors to punctate sites in the interphase nucleus mechanistically couples chromatin remodelling and the execution of signalling cascades that mediate gene expression with the combinatorial assembly of the regulatory machinery for biological control. In addition, a mitotic cycle of selective partitioning and sequential restoration of the transcriptional machinery provides a basis for the reassembly of regulatory complexes to render progeny cells competent for phenotypic gene expression. When this intranuclear targeting and localization of regulatory proteins is compromised, diseases, such as cancer, can occur. A detailed understanding of this process will provide further options for diagnosis and treatment.

Animals↗

PubMed2004

Mutation of the highly conserved Arg165 and Glu168 residues of human Gsalpha disrupts the alphaD-alphaE loop and enhances basal GDP/GTP exchange rate.

G protein signalling regulates a wide range of cellular processes such as motility, differentiation, secretion, neurotransmission, and cell division. G proteins consist of three subunits organized as a Galpha monomer associated with a Gbetagamma heterodimer. Structural studies have shown that Galpha subunits are constituted by two domains: a Ras-like domain, also called the GTPase domain (GTPaseD), and an helical domain (HD), which is unique to heterotrimeric G-proteins. The HD display significantly higher primary structure diversity than the GTPaseD. Regardless of this diversity, there are small regions of the HD which show high degree of identity with residues that are 100% conserved. One of such regions is the alpha helixD-alpha helixE loop (alphaD-alphaE) in the HD, which contains the consensus aminoacid sequence R*-[RSA]-[RSAN]-E*-[YF]-[QH]-L in all mammalian Galpha subunits. Interestingly, the highly conserved arginine (R*) and glutamic acid (E*) residues form a salt bridge that stabilizes the alphaD-alphaE loop, that is localized in the top of the cleft formed between the GTPaseD and HD. Because the guanine nucleotide binding site is deeply buried in this cleft and those interdomain interactions are playing an important role in regulating the basal GDP/GTP nucleotide exchange rate of Galpha subunits, we studied the role of these highly conserved R and E residues in Galpha function. In the present study, we mutated the human Gsalpha R165 and E168 residues to alanine (A), thus generating the R165--> A, E168--> A, and R165/E168--> A mutants. We expressed these human Gsalpha (hGsalpha) mutants in bacteria as histidine tagged proteins, purified them by niquel-agarose chromatography and studied their nucleotide exchange properties. We show that the double R165/E168--> A mutant exhibited a fivefold increased GTP binding kinetics, a higher GDP dissociation rate, and an augmented capacity to activate adenylyl cyclase. Structure analysis showed that disruption of the salt bridge between R165 and E168 by the introduced mutations, caused important structural changes in the HD at the alphaD-alphaE loop (residues 160-175) and in the GTPaseD at a region required for Gsalpha activation by the receptor (residues 308-315). In addition, other two GTPaseD regions that surround the GTP binding site were also affected.

Adenylyl Cyclases↗

PubMed2004

The vitamin D response element in the distal osteocalcin promoter contributes to chromatin organization of the proximal regulatory domain.

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.

Animals↗

PubMed2004

Intranuclear trafficking: organization and assembly of regulatory machinery for combinatorial biological control.

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.

Cell Nucleus↗

PubMed2004

Runx2 control of organization, assembly and activity of the regulatory machinery for skeletal gene expression.

We present an overview of Runx involvement in regulatory mechanisms that are requisite for fidelity of bone cell growth and differentiation, as well as for skeletal homeostasis and the structural and functional integrity of skeletal tissue. Runx-mediated control is addressed from the perspective of support for biological parameters of skeletal gene expression. We review recent findings that are consistent with an active role for Runx proteins as scaffolds for integration, organization and combinatorial assembly of nucleic acids and regulatory factors within the three-dimensional context of nuclear architecture.

Bone and Bones↗

PubMed2004

Nuclear microenvironments support assembly and organization of the transcriptional regulatory machinery for cell proliferation and differentiation.

The temporal and spatial organization of transcriptional regulatory machinery provides microenvironments within the nucleus where threshold concentrations of genes and cognate factors facilitate functional interactions. Conventional biochemical, molecular, and in vivo genetic approaches, together with high throughput genomic and proteomic analysis are rapidly expanding our database of regulatory macromolecules and signaling pathways that are requisite for control of genes that govern proliferation and differentiation. There is accruing insight into the architectural organization of regulatory machinery for gene expression that suggests signatures for biological control. Localized scaffolding of regulatory macromolecules at strategic promoter sites and focal compartmentalization of genes, transcripts, and regulatory factors within intranuclear microenvironments provides an infrastructure for combinatorial control of transcription that is operative within the three dimensional context of nuclear architecture.

Cell Cycle↗

PubMed2004

The Runx2 transcription factor plays a key role in the 1alpha,25-dihydroxy Vitamin D3-dependent upregulation of the rat osteocalcin (OC) gene expression in osteoblastic cells.

Bone-specific transcription of the osteocalcin (OC) gene is principally regulated by the Runx2 transcription factor and further stimulated in response to 1alpha,25-dihydroxy Vitamin D3 via its specific receptor (VDR). The rat OC gene promoter contains three recognition sites for Runx2 (sites A-C). Mutation of sites A and B, which flank the 1alpha,25-dihydroxy Vitamin D3-responsive element (VDRE), abolishes 1alpha,25-dihydroxy Vitamin D3-dependent enhancement of OC transcription, indicating a tight functional relationship between VDR and Runx2 factors. Additionally, the transcriptional co-activator p300 is recruited to the OC promoter by Runx2 where it up-regulates both basal and 1alpha,25-dihydroxy Vitamin D3-enhanced OC expression. Here, we present an overview of how in osteoblastic cells expressing OC, Runx2 modulates the 1alpha,25-dihydroxy Vitamin D3-dependent stimulation of the OC promoter by first recruiting transcriptional co-activators and then by further stabilizing the interaction of the VDR with the VDRE.

Animals↗

PubMed2004

Bone-specific transcription factor Runx2 interacts with the 1alpha,25-dihydroxyvitamin D3 receptor to up-regulate rat osteocalcin gene expression in osteoblastic cells.

Bone-specific transcription of the osteocalcin (OC) gene is regulated principally by the Runx2 transcription factor and is further stimulated in response to 1alpha,25-dihydroxyvitamin D3 via its specific receptor (VDR). The rat OC gene promoter contains three recognition sites for Runx2 (sites A, B, and C). Mutation of sites A and B, which flank the 1alpha,25-dihydroxyvitamin D3-responsive element (VDRE), abolishes 1alpha,25-dihydroxyvitamin D3-dependent enhancement of OC transcription, indicating a tight functional relationship between the VDR and Runx2 factors. In contrast to most of the members of the nuclear receptor family, VDR possesses a very short N-terminal A/B domain, which has led to the suggestion that its N-terminal region does not contribute to transcriptional enhancement. Here, we have combined transient-overexpression, coimmunoprecipitation, in situ colocalization, chromatin immunoprecipitation, and glutathione S-transferase pull-down analyses to demonstrate that in osteoblastic cells expressing OC, VDR interacts directly with Runx2 bound to site B, which is located immediately adjacent to the VDRE. This interaction contributes significantly to 1alpha,25-dihydroxyvitamin D3-dependent enhancement of the OC promoter and requires a region located C terminal to the runt homology DNA binding domain of Runx2 and the N-terminal region of VDR. Together, our results indicate that Runx2 plays a key role in the 1alpha,25-dihydroxyvitamin D3-dependent stimulation of the OC promoter in osteoblastic cells by further stabilizing the interaction of the VDR with the VDRE. These studies demonstrate a novel mechanism for combinatorial control of bone tissue-specific gene expression. This mechanism involves the intersection of two major pathways: Runx2, a "master" transcriptional regulator of osteoblast differentiation, and 1alpha,25-dihydroxyvitamin D3, a hormone that promotes expression of genes associated with these terminally differentiated bone cells.

Animals↗

PubMed2004

Dlx3 transcriptional regulation of osteoblast differentiation: temporal recruitment of Msx2, Dlx3, and Dlx5 homeodomain proteins to chromatin of the osteocalcin gene.

Genetic studies show that Msx2 and Dlx5 homeodomain (HD) proteins support skeletal development, but null mutation of the closely related Dlx3 gene results in early embryonic lethality. Here we find that expression of Dlx3 in the mouse embryo is associated with new bone formation and regulation of osteoblast differentiation. Dlx3 is expressed in osteoblasts, and overexpression of Dlx3 in osteoprogenitor cells promotes, while specific knock-down of Dlx3 by RNA interference inhibits, induction of osteogenic markers. We characterized gene regulation by Dlx3 in relation to that of Msx2 and Dlx5 during osteoblast differentiation. Chromatin immunoprecipitation assays revealed a molecular switch in HD protein association with the bone-specific osteocalcin (OC) gene. The transcriptionally repressed OC gene was occupied by Msx2 in proliferating osteoblasts, while Dlx3, Dlx5, and Runx2 were recruited postproliferatively to initiate transcription. Dlx5 occupancy increased over Dlx3 in mature osteoblasts at the mineralization stage of differentiation, coincident with increased RNA polymerase II occupancy. Dlx3 protein-DNA interactions stimulated OC promoter activity, while Dlx3-Runx2 protein-protein interaction reduced Runx2-mediated transcription. Deletion analysis showed that the Dlx3 interacting domain of Runx2 is from amino acids 376 to 432, which also include the transcriptionally active subnuclear targeting sequence (376 to 432). Thus, we provide cellular and molecular evidence for Dlx3 in regulating osteoprogenitor cell differentiation and for both positive and negative regulation of gene transcription. We propose that multiple HD proteins in osteoblasts constitute a regulatory network that mediates development of the bone phenotype through the sequential association of distinct HD proteins with promoter regulatory elements.

Amino Acid Sequence↗