Protein-deoxyribonucleic acid interactions linked to gene expression: DNase I digestion.
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Animals↗
Biomedical subjects
Jitesh Pratap
Publications and source records attributed to Jitesh Pratap.
Protein-deoxyribonucleic acid interactions linked to gene expression: ligation-mediated polymerase chain reaction.
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DNA↗
Cell growth regulatory role of Runx2 during proliferative expansion of preosteoblasts.
The Runx2 (CBFA1/AML3/PEBP2alphaA) transcription factor promotes lineage commitment and differentiation by activating bone phenotypic genes in postproliferative osteoblasts. However, the presence of Runx2 in actively dividing osteoprogenitor cells suggests that the protein may also participate in control of osteoblast growth. Here, we show that Runx2 is stringently regulated with respect to cell cycle entry and exit in osteoblasts. We addressed directly the contribution of Runx2 to bone cell proliferation using calvarial osteoblasts from wild-type and Runx2-deficient mice (i.e., Runx2(-/-) and Runx2(DeltaC/DeltaC)). Runx2(DeltaC/DeltaC) mice express a protein lacking the Runx2 COOH terminus, which integrates several cell proliferation-related signaling pathways (e.g., Smad, Yes/Src, mitogen-activated protein kinase, and retinoblastoma protein). Calvarial cells but not embryonic fibroblasts from Runx2(-/-) or Runx2(DeltaC/DeltaC) mutant mice exhibit increased cell growth rates as reflected by elevations of DNA synthesis and G(1)-S phase markers (e.g., cyclin E). Reintroduction of Runx2 into Runx2(-/-) calvarial cells by adenoviral delivery restores stringent cell growth control. Thus, Runx2 regulates normal osteoblast proliferation, and the COOH-terminal region is required for this biological function. We propose that Runx2 promotes osteoblast maturation at a key developmental transition by supporting exit from the cell cycle and activating genes that facilitate bone cell phenotype development.
Animals↗
Intranuclear trafficking of transcription factors: Requirements for vitamin D-mediated biological control of gene expression.
The architecturally associated subnuclear organization of nucleic acids and cognate regulatory factors suggest 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 of regulatory information as well as the assembly and activities of sites within the nucleus that support gene expression. Vitamin D control of gene expression serves as a paradigm for experimentally addressing mechanisms that govern the intranuclear targeting of regulatory factors to nuclear domains where transcription of developmental and tissue-specific genes occur. We will present an overview of molecular, cellular, genetic, and biochemical approaches that provide insight into the trafficking of regulatory factors that mediate vitamin D control of gene expression to transcriptionally active subnuclear sites. Examples will be presented that suggest modifications in the intranuclear targeting of transcription factors abrogate competency for vitamin D control of skeletal gene expression during development and fidelity of gene expression in tumor cells.
Animals↗
Nuclear microenvironments support physiological control of gene expression.
There is growing recognition that the organization of nucleic acids and regulatory proteins is functionally linked to the assembly, localization and activity of gene regulatory machinery. Cellular, molecular, biochemical and in-vivo genetic evidence support an obligatory relationship between nuclear microenvironments where regulatory complexes reside and fidelity of transcriptional control. Perturbations in mechanisms governing the intranuclear trafficking of transcription factors and the temporal/spatial organization of regulatory proteins within the nucleus occur with compromised gene expression that abrogates skeletal development and mediates leukemogenesis.
Cell Nucleus↗
Temporal and spatial parameters of skeletal gene expression: targeting RUNX factors and their coregulatory proteins to subnuclear domains.
Key components of the basal transcription machinery and several tissue-specific transcription factor complexes are functionally compartmentalized as specialized subnuclear domains. We have identified a unique 31-38 amino acid targeting signal (NMTS) that directs the Runx (Cbfa/AML) transcription factors to distinct nuclear matrix-(NM) associated sites within the nucleus that support gene expression. Our determination of the NMTS crystal structure, yeast 2 hybrid screens to identify NM interacting proteins, and in situ colocalization studies with Runx interacting factors (YAP, Smad, TLE) suggest that localization of Runx transcription factors at intranuclear sites facilitates the assembly and activity of regulatory complexes that mediate activation and suppression of target genes. Mice homozygous for the deletion of the intranuclear Runx2 targeting signal in a homologous recombination (Runx2 deltaC) do not form bone due to maturational arrest of osteoblasts, demonstrating the importance of fidelity of subnuclear localization for tissue-differentiating activity. These results provide evidence that Runx2 subnuclear targeting and the associated regulatory functions are essential for a spatiotemporal placement that facilitates activation of Runx-dependent genes involved in tissue differentiation during embryonic development.
Animals↗
Intranuclear organization of RUNX transcriptional regulatory machinery in biological control of skeletogenesis and cancer.
RUNX (AML/CBFA/PEBP2) transcription factors serve as paradigms for obligatory relationships between nuclear structure and physiological control of phenotypic gene expression. The RUNX proteins contribute to tissue restricted transcription by sequence-specific binding to promoter elements of target genes and serving as scaffolds for the assembly of coregulatory complexes that mediate biochemical and architectural control of activity. We will present an overview of approaches we are pursuing to address: (1) the involvement of RUNX proteins in governing competency for protein/DNA and protein/protein interactions at promoter regulatory sequences; (2) the recruitment of RUNX factors to subnuclear sites where the machinery for expression or repression of target genes is organized; and (3) the trafficking and integration of regulatory signals that control RUNX-mediated transcription.
Animals↗