S. Kaleem Zaidi

Runx2 (Cbfa1, AML-3) Interacts with Histone Deacetylase 6 and Represses the p21 CIP1/WAF1 Promoter

ABSTRACT Runx2 (Cbfa1, AML-3) is multifunctional transcription factor that is essential for osteoblast development. Runx2 binds specific DNA sequences and interacts with transcriptional coactivators and corepressors to either activate or repress transcription of tissue-specific genes. In this study, the p21 CIP/WAF1 promoter was identified as a repressible target of Runx2. A carboxy-terminal repression domain distinct from the well-characterized TLE/Groucho-binding domain contributed to Runx2-mediated p21 repression. This carboxy-terminal domain was sufficient to repress a heterologous GAL reporter. The repressive activity of this domain was sensitive to the histone deacetylase inhibitor trichostatin A but not to trapoxin B. HDAC6, which is insensitive to trapoxin B, specifically interacted with the carboxy terminus of Runx2. The HDAC6 interaction domain of Runx2 was mapped to a region overlapping the nuclear matrix-targeting signal. The Runx2 carboxy terminus was necessary for recruitment of HDAC6 from the cytoplasm to chromatin. HDAC6 also colocalized and coimmunoprecipitated with the nuclear matrix-associated protein Runx2 in osteoblasts. Finally, we show that HDAC6 is expressed in differentiating osteoblasts and that the Runx2 carboxy terminus is necessary for maximal repression of the p21 promoter in preosteoblasts. These data identify Runx2 as the first transcription factor to interact with HDAC6 and suggest that HDAC6 may bind to Runx2 in differentiating osteoblasts to regulate tissue-specific gene expression.

Subnuclear targeting of Runx/Cbfa/AML factors is essential for tissue-specific differentiation during embryonic development

Runx (Cbfa/AML) transcription factors are critical for tissue-specific gene expression. A unique targeting signal in the C terminus directs Runx factors to discrete foci within the nucleus. Using Runx2/CBFA1/AML3 and its essential role in osteogenesis as a model, we investigated the fundamental importance of fidelity of subnuclear localization for tissue differentiating activity by deleting the intranuclear targeting signal via homologous recombination. Mice homozygous for the deletion (Runx2ΔC) do not form bone due to maturational arrest of osteoblasts. Heterozygotes do not develop clavicles, but are otherwise normal. These phenotypes are indistinguishable from those of the homozygous and heterozygous null mutants, indicating that the intranuclear targeting signal is a critical determinant for function. The expressed truncated Runx2ΔC protein enters the nucleus and retains normal DNA binding activity, but shows complete loss of intranuclear targeting. These results demonstrate that the multifunctional N-terminal region of the Runx2 protein is not sufficient for biological activity. We conclude that subnuclear localization of Runx factors in specific foci together with associated regulatory functions is essential for control of Runx-dependent genes involved in tissue differentiation during embryonic development.

Runx2/Cbfa1 functions: diverse regulation of gene transcription by chromatin remodeling and co-regulatory protein interactions

Development of the osteoblast phenotype requires transcriptional mechanisms that regulate induction of a program of temporally expressed genes. Key components of gene activation, repression, and responsiveness to physiologic mediators require remodeling of the chromatin structure of a gene that renders promoter elements competent for the assembly of macromolecular transcriptional complexes. Here we review evidence that the Runx transcription factors support tissue-specific gene expression and bone formation by contributing to promoter structure, chromatin remodeling, and the integration of independent signaling pathways. In addition, we discuss the role of Runx2 in both activation and negative regulation of gene promoters (osteocalcin, bone sialoprotein, and Runx2/Cbfa1) in relation to the interaction of Runx with co-regulatory proteins in distinct subnuclear foci. The modifications in chromatin organization and transcription of the osteocalcin gene that are influenced by the activities of Runx2/Cbfa1 mediated by interacting proteins (YAP, TLE, SMAD, C/EBP) are emphasized. These functional properties of Runx2 provide novel insights into the requirements for multiple levels of transcriptional control within the context of nuclear architecture to support the convergence of regulatory signals that control tissue-specific gene expression.

Identification of HiNF-P, a Key Activator of Cell Cycle-Controlled Histone H4 Genes at the Onset of S Phase

ABSTRACT At the G1/S phase cell cycle transition, multiple histone genes are expressed to ensure that newly synthesized DNA is immediately packaged as chromatin. Here we have purified and functionally characterized the critical transcription factor HiNF-P, which is required for E2F-independent activation of the histone H4 multigene family. Using chromatin immunoprecipitation analysis and ligation-mediated PCR-assisted genomic sequencing, we show that HiNF-P interacts with conserved H4 cell cycle regulatory sequences in vivo. Antisense inhibition of HiNF-P reduces endogenous histone H4 gene expression. Furthermore, we find that HiNF-P utilizes NPAT/p220, a substrate of the cyclin E/cyclin-dependent kinase 2 (CDK2) kinase complex, as a key coactivator to enhance histone H4 gene transcription. The biological role of HiNF-P is reflected by impeded cell cycle progression into S phase upon antisense-mediated reduction of HiNF-P levels. Our results establish that HiNF-P is the ultimate link in a linear signaling pathway that is initiated with the growth factor-dependent induction of cyclin E/CDK2 kinase activity at the restriction point and culminates in the activation of histone H4 genes through HiNF-P at the G1/S phase transition.

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.

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 j C) 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.

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.

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.

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. J. Cell. Biochem. 88: 340–355, 2003.