Sayyed K. Zaidi

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.

Tyrosine phosphorylation controls Runx2‐mediated subnuclear targeting of YAP to repress transcription

Src/Yes tyrosine kinase signaling contributes to the regulation of bone homeostasis and inhibits osteoblast activity. Here we show that the endogenous Yes‐associated protein (YAP), a mediator of Src/Yes signaling, interacts with the native Runx2 protein, an osteoblast‐related transcription factor, and suppresses Runx2 transcriptional activity in a dose‐dependent manner. Runx2, through its PY motif, recruits YAP to subnuclear domains in situ and to the osteocalcin (OC) gene promoter in vivo. Inhibition of Src/Yes kinase blocks tyrosine phosphorylation of YAP and dissociates endogenous Runx2–YAP complexes. Consequently, recruitment of the YAP co‐repressor to subnuclear domains is abrogated and expression of the endogenous OC gene is induced. Our results suggest that Src/Yes signals are integrated through organization of Runx2–YAP transcriptional complexes at subnuclear sites to attenuate skeletal gene expression.

Mitotic occupancy and lineage-specific transcriptional control of rRNA genes by Runx2

Regulation of ribosomal RNA genes is a fundamental process that supports the growth of cells and is tightly coupled with cell differentiation. Although rRNA transcriptional control by RNA polymerase I (Pol I) and associated factors is well studied, the lineage-specific mechanisms governing rRNA expression remain elusive. Runt-related transcription factors Runx1, Runx2 and Runx3 establish and maintain cell identity, and convey phenotypic information through successive cell divisions for regulatory events that determine cell cycle progression or exit in progeny cells. Here we establish that mammalian Runx2 not only controls lineage commitment and cell proliferation by regulating genes transcribed by RNA Pol II, but also acts as a repressor of RNA Pol I mediated rRNA synthesis. Within the condensed mitotic chromosomes we find that Runx2 is retained in large discrete foci at nucleolar organizing regions where rRNA genes reside. These Runx2 chromosomal foci are associated with open chromatin, co-localize with the RNA Pol I transcription factor UBF1, and undergo transition into nucleoli at sites of rRNA synthesis during interphase. Ribosomal RNA transcription and protein synthesis are enhanced by Runx2 deficiency that results from gene ablation or RNA interference, whereas induction of Runx2 specifically and directly represses rDNA promoter activity. Runx2 forms complexes containing the RNA Pol I transcription factors UBF1 and SL1, co-occupies the rRNA gene promoter with these factors in vivo, and affects local chromatin histone modifications at rDNA regulatory regions. Thus Runx2 is a critical mechanistic link between cell fate, proliferation and growth control. Our results suggest that lineage-specific control of ribosomal biogenesis may be a fundamental function of transcription factors that govern cell fate.

Integration of Runx and Smad regulatory signals at transcriptionally active subnuclear sites

Runx factors control lineage commitment and are transcriptional effectors of Smad signaling. Genetic defects in these pathways interfere with normal development. The in situ localization of Runx and Smad proteins must impact the mechanisms by which these proteins function together in gene regulation. We show that the integration of Runx and Smad signals is mediated by in situ interactions at specific foci within the nucleus. Activated Smads are directed to these subnuclear foci only in the presence of Runx proteins. Smad–Runx complexes are associated in situ with the nuclear matrix, and this association requires the intranuclear targeting signal of Runx factors. The convergence of Smad and Runx proteins at these sites supports transcription as reflected by BrUTP labeling and functional cooperativity between the proteins. Thus, Runx-mediated intranuclear targeting of Smads is critical for the integration of two distinct pathways essential for fetal development.

Structural Coupling of Smad and Runx2 for Execution of the BMP2 Osteogenic Signal

Two regulatory pathways, bone morphogenetic protein (BMP)/transforming growth factor-β (TGFβ) and the transcription factor RUNX2, are required for bone formation in vivo. Here we show the interdependent requirement of these pathways to induce an osteogenic program. A panel of Runx2 deletion and point mutants was used to examine RUNX2-SMAD protein-protein interaction and the biological consequences on BMP2-induced osteogenic signaling determined in Runx2 null cells. These cells do not respond to BMP2 signal in the absence of Runx2. We established that a triple mutation in the C-terminal domain of RUNX2, HTY (426-428), disrupts the RUNX2-SMAD interaction, is deficient in its ability to integrate the BMP2/TGFβ signal on promoter reporter assays, and is only marginally functional in promoting early stages of osteoblast differentiation. Furthermore, the HTY mutation overlaps the unique nuclear matrix targeting signal of Runx factors and exhibits reduced subnuclear targeting. Thus, formation of a RUNX2-SMAD osteogenic complex and subnuclear targeting are structurally and functionally inseparable. Our results establish the critical residues of RUNX2 for execution and completion of BMP2 signaling for osteoblastogenesis through a mechanism that requires RUNX2-SMAD transcriptional activity.

Mitotic retention of gene expression patterns by the cell fate-determining transcription factor Runx2

During cell division, cessation of transcription is coupled with mitotic chromosome condensation. A fundamental biological question is how gene expression patterns are retained during mitosis to ensure the phenotype of progeny cells. We suggest that cell fate-determining transcription factors provide an epigenetic mechanism for the retention of gene expression patterns during cell division. Runx proteins are lineage-specific transcription factors that are essential for hematopoietic, neuronal, gastrointestinal, and osteogenic cell fates. Here we show that Runx2 protein is stable during cell division and remains associated with chromosomes during mitosis through sequence-specific DNA binding. Using siRNA-mediated silencing, mitotic cell synchronization, and expression profiling, we identify Runx2-regulated genes that are modulated postmitotically. Novel target genes involved in cell growth and differentiation were validated by chromatin immunoprecipitation. Importantly, we find that during mitosis, when transcription is shut down, Runx2 selectively occupies target gene promoters, and Runx2 deficiency alters mitotic histone modifications. We conclude that Runx proteins have an active role in retaining phenotype during cell division to support lineage-specific control of gene expression in progeny cells.

Nuclear microenvironments in biological control and cancer

Nucleic acids and regulatory proteins are compartmentalized in microenvironments within the nucleus. This subnuclear organization may support convergence and the integration of physiological signals for the combinatorial control of gene expression, DNA replication and repair. Nuclear organization is modified in many cancers. There are cancer-related changes in the composition, organization and assembly of regulatory complexes at intranuclear sites. Mechanistic insights into the temporal and spatial organization of machinery for gene expression within the nucleus, which is compromised in tumours, provide a novel platform for diagnosis and therapy.

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.

Mitotic bookmarking of genes: a novel dimension to epigenetic control

Regulatory machinery is focally organized in the interphase nucleus. The information contained in these focal nuclear microenvironments must be inherited during cell division to sustain physiologically responsive gene expression in progeny cells. Recent results suggest that focal mitotic retention of phenotypic transcription factors at promoters together with histone modifications and DNA methylation — a mechanism collectively known as gene bookmarking — is a novel parameter of inherited epigenetic control that sustains cellular identity after mitosis. The epigenetic signatures imposed by bookmarking poise genes for activation or suppression following mitosis. We discuss the implications of phenotypic transcription factor retention on mitotic chromosomes in biological control and disease.

Altered Runx1 subnuclear targeting enhances myeloid cell proliferation and blocks differentiation by activating a miR-24/MKP-7/MAPK network

Disruption of Runx1/AML1 subnuclear localization, either by a single amino acid substitution or by a chromosomal translocation [e.g., t(8;21)], is linked to the etiology of acute myeloid leukemia (AML). Here, we show that this defect induces a select set of micro-RNAs (miR) in myeloid progenitor cells and AML patients with t(8;21). Both Runx1 and the t(8;21)-encoded AML1-ETO occupy the miR-24-23-27 locus and reciprocally control miR-24 transcription. miR-24 directly downregulates mitogen-activated protein kinase (MAPK) phosphatase-7 and enhances phosphorylation of both c-jun-NH(2)-kinase and p38 kinases. Expression of miR-24 stimulates myeloid cell growth, renders proliferation independent of interleukin-3, and blocks granulocytic differentiation. Thus, compromised Runx1 function induces a miR-dependent mechanism that, through MAPK signaling, enhances myeloid proliferation but blocks differentiation--key steps that contribute to leukemia.