Subnuclear targeting of Runx1 is required for synergistic activation of the myeloid specific M-CSF receptor promoter by PU.1.

Many types of acute myelogenous leukemia involve chromosomal translocations that target the C-terminus of Runx1/AML1 transcription factor, a master regulator of hematopoiesis. The C-terminus of Runx1/AML1 that includes the nuclear matrix targeting signal (NMTS) is essential for embryonic development, hematopoiesis, and target gene regulation. During the onset and normal progression of hematopoiesis, several lineage-specific factors such as C/EBPalpha and PU.

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.

Coordinate control and selective expression of the full complement of replication-dependent histone H4 genes in normal and cancer cells.

The replication of eukaryotic genomes necessitates the coordination of histone biosynthesis with DNA replication at the onset of S phase. The multiple histone H4 genes encode identical proteins, but their regulatory sequences differ. The contributions of these individual genes to histone H4 mRNA expression have not been described.

Microtubule-dependent nuclear-cytoplasmic shuttling of Runx2.

RUNX/AML transcription factors are critical regulators of cell growth and differentiation in multiple lineages and have been linked to human cancers including acute myelogenous leukemia (RUNX1), as well as breast (RUNX2) and gastric cancers (RUNX3). RUNX proteins are targeted to gene regulatory micro-environments within the nucleus via a specific subnuclear targeting signal. However, the dynamics of RUNX distribution and compartmentalization between the cytoplasm and nucleus is minimally understood.

Canonical WNT signaling promotes osteogenesis by directly stimulating Runx2 gene expression.

Both activating and null mutations of proteins required for canonical WNT signaling have revealed the importance of this pathway for normal skeletal development. However, tissue-specific transcriptional mechanisms through which WNT signaling promotes the differentiation of bone-forming cells have yet to be identified. Here, we address the hypothesis that canonical WNT signaling and the bone-related transcription factor RUNX2/CBFA1/AML3 are functionally linked components of a pathway required for the onset of osteoblast differentiation.

HiNF-P directly links the cyclin E/CDK2/p220NPAT pathway to histone H4 gene regulation at the G1/S phase cell cycle transition.

Genome replication in eukaryotic cells necessitates the stringent coupling of histone biosynthesis with the onset of DNA replication at the G1/S phase transition. A fundamental question is the mechanism that links the restriction (R) point late in G1 with histone gene expression at the onset of S phase. Here we demonstrate that HiNF-P, a transcriptional regulator of replication-dependent histone H4 genes, interacts directly with p220(NPAT), a substrate of cyclin E/CDK2, to coactivate histone genes during S phase.

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.

Point mutation in AML1 disrupts subnuclear targeting, prevents myeloid differentiation, and effects a transformation-like phenotype.

The multifunctional C terminus of the hematopoietic AML1 transcription factor interacts with coregulatory proteins, supports the convergence and integration of physiological signals, and contains the nuclear matrix targeting signal, the protein motif that is necessary and sufficient to target AML1 to subnuclear sites. The (8;21) chromosomal translocation, which replaces the C terminus of AML1 with the ETO protein, modifies subnuclear targeting of AML1 in acute myeloid leukemia (AML) and results in defective myelopoiesis. We therefore addressed the relevance of AML1 subnuclear targeting and associated functions that reside in the C terminus to myeloid differentiation.

Organization of transcriptional regulatory machinery in osteoclast nuclei: compartmentalization of Runx1.

The osteoclast is a highly polarized multinucleated cell that resorbs bone. Using high resolution immunofluorescence microscopy, we demonstrated that all nuclei of an osteoclast are transcriptionally active. Each nucleus within the osteoclast contains punctately organized microenvironments where regulatory complexes that support transcriptional and post-transcriptional control reside.

The bone-specific expression of Runx2 oscillates during the cell cycle to support a G1-related antiproliferative function in osteoblasts.

The Runx2 (CBFA1/AML3/PEBP2alphaA) transcription factor promotes skeletal cell differentiation, but it also has a novel cell growth regulatory activity in osteoblasts. We addressed here whether Runx2 activity is functionally linked to cell cycle-related mechanisms that control normal osteoblast proliferation and differentiation. We found that the levels of Runx2 gene transcription, mRNA and protein, are each up-regulated with cessation of cell growth (i.

Nkx3.2-mediated repression of Runx2 promotes chondrogenic differentiation.

Runx2, a transcription factor known to be essential for osteoblast maturation and skeletogenesis, is also expressed in pre-cartilaginous mesenchymal condensations in the developing embryo. It is therefore necessary to understand the control and consequential regulatory activity of the Runx2 gene within the context of chondrogenic differentiation of a mesenchymal progenitor cell. We identify the homeodomain protein Nkx3.

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.

Impaired intranuclear trafficking of Runx2 (AML3/CBFA1) transcription factors in breast cancer cells inhibits osteolysis in vivo.

Runx transcription factors comprise a family of proteins that are essential for organogenesis. A unique nuclear matrix-targeting signal in the C terminus directs these factors to their appropriate subnuclear domains. At these sites, they interact with coregulatory proteins and target genes.

Smad function and intranuclear targeting share a Runx2 motif required for osteogenic lineage induction and BMP2 responsive transcription.

The coordinated activity of Runx2 and BMP/TGFbeta-activated Smads is critical for formation of the skeleton, but the precise structural basis for the Runx2/Smad interaction has not been resolved. By deletion mutagenesis, we have defined the Runx2 motif required for physical and functional interaction with either BMP or TGFbeta responsive Smads. Smad responsive transcriptional activity was retained upon deletion of the C-terminus to amino acid (aa) 432 but lost with deletion to aa 391.

Functional characterization of a human histone gene cluster duplication.

Histones are the major protein component of nucleosomes, and de novo histone synthesis is essential for packaging newly replicated DNA into chromatin. As a result, histone gene expression is exquisitely and functionally coupled with DNA replication. Vastly divergent organisms such as yeast, fly and human all demonstrate the phylogenetically conserved propensity to maintain clustering of histone genes at one or more genomic loci.

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.

Overlapping expression of Runx1(Cbfa2) and Runx2(Cbfa1) transcription factors supports cooperative induction of skeletal development.

Identifying the genetic pathways that regulate skeletal development is necessary to correct a variety of cartilage and bone abnormalities. The Runx family of transcription factors play a fundamental role in organ development and cell differentiation. Initial studies have shown that both Runx1 and Runx2 are expressed in pre-chondrogenic mesenchyme of the developing embryo at E12.

Quantitative signature for architectural organization of regulatory factors using intranuclear informatics.

Regulatory machinery for replication and gene expression is punctately organized in supramolecular complexes that are compartmentalized in nuclear microenvironments. Quantitative approaches are required to understand the assembly of regulatory machinery within the context of nuclear architecture and to provide a mechanistic link with biological control. We have developed 'intranuclear informatics' to quantify functionally relevant parameters of spatially organized nuclear domains.

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.