The ATP-dependent chromatin-remodeling enzyme brahma-related gene 1 (BRG1) regulates transcription of specific target genes during embryonic and postnatal development. Deletion of Brg1 from embryonic blood vessels results in yolk sac vascular remodeling defects. We now report that misregulation of the canonical Wnt signaling pathway underlies many Brg1 mutant vascular phenotypes. Brg1 deletion resulted in down-regulation of several Wnt receptors of the frizzled family, degradation of the intracellular Wnt signaling molecule β-catenin, and an overall decrease in Wnt signaling in endothelial cells. Pharmacological stabilization of β-catenin significantly rescued Brg1 mutant vessel morphology and transcription of Wnt target genes. Our data demonstrate that BRG1 impacts the canonical Wnt pathway at two different levels in vascular endothelium: through transcriptional regulation of both Wnt receptor genes and Wnt target genes. These findings establish an epigenetic mechanism for the modulation of Wnt signaling during embryonic vascular development.ukaryotes fit a large quantity of DNA into the nuclei of their cells by packaging it into a compact structure called chromatin. This packaging presents a barrier to transcription factors that must access DNA to bring about changes in gene expression. ATP-dependent chromatin-remodeling complexes alleviate this problem by temporarily unraveling and reorganizing chromatin, thereby making DNA more accessible to proteins that are required for transcription (1, 2). Once considered to be ubiquitous mediators of transcription, chromatin-remodeling complexes are increasingly recognized for their specialized functions and specific genomic targets during development (3, 4). Much of this specificity has been shown to stem from variations in the subunit composition of these large, multiprotein complexes (5-8).One family of ATP-dependent chromatin-remodeling complexes, the mammalian SWI/SNF (SWItch/Sucrose NonFermentable)-like complexes, promotes or represses transcription of genes by increasing or decreasing accessibility of DNA to large transcriptional machinery at specific loci (9, 10). Mammalian SWI/SNF complexes contain one of two central ATPase catalytic subunits: brahma (BRM) or brahma-related gene 1 (BRG1). Global deletion of Brg1 in mice leads to embryonic lethality at peri-implantation (11). Tissue-specific conditional mutations have elucidated numerous developmental roles for BRG1, including zygotic genome activation, erythropoiesis, T-cell, cardiac, and neuronal development (8,(12)(13)(14)(15). Deletion of Brg1 in developing endothelial cells results in defective yolk sac angiogenesis, although the mechanism by which BRG1 affects vascular development is unclear (16,17).The canonical Wnt/wingless signaling pathway is one of several signaling pathways known to contribute to embryonic vascular development (18-20). Canonical Wnt signaling occurs when soluble Wnt ligands interact with a cell-surface receptor complex consisting of the lipoprotein receptor-related 5/6 (Lrp5/6) prote...

SUMMARYArteries and veins acquire distinct molecular identities prior to the onset of embryonic blood circulation, and their specification is crucial for vascular development. The transcription factor COUP-TFII currently functions at the top of a signaling pathway governing venous fate. It promotes venous identity by inhibiting Notch signaling and subsequent arterialization of endothelial cells, yet nothing is known about what regulates COUP-TFII expression in veins. We now report that the chromatin-remodeling enzyme BRG1 promotes COUP-TFII expression in venous endothelial cells during murine embryonic development. Conditional deletion of Brg1 from vascular endothelial cells resulted in downregulated COUP-TFII expression and aberrant expression of arterial markers on veins. BRG1 promotes COUP-TFII expression by binding conserved regulatory elements within the COUP-TFII promoter and remodeling chromatin to make the promoter accessible to transcriptional machinery. This study provides the first description of a factor promoting COUP-TFII expression in vascular endothelium and highlights a novel role for chromatin remodeling in venous specification.

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Estrogen receptor alpha (ERalpha) binds to specific target DNA sequences, estrogen response elements (EREs), to regulate estrogen-responsive gene expression. The progesterone receptor (PR) gene has been used extensively as a marker of estrogen responsiveness. Although we previously identified cis elements within 1 kb of the PR-B transcription start site that are associated with ERalpha and help to confer estrogen responsiveness, the identification of ERalpha binding sites far removed from the transcription start site suggested that long-range regulation of this gene may occur. We now show that eight regions of the PR gene from 311 kb upstream to 4 kb downstream of the PR-B transcription start site interact with ERalpha and that coactivator proteins and acetylated histones are selectively associated with these gene regions. Specific PR gene regions confer estrogen responsiveness to a heterologous reporter plasmid, and mutation of EREs within these regions diminishes estrogen-induced transactivation. Importantly, chromosome conformation capture assays reveal ERalpha- and ligand-dependent interactions between proximal and distal PR gene regions. Taken together, our studies suggest that distal regions of the PR gene participate in the dynamic regulation of this gene and that the coordinated action of proximal and distal PR gene regions allows cells to respond to changes in hormone levels with extraordinary versatility and sensitivity.

Metastasis of cancer cells from the primary tumor is associated with poor prognosis and decreased overall survival. One protein implicated in inhibiting metastasis is the tumor metastasis suppressor nonmetastatic protein 23 homologue 1 (NM23-H1). NM23-H1 is a multifunctional protein, which, in addition to limiting metastasis, has DNase and histidine protein kinase activities. We have identified new functions for NM23-H1 in influencing estrogen receptor A (ERA)-mediated gene expression. Using a battery of molecular and biochemical techniques, we show that NM23-H1 interacts with ERA and increases the ERA-estrogen response element (ERE) interaction. When NM23-H1 expression is increased in U2 osteosarcoma and MDA-MB-231 breast cancer cells, transcription of a transiently transfected, estrogen-responsive reporter plasmid is decreased. More importantly, when endogenous NM23-H1 expression is knocked down in MCF-7 human breast cancer cells using small interfering RNA, estrogen responsiveness of the progesterone receptor (PR), Bcl-2, cathepsin D, and cyclin D1 genes, but not the pS2 gene, is enhanced. Furthermore, NM23-H1 associates with the region of the PR gene containing the +90 activator protein 1 site, but not with the ERE-containing region of the pS2 gene, indicating that NM23-H1 mediates gene-specific effects by association with endogenous chromatin. Our studies suggest that the capacity of NM23-H1 to limit the expression of estrogen-responsive genes such as cathepsin D and Bcl-2, which are involved in cell migration, apoptosis, and angiogenesis, may help to explain the metastasis-suppressive effects of this protein. The complementary abilities of ERA and NM23-H1 together to influence gene expression, cell migration, and apoptosis could be key factors in helping to determine tumor cell fate. [Cancer Res 2007;67(21):10600-7]