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...

Receptor-activity–modifying proteins (RAMPs) are single transmembrane-spanning proteins which serve as molecular chaperones and allosteric modulators of G-protein–coupled receptors (GPCRs) and their signaling pathways. Although RAMPs have been previously studied in the context of their effects on Family B GPCRs, the coevolution of RAMPs with many GPCR families suggests an expanded repertoire of potential interactions. Using bioluminescence resonance energy transfer-based and cell-surface expression approaches, we comprehensively screen for RAMP interactions within the chemokine receptor family and identify robust interactions between RAMPs and nearly all chemokine receptors. Most notably, we identify robust RAMP interaction with atypical chemokine receptors (ACKRs), which function to establish chemotactic gradients for directed cell migration. Specifically, RAMP3 association with atypical chemokine receptor 3 (ACKR3) diminishes adrenomedullin (AM) ligand availability without changing G-protein coupling. Instead, RAMP3 is required for the rapid recycling of ACKR3 to the plasma membrane through Rab4-positive vesicles following either AM or SDF-1/CXCL12 binding, thereby enabling formation of dynamic spatiotemporal chemotactic gradients. Consequently, genetic deletion of either ACKR3 or RAMP3 in mice abolishes directed cell migration of retinal angiogenesis. Thus, RAMP association with chemokine receptor family members represents a molecular interaction to control receptor signaling and trafficking properties.

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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.