The chromatin-remodeling enzyme BRG1 modulates vascular Wnt signaling at two levels

Abstract

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...

BRG1 promotesCOUP-TFIIexpression and venous specification during embryonic vascular development

“…Endothelial cells from individual murine yolk sacs and embryos were isolated using anti-PECAM-1-conjugated magnetic beads, as described previously (Griffin et al, 2011). Primary human umbilical vein endothelial cells (HUVECs) were isolated and maintained as previously described (Yao et al, 1996).…”

“…PCR genotyping of Brg1-floxed, Tie2-Cre + and BAT-gal transgenic embryos and mice was performed as described previously (Griffin et al, 2011). β-catenin-floxed mice and embryos were PCR genotyped using the following primers: forward (5Ј-AAGGTAGAGTGATGAAAGTTGTT-3Ј) and reverse (5Ј-CACCATGTCCTCTGTCTATTC-3Ј).…”

“…For whole-mount yolk sac immunostaining, E9.5 Brg1 fl/fl :Tie2-Cre + embryos with attached yolk sacs were dissected from maternal tissue, fixed for 10 minutes in 3% paraformaldehyde (PFA), permeabilized, blocked and stained as described (Griffin et al, 2011). Primary anti-PECAM-1 (1:100; BD Biosciences, San Jose, CA, USA; 557355) and secondary donkey-antirat-Cy3 IgG (1:250; Jackson ImmunoResearch, West Grove, PA, USA; 9712-165-153) antibodies were used.…”

“…C166 yolk sac endothelial cells (ATCC; CRL-2581) were maintained and transfected with 100 nM BRG1 siGENOME SMART pool or nonspecific control small interfering RNA (siRNA) oligonucleotides (Dharmacon, Lafayette, CO, USA; M-041135-01 and D-001210-01, respectively), as described previously (Griffin et al, 2011). After 24 hours, protein was harvested in Laemmli buffer [62.5 mM Tris (pH 6.8)/10% glycerol/5% SDS/0.01% bromophenol blue] plus 0.2 M dithiothreitol for western blotting.…”

Brg1 is required for stem cell maintenance in the murine intestinal epithelium in a tissue-specific manner

“…Brg1 has also been reported to interact with b-catenin and mediate transcription of Wnt target genes both in vitro [13,14] and in vivo [15]. The Wnt pathway is crucial in a number of processes involved in the intestinal development and homeostasis, such as maintenance of the stem cell identity, proliferation, epithelial segregation along the crypt-villus axis, and secretory lineage differentiation (reviewed in [16]).…”