Phosphorylation Regulates FOXC2-Mediated Transcription in Lymphatic Endothelial Cells

Abstract

One of the key mechanisms linking cell signaling and control of gene expression is reversible phosphorylation of transcription factors. FOXC2 is a forkhead transcription factor that is mutated in the human vascular disease lymphedema-distichiasis and plays an essential role in lymphatic vascular development. However, the mechanisms regulating FOXC2 transcriptional activity are not well understood. We report here that FOXC2 is phosphorylated on eight evolutionarily conserved proline-directed serine/threonine residues. Loss of phosphorylation at these sites triggers substantial changes in the FOXC2 transcriptional program. Through genome-wide location analysis in lymphatic endothelial cells, we demonstrate that the changes are due to selective inhibition of FOXC2 recruitment to chromatin. The extent of the inhibition varied between individual binding sites, suggesting a novel rheostat-like mechanism by which expression of specific genes can be differentially regulated by FOXC2 phosphorylation. Furthermore, unlike the wild-type protein, the phosphorylation-deficient mutant of FOXC2 failed to induce vascular remodeling in vivo. Collectively, our results point to the pivotal role of phosphorylation in the regulation of FOXC2-mediated transcription in lymphatic endothelial cells and underscore the importance of FOXC2 phosphorylation in vascular development. Forkhead box (Fox) proteins are a family of transcription factors (TFs) that play an important role in development, cell cycle regulation, and other key biological processes (1). In mammals, more than 40 forkhead family members have been identified, all sharing the evolutionarily conserved forkhead DNA binding domain. Despite the similarity in their DNA-binding domains, different members of the forkhead family have evolved distinct functional roles. The forkhead transcription factor FOXC2 was first demonstrated to play a role in the morphogenesis of the cardiovascular system during embryonic development (2, 3). Subsequent studies revealed that FOXC2 is also implicated in lymphatic vascular development and disease. Mutations in FOXC2 cause lymphedema-distichiasis (LD; OMIM 153400) characterized by lymphedema and double rows of eyelashes (4). In both humans and mice, FOXC2 is highly expressed in the developing lymphatic vessels, as well as in the adult lymphatic valves (5, 6). The critical role of FOXC2 in lymphatic vascular development has been underscored by the demonstration of abnormal lymphatic patterning and failure to form lymphatic valves in Foxc2-deficient mice (5,7,8). LD patients develop similar defects characterized by lymph and venous reflux, indicating failure or absence of lymphatic and venous valves (9, 10). On a mechanistic level, FOXC2 genomic binding sites are enriched in NFATC1 consensus sequences, and the two transcription factors cooperate in vivo during lymphatic vascular morphogenesis (8).Phosphorylation of transcription factors represents a rapid and reversible mechanism for dynamic regulation of transcriptional networks (11). Most of the Fox...

Cdk5 controls lymphatic vessel development and function by phosphorylation of Foxc2

“…This suggests that Cdk5 does not regulate all but only specific Foxc2 phosphorylation site(s). Moreover, this indicates that Foxc2 most likely gets regulated by additional other kinases besides Cdk5, that is, ERK1/2 and cell cycle Cdks, as it was previously suggested 31 . We checked whether Cdk5 collaborates with these pathways that have been associated with Foxc2 regulation, that is, ERK1/2 and cell cycle-related Cdks 31 .…”

“…Recently, it has been shown that Foxc2 is phosphorylated at eight conserved serine/threonine residues, essential for Foxc2-dependent transcription but a kinase that mediates Foxc2 phosphorylation in vivo has not been identified yet 31 . Co-immunoprecipitation of overexpressed or endogenous Cdk5 and Foxc2 indicated their direct interaction in human LECs (Fig.…”

“…30). The transcription factors Ets1, Ets2 and Foxc2 are regulated by phosphorylation at serine/threonine residues 30,31 and therefore might be of special interest as potential Cdk5 targets. However, we did not find obvious changes in expression of Foxc2, Prox1, Ets1 and Ets2, podoplanin or VEGFR3 (Supplementary Fig.…”

“…11i-l), characteristic for loss of Foxc2 phosphorylation 31 . This suggests that Cdk5 does not regulate all but only specific Foxc2 phosphorylation site(s).…”

“…Foxc2 phosphorylation was shown to selectively recruit Foxc2 to chromatin, differentially regulating expression of specific Foxc2 target genes 31 . Thus, next we aimed to clarify whether Cdk5-mediated activation of Foxc2-dependent transcription is due to phosphorylation.…”