Dynamic regulation of VEGF-inducible genes by an ERK-ERG-p300 transcriptional network

Abstract

The transcriptional pathways activated downstream of vascular endothelial growth factor (VEGF) signaling during angiogenesis remain incompletely characterized. By assessing the signals responsible for induction of the Notch ligand delta-like 4 (DLL4) in endothelial cells, we find that activation of the MAPK/ERK pathway mirrors the rapid and dynamic induction of DLL4 transcription and that this pathway is required for DLL4 expression. Furthermore, VEGF/ERK signaling induces phosphorylation and activation of the ETS transcription factor ERG, a prerequisite for DLL4 induction. Transcription of DLL4 coincides with dynamic ERG-dependent recruitment of the transcriptional co-activator p300. Genome-wide gene expression profiling identified a network of VEGF-responsive and ERG-dependent genes, and ERG chromatin immunoprecipitation (ChIP)-seq revealed the presence of conserved ERG-bound putative enhancer elements near these target genes. Functional experiments performed in vitro and in vivo confirm that this network of genes requires ERK, ERG and p300 activity. Finally, genome-editing and transgenic approaches demonstrate that a highly conserved ERG-bound enhancer located upstream of HLX (which encodes a transcription factor implicated in sprouting angiogenesis) is required for its VEGFmediated induction. Collectively, these findings elucidate a novel transcriptional pathway contributing to VEGF-dependent angiogenesis.

Activation of the Arterial Program Drives Development of Definitive Hemogenic Endothelium with Lymphoid Potential

“…As shown in Figure S5D-S5G, DAPT treatment abrogated the enhancing effect of LY294002 on formation of DLL4 + CXCR4 +/− arterial HE and CD235a/CD41a − CD45 + multipotential hematopoietic progenitors, consistent with prior observations in the mouse system that MAPK pathway specifies arterial identity of non-HE through activation of NOTCH signaling (Wythe et al, 2013). It has also been demonstrated that MAPK/ERK pathway promotes phosphorylation of ETS1 and ETS2 (Petrovic et al, 2003; Yang et al, 1996) and other ETS factor members including ELK1 (Yang et al, 1998) and ERG (Fish et al, 2017). ERG phosphorylation is required for p300 recruitment to DLL4 arterial-specific enhancer and its activation (Fish et al, 2017; Wythe et al, 2013).…”

“…It has also been demonstrated that MAPK/ERK pathway promotes phosphorylation of ETS1 and ETS2 (Petrovic et al, 2003; Yang et al, 1996) and other ETS factor members including ELK1 (Yang et al, 1998) and ERG (Fish et al, 2017). ERG phosphorylation is required for p300 recruitment to DLL4 arterial-specific enhancer and its activation (Fish et al, 2017; Wythe et al, 2013). To determine, whether ETS1 activity in our system is controlled by MAPK/ERK signaling, we assessed how ERK inhibition affects HE specification and hematopoietic differentiation in the presence of DOX (Figure S6A).…”

“…In addition, epistasis experiments revealed a strong dependence of ETS1 activity on MAPK/ERK signaling during HE development. Prior studies in zebra fish revealed that VEGF/ERK signaling induces phosphorylation ETS transcription factor ERG in non-HE (Fish et al, 2017; Wythe et al, 2013). This phosphorylation enhances DNA binding activity of ERG leading to activation of arterial-specific DLL4 enhancer.…”

Distinct Vegfa isoforms control endothelial cell proliferation through PI3 kinase signalling mediated regulation of cdkn1a/p21

“…So far, animal models carrying mutations in components of the Vegf pathway showed simultaneous defects in EC proliferation and migration. For instance, zebrafish mutants in kdrl, plcg or treated with the Vegf pathway inhibitor SU5416 display an absence of ERK phosphorylation (Fish et al, 2017; Shin et al, 2016), together with a reduction in ISV outgrowth and cell numbers. Similarly, mutants in vegfaa show a severe reduction in EC numbers and migration during ISV formation (Jin et al, 2017), phenotypes also seen in VEGFA mutant mice (Carmeliet et al, 1996; Ferrara et al, 1996).…”

Positive-feedback defines the timing and robustness of angiogenesis

“…To support in silico observations we expanded upon our previous transcriptomic study [30] to identify putative positive-feedback regulators of Vegfr by defining genes transcriptionally activated by Vegfr and repressed by Notch signaling in zebrafish ECs (Figure 2A). Of only 10 candidate Vegfr/Notch-regulated transcripts we identified h2.0-like homeobox-1 (hlx1), a known transcriptional target of Vegfr activity in-vivo [30][31][32] and the atypical tetraspanin transmembrane 4 L6 family member 18 (tm4sf18). TM4SF family proteins are known membrane-associated adaptors that ligand-independently activate receptor tyrosine kinase activity [33,34].…”