Vegfa expression is activated through positive and negative transcriptional regulatory networks controlled by the ETS factor Etv6 in vivo

VEGFA signaling is crucial for physiological and pathological angiogenesis and hematopoiesis. Although many context-dependent signaling pathways downstream of VEGFA have been uncovered, vegfa transcriptional regulation in vivo remains unclear. Here we show that the ETS transcription factor, Etv6, positively regulates vegfa expression during Xenopus blood stem cell development through multiple transcriptional inputs. In agreement with its established repressive functions, Etv6 directly inhibits the expression of the vegfa repressor, foxo3. Surprisingly, it also directly activates the expression of the vegfa activator, klf4. Finally, it indirectly binds to the vegfa promoter where it co-localizes with Klf4. Klf4 deficiency downregulates vegfa expression and significantly decreases Etv6 binding to the vegfa promoter, indicating that Klf4 recruits Etv6 to the vegfa promoter. Thus, our work uncovers a dual function for Etv6, as both a transcriptional repressor and activator, in controlling a major signaling pathway involved in blood and endothelial development in vivo. Given the established relationships between development and cancer, this elaborate gene regulatory network may inform new strategies for the treatment of VEGFA-dependent tumorigenesis.


Vascular Endothelial Growth Factor A (VEGFA) signaling is critical for both
VEGFA signaling is also essential during embryogenesis where it plays pivotal roles in the development of the endothelial and hematopoietic systems. Knockout of VegfA or VegfA receptor (Vegfr1, 2 or 3) genes in mice results in early lethality owing to severe defects in vascular development 6 . Mice deficient in Vegfr2 (also known as Flk1 or Kdr) show an absence of yolk sac blood islands and reduction of CD34 + hematopoietic progenitors 7 . In vitro, VEGFA is required for the specification of FLK1 + mesoderm into hematopoietic and cardiovascular lineages in mouse embryonic stem cell differentiation models 8,9 . In embryos, vegfa is essential for HSCs emergence and has been shown to be required at several stages of their programming [10][11][12]

ETV6, a transcriptional repressor belonging to the ETS family of transcription factors
(TFs) [14][15][16] , is a key regulator of hematopoietic, angiogenic and tumorigenic processes, and has been linked to vegfa regulation. It is essential for bone marrow hematopoiesis 17,18 and is involved in many chromosomal rearrangements which lead to childhood leukaemias 19

Etv6 acts as a direct transcriptional activator of klf4
The klf4 gene is a candidate direct target of Etv6 in the somites (Figure 2f) We first validated binding of Etv6 in the promoter regions of the two Xenopus klf4 genes, klf4.L and klf4.S (Figure 4a-b), and confirmed by WISH (Figure 4c) (Figure 5b-c). This confirmed that Klf4 binding is indeed enriched in the promoter of vegfa and co-localises with Etv6 binding (Figure 5d), supporting the hypothesis that Klf4 is involved in Etv6 recruitment to the vegfa promoter. To further confirm this notion, Etv6 ChIP-qPCR was carried out on klf4-deficient somites (Figure 5e). Etv6 enrichment in the vegfa promoter was severely reduced in klf4-deficient embryos, a reduction similar to that observed in etv6-deficient embryos (Figure 5e). Importantly, etv6 expression was unaffected in the somites of klf4-depleted embryos (Supplementary Figure 7). Therefore, Klf4 is required for Etv6 recruitment to the vegfa promoter.

Members of the ETS family of TFs are defined by a highly conserved ETS domain that recognizes a core sequence 5'-GGA(A/T)-3' motif within the context of a 9-to 10-bp DNA sequence 42 . As all ETS TFs bind to the same motif, additional mechanisms regulating the selection of specific transcriptional targets within biological contexts are required. It has been suggested that high affinity ETS motifs found in the promoter of housekeeping genes can be bound by any ETS TF whereas lower-affinity ETS binding sites found in tissue-specific promoters and only bound by a subset of ETS TFs are flanked by binding
sites of other TFs 43,44 . Cooperative binding with other TFs in sequences with composite binding sites results in a higher affinity and stable binding to DNA, and in synergistic repression or activation of specific target genes 45,46 .  52,53 (Figure 6) (Figure 6).

Gene repression by ETV6 and Yan is mediated by the pointed (SAM) and linker domains.
These domains can repress transcription independently through different mechanisms.
The linker domain represses transcription by complexing with co-repressors which recruit histone deacetylases (HDACs) 14,24, (Figure 4e). As single MO blocking both foxo3.L and foxo3.S could not be designed (Supplementary Figure 3a), two MOs, one targeting foxo3.L (foxo3.L MO) and the other targeting foxo3.S (foxo3.S MO), were co-injected in a 1:1 ratio in order to generate foxo3-deficient embryos (Supplementary Figure 3c). Every MO was titrated in order to determine the optimal concentration for embryo injection. MO sequences are as indicated in Supplementary Table 9. A two-step Golden Gate assembly method using the Golden Gate TALEN and TAL effector kit 2.0 (Addgene) was used to construct the TALEN plasmids containing the homodimer-type FokI nuclease domain 58 Figure 1a). Short peptides corresponding to these epitopes were synthesized, conjugated to keyhole limpet hemocyanin (KLH) and used in immunizations at a purity ≥90%. Two rabbits were immunized with each peptide and six polyclonal antibodies (Etv6-1a, -1b, - ChIP-seq Etv6 ChIP-seq was performed as previously described 60