A Single-Nucleotide Polymorphism in a Half-Binding Site Creates p53 and Estrogen Receptor Control of Vascular Endothelial Growth Factor Receptor 1

Abstract

Interactions between master regulatory pathways provide higher-order controls for cellular regulation. Recently, we reported a C3T single-nucleotide polymorphism (SNP) in the vascular endothelial growth factor receptor 1 (VEGFR-1/Flt1) promoter that merges human VEGF and p53 pathways. This finding suggested a new layer in environmental controls of a pathway relevant to several diseases. The Flt1-T SNP created what appeared to be a half-site p53 target response element (RE). The absence of information about p53 gene responsiveness mediated by half-site REs led us to address how it influences Flt1 expression. We now identify a second regulatory sequence comprising a partial RE for estrogen receptors (ERs) upstream of the p53 binding site. Surprisingly, this provides for synergistic stimulation of transcription specifically at the Flt1-T allele through the combined action of ligand-bound ER and stress-induced p53. In addition to demonstrating direct control of Flt1 expression by ER and p53 proteins acting as sequence-specific transcription factors at half-site REs, we establish a new interaction between three master regulatory pathways, p53, ER, and VEGF. The mechanism of joint regulation through half-sites is likely relevant to transcriptional control of other targets and expands the number of genes that may be directly controlled in master regulatory networks.Sequence-specific transcription factors (TFs) can modulate the expression patterns of many target genes both temporally and quantitatively through dynamic interaction with unique, cis-regulatory response element (RE) sequences. These REs, which are typically 5 to 20 bases long and located in proximity to the transcription start sites of genes can differ considerably, as exemplified by consensus sequence degeneracy (6, 40).

Primate-specific RFPL1 gene controls cell-cycle progression through cyclin B1/Cdc2 degradation

“…Fitting with this hypothesis, it has been previously reported a transcriptional cooperation between p53 and a ligand-bound estrogen receptor to result in a regulatory cross talk synergistically transactivating VEGFR1/FLT1, whereas p53 alone has relatively no effect. 29,30 In addition, Pax6-downregulated FOXM1 and MCM5 genes were not affected by p53 RNA interference despite it has been reported in other cell systems that p53 could also repress their expression independently of Pax6 signaling. 31,32 Thus, cross talk between Pax6 signaling and p53 activity appears to regulate specific genes in a given environment as the identification of either Pax6 or p53 target genes greatly varies depending on the cell system used.…”

Interaction between p53 and estradiol pathways in transcriptional responses to chemotherapeutics

“…23,24 The functional interaction appeared to occur through noncanonical cis-promoter REs for both DOX, 5FU or nutlin-3a (Fig. 1B).…”

“…[25][26][27] Neither p53 nor ER alone could significantly upregulate FLT1, but the combination resulted in synergistic activation. 24 We proposed that noncanonical p53 REs consisting of ½ or ¾ sites can expand the p53 target network providing for moderate or weak p53 responsiveness, but at the same time providing the opportunity of conditional, context-dependent transactivation. 5,25,27 Also, in the case of ERs the structural organization of the response element (ERE) has been shown to influence the binding affinity as well as the modulation of the expression of target genes.…”

“…Given our earlier results with the FLT1 gene, 23,24 we examined three of the 16 genes for the possibility of cis interactions by assessing p53 and ER occupancy. p53 bound directly to p53-related target sequences in the promoters of the TLR5, INPP5D and KRT15 genes.…”

“…The procedure for crosslinking, sonication, IP and analysis followed a previously described protocol. 23,24,35 …”