Stroke-associated intergenic variants modulate a human FOXF2 transcriptional enhancer

Abstract

SNPs associated with human stroke risk have been identified in the intergenic region between Forkhead family transcription factors FOXF2 and FOXQ1, but we lack a mechanism for the association. FoxF2 is expressed in vascular mural pericytes and is important for maintaining pericyte number and stabilizing small vessels in zebrafish. The stroke-associated SNPs are located in a previously unknown transcriptional enhancer for FOXF2, functional in human cells and zebrafish. We identify critical enhancer regions for FOXF2 gene expression, including binding sites occupied by transcription factors ETS1, RBPJ, and CTCF. rs74564934, a stroke-associated SNP adjacent to the ETS1 binding site, decreases enhancer function, as does mutation of RPBJ sites. rs74564934 is significantly associated with the increased risk of any stroke, ischemic stroke, small vessel stroke, and elevated white matter hyperintensity burden in humans. Foxf2 has a conserved function cross-species and is expressed in vascular mural pericytes of the vessel wall. Thus, stroke-associated SNPs modulate enhancer activity and expression of a regulator of vascular stabilization, FOXF2, thereby modulating stroke risk.

Keywords: FOXF2; pericyte; stroke.

Fig. 1.

Stroke-associated intergenic variants are located in a transcriptional enhancer. (A) Schematic of the relative location of SNPs and how they may act in a previously unknown FOXF2 distal enhancer to modify expression of FOXF2. Other genes in the region are also shown. (B) Map of the intergenic region between FOXF2 and FOXQ1. Box marks the ∼7-kb subregion that contains SNPs associated with elevated risk of stroke. ENCODE ChIP-seq peaks of H3K27Ac and CTCF are shown. (C) Enlargement showing all SNPs in the region, a GWAS-led SNP, ENCODE candidate cis-regulatory elements (cCREs), and H3K27Ac marks. Locations of subconstructs A–F are indicated (numbers refer to length in base pairs). Schematic of expression constructs placing construct A–F upstream of a basal promoter and luciferase. (D) Fold increase in luminescence over empty vector when constructs A–F are transfected into cells. (E) Luciferase activity of constructs A–E containing either low-risk or high-risk SNPs to enhance transcription. (F) Example of construct E: GFP expression in a perivascular cell (PVC) adjacent to endothelium (EC) in zebrafish. One-way ANOVA with Tukey’s post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. n = 3 technical replicates of 3 independent biological replicates.

Fig. 2.

iCRISPRi targeting of sites in the intergenic enhancer reduces FoxF2 transcription. (A) Low-resolution map of the region containing the SNPs indicating ENCODE ChIP-seq peaks for the transcription factors ETS1, RBPJ, and CTCF. (B) Higher resolution view of the 750-bp enhancer (E) and locations of CRISPR guides (red) centered over putative sites of transcription factor binding. Note the 3 control sites in the FoxF2 exon in (A). (C) Schematic of the iCRISPRi dead Cas9 (dCas9) fused to a KRAB transcriptional repressor and resulting repression of local gene expression. (D and E) Relative expression of (D) FOXF2 and (E) FOXC1 as determined by qPCR after iCRISPRi. (ns = not significant, **P < 0.01, ****P < 0.0001 by 1-way ANOVA with Tukey’s post hoc test; FOXF2: 3 technical replicates of 7 to 11 biological replicates; FOXC1: 3 technical replicates of 3 biologically independent replicates).

Fig. 3.

RBPJ, CTCF, and ETS1 bind the FOXF2 enhancer. (A) Chromosome-level map and ChIP-seq peaks of CTCF, ETS1, and RBPJ from ENCODE marked with consensus transcription factor binding sequences under each peak. Note that the RBPJ peak that coincides with the ETS1 peak does not have RBPJ consensus binding sequences; however, there are adjacent RBPJ sites (1, 2) a short distance away. (B–D) Representative ChIP-qPCR analysis of binding of antibodies to (B) ETS1, (C) RBPJ, or (D) CTCF (blue bars) or immunoglobulin G control (orange bars) from a single experiment (replicates are in SI Appendix, Fig. S4). GAPDH and SAT2 are negative controls. Gray lines indicate site of PCR product. (E) Schematic of Notch inhibitor experiment. (F) Notch inhibitor treatment reduces expression of (F) human FOXF2 and (G) zebrafish foxf2b at 75 hpf using the indicated inhibitors and doses. *P < 0.05, **P < 0.01, ***P < 0.001 by 1-way ANOVA with Tukey’s post hoc test; human cells, n = 3 biological replicates; zebrafish, n = 4 biological replicates.

Fig. 4.

The FOXF2 enhancer is critical for FOXF2 transcription; risk variants adjacent to ETS1 decrease enhancer activity. (A and B) Relative expression of FOXF2, FOXQ1, and FOXC1 after targeted excision of the FOXF2 (A) enhancer or (B) exon 1. P values are from a Student’s t test for each gene pair (control and Δenhancer), 3 biological replicates. (C) Schematic of the locus, marking the CRISPR targets for enhancer and exon 1. (D) Model of regulatory relationships based on enhancer and exon 1 deletion. The enhancer regulates FOXF2, while FOXF2 transcription regulates FOXQ1 and LINC01394. (E) Sequences of mutated enhancer fragments. Boldface lettering indicates transcription factor binding sites, while red indicates SNPs. (F) Schematic of the luciferase construct used to test expression. (G) Maps of mutated constructs derived from construct E with the following modifications: low-risk SNPs (control), 2 high-risk SNPs, single high-risk C-T SNP, or single high-risk T-A SNP mutated, with high-risk SNPs both deleted, and a construct with 2 putative RBPJ binding sites mutated to sequences not predicted to bind RBPJ. (H) Luciferase activity reporting transcriptional activation of the constructs in B (**P < 0.01, ****P < 0.0001 by ANOVA and Dunnett’s post hoc test, n = 4 biological replicates). (I) Forest plot of association of rs74564934 with stroke subtypes (AS, any stroke; AIS, all ischemic stroke; SVS, small vessel stroke; CES, cardioembolic stroke; LAS, large artery stroke). (J) Model of how ETS1, CTCF, and RBPJ bind the FOXF2 enhancer to activate FOXF2 expression in conjunction with the promoter. High-risk SNPs adjacent to the ETS1 site reduce the ability of the enhancer to promote FOXF2 expression.