Enhancer Variants Synergistically Drive Dysfunction of a Gene Regulatory Network In Hirschsprung Disease

Abstract

Common sequence variants in cis-regulatory elements (CREs) are suspected etiological causes of complex disorders. We previously identified an intronic enhancer variant in the RET gene disrupting SOX10 binding and increasing Hirschsprung disease (HSCR) risk 4-fold. We now show that two other functionally independent CRE variants, one binding Gata2 and the other binding Rarb, also reduce Ret expression and increase risk 2- and 1.7-fold. By studying human and mouse fetal gut tissues and cell lines, we demonstrate that reduced RET expression propagates throughout its gene regulatory network, exerting effects on both its positive and negative feedback components. We also provide evidence that the presence of a combination of CRE variants synergistically reduces RET expression and its effects throughout the GRN. These studies show how the effects of functionally independent non-coding variants in a coordinated gene regulatory network amplify their individually small effects, providing a model for complex disorders.

Figure 1. Genomic map and biological activity of enhancers within the RET locus

(A) A 350 kb genomic segment annotated with topologically associated domains (TAD) in 9 human cell lines with experimentally tested (mouse Neuro2a cells) and ENCODE-predicted (DNaseI Hypersensitivity, DHS) enhancers, and H3K4me1 and H3K27ac marks, from a 108 day human fetal intestine. All enhancers lie within a core 225 kb TAD common to all cell lines. (B) Fine-mapping of a 153 kb sub-region containing all identifiable enhancers, 38 HSCR-associated SNPs and known transcription factor (TF) ChIP-seq sites in human SK-N-SH cells. The 8 SNPs in color disrupt a TF binding site and lie within an enhancer. (C) in vitro luciferase assays in Neuro2A cells showing enhancer activity in wildtype and risk allele constructs: red elements at RET−7 (containing rs2506030), RET−5.5 (containing rs7069590) and RET+3 (containing rs2435357) demonstrate statistically significant allelic differences in enhancer activity; green elements do not. (D) Luciferase assays of multiple-enhancer constructs in Neuro2a cells (Table 1) demonstrating an exponential relationship between loss of enhancer activity and increasing risk of HSCR from risk allele dosage. The risk alleles in each haplotype are marked in red. The error bars represent standard errors of the mean (** P<0.001).

Figure 2. Tissue-specific enhancer activities of three RET cis-regulatory elements (CRE) during mouse development

(A) Transgenic assays of the human wildtype element RET−7 in mouse embryos demonstrate lacZ-driven expression in the gut (black arrowhead) and the dorsal root ganglion (DRG, red arrow) at day E11.5 (A). Analogous assays of the wildtype elements RET−5.5 (B) and RET+3 (C) also show tissue-specific expression in the gut and DRG at E11.5. At E12.5, RET−7 (D) continues to drive expression in the DRG but gut expression is lost, as is also in RET−5.5 (E); RET+3 (F) continues to have strong activity at E12.5 in both the gut and DRG.

Figure 3. Loss of enhancer activity at risk alleles and identification of cognate transcription factors (TF)

(A) Gene expression of putative TFs in the mouse gut at E11.5, E12.5 and E14.5 shows declining expression of Rarb, Gata2 and Gata3 but no change in Sox10; pair-wise comparisons are relative to E11.5. (B) Genomic map of the RET locus showing DHS sites with respect to enhancers in human SK-N-SH cells. ChIP in SK-N-SH with a RAR antibody shows enrichment of binding to RET−7, as compared to the background signal; the specificity of binding is shown by siRNA knockdown of RARB with concomitant reduced binding. Analogous assays for RET−5.5 and GATA2, and RET+3 and SOX10 show specific binding of these TFs to their cognate enhancers. (C) siRNA-mediated down-regulation of Sox10, Gata2, and Rarb expression in Neuro2A cells affects activity of wildtype but not risk alleles at enhancers, demonstrating specificity. All pairwise comparisons are with wildtype or risk alleles co-transfected with control siRNAs. The error bars represent standard errors of the mean (*P<0.01, ** P<0.001).

Figure 4. Loss-of-function of Ret and genes in its regulatory network (GRN)

(A) siRNA-mediated down regulation of Sox10, Gata2, and Rarb in Neuro2a cells attenuates Ret transcription, as does siRNA against Ret. (B) Additionally, loss of Ret expression leads to reduced expression of its transcription factors Sox10 and Gata2, but not Rarb, thus showing positive feedback. All pairwise comparisons are to control siRNA values. (C) Gene expression of the Ret GRN in the developing gut of wildtype and Ret null embryos show significant up-regulation of Gdnf and Gfra1 and down-regulation of Sox10 with Ret loss-of-function at E11.5 and E12.5; Cbl and Gata2 show loss of expression at E11.5 only; other components of canonical Ret downstream signaling, and Rarb, are unaffected by loss of Ret in vivo. All pairwise comparisons are between wildtype and null embryos at each stage. The error bars represent standard errors of the mean (*P<0.01, ** P<0.001).

Figure 5. Feedback between Ret and its transcription factors (TF)

(A) Ret gene expression in mouse Neuro2a cells with increasing doses of Ret siRNA (12–25 μM), with assays of transcript and protein levels of Ret, Sox10, Rarb and Gata2. Ret expression declines steadily followed by decreasing Sox10 expression only when Ret falls below 50% (17 μM); an analogous decline is observed for Gata2 but not Rarb. All pairwise comparisons are with transfections with control siRNAs. (B) Protein expression changes assessed by western blotting. The error bars represent standard errors of the mean (*P<0.01, ** P<0.001).

Figure 6. Dysregulation of the RET gene regulatory network (GRN) in the human fetal gut

(A) Combined genotypes of 3 enhancer variants in 8 fetal samples represented in terms of Hirschsprung disease (HSCR) resistant (R) and susceptible (S) haplotypes (Table 1). The risk alleles have been highlighted in red. (B) Average gene expression by genotype shows loss of RET expression by S haplotype dosage, and analogous effects on SOX10, GATA2 and CBL; GDNF and GFRA1 show the opposite effect, as in the mouse (Figure 4). All pairwise comparisons are with reference to the R/R haplotype. The error bars represent standard errors of the mean (*P<0.01, ** P<0.001).