Multiple renal cancer susceptibility polymorphisms modulate the HIF pathway

<div><p>Un-physiological activation of hypoxia inducible factor (HIF) is an early event in most renal cell cancers (RCC) following inactivation of the von Hippel-Lindau tumor suppressor. Despite intense study, how this impinges on cancer development is incompletely understood. To test for the impact of genetic signals on this pathway, we aligned human RCC-susceptibility polymorphisms with genome-wide assays of HIF-binding and observed highly significant overlap. Allele-specific assays of HIF binding, chromatin conformation and gene expression together with eQTL analyses in human tumors were applied to mechanistic analysis of one such overlapping site at chromosome 12p12.1. This defined a novel stage-specific mechanism in which the risk polymorphism, rs12814794, directly creates a new HIF-binding site that mediates HIF-1α isoform specific upregulation of its target <i>BHLHE41</i>. The alignment of multiple sites in the HIF <i>cis</i>-acting apparatus with RCC-susceptibility polymorphisms strongly supports a causal model in which minor variation in this pathway exerts significant effects on RCC development.</p></div

Allele-specific expression of <i>BHLHE41</i> is dependent on HIF-binding to rs12814794.

<p><b>A)</b> Schematic view of the chr 12p12.1 locus. The SNP rs12814794 at the HIF-binding enhancer and the intragenic SNP rs1048155 in the 3’ UTR of <i>BHLHE41</i> are indicated. <b>B)</b> Allele-specific qPCR experiments for rs1048155 using genomic DNA (gDNA) or complementary DNA (cDNA) derived from primary tubular cells of an individual heterozygous for rs1048155 and homozygous (AA) for rs12814794. Cells were exposed to 1 mM DMOG or left untreated. The allelic ratios B/A for signals from untreated or DMOG treated cDNA normalized to gDNA are shown on the right. No difference in allelic expression is measured at the intragenic SNP (ns, non-significant). The blue bar indicates the allelic ratio of gDNA. <b>C)</b> In these cells <i>BHLHE41</i> expression is not induced by DMOG. Expression levels of <i>BHLHE41</i> were normalized to the housekeeping gene HPRT and to values from the untreated control. Expression qPCR was performed in technical quadruplicates on one biological sample. Values are mean ± standard deviation. <b>D)</b> Allele-specific qPCR using gDNA and cDNA from an individual homozygous for the G allele at the HIF-binding enhancer. No difference in allelic expression is measured at the intragenic SNP. <b>E)</b> <i>BHLHE41</i> expression is induced in these cells by 1 mM DMOG. Expression qPCR was performed in technical quadruplicates on one biological sample. Values are mean ± standard deviation. <b>F)</b> Allele specific qPCR for gDNA and cDNA using PTC from an individual heterozygous for both SNPs. A significant shift in the allelic ratio of the intragenic SNP rs1048155 is detectable in DMOG treated cells. Mann-Whitney-Wilcoxon test: p<0.005. <b>G)</b> <i>BHLHE41</i> expression is induced by 1 mM DMOG. Expression qPCR was performed in technical quadruplicates on one biological sample. Values are mean ± standard deviation.</p

Published renal cancer-associated susceptibility loci.

<p>Published renal cancer-associated susceptibility loci.</p

Allele specific HIF-binding at the rs12814794 associated enhancer.

<p><b>A)</b> rs12814794 defines a one base pair exchange (A>G) which creates a HIF-binding motif (AC<u>G</u>TG). <b>B)</b> Allele-specific qPCR for rs12814794 from DNA fragments isolated in HIF-ChIP experiments (HIF-1α and HIF-1β) or input DNA from primary renal tubular cells (PTC, n = 4 individuals). DNA from individuals homozygous for the AA or GG genotype were used as positive controls. <b>C)</b> Quantification of the two different alleles (A or G) at rs12814794 in the ChIP-seq reads from HIF-1α and HIF-1β immunoprecipitations (PTC #1 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006872#pgen.1006872.g003" target="_blank">Fig 3</a>). <b>D)</b> Allele-specific qPCR for rs12814794 from DNA fragments isolated in FAIRE experiments or input DNA from primary renal tubular cells (PTC). DNA from individuals homozygous for the AA or GG genotype were used as positive controls. <b>E)</b> Quantification of the two different alleles (A or G) for rs12814794 present in reads from the FAIRE-seq experiment (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006872#pgen.1006872.g003" target="_blank">Fig 3a</a>: FAIRE track for PTC#1) and sequencing of the input. <b>F)</b> Allele-specific qPCR for rs12814794 from HIF-ChIP (HIF-1α, HIF-2α and HIF-1β) experiments in RCC L13 cells. DNA from control serum immunoprecipitations or input DNA from RCC L13 was used as controls. DNA from homozygous individuals (AA or GG) were used as controls for the two genotypes.</p

Interaction between HIF level, rs12814794 genotype and expression of <i>BHLHE41</i> mRNA.

<p><b>A)</b> 33 genotyped primary tubular cell cultures (3 GG, 12 AG, and 18 AA) were exposed to 1 mM DMOG to induce HIF. <i>BHLHE41</i> mRNA expression was assayed by qPCR. The mean (± standard deviation) inducibility of <i>BHLHE41</i> mRNA compared to the respective untreated cells (= 1) was then plotted for each of the different rs12814794 genotypes. *, p<0.05—Student’s t-test. <b>B)</b> The mean inducibility of <i>EGLN3</i> mRNA levels in the same samples was plotted as control. <b>C-F)</b> Box-and-whisker plots showing the correlation between <i>BHLHE41</i> mRNA levels and genotype at rs12814794 in TCGA analysis of ccRCC tumors (KIRC). Tumors were first divided into tertiles according to expression of previously described HIF-1 or HIF-2 metagenes <b>C)</b> lower tertile for HIF-1 metagene expression, <b>D)</b> upper tertile for HIF-1 metagene expression, <b>E)</b> lower tertile for HIF-2 metagene expression, <b>F)</b> upper tertile for HIF-2 metagene expression. <i>χ</i>²-test was used to calculate p values for higher expression in GG individuals compared to AA individuals. Whiskers extend to 1.5 times of the inter quartile range.</p

Non-random association between RCC GWAS loci and HIF-binding sites.

<p>4 RCC GWAS loci overlapped with 6 HIF ChIP-seq peaks. <b>A)</b> To assess the significance of this overlap, the expected number of co-localization events was calculated by randomly shuffling the GWAS loci around the genome. This was repeated 100,000 times and the frequency distribution for the number of shuffled GWAS sites that overlapped a HIF-binding site was plotted. The probability of observing 4 or more GWAS loci overlapping a HIF ChIP-seq peak is 1x10^-4 <b>B)</b> Conversely, HIF ChIP-seq peaks were randomly shuffled amongst all potential enhancer sites (as defined by the H3K27ac, H3K4me3, H3K4me1 in the same cell line 786-O), repeated 100,000 times and the frequency distribution for the number of shuffled HIF-binding sites that overlapped a GWAS site was plotted. The probability of observing 6 or more HIF-sites overlapping the GWAS sites is 9x10^-5.</p

Open chromatin at the chr 12p12.1 RCC susceptibility locus overlaps with HIF-binding in primary renal tubular cells.

<p><b>A)</b> HIF-1α ChIP-seq track for PTC isolated from individual #1 indicates binding of HIF at the chr 12p12.1 region in PTC (blue). HIF-1α was stabilized by 1 mM DMOG for 4h. FAIRE-seq data from primary renal tubular cells from three individuals (PTC#1–3, red) and ENCODE FAIRE-seq tracks of 786-O RCC and a selection of non-renal cells. Enhancer associated SNPs, which are in high LD with the tag SNP rs718314 are indicated below the tracks. <b>B)</b> FAIRE qPCR experiments confirm chromatin accessibility at the rs12814794 associated enhancer specifically in renal tubule derived cells (RCC and PTC) when compared to non-renal cells. Data is from one experiment per cell line. Average enrichment values compared to a putative non-enhancer region (control locus) are shown.</p

The chr 12p12.1 RCC risk locus encompasses a HIF-1-dependent enhancer that directly transactivates expression of <i>BHLHE41</i>.

<p><b>A)</b> High-throughput sequencing analysis of the chr 12p12.1 locus in 786-O renal cancer cells—SNPs in high LD (r²>0.8) with the reported RCC-associated SNP (blue), ChIP-seq analysis of HIF-binding (red), ChIP-seq analysis of histone H3K4me1, H3K4me3 and H3K27ac modification (blue), FAIRE-seq analysis of chromatin accessibility (purple) and Capture-C analysis of chromatin interactions (black). SNPs in high LD with the reported RCC-associated SNP overlap with 3 enhancer sites as defined by chromatin accessibility and histone modification (highlighted in yellow) that interact with the distant <i>BHLHE41</i> promoter (highlighted in green), including a HIF-bound enhancer (Capture site 1). <b>B)</b> RNA-seq analysis of VHL-reconstituted 786-O renal cancer cells that express HIF-2α, but not HIF-1α does not show HIF-dependent regulation of BHLHE41. Values are mean FPKM (Fragments per kilobase of exon per million fragments mapped) from 3 independent RNA-seq experiments ± standard deviation. <b>C)</b> Overexpression of HIF-1α, but not HIF-2α induces BHLHE41 mRNA in RNA-seq analysis of 786-O cells (VA, vector alone). Values are mean FPKM values from 3 three independent RNA-seq experiments ± standard deviation. <b>D & E)</b> siRNA mediated suppression of HIF-1α and HIF-1β, but not HIF-2α in VHL-defective RCC L13 and VHL re-expressing RCC4 renal cancer cells (untreated or treated with 1 mM of the HIF-stabilizer DMOG) reduces expression of <i>BHLHE41</i> mRNA. qPCR values were normalized to the housekeeping gene HPRT and values from the untreated control siRNA (dHIF) sample. Values are mean relative expression from triplicate qPCR experiments ± standard deviation.</p