A blog post from January last year described a paper by Purdue et al. (2011) in which a genome-wide association study (GWAS) identified two renal cell carcinoma (RCC) susceptibility loci within HIF2α (on chromosome 2) and an uncharacterised intergenic region (on chromosome 11). A recent study using VHL-deficient RCC cells has taken this work further by showing that this intergenic region contains a number of HIF2α and HIF1β binding sites within a previously unknown enhancer of cyclin D1 expression (Schödel et al., 2012).
Using chromatin immunoprecipitation (ChIP) with high-throughput sequencing, Schödel et al. (2012) detected HIF2α and HIF1β binding sites on chromosome 11 in the VHL-defective 786-O RCC cell line (but not in the pVHL-positive MCF-7 breast cancer cell line). ChIP with quantitative PCR (ChIP-qPCR) confirmed this binding in human RCC tissue, and hypoxia responsive elements (HREs) were also identified in this region.
This intergenic RCC susceptibility locus lacks the characteristics of a gene promoter. Consequently, a number of experiments were conducted to see if it was a transcriptional enhancer. Formaldehyde-assisted isolation of regulatory elements showed that this region had reduced nucleosome occupancy in 786-O cells (but not in MCF-7 cells). Similarly, ChIP-qPCR demonstrated that this locus had a variety of epigenetic marks that are characteristic of active enhancers (and not promoters) exclusively in the 786-O cells. Additionally, ChIP-qPCR in these cells showed that this locus had low levels of RNA polymerase II, which catalyses DNA transcription at promoters. Lastly, expression of a reporter gene was also enhanced by these sequences, and this effect was abrogated when the HREs were mutated. In total, these results suggest that the chromosome 11 RCC susceptibility locus contains a HIF-dependent transcriptional enhancer.
Further experiments showed that HIF binding and DNA accessibility was absent from a variety of cancerous and non-cancerous cell lines which expressed wild-type VHL, but was present in all VHL-deficient cell lines tested. In addition, genome-wide expression analysis in renal tumours and 786-O cells identified the nearby CCND1 gene (which encodes cyclin D1) as a HIF-regulated gene on chromosome 11. High-resolution fluorescent in situ hybridisation and chromosome conformation capture demonstrated that the HIF-binding enhancer and the CCND1 promoter physically associated in 786-O cells (but not in MCF-7 cells). These findings suggest that the chromosome 11 susceptibility locus is a cell type-specific long-range enhancer of the cyclin D1 gene.
The authors also saw that different polymorphisms in this region affect DNA accessibility and the binding of HIF2α, HIF1β and RNA polymerase II. Accordingly, these sequence variations could lead to the differential expression of cyclin D1, which offers a potential mechanism for the susceptibility effects observed in this region.
Given the connection between HIF signalling and BHD syndrome (Preston et al., 2010), a GWAS could uncover similar RCC susceptibility loci that are specific to FLCN-deficient cells. In particular, the potential links between FLCN, the HIF pathway and cell cycle control could help us to further understand the tumourigenic phenotypes observed in BHD syndrome.
- Preston RS, Philp A, Claessens T, Gijezen L, Dydensborg AB, Dunlop EA, Harper KT, Brinkhuizen T, Menko FH, Davies DM, Land SC, Pause A, Baar K, van Steensel MA, & Tee AR (2011). Absence of the Birt-Hogg-Dubé gene product is associated with increased hypoxia-inducible factor transcriptional activity and a loss of metabolic flexibility. Oncogene, 30 (10), 1159-73 PMID: 21057536
- Purdue MP, Johansson M, Zelenika D, Toro JR, Scelo G, Moore LE, Prokhortchouk E, Wu X, Kiemeney LA, Gaborieau V, Jacobs KB, Chow WH, Zaridze D, Matveev V, Lubinski J, Trubicka J, Szeszenia-Dabrowska N, Lissowska J, Rudnai P, Fabianova E, Bucur A, Bencko V, Foretova L, Janout V, Boffetta P, Colt JS, Davis FG, Schwartz KL, Banks RE, Selby PJ, Harnden P, Berg CD, Hsing AW, Grubb RL 3rd, Boeing H, Vineis P, Clavel-Chapelon F, Palli D, Tumino R, Krogh V, Panico S, Duell EJ, Quirós JR, Sanchez MJ, Navarro C, Ardanaz E, Dorronsoro M, Khaw KT, Allen NE, Bueno-de-Mesquita HB, Peeters PH, Trichopoulos D, Linseisen J, Ljungberg B, Overvad K, Tjønneland A, Romieu I, Riboli E, Mukeria A, Shangina O, Stevens VL, Thun MJ, Diver WR, Gapstur SM, Pharoah PD, Easton DF, Albanes D, Weinstein SJ, Virtamo J, Vatten L, Hveem K, Njølstad I, Tell GS, Stoltenberg C, Kumar R, Koppova K, Cussenot O, Benhamou S, Oosterwijk E, Vermeulen SH, Aben KK, van der Marel SL, Ye Y, Wood CG, Pu X, Mazur AM, Boulygina ES, Chekanov NN, Foglio M, Lechner D, Gut I, Heath S, Blanche H, Hutchinson A, Thomas G, Wang Z, Yeager M, Fraumeni JF Jr, Skryabin KG, McKay JD, Rothman N, Chanock SJ, Lathrop M, & Brennan P (2011). Genome-wide association study of renal cell carcinoma identifies two susceptibility loci on 2p21 and 11q13.3. Nature genetics, 43 (1), 60-5 PMID: 21131975
- Schödel J, Bardella C, Sciesielski LK, Brown JM, Pugh CW, Buckle V, Tomlinson IP, Ratcliffe PJ, & Mole DR (2012). Common genetic variants at the 11q13.3 renal cancer susceptibility locus influence binding of HIF to an enhancer of cyclin D1 expression. Nature genetics, 44 (4), 420-5 PMID: 22406644