An 8q24 gene desert variant associated with prostate cancer risk confers differential in vivo activity to a MYC enhancer

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Nora F. Wasserman, Ivy Aneas and Marcelo A. Nobrega

Genome Res. published online July 13, 2010| doi:10.1101/gr.105361.110

 

This paper demonstrates that one possible mechanism underlying disease association for non-coding risk variants(SNPs) discovered by GWAS is through regulating the activity of enhancer element, which controls the temporal and tissue-specific expression of its target genes. The researchers scan the five prostate-cancer associated regions spanning a 440 kb interval in a gene desert(contains few or no genes)on 8q24 which harbor risk variants(SNPs) to search for enhancers with BAC enhancer-trapping approach, and identify an enhancer within this region driving reporter gene expression in both prostate and mammary glands in transgenic mice. They further narrow down the location of this prostate enhancer into a 5kb region encompassing prostate-cancer risk SNP rs6983267. Results show enhancer with the risk allele, rs6983267-G , significantly increase the level of reporter gene's prostate and coagulating gland expression than non-risk allele, rs6983267-T. In addition, they find the tissue-specific reporter expression driven by this rs6983267-G containing enhancer is concordant with the expression pattern of proto-oncogene MYC, which lies immediately downstream of this gene desert and is overexpressed in aggressive prostate cancer, raising the possibility that this allele-specific enhancer regulate MYC expression.

 

Preceding this experimental characterization of SNP effect on enhancer activity, Genome-wide association study have indentified 620kb interval of a 1.2 Mb gene desert on 8q24 as hot spot for susceptibility loci associated with many epithelial cancer. Among these, prostate-cancer risk alleles reside in five distinct linkage disequilibrium(LD) blocks("non-random association of alleles at two or more loci"), where there is no well-annotated genes. The closest characterized gene locus is the oncogene MYC that is 200-kb downstream from the nearest prostate-cancer associated LD block. For the five LD blocks, 3 are independent region for prostate cancer solely, one is also breast-cancer associated and the other colorectal-cancer associated. The author hypothesizes that the disease etiology is that regulatory elements like enhancers lie within these regions which function over long distance to regulate expression of a gene involved in tumorigenesis in a tissue-specific manner. Here MYC is a strong candidate target gene. In other words, risk alleles in each distinct LD block may be encompassed by long-range enhancers which control MYC expression in the corresponding organs or tissue for each associated cancer type.

 

To test this hypothesis, they use bacterial artificial chromosome(BAC) enhancer-trapping strategy. BACs are large DNA contructs which carry up to several hundred kilobases of sequence. In various sequencing projects, genomic DNA has been cut into large fragments and cloned into BACs, and then these sequenced BACs can be used for genome mapping. Here the researchers select three BACs covering the genomic regions of five prostate-cancer associated LD blocks, and tag each BACs with reporter gene cassettes through Tn7 transposon-mediated random insertion of lacZ gene and its driving promoter. Any enhancer located within a given BAC can be detected by the reporter assay system, and thus all the associated regions will be examined for any regulatory element. Each modified BACs are then injected into fertilized mouse oocytes to produce transgenic mice, allowing for in vivo study of enhancer activity.

 

Snapshots of X-gal staining of lacZ expression are captured during prostate development and after prostate maturation. Mice with BAC CTD-2506D10 and BAC RP11124F15 share the same spacial and temporal lacZ expression pattern. Enhancers within these two BACs upregulate lacZ expression in prostate and other structures of urogenital system both during and after prostate development. Because these two BACs are designed to share overlapping region, the chance is very low that two independent enhancers with sequence variations displaying similar regulatory activity. So researchers are interested in testing if this gene regulation pattern is dictated by one enhancer in the shared region. The fact that there is one strong risk SNP rs6983267 within this interval prompts them to test the regulatory potential of 5kb conserved element harboring rs6983267 by similar mouse transgenic reporter assays. And they find the reporter gene expression pattern driven by this element containing risk allele rs6983267-G mimics that of the two BAC transgenes, both of which encompass the risk variant, increasing the probability that this regulatory activity can be traced to rs6983267-containing enhancer in the 5kb interval. What's more interesting, different alleles in this variant confer distinguishable enhancer regulatory performance, with non-risk allele rs6983267-T gives much weaker reporter gene expression in prostate and coagulating glands than risk allele rs6983267-G, indicating risk variant could regulate cis-regulatory element and control gene expression.

 

This paper highlights the utility and the advantages of BAC enhancer trapping followed by in vivo BAC transgenic reporter assays. Compared to widely-used plasmid-based reporter assays, the method enables effecient screening of large genomic region for cis-regulatory elements and maintaining the broad genomic context which affects regulatory potential. In addition, in vivo reporter assay in transgenic mice allows for observation of in vivo enhancer activity in regulating temporal and spacial gene expression in development. This approach can be advocated for future experimental study on characterizing regulating capacity of non-coding DNA.

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