The Effect of rs77275268 on Gene Expression at the Transcriptional Hub at 6q25.1

Abstract

Breast cancer is a prevalent and harmful disease that is divided into subtypes based on tumour characteristics. The main subtype is oestrogen receptor positive (ER+), accounting for approximately 80% of all breast carcinomas. ER+ breast cancer presents with overexpression of the oestrogen receptor (ER) and is commonly treated with hormone therapies that block the ER from responding to oestrogen stimulus, theoretically blocking growth of the cancer. However, in many cases, this treatment is unsuccessful and the mechanisms behind this resistance remain elusive. Genetic variation in a transcriptional hub around the ER gene (ESR1) at 6q25.1, including three upstream genes (ARMT1, CCDC170 and RMND1) has been correlated with increased genetic susceptibility. Therefore, characterisation of the gene expression at this region is important for future breast cancer therapies. SNP variants at the 6q25.1 region have been studied through genome wide association studies and have been associated with increased breast cancer susceptibility. The SNP rs77275268 within this hub is present in the human genome as either a cytosine (C) to thymine (T) variant within a CpG island of a known CTCF binding region. This SNP could affect the methylation status of this site potentially altering CTCF binding in this region. CTCF proteins are transcriptional regulators that bind to specific sites of the DNA and have been found to be affected by DNA methylation. CTCF can play a role in 3D chromatin structure and therefore altered binding of the protein can influence the expression of genes such as those in the ESR1 transcriptional hub. This project therefore aimed to elucidate the expression of the genes at the transcriptional hub with the rs77275268 SNP C and T variants. Expression and correlation of these genes at 6q25.1 (ESR1, CCDC170, ARMT1 and RMND1) in relation to the T variant of the rs77275268 SNP was analysed using quantitative real-time PCR (qPCR). These results showed co-expression of ESR1 and CCDC170 and also RMND1 and ARMT1 supporting the hypothesis that CTCF binding could be altered at this genomic region. Chromatin immunoprecipitation (ChIP) was then utilised to determine potential changes in binding at the SNP dependent on the variant. This was limited by the fact that only one biological replicate was generated and therefore initial results produced are not conclusive, however suggest a change in CTCF binding dependent on the SNP variant present. This project also sought to produce an alternative ER+ breast cancer cell line containing the rs77275268 SNP T variant for further CTCF investigation through CRISPR editing. However, these attempts were unsuccessful with the rate of homology directed repair to insert the specific mutation occurring at a very low rate. In summary, this study demonstrated that the rs77275268 SNP T variant changes how the genes at the transcriptional hub 6q25.1 are co-expressed. Preliminary investigation suggests that the altered expression could be influenced by changes in CTCF binding resulting in a change in chromatin looping. Further attempts to create a SNP edited ER+ breast cancer cell line with higher constitutive CTCF binding are needed to confirm this hypothesis.