Investigating BRCA1 Promoter Methylation using Molecular and Computational Approaches

Shivam Goel

Document Type

Article

Publication Date

2025

Keywords

JGM

Abstract

Breast Cancer is one of the most common types of cancer for women as it accounts for about 30% of all new female cancers every year. Breast cancer has many different subtypes, one of whom is Triple-Negative Breast Cancer (TNBC). Due to the lack of three main receptors, Estrogen, Progesterone, and Her2, it is one of the deadliest types of breast cancer.

BRCA1 is a critical component of the homologous recombination pathway, which is responsible for the error-free repair of DNA double stranded breaks. To put into context the importance of BRCA1, roughly half of all TNBC are linked to loss of BRCA1 activity.

Epigenetic silencing through BRCA1 promoter methylation is a common mode of BRCA1 gene inactivation as it accounts for half the BRCA1 deficient TNBC cases or around 25% of total TNBC cases. Unfortunately, the mechanism regarding how BRCA1 methylation spontaneously occurs is not very well studied due to the lack of biological models. To address this issue, the Liu lab took advantage of a rare but naturally occurring BRCA1 promoter mutation (c.-107A>T), which was found in two cases of familial breast and ovarian cancer, and was associated with BRCA1 promoter methylation in cis. The Liu lab introduced the c.-107A>T mutation in the induced pluripotent stem cell (iPSC) line WIBJ2 and the human mammary epithelial cell (HMEC) line AR7HT. When this mutation was introduced in WIBJ2 cells, DNA methylation at the BRCA1 promoter was observed. However, in the AR7HT cells, there was no significant gain of methylation at the BRCA1 promoter. Based on this observation, I investigated the binding activity of RNA Polymerase II to the BRCA1 promoter by performing the ChIP assay followed by a qPCR, which showed that there was RNA Polymerase II binding at the BRCA1 promoter region in both WIBJ2 and AR7HT cells. Sanger Sequencing analysis performed on the WIBJ2 ChIP-qPCR amplification products showed a relative enrichment of the wildtype BRCA1 allele in the Pol2 pulldown sample, relative to both input DNA and the igG control pulled down sample, supporting our hypothesis that RNA Polymerase II is preferentially bound to the wild-type (WT) promoter over the mutant promoter, in WIBJ2 cells.

ZNF680 was originally identified as a transcription factor critical for induction of BRCA1 methylation at the c.-107A>T mutant promoter in WIBJ2 cells. Using several in silico techniques and databases, I showed that ZNF680 preferentially binds to promoter, TSS, enhancer, and heterochromatin regions of the genome. However, when compared to other transcription factors such as ZNF610 and ZNF785 who also bind at the BRCA1 promoter, ZNF680 ChIP-seq peak distribution pattern is unusual, in that the percentage of peaks mapping to TSS or promoter regions is much lower. Furthermore, when looking at DNA methylation via EPIC Array analysis and RNA Sequencing data, it appears that ZNF680 is not a master regulator of methylation on a genome-wide level, but that it may be regulating only a small number of genes. This suggests that ZNF680 effect at the BRCA1 promoter may be a very specific occurrence.

Please contact the Joan Staats Library for information regarding this document.

COinS