Estrogen Receptor a Mediated Long Rang Chromatin Interactions at the Ret Gene Locus in Breast Cancer

Abstract

Estrogens function as the primary female sex hormones in women of reproductive age. It promotes the development of female secondary sexual characteristics, and is an essential part of a woman?s reproductive process. Recent studies indicate that about 80% of breast cancers, once established, are estrogen dependent. These are known as hormone-sensitive or hormone-receptor-positive cancers. ERa is the key transcription regulator of this breast cancer progression. It is over-expressed in around 70% of breast cancer cases and estrogen has been shown to stimulate proliferation of mammary cells.

Using high-throughput ChIP (Chromatin ImmunoPrecipitation)-based technology, such as ChIP-Seq (ChIP process followed by sequencing), we generated a global map of ERa binding sites in the genome of MCF7 breast cancer cell line. From this dataset, we identified two ERa binding sites near the RET (rearrangement during transfection) gene. RET is a proto-oncogene that encodes a receptor tyrosine kinase. It has been shown to be involved in human papillary thyroid tumors and in multiple endocrine neoplasia type 2. Previous studies have shown the RET gene is expressed in primary breast tumors and cell lines. In MCF7 cells, the expression of RET, the activation of its downstream signaling pathways, and the increase of anchor-independent proliferation have been shown to be estrogen dependent. In addition, RET expression is up-regulated by estrogen treatment and knock down of ERa down-regulates RET.

We verified that two ERa binding sites within the RET locus, which are located 50kb upstream and 35kb downstream of the transcription start site, are recruited upon estrogen stimuli. Using Chromosome Conformation Capture assay, we showed that these two ERa binding sites are brought in close proximity with each other and with the promoter region of the RET gene in an estrogen dependent manner. Knock down of ERa disrupted this long-range interaction. In addition, we showed that co-regulatory factors, such as FoxA1, cJun, and AP2? are recruited to these two ERa binding sites. Among them, AP2? knock-down resulted in a decrease of RET expression and a concomitant decrease in long range chromatin interaction. Taken together, these results suggest that ERa collaborates with other DNA binding transcription factors to form chromatin loops which directly regulate the transcription of the RET gene in breast cancer cells.