부산대학교 도서관

Enhancer sequences have been well documented for over a decade, and whilst their function as gene expression regulators is widely appreciated, the mechanism by which they exert their control is not yet understood. Transcription of enhancer regions is linked to enhancer activity, but it is unclear if the enhancer RNA (eRNA) transcript is necessary for cis regulation or merely a by-product of transcription. Through RNA-Sequencing of estradiol (E2) treated MCF7 cells and the use of publicly available sequencing data, we have identified a region neighbouring the CCND1 gene locus which contains at least one enhancer whose bi-directional transcription is upregulated with E2 treatment in estrogen receptor (ER) positive breast cancer cell lines. This enhancer region is known to have cis-regulatory effects on the neighbouring CCND1 gene, whose amplification and overexpression is linked to a poorer prognosis and treatment resistance in ER positive breast cancers. To determine the role of the bi-directionally transcribed eRNAs arising from this enhancer region, we have identified their cellular location and used appropriate siRNA techniques to knockdown both transcripts. We show that siRNA knockdown of either eRNA does not affect regulation of the neighbouring CCND1 gene but premature termination of transcription of the antisense enhancer not only knocks-down the eRNA but also down regulates CCND1 and may have a more global effect on ER regulation. We discuss the challenges encountered in CRISPR/Cas9 mediated knock-in of a polyadenylation signal and compare the resultant effects of knockdown of these eRNA with the premature transcription termination of the enhancer from which they arise and discuss these findings in the context of alternative possible roles for eRNAs.