Defining the regulation and function of SAFB1 and SAFB2 in human breast cancer cells

Abstract

Scaffold attachment factor B1 (SAFB1) and SAFB2 are oestrogen receptor (ER) corepressors that bind and modulate ER activity through chromatin remodelling or interaction with the basal transcription machinery. However, little is known about the fundamental characteristics and function of SAFB1 and SAFB2 proteins in breast cancer. In this study, an investigation of the characteristics and function(s) of SAFB1 and SAFB2 was undertaken; their expression profile was first assessed in ER-positive (MCF-7) and ER-negative (MDA-MB-231) breast cancer cell lines. Results show that SAFB1 and SAFB2 are themselves regulated by an active metabolite of oestrogen, 17β-oestradiol, in both ER positive and ER negative breast cancer cells. Using a combined approach of RNA interference and gene expression profile studies, 12 novel targets closely linked to tumour progression were identified for SAFB1 and SAFB2. Expression levels of the following genes, CDKN2A, CLU, ESR1, IGFBP2, IL2RA, ITGB4, KIT, KLK5, MT3, NGFR and SPRR1B increased while IL-6 expression and secretion decreased when cells were depleted of SAFB proteins. This observation supports their primary role as transcriptional repressors with SAFB2 playing a prominent role in transcriptional regulation in MDA-MB-231 cells. This study has also established a novel link between SAFB proteins and ITGB4 and IL-6 expression. Both SAFB proteins have an internal RNA-recognition motif but little is known about the RNA-binding properties of SAFB1 or SAFB2. To investigate this, the concluding part of the project utilised crosslinking and immunoprecipitation (CLIP) coupled with high-throughput sequencing. This experimental approach enabled a transcriptome-wide mapping of SAFB1 protein-RNA interactions in breast cancer cells. SAFB1 crosslink sites are significantly enriched in ncRNAs, particularly within snRNAs. A putative RNA-binding motif for SAFB1 that contains adenine-rich sequences, highly similar to the RNA-binding motifs for SR proteins was also identified. In summary, this study has defined the characteristics of SAFB1 and SAFB2 in both ER positive and ER negative breast cancer cell lines. It has also identified transcriptional targets and the RNA-binding ability of SAFB1 and SAFB2 in human breast cancer cells.