Functional role of SETD2, BAP1, PARP-3 and PBRM1 candidate genes on the regulation of hTERT gene expression

// Hannah Linne 1 , Hemad Yasaei 1, 4 , Alison Marriott 1 , Amanda Harvey 1, 2 , Kefah Mokbel 1, 3 , Robert Newbold 1, 2 and Terry Roberts 1, 2 1 College of Health and Life Sciences, Department of Life Sciences, Biosciences, Brunel University London, Middlesex, UK 2 Institute of Environment, Health and Societies, Brunel University London, Middlesex, UK 3 London Breast Institute, The Princess Grace Hospital, London, UK 4 Current address: Dubai Genetics Centre, Dubai Health Authority, Dubai, United Arab Emirates Correspondence to: Terry Roberts, email: terry.roberts@brunel.ac.uk Keywords: telomerase, breast cancer, epigenetic, chromosome 3, microcell-mediated chromosome transfer Received: January 25, 2017     Accepted: May 15, 2017     Published: June 27, 2017 ABSTRACT Narrowing the search for the critical hTERT repressor sequence(s) has identified three regions on chromosome 3p (3p12-p21.1, 3p21.2 and 3p21.3-p22). However, the precise location and identity of the sequence(s) responsible for hTERT transcriptional repression remains elusive. In order to identify critical hTERT repressor sequences located within human chromosome 3p12-p22, we investigated hTERT transcriptional activity within 21NT microcell hybrid clones containing chromosome 3 fragments. Mapping of chromosome 3 structure in a single hTERT- repressed 21NT-#3fragment hybrid clone, revealed a 490kb region of deletion localised to 3p21.3 and encompassing the histone H3, lysine 36 (H3K36) trimethyltransferase enzyme SETD2; a putative tumour suppressor gene in breast cancer. Three additional genes, BAP1, PARP-3 and PBRM1, were also selected for further investigation based on their location within the 3p21.1-p21.3 region, together with their documented role in the epigenetic regulation of target gene expression or hTERT regulation. All four genes (SETD2, BAP1, PARP-3 and PBRM1) were found to be expressed at low levels in 21NT. Gene copy number variation (CNV) analysis of SETD2, BAP1, PARP-3 and PBRM1 within a panel of nine breast cancer cell lines demonstrated single copy number loss of all candidate genes within five (56%) cell lines (including 21NT cells). Stable, forced overexpression of BAP1, but not PARP2, SETD2 or PBRM1, within 21NT cells was associated with a significant reduction in hTERT expression levels relative to wild-type controls. We propose that at least two sequences exist on human chromosome 3p, that function to regulate hTERT transcription within human breast cancer cells.