Transcriptional control of the B3GALT5 gene by a retroviral promoter and methylation of distant regulatory elements

We focused on transcription factors and epigenetic marks that regulate the B3GALT5 gene through its retroviral long terminal repeat (LTR) promoter. We compared the expression levels of the B3GALT5 LTR transcript, quantitated by competitive RT-PCR, with those of the candidate transcription factors HNF1alpha/beta and Cdx1/2, determined by Western blot analysis, in colon cancer biopsies, various cell lines, and cell models serving as controls. We found that HNF1alpha/beta were easily detected, irrespective of the amount of LTR transcript expressed by the source, whereas Cdx1/2 were undetectable, and no sample lacking HNF1alpha/beta expressed the LTR transcript. On transfection in proper host cells, both HNF1alpha and HNF1beta provided detectable LTR transcript, whereas shRNA-mediated silencing of HNF1beta impaired transcription. Treating cells with 5'-aza-2'-deoxycytidine (5AZA) strongly reduced expression, without affecting HNF1alpha/beta, despite the lack of CpG islands in the LTR and proximal sequences. By electrophoresis mobility shift and luciferase reporter assays, the LTR promoter binding and activity did not correlate with the amounts of LTR transcript expressed in the cells and depended on the levels of the transcription factors. We conclude that HNF1alpha/beta are necessary but insufficient to activate and regulate B3GALT5 LTR transcription, which depends on unknown regulatory elements that are active when methylated and located outside of and far from the LTR promoter.-Zulueta, A., Caretti, A., Signorelli, P., Dall'Olio, F., Trinchera, M. Transcriptional control of the B3GALT5 gene by a retroviral promoter and methylation of distant regulatory elements