Regulatory dissection of the CBX5 and hnRNPA1 bi-directional promoter in human breast cancer cells reveals novel transcript variants differentially associated with HP1α down-regulation in metastatic cells

Johan Vad-Nielsen,Rune Thomsen,Tina Fuglsang Daugaard,Anders Lade Nielsen,Boe Sandahl Sørensen,Kristine Raaby Jakobsen,Anja Brügmann

doi:10.1186/s12885-016-2059-x

Johan Vad-Nielsen, Rune Thomsen + Show 5 more

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https://doi.org/10.1186/s12885-016-2059-x

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Abstract

BackgroundThe three members of the human heterochromatin protein 1 (HP1) family of proteins, HP1α, HP1β, and HPγ, are involved in chromatin packing and epigenetic gene regulation. HP1α is encoded from the CBX5 gene and is a suppressor of metastasis. CBX5 is down-regulated at the transcriptional and protein level in metastatic compared to non-metastatic breast cancer. CBX5 shares a bi-directional promoter structure with the hnRNPA1 gene. But whereas CBX5 expression is down-regulated in metastatic cells, hnRNAP1 expression is constant. Here, we address the regulation of CBX5 in human breast cancer.MethodsTransient transfection and transposon mediated integration of dual-reporter mini-genes containing the bi-directional hnRNPA1 and CBX5 promoter was performed to investigate transcriptional regulation in breast cancer cell lines. Bioinformatics and functional analysis were performed to characterize transcriptional events specifically regulating CBX5 expression. TSA treatment and Chromatin Immunoprecipitation (ChIP) were performed to investigate the chromatin structure along CBX5 in breast cancer cells. Finally, expression of hnRNPA1 and CBX5 mRNA isoforms were measured by quantitative reverse transcriptase PCR (qRT-PCR) in breast cancer tissue samples.ResultsWe demonstrate that an hnRNPA1 and CBX5 bi-directional core promoter fragment does not comprise intrinsic capacity for specific CBX5 down-regulation in metastatic cells. Characterization of transcriptional events in the 20 kb CBX5 intron 1 revealed existence of several novel CBX5 transcripts. Two of these encode consensus HP1α protein but used autonomous promoters in intron 1 by which HP1α expression could be de-coupled from the bi-directional promoter. In addition, another CBX5 transcriptional isoform, STET, was discovered. This transcript includes CBX5 exon 1 and part of intron 1 sequences but lacks inclusion of HP1α encoding exons. Inverse correlation between STET and HP1α coding CBX5 mRNA expression was observed in breast cancer cell lines and tissue samples from breast cancer patients.ConclusionWe find that HP1α is down-regulated in a mechanism involving CBX5 promoter downstream sequences and that regulation through alternative polyadenylation and splicing generates a transcript, STET, with potential importance in carcinogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2059-x) contains supplementary material, which is available to authorized users.

Highlights

The three members of the human heterochromatin protein 1 (HP1) family of proteins, HP1α, HP1β, and HPγ, are involved in chromatin packing and epigenetic gene regulation
We showed that the CBX5 promoter is less occupied by basal transcription factors such as TBP, TFIIB, TFIIH as well as polymerase II (Pol-II) in MDA-MB-231 cells when compared to MCF7 cells [37]
We conclude that the bi-directional promoter region per se is not sufficient to mediate preferential CBX5 downregulation compared to hnRNPA1 in MDA-MB-231 cells versus MCF7 cells

Summary

Introduction

The three members of the human heterochromatin protein 1 (HP1) family of proteins, HP1α, HP1β, and HPγ, are involved in chromatin packing and epigenetic gene regulation. The hinge region of HP1 contributes to chromatin association through interactions with histone H1 and RNA Through this interaction, RNA components are thought to be important in the maintenance and localization of HP1 proteins along specific sites at the genome, e.g. for HP1α pericentric heterochromatin localization [8, 27,28,29,30]. When HP1 is bound to di- or trimethylated H3K9 through the CD, subsequent recruitment of SUV39h1 causes adjacent H3K9 residues to become methylated This creates new binding sites for additional HP1 proteins, which, in turn, will further recruit SUV39h1 proteins. PEV is suppressed with decreased HP1 expression and enhanced with increased HP1 expression [32, 33]

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