Treatment of breast cancer cells with DNA demethylating agents leads to a release of Pol II stalling at genes with DNA-hypermethylated regions upstream of TSS

Abstract

Inactivation of tumor suppressor genes plays an important role in tumorigenesis, and epigenetic modifications such as DNA methylation are frequently associated with transcriptional repression. Here, we show that gene silencing at selected genes with signs of DNA hypermethylation in breast cancer cells involves Pol II stalling. We studied several repressed genes with DNA hypermethylation within a region 1-kb upstream of the transcriptional start site that were upregulated after treatment with DNA demethylating agents, such as Azacytidine and several natural products. All those selected genes had stalled Pol II at their transcriptional start site and showed enhanced ser2 phosphorylated Pol II and elevated transcripts after drug treatment indicating successful elongation. In addition, a decrease of the epigenetic regulator LSH in a breast cancer cell line by siRNA treatment reduced DNA methylation and overcame Pol II stalling, whereas overexpression of LSH in a normal breast epithelial cell line increased DNA methylation and resulted in repression. Decrease of LSH was associated with reduced DNMT3b binding to promoter sequences, and depletion of DNMT3b by siRNA could release Pol II suggesting that DNMT3b is functionally involved. The release of paused Pol II was accompanied by a dynamic switch from repressive to active chromatin marks. Thus release of Pol II stalling can act as a mechanism for gene reactivation at specific target genes after DNA demethylating treatment in cancer cells.

Chromatin Remodeling Factor LSH Drives Cancer Progression by Suppressing the Activity of Fumarate Hydratase

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Transcriptome sequencing of human breast cancer reveals aberrant intronic transcription in amplicons and dysregulation of alternative splicing with major therapeutic implications

“…These are characterised by detection of amplification/overexpression, not genetic analysis of mutations or rearrangements revealed by exome sequencing (9)(10)(11)(12)(13)(14). Recent demonstrations of the expression of novel (non-RefSeq) splice variants in cell lines (15)(16)(17)(18)(19), suggest amplification may not simply involve overexpression of exonic sequences alone but also non-exonic sequences.…”

The Chromodomain Helicase DNA-Binding Chromatin Remodelers: Family Traits that Protect from and Promote Cancer

“…Exposure to cigarette smoke correlates with hypermethylation of the CHD1 promoter (Lyn-Cook et al 2014). Promoter methylation is associated with RNA polymerase II (Pol II) stalling and compromised transcriptional activation, whereas demethylating agents abrogate this effect by inducing Pol II phosphorylation at serine 2 to promote transcriptional elongation (Tao et al 2011). Estrogen signaling plays a key role in promoting proliferation of estrogen receptor positive breast cancer.…”