Inactivation of tumor suppressor genes plays an important role in tumorigenesis, and epigenetic modifications such as DNA methylation can be associated with transcriptional repression. Although CG methylation is thought to repress transcription it had been not addressed as yet, if CG methylation alters transcriptional elongation as opposed to transcriptional initiation. To understand more about the molecular mechanisms involved in transcriptional silencing associated with DNA methylation, we selected several target genes that were silenced in breast cancer cell lines but not in normal breast cells. Those silenced genes became re-activated after treatment of breast cancer cells with demethylating drugs such as Azacytidine, or treatment with natural products such as (-)-epigallocatechin-3-gallate, Parthenolide and Quercitrin hydrate. Up-regulation of transcription was accompanied by de-methylation of sequences in the upstream promoter region. To explore the mechanism of gene silencing and transcriptional activation, we examined the association of RNA polymerase II at promoter levels using chromatin immunoprecipitation. Serine 5 phosphorylated Pol II, indicative of Pol II initiation, was present at actively transcribed genes, but was also detected at the promoter region of silenced genes suggesting a paused Pol II. Modulation of cytosine methylation with diverse drugs altered stalled Pol II into the ser2 phosphorylated form indicating successful transition into the elongation phase of transcription. The release of paused Pol II was accompanied by a dynamic switch from repressive chromatin marks, including H3K27me3 , H3K9me3 , the polycomb complex proteins EZH2 and SUZ12, to active chromatin marks, including H3K4me3 serving as a binding domain for CHD1, a factor capable to promote the elongation phase of transcription. In addition, we found the chromatin remodeling protein involved in cytosine methylation and repression of silenced genes. Depletion of LSH by RNA interference in breast cancer cell lines reduced DNA methylation in the promoter region and overcame Pol II stalling. On the other hand, 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. Furthermore depletion of DNMT3B by RNA interference could reduce DNA methylation and release Pol II stalling at those silenced genes. These results suggest that LSH and DNMT3B are functionally involved in setting methylation patterns in breast cancer cell lines and lead to transcriptional repression. 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.To understand the physiological role of LSH in breast cancer, we depleted LSH in breast cancer cell lines using RNA interference. We observed reduced growth in culture and a decrease in the formation of colonies in soft agar colonies. In addition, we detected impaired migration activity in a ?wound healing assay? suggesting a physiologic role of LSH mediated DNA methylation in the growth characteristics of cancer cells. Silencing of tumor suppressor genes through epigenetic repression is one of the hallmarks of human cancer. LSH controls DNA methylation and gene repression at selected target genes in cancer cells which is in part mediated by Pol II stalling Understanding the distinct molecular pathways and identifying novel molecular targets can be helpful in devising future strategies to reverse epigenetic gene silencing as supportive cancer therapy.
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