Limits...
Lsh mediated RNA polymerase II stalling at HoxC6 and HoxC8 involves DNA methylation.

Tao Y, Xi S, Briones V, Muegge K - PLoS ONE (2010)

Bottom Line: Hypomethylation in Lsh-/- cells is associated with efficient transcriptional elongation and splicing, in part mediated by the chromodomain protein Chd1.Dynamic modulation of DNA methylation in Lsh-/- and wild type cells demonstrates that catalytically active DNA methyltransferase activity is required for Pol II stalling.Taken together, the data suggests that DNA methylation can be compatible with Pol II binding at selected genes and Pol II stalling can act as alternate mechanism to explain transcriptional silencing associated with DNA methylation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cancer Prevention, SAIC-Frederick, National Cancer Institute, Frederick, Maryland, United States of America.

ABSTRACT
DNA cytosine methylation is an important epigenetic mechanism that is involved in transcriptional silencing of developmental genes. Several molecular pathways have been described that interfere with Pol II initiation, but at individual genes the molecular mechanism of repression remains uncertain. Here, we study the molecular mechanism of transcriptional regulation at Hox genes in dependence of the epigenetic regulator Lsh that controls CpG methylation at selected Hox genes. Wild type cells show a nucleosomal deprived region around the transcriptional start site at methylated Hox genes and mediate gene silencing via Pol II stalling. Hypomethylation in Lsh-/- cells is associated with efficient transcriptional elongation and splicing, in part mediated by the chromodomain protein Chd1. Dynamic modulation of DNA methylation in Lsh-/- and wild type cells demonstrates that catalytically active DNA methyltransferase activity is required for Pol II stalling. Taken together, the data suggests that DNA methylation can be compatible with Pol II binding at selected genes and Pol II stalling can act as alternate mechanism to explain transcriptional silencing associated with DNA methylation.

Show MeSH

Related in: MedlinePlus

Chromatin marks associated with transcriptional elongation are increased in the absence of Lsh.(A) Schematic diagram depicting the position of the primer pairs (black lines) used for ChIP analysis. Comparison of the ratio of H3K36Me3 (B), H3K79Me2 (C) and H3K4Me3 (E) enrichment to total H3 occupancies (Fig. S6) between Lsh WT and Lsh−/−MEFs at up, TSS, down and 3′UTR regions of HoxC6 and HoxC8 genes. (D) ChIP analysis for the detection of Ser 2 RNA Pol II (H5). (F) ChIP analysis for detection of Chd1. (G) Western analysis for detection of Chd1 protein comparing whole cell extracts derived from Lsh−/−MEFs targeted by Chd1 siRNA or control siRNA (H) Real-time RT-PCR analysis detecting HoxC6 and HoxC8 mRNA in Lsh−/− MEFs after targeting by Chd1 siRNA compared to control treated cells. Results represent standard deviations for the mean of three independent experiments. Asterix indicates a p value p<0.05.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2820093&req=5

pone-0009163-g004: Chromatin marks associated with transcriptional elongation are increased in the absence of Lsh.(A) Schematic diagram depicting the position of the primer pairs (black lines) used for ChIP analysis. Comparison of the ratio of H3K36Me3 (B), H3K79Me2 (C) and H3K4Me3 (E) enrichment to total H3 occupancies (Fig. S6) between Lsh WT and Lsh−/−MEFs at up, TSS, down and 3′UTR regions of HoxC6 and HoxC8 genes. (D) ChIP analysis for the detection of Ser 2 RNA Pol II (H5). (F) ChIP analysis for detection of Chd1. (G) Western analysis for detection of Chd1 protein comparing whole cell extracts derived from Lsh−/−MEFs targeted by Chd1 siRNA or control siRNA (H) Real-time RT-PCR analysis detecting HoxC6 and HoxC8 mRNA in Lsh−/− MEFs after targeting by Chd1 siRNA compared to control treated cells. Results represent standard deviations for the mean of three independent experiments. Asterix indicates a p value p<0.05.

Mentions: To support further the notion of Pol II stalling versus elongation, histone modifications that are hallmarks of RNA Pol II elongation and enriched at the gene body were assessed [35], [39], [40]. Histone 3 lysine 36 trimethylation (H3K36me3) and H3K79 dimethylation (H3K79me2) are present at fairly equal levels in cellular extracts of Lsh−/− and WT cells (Fig. S5). Both H3K36me3 and H3K79me2 showed enrichment at the gene body of HoxC6 and HoxC8 genes in Lsh−/− cells compared to WT cells (Fig. 4A, B, C). Furthermore, Ser2 Pol II enrichment at the 3′ end, another hallmark of successful transcriptional elongation [36] was increased at HoxC6 and HoxC8 genes in Lsh−/− cells compared to WT samples (Fig. 4D). Taken together, the change in histone modifications and Ser2 Pol II further supported the observation of Pol II stalling at HoxC6 and HoxC8 genes in WT cells and successful Pol II elongation and transcription in Lsh−/− MEFs.


Lsh mediated RNA polymerase II stalling at HoxC6 and HoxC8 involves DNA methylation.

Tao Y, Xi S, Briones V, Muegge K - PLoS ONE (2010)

Chromatin marks associated with transcriptional elongation are increased in the absence of Lsh.(A) Schematic diagram depicting the position of the primer pairs (black lines) used for ChIP analysis. Comparison of the ratio of H3K36Me3 (B), H3K79Me2 (C) and H3K4Me3 (E) enrichment to total H3 occupancies (Fig. S6) between Lsh WT and Lsh−/−MEFs at up, TSS, down and 3′UTR regions of HoxC6 and HoxC8 genes. (D) ChIP analysis for the detection of Ser 2 RNA Pol II (H5). (F) ChIP analysis for detection of Chd1. (G) Western analysis for detection of Chd1 protein comparing whole cell extracts derived from Lsh−/−MEFs targeted by Chd1 siRNA or control siRNA (H) Real-time RT-PCR analysis detecting HoxC6 and HoxC8 mRNA in Lsh−/− MEFs after targeting by Chd1 siRNA compared to control treated cells. Results represent standard deviations for the mean of three independent experiments. Asterix indicates a p value p<0.05.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2820093&req=5

pone-0009163-g004: Chromatin marks associated with transcriptional elongation are increased in the absence of Lsh.(A) Schematic diagram depicting the position of the primer pairs (black lines) used for ChIP analysis. Comparison of the ratio of H3K36Me3 (B), H3K79Me2 (C) and H3K4Me3 (E) enrichment to total H3 occupancies (Fig. S6) between Lsh WT and Lsh−/−MEFs at up, TSS, down and 3′UTR regions of HoxC6 and HoxC8 genes. (D) ChIP analysis for the detection of Ser 2 RNA Pol II (H5). (F) ChIP analysis for detection of Chd1. (G) Western analysis for detection of Chd1 protein comparing whole cell extracts derived from Lsh−/−MEFs targeted by Chd1 siRNA or control siRNA (H) Real-time RT-PCR analysis detecting HoxC6 and HoxC8 mRNA in Lsh−/− MEFs after targeting by Chd1 siRNA compared to control treated cells. Results represent standard deviations for the mean of three independent experiments. Asterix indicates a p value p<0.05.
Mentions: To support further the notion of Pol II stalling versus elongation, histone modifications that are hallmarks of RNA Pol II elongation and enriched at the gene body were assessed [35], [39], [40]. Histone 3 lysine 36 trimethylation (H3K36me3) and H3K79 dimethylation (H3K79me2) are present at fairly equal levels in cellular extracts of Lsh−/− and WT cells (Fig. S5). Both H3K36me3 and H3K79me2 showed enrichment at the gene body of HoxC6 and HoxC8 genes in Lsh−/− cells compared to WT cells (Fig. 4A, B, C). Furthermore, Ser2 Pol II enrichment at the 3′ end, another hallmark of successful transcriptional elongation [36] was increased at HoxC6 and HoxC8 genes in Lsh−/− cells compared to WT samples (Fig. 4D). Taken together, the change in histone modifications and Ser2 Pol II further supported the observation of Pol II stalling at HoxC6 and HoxC8 genes in WT cells and successful Pol II elongation and transcription in Lsh−/− MEFs.

Bottom Line: Hypomethylation in Lsh-/- cells is associated with efficient transcriptional elongation and splicing, in part mediated by the chromodomain protein Chd1.Dynamic modulation of DNA methylation in Lsh-/- and wild type cells demonstrates that catalytically active DNA methyltransferase activity is required for Pol II stalling.Taken together, the data suggests that DNA methylation can be compatible with Pol II binding at selected genes and Pol II stalling can act as alternate mechanism to explain transcriptional silencing associated with DNA methylation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cancer Prevention, SAIC-Frederick, National Cancer Institute, Frederick, Maryland, United States of America.

ABSTRACT
DNA cytosine methylation is an important epigenetic mechanism that is involved in transcriptional silencing of developmental genes. Several molecular pathways have been described that interfere with Pol II initiation, but at individual genes the molecular mechanism of repression remains uncertain. Here, we study the molecular mechanism of transcriptional regulation at Hox genes in dependence of the epigenetic regulator Lsh that controls CpG methylation at selected Hox genes. Wild type cells show a nucleosomal deprived region around the transcriptional start site at methylated Hox genes and mediate gene silencing via Pol II stalling. Hypomethylation in Lsh-/- cells is associated with efficient transcriptional elongation and splicing, in part mediated by the chromodomain protein Chd1. Dynamic modulation of DNA methylation in Lsh-/- and wild type cells demonstrates that catalytically active DNA methyltransferase activity is required for Pol II stalling. Taken together, the data suggests that DNA methylation can be compatible with Pol II binding at selected genes and Pol II stalling can act as alternate mechanism to explain transcriptional silencing associated with DNA methylation.

Show MeSH
Related in: MedlinePlus