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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.

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MNase protected sites are similar at methylated and hypomethylated Hox genes.(A) Diagram depicting the procedure for the MNase assay and the PCR amplicons used for the detection of Hox genes. The chromatin derived from Lsh+/+ and Lsh−/− MEFs was treated with MNase. The DNA was then gel-purified and fragments less than 200 bp were used for real-time PCR. The amplicons were about 100±5 bp in size and were spaced 60±5 bp apart. The MNase protected profile of HoxC6(B) and HoxC8(C) genes was determined by normalizing the amount of the MNase derived PCR products to those of untreated DNA. Error bars represent the mean standard deviation of three to four independent experiments. (D) MeDIP analysis to enrich for methylated DNA at HoxC6 (left) and HoxC8 (right) genes of Lsh+/+ and Lsh−/−samples after MNase treatment.
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pone-0009163-g002: MNase protected sites are similar at methylated and hypomethylated Hox genes.(A) Diagram depicting the procedure for the MNase assay and the PCR amplicons used for the detection of Hox genes. The chromatin derived from Lsh+/+ and Lsh−/− MEFs was treated with MNase. The DNA was then gel-purified and fragments less than 200 bp were used for real-time PCR. The amplicons were about 100±5 bp in size and were spaced 60±5 bp apart. The MNase protected profile of HoxC6(B) and HoxC8(C) genes was determined by normalizing the amount of the MNase derived PCR products to those of untreated DNA. Error bars represent the mean standard deviation of three to four independent experiments. (D) MeDIP analysis to enrich for methylated DNA at HoxC6 (left) and HoxC8 (right) genes of Lsh+/+ and Lsh−/−samples after MNase treatment.

Mentions: Phased nucleosomal positioning at TSS or enhancer elements is associated with active transcription, and regular nucleosomal pattern have been found at Hox clusters [31], [32], [33], [34]. Furthermore, Lsh is a homologue of SNF2 factors and may affect nucleosomal positioning at HoxC genes; although Lsh has not been demonstrated, as yet, to posses nucleosomal remodeling activity. To examine nucleosomal positioning at Hox genes, we performed a MNase protection assay [33] comparing mononucleosomes prepared from wild type or Lsh−/− samples by tiling PCR analysis (Fig. 2A). As shown in Fig. 2B,C, a similar pattern of MNase sensitivity around the promoter regions of HoxC6 and HoxC8 was observed comparing WT and Lsh−/− samples (for HoxA6 see Fig. S3A). The pattern suggested a fixed +1 nucleosome with a less pronounced −1 nucleosome and revealed a MNase unprotected region around the TSS. In further support, mononucleosomal DNA was subjected to MeDIP analysis to enrich for methylated DNA around the TSS of WT cells. Wild types samples revealed a MNase unprotected region around the TSS (Fig. 2D). Taken together, the results suggested that DNA hyper- or hypomethylation in WT or Lsh−/− cells had no detectable effect on nucleosomal positioning at those genes and wild type samples revealed a MNase unprotected region around the TSS similar to that in Lsh−/− samples.


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

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

MNase protected sites are similar at methylated and hypomethylated Hox genes.(A) Diagram depicting the procedure for the MNase assay and the PCR amplicons used for the detection of Hox genes. The chromatin derived from Lsh+/+ and Lsh−/− MEFs was treated with MNase. The DNA was then gel-purified and fragments less than 200 bp were used for real-time PCR. The amplicons were about 100±5 bp in size and were spaced 60±5 bp apart. The MNase protected profile of HoxC6(B) and HoxC8(C) genes was determined by normalizing the amount of the MNase derived PCR products to those of untreated DNA. Error bars represent the mean standard deviation of three to four independent experiments. (D) MeDIP analysis to enrich for methylated DNA at HoxC6 (left) and HoxC8 (right) genes of Lsh+/+ and Lsh−/−samples after MNase treatment.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2820093&req=5

pone-0009163-g002: MNase protected sites are similar at methylated and hypomethylated Hox genes.(A) Diagram depicting the procedure for the MNase assay and the PCR amplicons used for the detection of Hox genes. The chromatin derived from Lsh+/+ and Lsh−/− MEFs was treated with MNase. The DNA was then gel-purified and fragments less than 200 bp were used for real-time PCR. The amplicons were about 100±5 bp in size and were spaced 60±5 bp apart. The MNase protected profile of HoxC6(B) and HoxC8(C) genes was determined by normalizing the amount of the MNase derived PCR products to those of untreated DNA. Error bars represent the mean standard deviation of three to four independent experiments. (D) MeDIP analysis to enrich for methylated DNA at HoxC6 (left) and HoxC8 (right) genes of Lsh+/+ and Lsh−/−samples after MNase treatment.
Mentions: Phased nucleosomal positioning at TSS or enhancer elements is associated with active transcription, and regular nucleosomal pattern have been found at Hox clusters [31], [32], [33], [34]. Furthermore, Lsh is a homologue of SNF2 factors and may affect nucleosomal positioning at HoxC genes; although Lsh has not been demonstrated, as yet, to posses nucleosomal remodeling activity. To examine nucleosomal positioning at Hox genes, we performed a MNase protection assay [33] comparing mononucleosomes prepared from wild type or Lsh−/− samples by tiling PCR analysis (Fig. 2A). As shown in Fig. 2B,C, a similar pattern of MNase sensitivity around the promoter regions of HoxC6 and HoxC8 was observed comparing WT and Lsh−/− samples (for HoxA6 see Fig. S3A). The pattern suggested a fixed +1 nucleosome with a less pronounced −1 nucleosome and revealed a MNase unprotected region around the TSS. In further support, mononucleosomal DNA was subjected to MeDIP analysis to enrich for methylated DNA around the TSS of WT cells. Wild types samples revealed a MNase unprotected region around the TSS (Fig. 2D). Taken together, the results suggested that DNA hyper- or hypomethylation in WT or Lsh−/− cells had no detectable effect on nucleosomal positioning at those genes and wild type samples revealed a MNase unprotected region around the TSS similar to that in Lsh−/− samples.

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