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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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The catalytic domain of Dnmt3b is required for Pol II stalling.(A) RT-PCR analysis for the detection of HoxC6 and HoxC8 mRNA from Lsh+/+ and Lsh−/− MEFs transfected with wild type Dnmt3b (Wt 3b) and Mutant Dnmt3b (Mut 3b) for 72 hrs. (B) Bisulphite sequencing results to illustrate reduced CpG methylation at the first 10 (HoxC6, Left) or (HoxC8, Right) in samples prepared from WT MEF cells transfected with Mut 3b for 72 hrs. (C) Ratio of Ser 5 Pol II ChIP signals for the TSS region over the downstream region and the ratio of Ser 5 Pol II signal using a primer set located 5′ and 3′ of exon1 comparing chromatin from WT MEFs after the transfection with Mut 3b. (D) Real time RT-PCR analysis detecting the ratio of unspliced to spliced mRNA of the HoxC6 (left) and HoxC8 (right) gene in WT MEFs after the transfection with Mut 3b.
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pone-0009163-g008: The catalytic domain of Dnmt3b is required for Pol II stalling.(A) RT-PCR analysis for the detection of HoxC6 and HoxC8 mRNA from Lsh+/+ and Lsh−/− MEFs transfected with wild type Dnmt3b (Wt 3b) and Mutant Dnmt3b (Mut 3b) for 72 hrs. (B) Bisulphite sequencing results to illustrate reduced CpG methylation at the first 10 (HoxC6, Left) or (HoxC8, Right) in samples prepared from WT MEF cells transfected with Mut 3b for 72 hrs. (C) Ratio of Ser 5 Pol II ChIP signals for the TSS region over the downstream region and the ratio of Ser 5 Pol II signal using a primer set located 5′ and 3′ of exon1 comparing chromatin from WT MEFs after the transfection with Mut 3b. (D) Real time RT-PCR analysis detecting the ratio of unspliced to spliced mRNA of the HoxC6 (left) and HoxC8 (right) gene in WT MEFs after the transfection with Mut 3b.

Mentions: Finally, we examined the role of catalytically active Dnmt3b in HoxC6 and HoxC8 gene silencing. We transfected WT MEFs with a mutant form of Dnmt3b (a kind gift of Dr Hsieh) that had been previously shown to be catalytically inactive [45]. RT-PCR analysis showed an increase in HoxC6 and HoxC8 mRNA in WT cells that had been transfected with mutant Dnmt3b, in contrast to control WT cells (mock transfected or transfected with wild type Dnmt3b) (Fig. 8A). Bisulphite sequencing indicated effective reduction of CpG methylation at HoxC6 and HoxC8 TSS regions after introduction of mutant Dnmt3b (Fig. 8B). Calculation of the Pol II stalling index showed that transfection of mutant Dnmt3b reduced Pol II stalling in WT cells at HoxC6 and HoxC8 genes (Fig. 8C). Furthermore, the ratio of pre-mRNA to spliced transcripts shifted towards a profile comparable to that generated by Lsh−/− deletion (Fig. 8D). Taken together, the data suggests that catalytically active Dnmt3b is functionally involved in Pol II stalling at HoxC6 and HoxC8 genes.


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

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

The catalytic domain of Dnmt3b is required for Pol II stalling.(A) RT-PCR analysis for the detection of HoxC6 and HoxC8 mRNA from Lsh+/+ and Lsh−/− MEFs transfected with wild type Dnmt3b (Wt 3b) and Mutant Dnmt3b (Mut 3b) for 72 hrs. (B) Bisulphite sequencing results to illustrate reduced CpG methylation at the first 10 (HoxC6, Left) or (HoxC8, Right) in samples prepared from WT MEF cells transfected with Mut 3b for 72 hrs. (C) Ratio of Ser 5 Pol II ChIP signals for the TSS region over the downstream region and the ratio of Ser 5 Pol II signal using a primer set located 5′ and 3′ of exon1 comparing chromatin from WT MEFs after the transfection with Mut 3b. (D) Real time RT-PCR analysis detecting the ratio of unspliced to spliced mRNA of the HoxC6 (left) and HoxC8 (right) gene in WT MEFs after the transfection with Mut 3b.
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Related In: Results  -  Collection

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

pone-0009163-g008: The catalytic domain of Dnmt3b is required for Pol II stalling.(A) RT-PCR analysis for the detection of HoxC6 and HoxC8 mRNA from Lsh+/+ and Lsh−/− MEFs transfected with wild type Dnmt3b (Wt 3b) and Mutant Dnmt3b (Mut 3b) for 72 hrs. (B) Bisulphite sequencing results to illustrate reduced CpG methylation at the first 10 (HoxC6, Left) or (HoxC8, Right) in samples prepared from WT MEF cells transfected with Mut 3b for 72 hrs. (C) Ratio of Ser 5 Pol II ChIP signals for the TSS region over the downstream region and the ratio of Ser 5 Pol II signal using a primer set located 5′ and 3′ of exon1 comparing chromatin from WT MEFs after the transfection with Mut 3b. (D) Real time RT-PCR analysis detecting the ratio of unspliced to spliced mRNA of the HoxC6 (left) and HoxC8 (right) gene in WT MEFs after the transfection with Mut 3b.
Mentions: Finally, we examined the role of catalytically active Dnmt3b in HoxC6 and HoxC8 gene silencing. We transfected WT MEFs with a mutant form of Dnmt3b (a kind gift of Dr Hsieh) that had been previously shown to be catalytically inactive [45]. RT-PCR analysis showed an increase in HoxC6 and HoxC8 mRNA in WT cells that had been transfected with mutant Dnmt3b, in contrast to control WT cells (mock transfected or transfected with wild type Dnmt3b) (Fig. 8A). Bisulphite sequencing indicated effective reduction of CpG methylation at HoxC6 and HoxC8 TSS regions after introduction of mutant Dnmt3b (Fig. 8B). Calculation of the Pol II stalling index showed that transfection of mutant Dnmt3b reduced Pol II stalling in WT cells at HoxC6 and HoxC8 genes (Fig. 8C). Furthermore, the ratio of pre-mRNA to spliced transcripts shifted towards a profile comparable to that generated by Lsh−/− deletion (Fig. 8D). Taken together, the data suggests that catalytically active Dnmt3b is functionally involved in Pol II stalling at HoxC6 and HoxC8 genes.

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