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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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5-Azacytidine treatment overcomes Pol II stalling.(A) Summary of bisulphite sequencing results derived from ten clones (Figure S8) to illustrate the percentage of reduced CpG methylation after 5-Azacytidine treatment at the first 10 (HoxC6, Up) or 11 (HoxC8, Down) CpG sites downstream of TSS (region 1) or 10 (HoxC6) or 12 (HoxC8) CpG sites at the second exon (region 2) (B) Real-time PCR analysis for detection of HoxC6 and HoxC8 genes in Lsh+/+ MEFs before and after five days of 5-Azacytidine (Aza) treatment (1 µM). The mock treated samples were set to one for better comparison. (C) Ratio of Ser 5 Pol II ChIP signals of the TSS region over the downstream region and the ratio of 5′r and 3′regions of exon1 comparing chromatin derived from Lsh +/+ MEFs of mock and 5-Azacytidine treated cells. (D) ChIPs analysis for detection of Chd1 in the TSS region comparing chromatin derived from WT MEFs of mock and 5-Azacytidine treated cells. IgG serves as a negative control. (E) Real time RT-PCR analyses detecting the ratio of unspliced to spliced mRNA of the HoxC6 and HoxC8 gene from Lsh+/+ and Lsh−/− MEFs after the treatment of 5-Azacytidine.
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pone-0009163-g006: 5-Azacytidine treatment overcomes Pol II stalling.(A) Summary of bisulphite sequencing results derived from ten clones (Figure S8) to illustrate the percentage of reduced CpG methylation after 5-Azacytidine treatment at the first 10 (HoxC6, Up) or 11 (HoxC8, Down) CpG sites downstream of TSS (region 1) or 10 (HoxC6) or 12 (HoxC8) CpG sites at the second exon (region 2) (B) Real-time PCR analysis for detection of HoxC6 and HoxC8 genes in Lsh+/+ MEFs before and after five days of 5-Azacytidine (Aza) treatment (1 µM). The mock treated samples were set to one for better comparison. (C) Ratio of Ser 5 Pol II ChIP signals of the TSS region over the downstream region and the ratio of 5′r and 3′regions of exon1 comparing chromatin derived from Lsh +/+ MEFs of mock and 5-Azacytidine treated cells. (D) ChIPs analysis for detection of Chd1 in the TSS region comparing chromatin derived from WT MEFs of mock and 5-Azacytidine treated cells. IgG serves as a negative control. (E) Real time RT-PCR analyses detecting the ratio of unspliced to spliced mRNA of the HoxC6 and HoxC8 gene from Lsh+/+ and Lsh−/− MEFs after the treatment of 5-Azacytidine.

Mentions: To address the question, whether changes in DNA methylation by other means than Lsh depletion can affect Pol II stalling, we treated WT MEFs with 5-Azacytidine. Bisulphite sequencing indicated reduced CpG methylation at the TSS region (region1) and further downstream at the second exon comprising the splicing signal (region2) after 5-Azacytidine treatment (Fig. 6A and Fig. S7). Also, hypomethylated WT cells showed re-activation of repressed HoxC6 and HoxC8 transcripts (Fig. 6B). Moreover, the Pol II stalling index changed 3 to 4 fold at HoxC6 and HoxC8 genes after 5-Azacytidine treatment (Fig. 6C). In addition, ChIPs analysis revealed an increase of Chd1 binding at HoxC6 and the HoxC8 genes after 5-Azacytidine treatment, similar to enhanced Chd1 binding observed in Lsh−/−samples (Fig. 6D). Finally, the ratio of total RNA to spliced transcripts (a/c) and pre-mRNA to spliced transcripts (b/c) were reduced after treatment of WT cells with 5-Azacytidine suggesting an improved conversion of pre-mRNA into mature transcripts similar to that seen in Lsh−/− samples (Fig. 6E). Taken together, the data suggested that 5-Azacytidine treatment can overcame Pol II stalling in WT cells.


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

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

5-Azacytidine treatment overcomes Pol II stalling.(A) Summary of bisulphite sequencing results derived from ten clones (Figure S8) to illustrate the percentage of reduced CpG methylation after 5-Azacytidine treatment at the first 10 (HoxC6, Up) or 11 (HoxC8, Down) CpG sites downstream of TSS (region 1) or 10 (HoxC6) or 12 (HoxC8) CpG sites at the second exon (region 2) (B) Real-time PCR analysis for detection of HoxC6 and HoxC8 genes in Lsh+/+ MEFs before and after five days of 5-Azacytidine (Aza) treatment (1 µM). The mock treated samples were set to one for better comparison. (C) Ratio of Ser 5 Pol II ChIP signals of the TSS region over the downstream region and the ratio of 5′r and 3′regions of exon1 comparing chromatin derived from Lsh +/+ MEFs of mock and 5-Azacytidine treated cells. (D) ChIPs analysis for detection of Chd1 in the TSS region comparing chromatin derived from WT MEFs of mock and 5-Azacytidine treated cells. IgG serves as a negative control. (E) Real time RT-PCR analyses detecting the ratio of unspliced to spliced mRNA of the HoxC6 and HoxC8 gene from Lsh+/+ and Lsh−/− MEFs after the treatment of 5-Azacytidine.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2820093&req=5

pone-0009163-g006: 5-Azacytidine treatment overcomes Pol II stalling.(A) Summary of bisulphite sequencing results derived from ten clones (Figure S8) to illustrate the percentage of reduced CpG methylation after 5-Azacytidine treatment at the first 10 (HoxC6, Up) or 11 (HoxC8, Down) CpG sites downstream of TSS (region 1) or 10 (HoxC6) or 12 (HoxC8) CpG sites at the second exon (region 2) (B) Real-time PCR analysis for detection of HoxC6 and HoxC8 genes in Lsh+/+ MEFs before and after five days of 5-Azacytidine (Aza) treatment (1 µM). The mock treated samples were set to one for better comparison. (C) Ratio of Ser 5 Pol II ChIP signals of the TSS region over the downstream region and the ratio of 5′r and 3′regions of exon1 comparing chromatin derived from Lsh +/+ MEFs of mock and 5-Azacytidine treated cells. (D) ChIPs analysis for detection of Chd1 in the TSS region comparing chromatin derived from WT MEFs of mock and 5-Azacytidine treated cells. IgG serves as a negative control. (E) Real time RT-PCR analyses detecting the ratio of unspliced to spliced mRNA of the HoxC6 and HoxC8 gene from Lsh+/+ and Lsh−/− MEFs after the treatment of 5-Azacytidine.
Mentions: To address the question, whether changes in DNA methylation by other means than Lsh depletion can affect Pol II stalling, we treated WT MEFs with 5-Azacytidine. Bisulphite sequencing indicated reduced CpG methylation at the TSS region (region1) and further downstream at the second exon comprising the splicing signal (region2) after 5-Azacytidine treatment (Fig. 6A and Fig. S7). Also, hypomethylated WT cells showed re-activation of repressed HoxC6 and HoxC8 transcripts (Fig. 6B). Moreover, the Pol II stalling index changed 3 to 4 fold at HoxC6 and HoxC8 genes after 5-Azacytidine treatment (Fig. 6C). In addition, ChIPs analysis revealed an increase of Chd1 binding at HoxC6 and the HoxC8 genes after 5-Azacytidine treatment, similar to enhanced Chd1 binding observed in Lsh−/−samples (Fig. 6D). Finally, the ratio of total RNA to spliced transcripts (a/c) and pre-mRNA to spliced transcripts (b/c) were reduced after treatment of WT cells with 5-Azacytidine suggesting an improved conversion of pre-mRNA into mature transcripts similar to that seen in Lsh−/− samples (Fig. 6E). Taken together, the data suggested that 5-Azacytidine treatment can overcame Pol II stalling in WT cells.

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