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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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Re-introduction of Lsh into Lsh−/− cells restores Pol II stalling and requires the presence of Dnmt3b.(A) Western analysis using anti-Lsh antibodies or anti-Flag antibodies to detect Lsh protein expression in Lsh−/− MEFs stably transfected with the tet-off repressor Lsh vector before and after 24 hrs of tetracycline treatment. Note, that traces of Lsh protein were visible after longer exposure of the blot indicating a slight leakiness of the repressor system. (B) RT-PCR analysis for detection of HoxC6 and HoxC8 and Lsh mRNA comparing extracts derived from Lsh−/−MEFs before and after 24 hours of tetracycline treatment. (C) Genomic DNA derived from Lsh−/− MEFs (before and after 24 hrs of tetracycline treatment) was examined by bisulphite sequencing at regions downstream of TSS at the HoxC6 (Left) and the HoxC8 (Right) genes. (D) Ratio of Ser 5 Pol II ChIP signals for the TSS region to the downstream region and the ratio of Ser 5 Pol II signal using a primer set located 5′ and 3′ of exon1 comparing chromatin derived from Lsh−/− MEFs before and after 24 hours of tetracycline treatment to induce Lsh. Error bars represented the standard errors of the mean of three independent experiments. Western Blot (E) and Real-time RT-PCR analysis (F) for detection of Dnmt3b mRNA from Lsh inducible cells treated with control siRNA (siControl) or siRNA targeted against Dnmt3b (siDnmt3b) for 48 hrs. (G) Real time RT-PCR analyses detecting the ratio of unspliced to spliced mRNA of the HoxC6 and HoxC8 gene in Lsh−/− MEFs after targeting by Dnmt3b siRNA or control siRNA before and after 24 hours of tetracycline treatment to induce Lsh. (H) Summary of bisulphite sequencing results derived from ten clones for each sample (Figure S10) to illustrate the percentage of CpG methylation loss at the first 10 (HoxC6, left) or 10 (HoxC8, right) CpG sites downstream of TSS in Lsh−/− MEFs after targeting by Dnmt3b siRNA or control siRNA before and after 24 hours of tetracycline treatment to induce Lsh. (I) Real time RT-PCR analyses detecting the ratio of unspliced to spliced mRNA of the HoxC6 and HoxC8 genes in Lsh−/− MEFs after targeting by Dnmt3b siRNA or control siRNA before and after 24 hours of tetracycline treatment to induce Lsh.
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pone-0009163-g007: Re-introduction of Lsh into Lsh−/− cells restores Pol II stalling and requires the presence of Dnmt3b.(A) Western analysis using anti-Lsh antibodies or anti-Flag antibodies to detect Lsh protein expression in Lsh−/− MEFs stably transfected with the tet-off repressor Lsh vector before and after 24 hrs of tetracycline treatment. Note, that traces of Lsh protein were visible after longer exposure of the blot indicating a slight leakiness of the repressor system. (B) RT-PCR analysis for detection of HoxC6 and HoxC8 and Lsh mRNA comparing extracts derived from Lsh−/−MEFs before and after 24 hours of tetracycline treatment. (C) Genomic DNA derived from Lsh−/− MEFs (before and after 24 hrs of tetracycline treatment) was examined by bisulphite sequencing at regions downstream of TSS at the HoxC6 (Left) and the HoxC8 (Right) genes. (D) Ratio of Ser 5 Pol II ChIP signals for the TSS region to the downstream region and the ratio of Ser 5 Pol II signal using a primer set located 5′ and 3′ of exon1 comparing chromatin derived from Lsh−/− MEFs before and after 24 hours of tetracycline treatment to induce Lsh. Error bars represented the standard errors of the mean of three independent experiments. Western Blot (E) and Real-time RT-PCR analysis (F) for detection of Dnmt3b mRNA from Lsh inducible cells treated with control siRNA (siControl) or siRNA targeted against Dnmt3b (siDnmt3b) for 48 hrs. (G) Real time RT-PCR analyses detecting the ratio of unspliced to spliced mRNA of the HoxC6 and HoxC8 gene in Lsh−/− MEFs after targeting by Dnmt3b siRNA or control siRNA before and after 24 hours of tetracycline treatment to induce Lsh. (H) Summary of bisulphite sequencing results derived from ten clones for each sample (Figure S10) to illustrate the percentage of CpG methylation loss at the first 10 (HoxC6, left) or 10 (HoxC8, right) CpG sites downstream of TSS in Lsh−/− MEFs after targeting by Dnmt3b siRNA or control siRNA before and after 24 hours of tetracycline treatment to induce Lsh. (I) Real time RT-PCR analyses detecting the ratio of unspliced to spliced mRNA of the HoxC6 and HoxC8 genes in Lsh−/− MEFs after targeting by Dnmt3b siRNA or control siRNA before and after 24 hours of tetracycline treatment to induce Lsh.

Mentions: To understand whether dynamic regulation of DNA methylation in MEFs can cause Pol II stalling in hypomethylated Lsh−/− cells, we attempted to restore Lsh function. Lsh was introduced into Lsh−/− MEFs using an inducible tetracycline dependent expression system. As shown in Fig. 7A, Lsh protein levels were enhanced within 24 hrs of tetracycline treatment. Also, HoxC6 and HoxC8 mRNA expression was reduced after Lsh induction, as demonstrated by RT-PCR analysis (Fig. 7B). This change in gene expression was accompanied by some gain in CpG methylation at the TSS regions of HoxC6 and HoxC8 genes (from 11 to 35% at HoxC6 and from 8 to 47% for HoxC8) and at the second exon next to the splicing signal (Fig. 7C and Fig. S8A,B). This result was consistent with our previous observations that Lsh can directly associate with Hox genes (Fig. S1) and that Lsh is required for targeting of Dnmt3b to HoxA genes [16]. Moreover, Lsh re-expression in Lsh−/− cells increased the Pol II stalling index (Fig. 7D), further supporting a close connection of Lsh mediated DNA methylation and Pol II stalling.


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

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

Re-introduction of Lsh into Lsh−/− cells restores Pol II stalling and requires the presence of Dnmt3b.(A) Western analysis using anti-Lsh antibodies or anti-Flag antibodies to detect Lsh protein expression in Lsh−/− MEFs stably transfected with the tet-off repressor Lsh vector before and after 24 hrs of tetracycline treatment. Note, that traces of Lsh protein were visible after longer exposure of the blot indicating a slight leakiness of the repressor system. (B) RT-PCR analysis for detection of HoxC6 and HoxC8 and Lsh mRNA comparing extracts derived from Lsh−/−MEFs before and after 24 hours of tetracycline treatment. (C) Genomic DNA derived from Lsh−/− MEFs (before and after 24 hrs of tetracycline treatment) was examined by bisulphite sequencing at regions downstream of TSS at the HoxC6 (Left) and the HoxC8 (Right) genes. (D) Ratio of Ser 5 Pol II ChIP signals for the TSS region to the downstream region and the ratio of Ser 5 Pol II signal using a primer set located 5′ and 3′ of exon1 comparing chromatin derived from Lsh−/− MEFs before and after 24 hours of tetracycline treatment to induce Lsh. Error bars represented the standard errors of the mean of three independent experiments. Western Blot (E) and Real-time RT-PCR analysis (F) for detection of Dnmt3b mRNA from Lsh inducible cells treated with control siRNA (siControl) or siRNA targeted against Dnmt3b (siDnmt3b) for 48 hrs. (G) Real time RT-PCR analyses detecting the ratio of unspliced to spliced mRNA of the HoxC6 and HoxC8 gene in Lsh−/− MEFs after targeting by Dnmt3b siRNA or control siRNA before and after 24 hours of tetracycline treatment to induce Lsh. (H) Summary of bisulphite sequencing results derived from ten clones for each sample (Figure S10) to illustrate the percentage of CpG methylation loss at the first 10 (HoxC6, left) or 10 (HoxC8, right) CpG sites downstream of TSS in Lsh−/− MEFs after targeting by Dnmt3b siRNA or control siRNA before and after 24 hours of tetracycline treatment to induce Lsh. (I) Real time RT-PCR analyses detecting the ratio of unspliced to spliced mRNA of the HoxC6 and HoxC8 genes in Lsh−/− MEFs after targeting by Dnmt3b siRNA or control siRNA before and after 24 hours of tetracycline treatment to induce Lsh.
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pone-0009163-g007: Re-introduction of Lsh into Lsh−/− cells restores Pol II stalling and requires the presence of Dnmt3b.(A) Western analysis using anti-Lsh antibodies or anti-Flag antibodies to detect Lsh protein expression in Lsh−/− MEFs stably transfected with the tet-off repressor Lsh vector before and after 24 hrs of tetracycline treatment. Note, that traces of Lsh protein were visible after longer exposure of the blot indicating a slight leakiness of the repressor system. (B) RT-PCR analysis for detection of HoxC6 and HoxC8 and Lsh mRNA comparing extracts derived from Lsh−/−MEFs before and after 24 hours of tetracycline treatment. (C) Genomic DNA derived from Lsh−/− MEFs (before and after 24 hrs of tetracycline treatment) was examined by bisulphite sequencing at regions downstream of TSS at the HoxC6 (Left) and the HoxC8 (Right) genes. (D) Ratio of Ser 5 Pol II ChIP signals for the TSS region to the downstream region and the ratio of Ser 5 Pol II signal using a primer set located 5′ and 3′ of exon1 comparing chromatin derived from Lsh−/− MEFs before and after 24 hours of tetracycline treatment to induce Lsh. Error bars represented the standard errors of the mean of three independent experiments. Western Blot (E) and Real-time RT-PCR analysis (F) for detection of Dnmt3b mRNA from Lsh inducible cells treated with control siRNA (siControl) or siRNA targeted against Dnmt3b (siDnmt3b) for 48 hrs. (G) Real time RT-PCR analyses detecting the ratio of unspliced to spliced mRNA of the HoxC6 and HoxC8 gene in Lsh−/− MEFs after targeting by Dnmt3b siRNA or control siRNA before and after 24 hours of tetracycline treatment to induce Lsh. (H) Summary of bisulphite sequencing results derived from ten clones for each sample (Figure S10) to illustrate the percentage of CpG methylation loss at the first 10 (HoxC6, left) or 10 (HoxC8, right) CpG sites downstream of TSS in Lsh−/− MEFs after targeting by Dnmt3b siRNA or control siRNA before and after 24 hours of tetracycline treatment to induce Lsh. (I) Real time RT-PCR analyses detecting the ratio of unspliced to spliced mRNA of the HoxC6 and HoxC8 genes in Lsh−/− MEFs after targeting by Dnmt3b siRNA or control siRNA before and after 24 hours of tetracycline treatment to induce Lsh.
Mentions: To understand whether dynamic regulation of DNA methylation in MEFs can cause Pol II stalling in hypomethylated Lsh−/− cells, we attempted to restore Lsh function. Lsh was introduced into Lsh−/− MEFs using an inducible tetracycline dependent expression system. As shown in Fig. 7A, Lsh protein levels were enhanced within 24 hrs of tetracycline treatment. Also, HoxC6 and HoxC8 mRNA expression was reduced after Lsh induction, as demonstrated by RT-PCR analysis (Fig. 7B). This change in gene expression was accompanied by some gain in CpG methylation at the TSS regions of HoxC6 and HoxC8 genes (from 11 to 35% at HoxC6 and from 8 to 47% for HoxC8) and at the second exon next to the splicing signal (Fig. 7C and Fig. S8A,B). This result was consistent with our previous observations that Lsh can directly associate with Hox genes (Fig. S1) and that Lsh is required for targeting of Dnmt3b to HoxA genes [16]. Moreover, Lsh re-expression in Lsh−/− cells increased the Pol II stalling index (Fig. 7D), further supporting a close connection of Lsh mediated DNA methylation and Pol II stalling.

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