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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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Stalling of RNA polymerase II at Hox genes.(A) Enrichment of unphosphorylated Pol II at HoxC6 (top) and HoxC8 (bottom) genes using ChIPs. Primers for upstream and downstream regions were positioned within 1000 bp of TSS (five primer sets for each group). TSS primers (two primer sets) covered the promoter-proximal region. The enrichment of unphosphorylated RNA Pol II (8WG16) binding in Lsh+/+ and Lsh−/− MEFs was determined and normalized for better comparison to the binding at TSS in Lsh+/+ (the value of the TSS region was set to 1). (B) Binding of Ser 5 Pol II (H14) using ChIPs at HoxC6 (top), and HoxC8 (bottom) at upstream, downstream regions and the TSS region as in (A). (C) Ratios of Ser 5 Pol II ChIP signals for the TSS region to the downstream region and the ratios of Ser 5 Pol II signal using a primer set located 5′ and 3′ of exon1. Error bars represent the standard deviation for the mean of three independent experiments. (D,E) Binding of Ser 5 Pol II (H14) at HoxC6 (D), and HoxC8 (E) at upstream, downstream regions and the TSS region in hindlimb (top) and liver tissues (bottom).
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pone-0009163-g003: Stalling of RNA polymerase II at Hox genes.(A) Enrichment of unphosphorylated Pol II at HoxC6 (top) and HoxC8 (bottom) genes using ChIPs. Primers for upstream and downstream regions were positioned within 1000 bp of TSS (five primer sets for each group). TSS primers (two primer sets) covered the promoter-proximal region. The enrichment of unphosphorylated RNA Pol II (8WG16) binding in Lsh+/+ and Lsh−/− MEFs was determined and normalized for better comparison to the binding at TSS in Lsh+/+ (the value of the TSS region was set to 1). (B) Binding of Ser 5 Pol II (H14) using ChIPs at HoxC6 (top), and HoxC8 (bottom) at upstream, downstream regions and the TSS region as in (A). (C) Ratios of Ser 5 Pol II ChIP signals for the TSS region to the downstream region and the ratios of Ser 5 Pol II signal using a primer set located 5′ and 3′ of exon1. Error bars represent the standard deviation for the mean of three independent experiments. (D,E) Binding of Ser 5 Pol II (H14) at HoxC6 (D), and HoxC8 (E) at upstream, downstream regions and the TSS region in hindlimb (top) and liver tissues (bottom).

Mentions: Nucleosomal deprived regions can be associated with active promoter regions and engaged RNA Pol II [33]. Thus, we tested the idea whether the MNase unprotected region around the TSS of Hox genes in WT or Lsh−/− samples could be linked to Pol II binding. First, we confirmed the position of the major TSS sites at either HoxC6 and HoxC8 genes by using 5′RACE and site specific RT-PCR analysis (Fig. S4). Then, ChIPs analysis was performed using specific antibodies raised against the heptapeptide repeat of the carboxyterminal domain (CTD) of Pol II. Chromatin samples derived from Lsh−/− MEFs showed enrichment of Pol II at the TSS of HoxC6 and HoxC8 genes (Fig. 3A, and for HoxA6 Fig. S3B). Likewise, WT samples showed a peak of unphosphorylated Pol II binding at TSS consistent with the hypothesis that Pol II binding may interfere with nucleosomal occupancy and may determine phasing of the +1 nucleosome [33]. These findings suggest that CpG island methylation at HoxC6 and HoxC8 genes does not inevitably hinder Pol II binding. Moreover, repression of HoxC6 and HoxC8 transcription was not simply caused by a lack of Pol II binding but by a molecular mechanism following Pol II engagement.


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

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

Stalling of RNA polymerase II at Hox genes.(A) Enrichment of unphosphorylated Pol II at HoxC6 (top) and HoxC8 (bottom) genes using ChIPs. Primers for upstream and downstream regions were positioned within 1000 bp of TSS (five primer sets for each group). TSS primers (two primer sets) covered the promoter-proximal region. The enrichment of unphosphorylated RNA Pol II (8WG16) binding in Lsh+/+ and Lsh−/− MEFs was determined and normalized for better comparison to the binding at TSS in Lsh+/+ (the value of the TSS region was set to 1). (B) Binding of Ser 5 Pol II (H14) using ChIPs at HoxC6 (top), and HoxC8 (bottom) at upstream, downstream regions and the TSS region as in (A). (C) Ratios of Ser 5 Pol II ChIP signals for the TSS region to the downstream region and the ratios of Ser 5 Pol II signal using a primer set located 5′ and 3′ of exon1. Error bars represent the standard deviation for the mean of three independent experiments. (D,E) Binding of Ser 5 Pol II (H14) at HoxC6 (D), and HoxC8 (E) at upstream, downstream regions and the TSS region in hindlimb (top) and liver tissues (bottom).
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Related In: Results  -  Collection

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pone-0009163-g003: Stalling of RNA polymerase II at Hox genes.(A) Enrichment of unphosphorylated Pol II at HoxC6 (top) and HoxC8 (bottom) genes using ChIPs. Primers for upstream and downstream regions were positioned within 1000 bp of TSS (five primer sets for each group). TSS primers (two primer sets) covered the promoter-proximal region. The enrichment of unphosphorylated RNA Pol II (8WG16) binding in Lsh+/+ and Lsh−/− MEFs was determined and normalized for better comparison to the binding at TSS in Lsh+/+ (the value of the TSS region was set to 1). (B) Binding of Ser 5 Pol II (H14) using ChIPs at HoxC6 (top), and HoxC8 (bottom) at upstream, downstream regions and the TSS region as in (A). (C) Ratios of Ser 5 Pol II ChIP signals for the TSS region to the downstream region and the ratios of Ser 5 Pol II signal using a primer set located 5′ and 3′ of exon1. Error bars represent the standard deviation for the mean of three independent experiments. (D,E) Binding of Ser 5 Pol II (H14) at HoxC6 (D), and HoxC8 (E) at upstream, downstream regions and the TSS region in hindlimb (top) and liver tissues (bottom).
Mentions: Nucleosomal deprived regions can be associated with active promoter regions and engaged RNA Pol II [33]. Thus, we tested the idea whether the MNase unprotected region around the TSS of Hox genes in WT or Lsh−/− samples could be linked to Pol II binding. First, we confirmed the position of the major TSS sites at either HoxC6 and HoxC8 genes by using 5′RACE and site specific RT-PCR analysis (Fig. S4). Then, ChIPs analysis was performed using specific antibodies raised against the heptapeptide repeat of the carboxyterminal domain (CTD) of Pol II. Chromatin samples derived from Lsh−/− MEFs showed enrichment of Pol II at the TSS of HoxC6 and HoxC8 genes (Fig. 3A, and for HoxA6 Fig. S3B). Likewise, WT samples showed a peak of unphosphorylated Pol II binding at TSS consistent with the hypothesis that Pol II binding may interfere with nucleosomal occupancy and may determine phasing of the +1 nucleosome [33]. These findings suggest that CpG island methylation at HoxC6 and HoxC8 genes does not inevitably hinder Pol II binding. Moreover, repression of HoxC6 and HoxC8 transcription was not simply caused by a lack of Pol II binding but by a molecular mechanism following Pol II engagement.

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