Limits...
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.

Show MeSH

Related in: MedlinePlus

Enhanced transcriptional elongation and generation of spliced mature transcripts after Lsh depletion.(A) Schematic representation of the probe and primer pairs used to detect 5′ primary transcripts, unspliced transcripts and spliced mRNA. (B) Real time RT-PCR analysis detecting the ratio of unspliced to spliced mRNA of the HoxC6 and HoxC8 gene from Lsh+/+ and Lsh−/− MEFs. Results represent the mean and standard deviations of three independent experiments. (C) RT-PCR analysis for detection of HoxC6 and HoxC8 transcripts derived from nuclei of Lsh+/+ and Lsh−/− MEFs. U2 small nuclear RNA (snRNA) was unaffected by Lsh and served as a control. (D) Real-time RT-PCR analysis from four independent experiments to detect the ratio of unspliced to spliced nascent transcripts of HoxC6 and HoxC8 genes by nuclear run on assay. (E) Real time RT-PCR analyses detecting the ratio of unspliced to spliced mRNA of the HoxC6 and HoxC8 genes in Lsh−/− MEFs after Chd1 siRNA treatment. Error bars represent the standard deviations for the mean of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2820093&req=5

pone-0009163-g005: Enhanced transcriptional elongation and generation of spliced mature transcripts after Lsh depletion.(A) Schematic representation of the probe and primer pairs used to detect 5′ primary transcripts, unspliced transcripts and spliced mRNA. (B) Real time RT-PCR analysis detecting the ratio of unspliced to spliced mRNA of the HoxC6 and HoxC8 gene from Lsh+/+ and Lsh−/− MEFs. Results represent the mean and standard deviations of three independent experiments. (C) RT-PCR analysis for detection of HoxC6 and HoxC8 transcripts derived from nuclei of Lsh+/+ and Lsh−/− MEFs. U2 small nuclear RNA (snRNA) was unaffected by Lsh and served as a control. (D) Real-time RT-PCR analysis from four independent experiments to detect the ratio of unspliced to spliced nascent transcripts of HoxC6 and HoxC8 genes by nuclear run on assay. (E) Real time RT-PCR analyses detecting the ratio of unspliced to spliced mRNA of the HoxC6 and HoxC8 genes in Lsh−/− MEFs after Chd1 siRNA treatment. Error bars represent the standard deviations for the mean of three independent experiments.

Mentions: To delineate further the role of Chd1 in transcription, we examined the maturation of pre-mRNA to spliced RNA since Chd1 associates with elongation factors as well as splicing components, and consequently, depletion of Chd1 impairs the splicing efficiency [41], [42]. Primers were designed to detect transcripts at HoxC6 and HoxC8 genes: for detection of ‘total’ transcripts (at the first exon,a), for pre-mRNA (at the exon/intron boundary,b), or for spliced RNA (c, spanning both exons)(Fig. 5A). The ratio of total transcripts to spliced RNA (a/c) and pre-mRNA to spliced RNA (b/c) was lower in Lsh−/− samples compared to WT samples suggesting that Lsh−/− cells were relatively more efficient in splicing than WT cells (Fig. 5B). Nuclear RNA samples derived from WT cells showed some evidence of transcripts close to the TSS (site a) and transcripts of pre-mRNA (site b) but barely detectable spliced transcripts (site c) in contrast to Lsh−/− samples (Fig. 5C). To rule out effects of RNA long-term stability and to quantify transcription rates, a nuclear run-on assay was performed using Br-UTP labeling for detection of nascent RNA [44]. Efficient immunoprecipitation was detected in cells that had been labeled with Br-UTP indicating the specificity of the assay (Fig. S6A,B). Using nascent transcripts, the ratio of pre-mRNA to spliced transcripts was less than one in Lsh−/− samples indicating efficient splicing (Fig. 5D). In contrast, WT samples showed a 3 to 8 fold higher ratio in relation to Lsh−/− when either total transcripts to mature mRNA (a/c) or pre-mRNA to spliced mRNA (b/c) were compared. Therefore, the data suggests that WT samples compared to Lsh−/− samples are impaired in converting HoxC6 and HoxC8 pre-mRNA templates into mature transcripts. To further support the notion that Chd1 is involved in splicing in Lsh−/− cells, we examined Chd1 depleted Lsh−/− cells (as shown in Fig. 4G). Upon Chd1 depletion, the ratio of pre-mRNA to spliced transcripts reverted back to ratios similar to those in WT samples pointing to a functional role for Chd1 in splicing in (Fig. 5E). Taken together, the data suggests an inadequacy in transcriptional elongation and generation of spliced mature RNA in WT cells, and successful elongation and splicing of pre-mRNA in Lsh−/− cells that involves Chd1.


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

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

Enhanced transcriptional elongation and generation of spliced mature transcripts after Lsh depletion.(A) Schematic representation of the probe and primer pairs used to detect 5′ primary transcripts, unspliced transcripts and spliced mRNA. (B) Real time RT-PCR analysis detecting the ratio of unspliced to spliced mRNA of the HoxC6 and HoxC8 gene from Lsh+/+ and Lsh−/− MEFs. Results represent the mean and standard deviations of three independent experiments. (C) RT-PCR analysis for detection of HoxC6 and HoxC8 transcripts derived from nuclei of Lsh+/+ and Lsh−/− MEFs. U2 small nuclear RNA (snRNA) was unaffected by Lsh and served as a control. (D) Real-time RT-PCR analysis from four independent experiments to detect the ratio of unspliced to spliced nascent transcripts of HoxC6 and HoxC8 genes by nuclear run on assay. (E) Real time RT-PCR analyses detecting the ratio of unspliced to spliced mRNA of the HoxC6 and HoxC8 genes in Lsh−/− MEFs after Chd1 siRNA treatment. Error bars represent the standard deviations for the mean of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0009163-g005: Enhanced transcriptional elongation and generation of spliced mature transcripts after Lsh depletion.(A) Schematic representation of the probe and primer pairs used to detect 5′ primary transcripts, unspliced transcripts and spliced mRNA. (B) Real time RT-PCR analysis detecting the ratio of unspliced to spliced mRNA of the HoxC6 and HoxC8 gene from Lsh+/+ and Lsh−/− MEFs. Results represent the mean and standard deviations of three independent experiments. (C) RT-PCR analysis for detection of HoxC6 and HoxC8 transcripts derived from nuclei of Lsh+/+ and Lsh−/− MEFs. U2 small nuclear RNA (snRNA) was unaffected by Lsh and served as a control. (D) Real-time RT-PCR analysis from four independent experiments to detect the ratio of unspliced to spliced nascent transcripts of HoxC6 and HoxC8 genes by nuclear run on assay. (E) Real time RT-PCR analyses detecting the ratio of unspliced to spliced mRNA of the HoxC6 and HoxC8 genes in Lsh−/− MEFs after Chd1 siRNA treatment. Error bars represent the standard deviations for the mean of three independent experiments.
Mentions: To delineate further the role of Chd1 in transcription, we examined the maturation of pre-mRNA to spliced RNA since Chd1 associates with elongation factors as well as splicing components, and consequently, depletion of Chd1 impairs the splicing efficiency [41], [42]. Primers were designed to detect transcripts at HoxC6 and HoxC8 genes: for detection of ‘total’ transcripts (at the first exon,a), for pre-mRNA (at the exon/intron boundary,b), or for spliced RNA (c, spanning both exons)(Fig. 5A). The ratio of total transcripts to spliced RNA (a/c) and pre-mRNA to spliced RNA (b/c) was lower in Lsh−/− samples compared to WT samples suggesting that Lsh−/− cells were relatively more efficient in splicing than WT cells (Fig. 5B). Nuclear RNA samples derived from WT cells showed some evidence of transcripts close to the TSS (site a) and transcripts of pre-mRNA (site b) but barely detectable spliced transcripts (site c) in contrast to Lsh−/− samples (Fig. 5C). To rule out effects of RNA long-term stability and to quantify transcription rates, a nuclear run-on assay was performed using Br-UTP labeling for detection of nascent RNA [44]. Efficient immunoprecipitation was detected in cells that had been labeled with Br-UTP indicating the specificity of the assay (Fig. S6A,B). Using nascent transcripts, the ratio of pre-mRNA to spliced transcripts was less than one in Lsh−/− samples indicating efficient splicing (Fig. 5D). In contrast, WT samples showed a 3 to 8 fold higher ratio in relation to Lsh−/− when either total transcripts to mature mRNA (a/c) or pre-mRNA to spliced mRNA (b/c) were compared. Therefore, the data suggests that WT samples compared to Lsh−/− samples are impaired in converting HoxC6 and HoxC8 pre-mRNA templates into mature transcripts. To further support the notion that Chd1 is involved in splicing in Lsh−/− cells, we examined Chd1 depleted Lsh−/− cells (as shown in Fig. 4G). Upon Chd1 depletion, the ratio of pre-mRNA to spliced transcripts reverted back to ratios similar to those in WT samples pointing to a functional role for Chd1 in splicing in (Fig. 5E). Taken together, the data suggests an inadequacy in transcriptional elongation and generation of spliced mature RNA in WT cells, and successful elongation and splicing of pre-mRNA in Lsh−/− cells that involves Chd1.

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