Limits...
Dicer is associated with ribosomal DNA chromatin in mammalian cells.

Sinkkonen L, Hugenschmidt T, Filipowicz W, Svoboda P - PLoS ONE (2010)

Bottom Line: We analyzed in detail chromatin structure of rDNA but did not find any epigenetic changes at rDNA loci in Dicer(-/-) ES cells.We found that Dicer, a key component of RNA silencing pathways, can be detected in association with rDNA chromatin in mammalian cells.The role of this particular localization of Dicer is not readily apparent since the enzyme is associated with rDNA genes regardless of their transcriptional activity.

View Article: PubMed Central - PubMed

Affiliation: Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

ABSTRACT

Background: RNA silencing is a common term for pathways utilizing small RNAs as sequence-specific guides to repress gene expression. Components of the RNA silencing machinery are involved in different aspects of chromatin function in numerous organisms. However, association of RNA silencing with chromatin in mammalian cells remains unclear.

Methodology/principal findings: Immunostaining of mitotic chromosomes with antibodies visualizing either endogenous or ectopically expressed Dicer in mammalian cells revealed association of the protein with ribosomal DNA (rDNA) repeats. Chromatin immunoprecipitations and bisulfite sequencing experiments indicated that Dicer is associated with transcribed regions of both active and silenced genes in rDNA arrays of interphase chromosomes. Metabolic labeling of the mouse embryonic stem (ES) cells lacking Dicer did not reveal apparent defect in rRNA biogenesis though pre-rRNA synthesis in these cells was decreased, likely as a consequence of their slower growth caused by the loss of miRNAs. We analyzed in detail chromatin structure of rDNA but did not find any epigenetic changes at rDNA loci in Dicer(-/-) ES cells. Instead, we found that rDNA methylation is rather low in primary tissues, contrasting with rDNA methylation patterns in transformed cell lines.

Conclusion/significance: We found that Dicer, a key component of RNA silencing pathways, can be detected in association with rDNA chromatin in mammalian cells. The role of this particular localization of Dicer is not readily apparent since the enzyme is associated with rDNA genes regardless of their transcriptional activity. However, localization of Dicer to the transcribed region suggests that transcription may contribute to the Dicer deposition at rDNA chromatin. We hypothesize that Dicer functions in maintaining integrity of rDNA arrays.

Show MeSH

Related in: MedlinePlus

Methylation status of rDNA in mouse ES cells and selected tissues.(A) Schematic representation of mouse rRNA promoter showing position of bisulfite-sequenced region. Black arrows represent enhancer repeats, black rectangles indicate position of the UCE and core promoter elements. (B) Methylation status of rRNA promoter in mouse Dicer+/− and Dicer−/− ES cells, oocytes, blastocysts, liver, testis, and brain. Black dots represent methylated CpG nucleotides. Each row of dots represents one bisulfite-sequenced clone. Oocyte data were pooled from 2 independent amplifications. The redundancy of the bisulfite-sequenced clones is very low as evident from low, if any, similarity of the methylation pattern between clones containing at least one methylated cytosine.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2921364&req=5

pone-0012175-g008: Methylation status of rDNA in mouse ES cells and selected tissues.(A) Schematic representation of mouse rRNA promoter showing position of bisulfite-sequenced region. Black arrows represent enhancer repeats, black rectangles indicate position of the UCE and core promoter elements. (B) Methylation status of rRNA promoter in mouse Dicer+/− and Dicer−/− ES cells, oocytes, blastocysts, liver, testis, and brain. Black dots represent methylated CpG nucleotides. Each row of dots represents one bisulfite-sequenced clone. Oocyte data were pooled from 2 independent amplifications. The redundancy of the bisulfite-sequenced clones is very low as evident from low, if any, similarity of the methylation pattern between clones containing at least one methylated cytosine.

Mentions: Finally, analysis of rDNA from Dicer+/− and Dicer−/− ES cells by bisulfite sequencing revealed that it was largely hypomethylated, irrespective of the Dicer expression status. This contrasts with the situation in HEK293 cells in which approximately half of rDNA genes were methylated at the promoter (Fig. 5); depletion of Dicer in HEK293 cells by RNAi [33] had no effect on the DNA methylation status of rDNA (Fig. S2). Interestingly, the rRNA promoter hypomethylation was not specific to ES cells, as it was also found in primary tissues, including blastocysts (from which ES cells are derived), oocytes, and testis, brain and liver (Fig. 8). This rather unexpected finding is discussed in more detail in the Discussion. Taken together, analysis of epigenetic marks and pulse-chase analyses make it improbable that Dicer plays a role in transcriptional regulation or processing of rRNA.


Dicer is associated with ribosomal DNA chromatin in mammalian cells.

Sinkkonen L, Hugenschmidt T, Filipowicz W, Svoboda P - PLoS ONE (2010)

Methylation status of rDNA in mouse ES cells and selected tissues.(A) Schematic representation of mouse rRNA promoter showing position of bisulfite-sequenced region. Black arrows represent enhancer repeats, black rectangles indicate position of the UCE and core promoter elements. (B) Methylation status of rRNA promoter in mouse Dicer+/− and Dicer−/− ES cells, oocytes, blastocysts, liver, testis, and brain. Black dots represent methylated CpG nucleotides. Each row of dots represents one bisulfite-sequenced clone. Oocyte data were pooled from 2 independent amplifications. The redundancy of the bisulfite-sequenced clones is very low as evident from low, if any, similarity of the methylation pattern between clones containing at least one methylated cytosine.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2921364&req=5

pone-0012175-g008: Methylation status of rDNA in mouse ES cells and selected tissues.(A) Schematic representation of mouse rRNA promoter showing position of bisulfite-sequenced region. Black arrows represent enhancer repeats, black rectangles indicate position of the UCE and core promoter elements. (B) Methylation status of rRNA promoter in mouse Dicer+/− and Dicer−/− ES cells, oocytes, blastocysts, liver, testis, and brain. Black dots represent methylated CpG nucleotides. Each row of dots represents one bisulfite-sequenced clone. Oocyte data were pooled from 2 independent amplifications. The redundancy of the bisulfite-sequenced clones is very low as evident from low, if any, similarity of the methylation pattern between clones containing at least one methylated cytosine.
Mentions: Finally, analysis of rDNA from Dicer+/− and Dicer−/− ES cells by bisulfite sequencing revealed that it was largely hypomethylated, irrespective of the Dicer expression status. This contrasts with the situation in HEK293 cells in which approximately half of rDNA genes were methylated at the promoter (Fig. 5); depletion of Dicer in HEK293 cells by RNAi [33] had no effect on the DNA methylation status of rDNA (Fig. S2). Interestingly, the rRNA promoter hypomethylation was not specific to ES cells, as it was also found in primary tissues, including blastocysts (from which ES cells are derived), oocytes, and testis, brain and liver (Fig. 8). This rather unexpected finding is discussed in more detail in the Discussion. Taken together, analysis of epigenetic marks and pulse-chase analyses make it improbable that Dicer plays a role in transcriptional regulation or processing of rRNA.

Bottom Line: We analyzed in detail chromatin structure of rDNA but did not find any epigenetic changes at rDNA loci in Dicer(-/-) ES cells.We found that Dicer, a key component of RNA silencing pathways, can be detected in association with rDNA chromatin in mammalian cells.The role of this particular localization of Dicer is not readily apparent since the enzyme is associated with rDNA genes regardless of their transcriptional activity.

View Article: PubMed Central - PubMed

Affiliation: Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

ABSTRACT

Background: RNA silencing is a common term for pathways utilizing small RNAs as sequence-specific guides to repress gene expression. Components of the RNA silencing machinery are involved in different aspects of chromatin function in numerous organisms. However, association of RNA silencing with chromatin in mammalian cells remains unclear.

Methodology/principal findings: Immunostaining of mitotic chromosomes with antibodies visualizing either endogenous or ectopically expressed Dicer in mammalian cells revealed association of the protein with ribosomal DNA (rDNA) repeats. Chromatin immunoprecipitations and bisulfite sequencing experiments indicated that Dicer is associated with transcribed regions of both active and silenced genes in rDNA arrays of interphase chromosomes. Metabolic labeling of the mouse embryonic stem (ES) cells lacking Dicer did not reveal apparent defect in rRNA biogenesis though pre-rRNA synthesis in these cells was decreased, likely as a consequence of their slower growth caused by the loss of miRNAs. We analyzed in detail chromatin structure of rDNA but did not find any epigenetic changes at rDNA loci in Dicer(-/-) ES cells. Instead, we found that rDNA methylation is rather low in primary tissues, contrasting with rDNA methylation patterns in transformed cell lines.

Conclusion/significance: We found that Dicer, a key component of RNA silencing pathways, can be detected in association with rDNA chromatin in mammalian cells. The role of this particular localization of Dicer is not readily apparent since the enzyme is associated with rDNA genes regardless of their transcriptional activity. However, localization of Dicer to the transcribed region suggests that transcription may contribute to the Dicer deposition at rDNA chromatin. We hypothesize that Dicer functions in maintaining integrity of rDNA arrays.

Show MeSH
Related in: MedlinePlus