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 the Dicer associated rDNA as revealed by bisulfite sequencing.The sequenced region, spanning positions –186 to +20 of the rDNA repeat [38], is divided into three domains: core promoter (core p), upstream control region (UCE) and sequences upstream of UCE. Black dots represent methylated CpG nucleotides. Each row of dots represents one bisulfite-sequenced clone. The clones originate from two independent experiments. HEK293 (input), bisulfite sequencing of the rRNA promoter region of total HEK293 DNA. Dicer (D349), DNA immunoprecipitated with anti-Dicer antibody D349. H3K9me2, DNA associated with histone H3 dimethylated on lysine 9 (marker for inactive genes). H4ac, DNA associated with acetylated histone H4 (marker for active genes).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2921364&req=5

pone-0012175-g004: Methylation status of the Dicer associated rDNA as revealed by bisulfite sequencing.The sequenced region, spanning positions –186 to +20 of the rDNA repeat [38], is divided into three domains: core promoter (core p), upstream control region (UCE) and sequences upstream of UCE. Black dots represent methylated CpG nucleotides. Each row of dots represents one bisulfite-sequenced clone. The clones originate from two independent experiments. HEK293 (input), bisulfite sequencing of the rRNA promoter region of total HEK293 DNA. Dicer (D349), DNA immunoprecipitated with anti-Dicer antibody D349. H3K9me2, DNA associated with histone H3 dimethylated on lysine 9 (marker for inactive genes). H4ac, DNA associated with acetylated histone H4 (marker for active genes).

Mentions: To find out whether Dicer is preferentially associated with active or inactive rRNA genes, we used bisulfite sequencing to analyze promoter methylation in rDNA co-precipitated with D349 antibody from HEK293 cells. For comparison, we bisulfite-sequenced rDNA immunoprecipitated with antibodies against pan-acetylated histone H4 (H4ac, a marker for active genes) and dimethylated histone H3K9 (H3K9me2, a marker for inactive genes). As expected, the anti-H4ac antibody immunoprecipitated hypomethylated rDNA while the anti-H3K9me2 antibody immunoprecipitated hypermethylated rDNA. Interestingly, D349 antibody enriched both hypo- and hypermethylated rDNA, suggesting that Dicer association with chromatin does not discriminate between active (hypomethylated) and silenced (hypermethylated) rRNA genes (Fig. 4). Although we can not rule out that hypomethylated DNA associated with Dicer represents silenced genes, this possibility is unlikely since no similar fraction of hypomethylated DNA was found in DNA associated with H3K9me2.


Dicer is associated with ribosomal DNA chromatin in mammalian cells.

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

Methylation status of the Dicer associated rDNA as revealed by bisulfite sequencing.The sequenced region, spanning positions –186 to +20 of the rDNA repeat [38], is divided into three domains: core promoter (core p), upstream control region (UCE) and sequences upstream of UCE. Black dots represent methylated CpG nucleotides. Each row of dots represents one bisulfite-sequenced clone. The clones originate from two independent experiments. HEK293 (input), bisulfite sequencing of the rRNA promoter region of total HEK293 DNA. Dicer (D349), DNA immunoprecipitated with anti-Dicer antibody D349. H3K9me2, DNA associated with histone H3 dimethylated on lysine 9 (marker for inactive genes). H4ac, DNA associated with acetylated histone H4 (marker for active genes).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0012175-g004: Methylation status of the Dicer associated rDNA as revealed by bisulfite sequencing.The sequenced region, spanning positions –186 to +20 of the rDNA repeat [38], is divided into three domains: core promoter (core p), upstream control region (UCE) and sequences upstream of UCE. Black dots represent methylated CpG nucleotides. Each row of dots represents one bisulfite-sequenced clone. The clones originate from two independent experiments. HEK293 (input), bisulfite sequencing of the rRNA promoter region of total HEK293 DNA. Dicer (D349), DNA immunoprecipitated with anti-Dicer antibody D349. H3K9me2, DNA associated with histone H3 dimethylated on lysine 9 (marker for inactive genes). H4ac, DNA associated with acetylated histone H4 (marker for active genes).
Mentions: To find out whether Dicer is preferentially associated with active or inactive rRNA genes, we used bisulfite sequencing to analyze promoter methylation in rDNA co-precipitated with D349 antibody from HEK293 cells. For comparison, we bisulfite-sequenced rDNA immunoprecipitated with antibodies against pan-acetylated histone H4 (H4ac, a marker for active genes) and dimethylated histone H3K9 (H3K9me2, a marker for inactive genes). As expected, the anti-H4ac antibody immunoprecipitated hypomethylated rDNA while the anti-H3K9me2 antibody immunoprecipitated hypermethylated rDNA. Interestingly, D349 antibody enriched both hypo- and hypermethylated rDNA, suggesting that Dicer association with chromatin does not discriminate between active (hypomethylated) and silenced (hypermethylated) rRNA genes (Fig. 4). Although we can not rule out that hypomethylated DNA associated with Dicer represents silenced genes, this possibility is unlikely since no similar fraction of hypomethylated DNA was found in DNA associated with H3K9me2.

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