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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.

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Localization of Dicer on mammalian mitotic chromosomes.Chromosome spreads from human or mouse cells were stained with anti-Dicer and other indicated antibodies. (A) Staining of HEK293 chromosomes with anti-CENP-A (red) and anti-Dicer D349 (green) antibodies. The p-arms of acrocentric chromosomes stained by D349 antibody are indicated by arrows. The inset shows an example of a distinctly shaped acrocentric chromosome with a pair of dots Dicer staining on p-arms. (B) Pairs of dots stained with anti-Dicer antibodies D349 (left column) and D350 (right column) (red) co-localize with dots stained by anti-UBF antibody (green). The UBF staining co-localized with the D349 and D350 antibody stainings in 95,4% (n = 65) and 93,5% (n = 62) of all inspected HEK293 chromosomes, respectively. Likewise, pairs of dots stained with anti-Dicer antibodies D349 or D350 (red) co-localize with dots stained by anti-UBF antibody (green) on mitotic chromosomes prepared from HeLa cells, human primary lymphocytes, and mouse teratocarcinoma P19 cells.
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pone-0012175-g001: Localization of Dicer on mammalian mitotic chromosomes.Chromosome spreads from human or mouse cells were stained with anti-Dicer and other indicated antibodies. (A) Staining of HEK293 chromosomes with anti-CENP-A (red) and anti-Dicer D349 (green) antibodies. The p-arms of acrocentric chromosomes stained by D349 antibody are indicated by arrows. The inset shows an example of a distinctly shaped acrocentric chromosome with a pair of dots Dicer staining on p-arms. (B) Pairs of dots stained with anti-Dicer antibodies D349 (left column) and D350 (right column) (red) co-localize with dots stained by anti-UBF antibody (green). The UBF staining co-localized with the D349 and D350 antibody stainings in 95,4% (n = 65) and 93,5% (n = 62) of all inspected HEK293 chromosomes, respectively. Likewise, pairs of dots stained with anti-Dicer antibodies D349 or D350 (red) co-localize with dots stained by anti-UBF antibody (green) on mitotic chromosomes prepared from HeLa cells, human primary lymphocytes, and mouse teratocarcinoma P19 cells.

Mentions: We have immunostained mitotic chromosomes of human HEK293 cells using two polyclonal antibodies, D349 and D350, raised against different epitopes of human Dicer (Fig. S1). Both antibodies produced characteristic “pair of dots” signals at short (p) arms of a set of distinctly shaped chromosomes, recognized as acrocentric chromosomes 13, 14, 15, 21, and 22 (Fig. 1). Co-staining for CENP-A, a centromere-specific protein, demonstrated that signals visualized with anti-Dicer antibodies were close to but not overlapping with the pairs of CENP-A-specific signals, consistent with the Dicer localization on p-arms of acrocentric chromosomes (Fig. 1A).


Dicer is associated with ribosomal DNA chromatin in mammalian cells.

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

Localization of Dicer on mammalian mitotic chromosomes.Chromosome spreads from human or mouse cells were stained with anti-Dicer and other indicated antibodies. (A) Staining of HEK293 chromosomes with anti-CENP-A (red) and anti-Dicer D349 (green) antibodies. The p-arms of acrocentric chromosomes stained by D349 antibody are indicated by arrows. The inset shows an example of a distinctly shaped acrocentric chromosome with a pair of dots Dicer staining on p-arms. (B) Pairs of dots stained with anti-Dicer antibodies D349 (left column) and D350 (right column) (red) co-localize with dots stained by anti-UBF antibody (green). The UBF staining co-localized with the D349 and D350 antibody stainings in 95,4% (n = 65) and 93,5% (n = 62) of all inspected HEK293 chromosomes, respectively. Likewise, pairs of dots stained with anti-Dicer antibodies D349 or D350 (red) co-localize with dots stained by anti-UBF antibody (green) on mitotic chromosomes prepared from HeLa cells, human primary lymphocytes, and mouse teratocarcinoma P19 cells.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0012175-g001: Localization of Dicer on mammalian mitotic chromosomes.Chromosome spreads from human or mouse cells were stained with anti-Dicer and other indicated antibodies. (A) Staining of HEK293 chromosomes with anti-CENP-A (red) and anti-Dicer D349 (green) antibodies. The p-arms of acrocentric chromosomes stained by D349 antibody are indicated by arrows. The inset shows an example of a distinctly shaped acrocentric chromosome with a pair of dots Dicer staining on p-arms. (B) Pairs of dots stained with anti-Dicer antibodies D349 (left column) and D350 (right column) (red) co-localize with dots stained by anti-UBF antibody (green). The UBF staining co-localized with the D349 and D350 antibody stainings in 95,4% (n = 65) and 93,5% (n = 62) of all inspected HEK293 chromosomes, respectively. Likewise, pairs of dots stained with anti-Dicer antibodies D349 or D350 (red) co-localize with dots stained by anti-UBF antibody (green) on mitotic chromosomes prepared from HeLa cells, human primary lymphocytes, and mouse teratocarcinoma P19 cells.
Mentions: We have immunostained mitotic chromosomes of human HEK293 cells using two polyclonal antibodies, D349 and D350, raised against different epitopes of human Dicer (Fig. S1). Both antibodies produced characteristic “pair of dots” signals at short (p) arms of a set of distinctly shaped chromosomes, recognized as acrocentric chromosomes 13, 14, 15, 21, and 22 (Fig. 1). Co-staining for CENP-A, a centromere-specific protein, demonstrated that signals visualized with anti-Dicer antibodies were close to but not overlapping with the pairs of CENP-A-specific signals, consistent with the Dicer localization on p-arms of acrocentric chromosomes (Fig. 1A).

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