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Overexpression of ribosomal RNA in prostate cancer is common but not linked to rDNA promoter hypomethylation.

Uemura M, Zheng Q, Koh CM, Nelson WG, Yegnasubramanian S, De Marzo AM - Oncogene (2011)

Bottom Line: Further, as a surrogate for nucleolar size and number, we examined the expression of fibrillarin, which did not correlate with rRNA levels.We conclude that rRNA levels are increased in human prostate cancer, but that hypomethylation of the rDNA promoter does not explain this increase, nor does hypomethylation explain alterations in nucleolar number and structure in prostate cancer cells.Rather, rRNA levels and nucleolar size and number relate more closely to MYC overexpression.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.

ABSTRACT
Alterations in nucleoli, including increased numbers, increased size, altered architecture and increased function are hallmarks of prostate cancer cells. The mechanisms that result in increased nucleolar size, number and function in prostate cancer have not been fully elucidated. The nucleolus is formed around repeats of a transcriptional unit encoding a 45S ribosomal RNA (rRNA) precursor that is then processed to yield the mature 18S, 5.8S and 28S RNA species. Although it has been generally accepted that tumor cells overexpress rRNA species, this has not been examined in clinical prostate cancer. We find that indeed levels of the 45S rRNA, 28S, 18S and 5.8S are overexpressed in the majority of human primary prostate cancer specimens as compared with matched benign tissues. One mechanism that can alter nucleolar function and structure in cancer cells is hypomethylation of CpG dinucleotides of the upstream rDNA promoter region. However, this mechanism has not been examined in prostate cancer. To determine whether rRNA overexpression could be explained by hypomethylation of these CpG sites, we also evaluated the DNA methylation status of the rDNA promoter in prostate cancer cell lines and the clinical specimens. Bisulfite sequencing of genomic DNA revealed two roughly equal populations of loci in cell lines consisting of those that contained densely methylated deoxycytidine residues within CpGs and those that were largely unmethylated. All clinical specimens also contained two populations with no marked changes in methylation of this region in cancer as compared with normal. We recently reported that MYC can regulate rRNA levels in human prostate cancer; here we show that MYC mRNA levels are correlated with 45S, 18S and 5.8S rRNA levels. Further, as a surrogate for nucleolar size and number, we examined the expression of fibrillarin, which did not correlate with rRNA levels. We conclude that rRNA levels are increased in human prostate cancer, but that hypomethylation of the rDNA promoter does not explain this increase, nor does hypomethylation explain alterations in nucleolar number and structure in prostate cancer cells. Rather, rRNA levels and nucleolar size and number relate more closely to MYC overexpression.

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The human rDNA promoter region. (a) Genotyping of rDNA promoter region. The sequence of the rDNA promoter as shown was identical for all prostate cancer tissues and normal tissues, as well as all the cell lines. In this map, primer sequences for genotyping are indicated by the underlined regions and the primer sequences for bisulfite sequencing are indicated as dotted underlines. CpG dinucleotides are boxed with the location marked relative to the transcription start site (arrow). Discrepancies with the published DNA sequence (U13369) are marked. Nucleotides that show base substitutions as compared with the reference U13369 seqeunce are illustrated with dots above the DNA sequence. Deletions (generally a single base), relative to the reference sequence, are marked by a ‘v ‘between nucleotides. (b) Schematic representation of a single human rDNA repeat. The approximate positions relative to the transcription start site are indicated (DNA base number in kb). (c) Vertical lines indicate locations of CpG dinucleotides on the 45S rRNA promoter relative to the transcription start site, indicated with the dashed curved arrow, with primer pairs used for bisulfite mapping marked by inward pointing arrows.
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fig1: The human rDNA promoter region. (a) Genotyping of rDNA promoter region. The sequence of the rDNA promoter as shown was identical for all prostate cancer tissues and normal tissues, as well as all the cell lines. In this map, primer sequences for genotyping are indicated by the underlined regions and the primer sequences for bisulfite sequencing are indicated as dotted underlines. CpG dinucleotides are boxed with the location marked relative to the transcription start site (arrow). Discrepancies with the published DNA sequence (U13369) are marked. Nucleotides that show base substitutions as compared with the reference U13369 seqeunce are illustrated with dots above the DNA sequence. Deletions (generally a single base), relative to the reference sequence, are marked by a ‘v ‘between nucleotides. (b) Schematic representation of a single human rDNA repeat. The approximate positions relative to the transcription start site are indicated (DNA base number in kb). (c) Vertical lines indicate locations of CpG dinucleotides on the 45S rRNA promoter relative to the transcription start site, indicated with the dashed curved arrow, with primer pairs used for bisulfite mapping marked by inward pointing arrows.

Mentions: The nucleolus is formed around tandem repeats of the ribosomal RNA genes (rDNA), each of which contains a promoter region (Figure 1). Human rDNA repeat units comprise ∼43 kb (Gonzalez and Sylvester, 1995). Sequences encoding pre-rRNA (18S, 5.8S and 28S) are ∼13 kb and these are separated by intergenic spacers of ∼30 kb, which are largely non-transcribed. rDNA repeats are generally found in two major chromatin states. In all species examined, including humans, ∼1/2 of the repeat units are present in densely compact ‘heterochromatin' that is not accessible to the cross-linking agent psoralen in vivo (McStay and Grummt, 2008). The other 1/2 of the repeats is generally accessible to psoralen and these contain a more open ‘euchromatic' chromatin structure. The rDNA promoter consists of a core promoter element adjacent to the transcription start site and a nearby upstream control element (Figures 1b and c) (Haltiner et al., 1986). In human cells, deoxycytidine methylation of the ∼25 CpGs residing within the promoter is present in two major patterns. For each repeat unit, either this entire region contains largely unmethylated CpGs or the entire region contains largely methylated CpGs. In general, ∼1/2 of the repeats show each type of configuration (McStay and Grummt, 2008), and the closed psoralen-inaccessible repeats correspond to those containing densely methylated CpG dinucleotides in the promoter regions, whereas the psoralen-accessible repeats are largely unmethylated in the promoter regions (McStay and Grummt, 2008). Further, the densely methylated psoralen inaccessible repeats are transcriptionally inactive. And, of the non-methylated repeats, it appears that only a limited number of these units are active at any one time.


Overexpression of ribosomal RNA in prostate cancer is common but not linked to rDNA promoter hypomethylation.

Uemura M, Zheng Q, Koh CM, Nelson WG, Yegnasubramanian S, De Marzo AM - Oncogene (2011)

The human rDNA promoter region. (a) Genotyping of rDNA promoter region. The sequence of the rDNA promoter as shown was identical for all prostate cancer tissues and normal tissues, as well as all the cell lines. In this map, primer sequences for genotyping are indicated by the underlined regions and the primer sequences for bisulfite sequencing are indicated as dotted underlines. CpG dinucleotides are boxed with the location marked relative to the transcription start site (arrow). Discrepancies with the published DNA sequence (U13369) are marked. Nucleotides that show base substitutions as compared with the reference U13369 seqeunce are illustrated with dots above the DNA sequence. Deletions (generally a single base), relative to the reference sequence, are marked by a ‘v ‘between nucleotides. (b) Schematic representation of a single human rDNA repeat. The approximate positions relative to the transcription start site are indicated (DNA base number in kb). (c) Vertical lines indicate locations of CpG dinucleotides on the 45S rRNA promoter relative to the transcription start site, indicated with the dashed curved arrow, with primer pairs used for bisulfite mapping marked by inward pointing arrows.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3298623&req=5

fig1: The human rDNA promoter region. (a) Genotyping of rDNA promoter region. The sequence of the rDNA promoter as shown was identical for all prostate cancer tissues and normal tissues, as well as all the cell lines. In this map, primer sequences for genotyping are indicated by the underlined regions and the primer sequences for bisulfite sequencing are indicated as dotted underlines. CpG dinucleotides are boxed with the location marked relative to the transcription start site (arrow). Discrepancies with the published DNA sequence (U13369) are marked. Nucleotides that show base substitutions as compared with the reference U13369 seqeunce are illustrated with dots above the DNA sequence. Deletions (generally a single base), relative to the reference sequence, are marked by a ‘v ‘between nucleotides. (b) Schematic representation of a single human rDNA repeat. The approximate positions relative to the transcription start site are indicated (DNA base number in kb). (c) Vertical lines indicate locations of CpG dinucleotides on the 45S rRNA promoter relative to the transcription start site, indicated with the dashed curved arrow, with primer pairs used for bisulfite mapping marked by inward pointing arrows.
Mentions: The nucleolus is formed around tandem repeats of the ribosomal RNA genes (rDNA), each of which contains a promoter region (Figure 1). Human rDNA repeat units comprise ∼43 kb (Gonzalez and Sylvester, 1995). Sequences encoding pre-rRNA (18S, 5.8S and 28S) are ∼13 kb and these are separated by intergenic spacers of ∼30 kb, which are largely non-transcribed. rDNA repeats are generally found in two major chromatin states. In all species examined, including humans, ∼1/2 of the repeat units are present in densely compact ‘heterochromatin' that is not accessible to the cross-linking agent psoralen in vivo (McStay and Grummt, 2008). The other 1/2 of the repeats is generally accessible to psoralen and these contain a more open ‘euchromatic' chromatin structure. The rDNA promoter consists of a core promoter element adjacent to the transcription start site and a nearby upstream control element (Figures 1b and c) (Haltiner et al., 1986). In human cells, deoxycytidine methylation of the ∼25 CpGs residing within the promoter is present in two major patterns. For each repeat unit, either this entire region contains largely unmethylated CpGs or the entire region contains largely methylated CpGs. In general, ∼1/2 of the repeats show each type of configuration (McStay and Grummt, 2008), and the closed psoralen-inaccessible repeats correspond to those containing densely methylated CpG dinucleotides in the promoter regions, whereas the psoralen-accessible repeats are largely unmethylated in the promoter regions (McStay and Grummt, 2008). Further, the densely methylated psoralen inaccessible repeats are transcriptionally inactive. And, of the non-methylated repeats, it appears that only a limited number of these units are active at any one time.

Bottom Line: Further, as a surrogate for nucleolar size and number, we examined the expression of fibrillarin, which did not correlate with rRNA levels.We conclude that rRNA levels are increased in human prostate cancer, but that hypomethylation of the rDNA promoter does not explain this increase, nor does hypomethylation explain alterations in nucleolar number and structure in prostate cancer cells.Rather, rRNA levels and nucleolar size and number relate more closely to MYC overexpression.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.

ABSTRACT
Alterations in nucleoli, including increased numbers, increased size, altered architecture and increased function are hallmarks of prostate cancer cells. The mechanisms that result in increased nucleolar size, number and function in prostate cancer have not been fully elucidated. The nucleolus is formed around repeats of a transcriptional unit encoding a 45S ribosomal RNA (rRNA) precursor that is then processed to yield the mature 18S, 5.8S and 28S RNA species. Although it has been generally accepted that tumor cells overexpress rRNA species, this has not been examined in clinical prostate cancer. We find that indeed levels of the 45S rRNA, 28S, 18S and 5.8S are overexpressed in the majority of human primary prostate cancer specimens as compared with matched benign tissues. One mechanism that can alter nucleolar function and structure in cancer cells is hypomethylation of CpG dinucleotides of the upstream rDNA promoter region. However, this mechanism has not been examined in prostate cancer. To determine whether rRNA overexpression could be explained by hypomethylation of these CpG sites, we also evaluated the DNA methylation status of the rDNA promoter in prostate cancer cell lines and the clinical specimens. Bisulfite sequencing of genomic DNA revealed two roughly equal populations of loci in cell lines consisting of those that contained densely methylated deoxycytidine residues within CpGs and those that were largely unmethylated. All clinical specimens also contained two populations with no marked changes in methylation of this region in cancer as compared with normal. We recently reported that MYC can regulate rRNA levels in human prostate cancer; here we show that MYC mRNA levels are correlated with 45S, 18S and 5.8S rRNA levels. Further, as a surrogate for nucleolar size and number, we examined the expression of fibrillarin, which did not correlate with rRNA levels. We conclude that rRNA levels are increased in human prostate cancer, but that hypomethylation of the rDNA promoter does not explain this increase, nor does hypomethylation explain alterations in nucleolar number and structure in prostate cancer cells. Rather, rRNA levels and nucleolar size and number relate more closely to MYC overexpression.

Show MeSH
Related in: MedlinePlus