Reduced rRNA expression and increased rDNA promoter methylation in CD34+ cells of patients with myelodysplastic syndromes

Abstract

IntroductionThe DNA-hypomethylating drugs 5-azacytidine (5AC) and 5-aza-2Ј-deoxycytidine (DAC) are in clinical use for the treatment of myelodysplastic syndrome (MDS) 1,2 and have been shown to improve overall response rates and increase the time to progression to acute myelogenous leukemia compared with best supportive care. 3,4 Several studies have documented an increase in genomewide promoter methylation in mononuclear cells and in CD34 ϩ hematopoietic progenitor cells (HPCs) derived from the BM of MDS patients. 5,6 However, drug-induced differences in genomewide promoter methylation signatures and in gene-expression profiles have not been found to be correlated with the clinical response to hypomethylating agents. 7 In addition, a correlation between changes in promoter methylation profiles and acquired resistance to 5AC or DAC has never been demonstrated 8,9 Therefore, the role of DNA hypermethylation in the pathogenesis of MDS remains to be determined, as does the question of whether the therapeutic responses to 5AC and DAC result from the induction of gene-specific DNA hypomethylation as opposed to other sequelae such as the induction of a DNA-damage response, the activation of an immune response, or the induction of senescence. 7 The nucleolus contains clusters of genes that encode the 45S pre-rRNA precursor of rRNA that is subsequently processed to generate the 18S, 5.8S, and 28S rRNA components. 10,11 In human cells, there are approximately 400 copies of rDNA genes arranged as tandem repeats within nucleolar organizer regions located on chromosomes 13-15, 21, and 22, although not all of the genes are actively transcribed. Transcription of the rRNA genes depends on the chromatin structure of the promoter region. 12-14 Promoters of active rRNA genes are devoid of CpG methylation and are associated with acetylated histones, whereas the reverse is found for silenced genes. 15 DNA methyltransferase 1 (DNMT1) controls rDNA gene transcription 16 and also regulates the nucleolar architecture by maintaining the heterochromatin state within intergenic repetitive sequences. 17,18 Several recent observations have suggested that ribosomal protein synthesis is an essential component of normal hematopoietic stem cell (HSC) function. Haploinsufficiency of the ribosomal gene RPS14 in 5qϪ syndrome, 19,20 the presence of RPS19 mutations in Diamond-Blackfan anemia, 21 and the aberrant expression of multiple ribosomal proteins in MDS 22-24 all suggest that altered ribosome biogenesis could play a major role in the pathogenesis of BM failure syndromes. To determine whether rRNA synthesis might be altered in HPCs from a broader spectrum of MDS patients, in the present study, we compared the level of rRNA expression and the extent of rDNA promoter methylation in CD34 ϩ cells derived from BM of MDS patients with those in normal CD34 ϩ cells. Our data demonstrate that rRNA expression is decreased in HPCs from MDS patients, whereas methylation within the rDNA upstream regulatory region is increased. We also show that rRNA expres...

Relative expression of rRNA transcripts and 45S rDNA promoter methylation status are dysregulated in tumors in comparison with matched-normal tissues in breast cancer

“…2) in breast cancer cell lines as proposed earlier by other studies (22)(23)(24)(25). Considering the repetitive nature of rRNA genes, this result may be due to the low number of clones (5 clones) analyzed for each cell line.…”

“…It is known that promoter DNA methylation has a repressive effect, particularly on Pol II transcribed genes in cancer (20,38) and increased methylation levels are implicated in decreased levels of rRNA transcription (22,23). Thus, we hypothesized that rRNA transcription levels may also be downregulated in these breast cancer cell lines with a hypermethylated 45S rDNA promoter.…”

“…Methylation at the 45S rDNA promoter region decreased the expression of rRNA genes in hepatocellular carcinoma (22) and in CD34 + cells of patients with myelodysplastic syndromes (23). On the other hand, other studies have shown no relationship or a positive correlation between promoter methylation and rRNA transcription (24,25).…”

“…TBP, GAPDH and ACTB have been used as reference genes to determine rRNA expression levels in several studies (23,24,35) but these RNA polymerase II (Pol II) transcribed genes are variably expressed in numerous types of cancer (36,37). However, several studies advise against using rRNA levels to determine mRNA levels (26,27).…”

The Epigenetic Pathways to Ribosomal DNA Silencing

“…In addition, the promoter and 5=-regulatory regions of rDNA are significantly hypermethylated in the brains of suicidal males, consistent with decreased rRNA expression in the hippocampus (193). In CD34 ϩ cells of patients with myelodysplastic syndrome, a clonal disorder of hematopoietic stem cells, increased DNA methylation at the rDNA promoter, together with reduced rRNA synthesis, is suggested to contribute to defective hematopoiesis and bone marrow failure in the majority of these patients (194). However, in white blood cells of patients with systemic lupus erythematosus, an autoimmune inflammatory disease, DNA methylation of rDNA is decreased (195).…”