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

Abstract

Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic stem cells characterized by ineffective hematopoiesis. The DNA-hypomethylating agents 5-azacytidine and 5-aza-2'-deoxycytidine are effective treatments for patients with MDS, increasing the time to progression to acute myelogenous leukemia and improving overall response rates. Although genome-wide increases in DNA methylation have been documented in BM cells from MDS patients, the methylation signatures of specific gene promoters have not been correlated with the clinical response to these therapies. Recently, attention has been drawn to the potential etiologic role of decreased expression of specific ribosomal proteins in MDS and in other BM failure states. Therefore, we investigated whether rRNA expression is dysregulated in MDS. We found significantly decreased rRNA expression and increased rDNA promoter methylation in CD34(+) hematopoietic progenitor cells from the majority of MDS patients compared with normal controls. Treatment of myeloid cell lines with 5-aza-2'-deoxycytidine resulted in a significant decrease in the methylation of the rDNA promoter and an increase in rRNA levels. These observations suggest that an increase in rDNA promoter methylation can result in decreased rRNA synthesis that may contribute to defective hematopoiesis and BM failure in some patients with MDS.

Figure 1

Pre-rRNA expression in CD34⁺ cells from healthy and MDS BM. (A) Levels of pre-rRNA expression were determined in CD34⁺ cells from 5 healthy controls (circles) and 6 MDS patients (squares) in a single representative experiment. GAPDH was used as the internal control and samples were run in triplicate. (B) Cumulative data showing levels of pre-rRNA in CD34⁺ cells from 10 healthy control and 22 MDS samples. GAPDH was used as the internal control. The ends of the whiskers represent minimum and maximum values and the bar indicates the median value (50th percentile). Significance was determined using the Mann-Whitney test.

Figure 2

Methylation of the rDNA promoter in MDS CD34⁺ cells using pyrosequencing. Heat map representation of the extent of rDNA gene promoter methylation at individual CpGs across the upstream core element and the core promoter region. A 247-bp DNA segment was amplified and the extent of methylation at 23 of 29 CpG sites (−195 to +52 bp) was determined by pyrosequencing using primers A1 to A3. Each square represents a single CpG and each row represents a sample. The extent of methylation is represented over the range of 0% (white) to 100% (dark blue). The heat map was constructed using R software.

Figure 3

rRNA promoter methylation by sodium bisulfite sequencing. DNA samples derived from CD34⁺ healthy subjects and MDS patients were treated with sodium bisulfite, PCR amplified, cloned, and sequenced. Each row represents an individual clone. The open squares represent unmethylated CpGs, closed squares represent methylated CpGs, and gray squares indicate that data were not obtained. One of the CpGs (CpG 6 in Figure 2) reported in the published rDNA sequence (accession number U13369) was found to be missing when individual clones were sequenced.

Figure 4

Hypermethylation of the rDNA promoter and decreased rRNA expression in MDS CD34⁺ cells. (A-B) The average percentage of DNA methylation across the 23 CpGs of the rDNA promoter was higher (P < .0001; A) and the pre-rRNA expression level was lower (P < .01; B) in the 13 MDS samples compared with the 8 control samples. The ends of the whiskers represent minimum and maximum values and the bar indicates the median value (50th percentile). Significance was determined using the Mann-Whitney test.

Figure 5

rRNA expression is correlated with the extent of rDNA promoter methylation. (A) As MDS samples had a higher average percentage of DNA methylation and lower rRNA expression, these variables were inversely correlated in all data (P < .01). (B) Pearson correlation estimates between pre-rRNA expression and rDNA promoter methylation at 23 CpGs in 8 control samples and 13 MDS samples are shown. Vertical lines represent 95% confidence intervals.

Figure 6

Effect of DAC on rRNA expression and rDNA promoter methylation in myeloid leukemia cell lines. (A-C) THP1, Mono Mac 6, and ML2 cells were treated with DMSO or 0.1μM DAC for 3 days. The level of expression of pre-rRNA is shown relative to that in DMSO-treated cells. GAPDH was used as an internal control and the samples were run in triplicate. (D-F) The cells were treated with DMSO or 0.1μM DAC for 3 days and the extent of CpG methylation was determined by pyrosequencing at CpGs 7-23 spanning the rDNA upstream core element and the core promoter.