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Blood First Edition Paper, prepublished online October 15, 2012; DOI 10.1182/blood-2012-04-423111
Reduced Ribosomal RNA Expression and Increased Ribosomal DNA
Promoter Methylation in CD34+ Cells of Patients with Myelodysplastic
Syndromes
Aparna Raval1, Kunju J. Sridhar2, Shripa Patel3, Brit B. Turnbull4, Peter L.
Greenberg2 and Beverly S. Mitchell1
Stanford Cancer Institute1, Division of Hematology2, Stanford Protein and Nucleic
Acid Facility3 and Department of Health Research and Policy4, Stanford University,
Stanford, CA 94305
Corresponding Author:
Beverly S. Mitchell M.D.
George E Becker Professor of Medicine
Stanford Cancer Institute,
LLSC Research Building (SIM1),
265 Campus Drive, Room G2167,
Stanford, CA 94305-5458.
Phone: 650-736-7716
Fax: 650-736-0607
E-mail: [email protected]
1
Copyright © 2012 American Society of Hematology
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Abstract
Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic
stem
cells
characterized
by
ineffective
hematopoiesis.
The
DNA
hypomethylating agents 5-azacytidine (5AC) and 5-aza-2’-deoxycytidine
(DAC) are effective treatments for patients with MDS, increasing time to
progression to acute myelogenous leukemia and improving overall response
rates. Although genome-wide increases in DNA methylation have been
documented in bone marrow cells from MDS patients, the methylation
signatures of specific gene promoters have not 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 bone marrow failure (BMF) states. We therefore asked
whether ribosomal RNA (rRNA) expression is dysregulated in MDS. We
found significantly decreased rRNA expression and increased rDNA
promoter methylation in CD34+ hematopoietic progenitor cells (HPC) from
the majority of MDS patients as compared to normal controls. Treatment of
myeloid cell lines with DAC 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 BMF in some individuals with MDS.
2
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Introduction:
The DNA hypomethylating drugs 5AC and DAC are in clinical use for
the treatment of MDS
1,2
and have been shown to improve overall response
rates and increase the time to progression to acute myelogenous leukemia
(AML) as compared with best supportive care
3,4
. A number of studies have
documented an increase in genome-wide promoter methylation in
mononuclear cells as well as in CD34+ HPC derived from the bone marrows
of MDS patients
5,6
. However, drug-induced differences in genome-wide
promoter methylation signatures and in gene expression profiles have not
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 prerRNA precursor of ribosomal RNA that is subsequently processed to generate
the 18S, 5.8S and 28S rRNA components 10,11. In human cells, there are ~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
3
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histones, while the reverse is found for silenced genes
methyltransferase 1 (DNMT1) controls rDNA gene transcription
15
. DNA
16
and also
regulates the nucleolar architecture by maintaining the heterochromatin state
within intergeneic repetitive sequences 17,18.
A number of recent observations have suggested that ribosomal protein
synthesis is an essential component of normal hematopoietic stem cell
function. Haploinsufficiency of the ribosomal gene RPS14 in 5q- 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 bone marrow failure syndromes. To determine whether rRNA synthesis
might be altered in HPC from a broader spectrum of MDS patients, we have
compared the level of rRNA expression and the extent of rDNA promoter
methylation in CD34+ cells derived from bone marrows of MDS patients to
those in normal CD34+ cells. Our data demonstrate that rRNA expression is
decreased in MDS hematopoietic progenitors while methylation within the
rDNA upstream regulatory region is increased. We show that rRNA
expression and rDNA methylation are inversely correlated in our combined
data obtained from healthy controls and MDS patients. In addition, treatment
of human myeloid leukemia cell lines with DAC resulted in both a decrease in
promoter methylation and an increase in rRNA expression. We suggest that
disruption of ribosomal biogenesis resulting from alterations in rRNA
synthesis may also underlie hematopoietic progenitor cell dysfunction in
some MDS patients.
4
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Materials and Methods
MDS Patient samples
CD34+ mononuclear cells were enriched from freshly obtained bone marrow
from 22 MDS patients and 11 healthy donors by magnetic bead separation
(Miltenyi Biotec). CD34+ purity was greater than 90% as assessed by flow
cytometry. Cells were immediately snap-frozen and stored at -80C until use.
Bone marrow samples were obtained from patients after informed consent per
the Declaration of Helsinki and according to an IRB-approved protocol.
Cell Culture Experiments:
The THP1, Mono Mac6 and ML-2 (human myelomonocytoc leukemia cell
lines) were incubated (37°C and 5% CO2) in RPMI 1640 supplemented with
10% FBS (HyClone Laboratories, Logan, UT), 100 U/ml penicillin, and 100
μg/ml streptomycin (Invitrogen, Carlsbad CA). Cells were treated with 0.1μM
DAC (Sigma-Aldrich, St. Louis MO) for a maximum of 3 days and drug was
added daily. Cells were harvested after three days of treatment and DNA and
RNA were extracted according to manufacturer’s instructions (Qiagen,
Valencia, CA).
Quantitative RT-PCR
Total RNA was extracted from cell lines and bone marrow-derived CD34+
cells using the RNAeasy micro kit (Qiagen, Valencia, CA). Total RNA was
treated with DNAse (Ambion, Life Technologies, Carlsbad, CA) according to
the manufacturer’s instructions and reverse transcription was performed with
random hexamer primers using the SUPERSCRIPT First-Strand Synthesis kit
(Invitrogen). PCR primers were located within the 5’ Externally Transcriped
Sequence of the rDNA gene and pre-rRNA expression was measured by
5
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quantitative RT-PCR carried out in triplicates using SYBR Green Master Mix
(Applied Biosystems, Foster City, CA). cDNA was amplified using primers
within the rDNA promoter and the absence of PCR product confirmed the
lack of rDNA amplification. The RT-PCR primers used were: pre-rRNA:
forward
primer
5'-
gaacggtggtgtgtcgttc
-3',
reverse
primer
5'-
gcgtctcgtctcgtctcact -3' (Murayama A et al Cell 2008); GAPDH: forward
primer 5'-ccccttcattgacctcaactacat-3', reverse primer 5'-cgctcctggaagatggtga3’.
Quantitative DNA Methylation using Pyrosequencing: Genomic DNA
from CD34+ cells from 14 MDS and 11 normal samples (100-200 ng) and
from myeloid cell lines (1 mg) was treated with sodium bisulfite using the
EZ DNA methylation kit (Zymo Research, Orange, CA). For 13/14 MDS and
8/11 control samples used for promoter methylation analysis, pre-rRNA
expression was also studied by quantitative RT-PCR. Quantitative DNA
methylation analysis was performed by pyrosequencing as described
25
.
Methylation of specific CpGs by pyrosequencing was evaluated using the
published human rDNA repeat sequence (accession number U13369). A 247
bp rDNA promoter region was amplified using rDNA specific primers;
forward: 5'-GTGTTTTTGGGTTGATTAGAGG-3' and reverse: 5'-biotin
CATCCCAAAACCCAACCTCTCC-3'. Three different primers (P1: 5'GGTTGATTAGAGGGATT-3'; P2 : 5'-TTTTGGGGATAGGTG-3' and P3 :
5'-TTYGGGGGAGGTATATTTT-3') were annealed to the purified singlestranded 247 bp PCR product and pyrosequencing was performed using
PyroMark Q24 (Qiagen). Determining the ratio of cytosine to thymidine
incorporation during pyrosequencing allows quantitation of the degree of
methylation of target CpG sequences. Data were analyzed using the
6
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PyroMark Q24 software. Of the 29 CpGs present within the 247bp amplified
PCR product, six were not included in the analysis because the observed to
expected methylation ratios (as compared to in vitro methylated standard
DNA) was not linear.
Bisulfite Sequencing:
The bisulfite treated DNA from CD34+ normal and MDS samples were
amplified using specific rDNA primers, as outlined above. The 247 bp PCR
products were purified from a 1.5% agarose gel using the Qiagen Gel
Extraction kit (Qiagen) and subcloned using the TOPO TA-Cloning kit
(Invitrogen). Five clones were sequenced from each samples and methylation
at 26 CpGs per clone was analyzed using SeqMan Pro software (DNASTAR).
Statistical Analysis: The Mann-Whitney test with two-sided alpha-level
0.05 was used to compare level of rRNA expression and average percent
promoter methylation between control and MDS samples. To examine the
relationship between rRNA expression and promoter methylation at each
individual CpG, Pearson correlation estimates were calculated with
confidence intervals approximated using Fisher’s Z transform. To
examine the relationship between rRNA expression and average percent
promoter methylation, the Pearson correlation estimate was calculated,
and a linear model was fit, controlling for disease status. In multiple
hypothesis testing, significance was determined by using the Bonferroni
correction to control family-wise error-rate at 0.05.
7
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Results:
Expression of pre-rRNA in MDS samples:
The level of expression of 48S precursor rRNA in CD34+ HPCs was
determined by measuring the abundance of the transcript processed at
externally transcribed regions, as described in Materials and Methods. Results
from a representative experiment (Figure 1A) showed that pre-rRNA
expression was significantly reduced (p value < 0.01) in CD34+ HPC from 6
MDS patients compared to that in cells from 5 normal controls. Overall, prerRNA expression was studied in CD34+ HPC from 22 MDS samples and 11
controls. The cumulative data from repetitive quantitative RT-PCR
determinations revealed that pre-rRNA expression was significantly
decreased (p value < 0.001) in 22 MDS samples as compared to control
samples (Figure 1B). These data demonstrate that rRNA synthesis is reduced
in bone marrow progenitor cells from the majority of MDS patients studied.
Methylation of the rDNA promoter in MDS CD34+ cells
The rRNA gene promoter is highly enriched in CpG dinucleotides
26-28
. We
postulated that reduced rRNA levels in MDS could result from promoter
hypermethylation. PCR amplification of sodium bisulfite-treated DNA and
subsequent pyrosequencing of the PCR products was used to quantitate
methylation at 23/29 CpG sites within the rDNA promoter. Six of the 29
CpGs were not included in the analysis as the observed to expected
methylation ratios observed by in vitro methylated standard DNA was not
linear. A 247 bp DNA segment was amplified using rRNA promoter-specific
primers that overlapped the upstream core element and the core promoter as
illustrated in Figure 2. This amplicon served as a template for analyzing the
degree of promoter methylation at 23 CpGs spanning rDNA promoter region
8
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(-195 to +52) using the primers P1 to P3. The heat map shown is a schematic
representation of the percent DNA methylation at each of these 23 CpGs. The
extent of methylation varies from 100% methylation (dark blue) to no
methylation (white) in CD34+ HPCs from normal controls (upper panel) and
14 MDS samples (lower panel). Densely methylated CpGs were present
across the 247 bp rDNA promoter in MDS samples. The percent methylation
at each CpG was significantly greater than that in control samples at 22/23
CpGs (p value < 0.05), (Supplementary Figure 1A-D). Bisulfite sequencing
was used to determine whether rDNA promoter methylation varied within
individual PCR clones obtained from three normal and four MDS CD34+
samples. As shown in Figure 3, sequencing of clones from control samples
showed that the majority of clones were either unmethylated or methylated at
low frequency. In contrast, the frequency of methylated CpGs per clone in
MDS samples was high in the majority of clones analyzed. None of the clones
obtained from patient samples was methylated at every CpGs and two clones
were unmethylation. These data further confirm the increased rDNA promoter
methylation observed in patient samples obtained using pyrosequencing and
also suggest that the number of transcriptionally active rDNA genes may be
significantly fewer in MDS CD34+ cells.
rDNA promoter methylation quantitated by pyrosequencing was then
correlated with rRNA expression levels in 8 controls and 13 MDS samples
(8/11 control and 13/22 MDS samples shown in Figure 1B). The average of
the percent methylation across 23 CpGs in CD34+ HPC was significantly
increased (p-value < 0.0001) in MDS samples as shown in Figure 4A and the
pre-rRNA expression levels were significantly decreased (Figure 4B) in the
same patient samples. When MDS and control samples are combined, the
9
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average percent methylation and expression is inversely correlated with a pvalue < 0.01. The Pearson correlation coefficient estimate is -0.6, with 95%
confidence interval (-0.2, -0.8). The relationship between the three variables
(average percent methylation, rRNA expression, and disease status) is shown
in Figure 5A. The estimates of correlation, with 95% confidence intervals,
between expression and methylation at each individual CpG (among the 13
MDS samples and 8 control samples) are shown in Figure 5B. All point
estimates are negative, suggesting inverse correlation across the 23 CpG’s,
although we cannot declare significance at individual CpG’s when correcting
for multiple hypothesis testing. These data are concordant with studies that
have shown an increase in overall methylation in MDS samples but are the
first to address specific methylation changes in the rDNA promoter region.
Effect of DAC treatment on pre-rRNA expression and promoter
methylation
To address the question of whether DAC treatment would decrease
rDNA promoter methylation and concordantly increase rRNA synthesis, we
treated leukemic cell lines with the drug. Cells treated with 0.1 μΜ DAC for
five days resulted in a gradual increase in pre-rRNA expression over days 2 to
5 as compared to control cells treated with DMSO over the same interval.
Increasing the DAC concentration from 0.1 to 0.5μM did not further increase
rRNA expression (data not shown), suggesting that 0.1μM DAC is the
optimal dose. We then treated three different myelomonocytic leukemia cell
lines, THP1, Mono Mac 6 and ML2, with 0.1μM DAC for 3 days and
observed an increase in pre-rRNA expression and a decrease in promoter
methylation as measured by pyrosequencing at CpGs 7-23 in each cell lines
10
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(Figure 6A-F). Due to limited number of cells obtained from the bone marrow
aspirates, we were unable to perform rRNA expression and promoter
methylation analysis in CD34+ enriched populations from MDS patients.
Discussion
The requirement for ribosomal biogenesis reflects the overall demand for
cellular protein synthesis. Formation of the mature 80S ribosome requires
coordinated expression of four rRNAs and approximately 80 ribosomal
proteins and perturbation at any stage of ribosomal biogenesis or assembly
would be expected to impair cellular proliferation
29
. Our data demonstrate
decreased rRNA levels that inversely correlate with rDNA promoter
methylation in control and MDS CD34+ hematopoietic progenitors. The high
throughput array-based published reports for gene expression (Affymetrix
gene-chip arrays)
7,30,31
or promoter methylation (Nimblegen oligonucleotide
arrays or Illumina bead arrays)
5,6
in MDS used arrays that lack probes for
rRNA or rDNA, respectively. Therefore, this is the first report to specifically
examine rRNA gene expression and rDNA promoter methylation in MDS.
Our results suggest that the increase in methylation of this CpG-rich promoter
is at least one of the mechanisms resulting in decreased rRNA gene
expression, potentially altering the synthesis of ribosomal proteins
32
, thus
hampering ribosomal biogenesis.
Impairment of ribosome biogenesis characterizes several BMF
syndromes
33
. Multiple mouse models have demonstrated that mutation of
genes including those encoding Rps19, Rps14 and Rps6 result in defective
hematopoiesis. Haploinsufficiency of Rps6 or of the Cd74-Nid67 region that
contains 6 genes including Rps14 results in macrocytic anemia, erythroid
11
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hypoplasia, megakaryocytic dysplasia and reduced number of hematopoietic
stem/progenitor cells, all of which are characteristic features of 5q- syndrome
20,24
.
Similarly,
conditional
expression
of
mutant
Rps19
or
its
haploinsufficiency in mice results in anemia, exhaustion of HSC and bone
marrow failure
34,35
. Bone marrows from these mice have high levels of p53
protein expression and increased apoptosis, while breeding them with p53deficient animals rescues the phenotype. A number of studies have
demonstrated that the release of specific ribosomal proteins, including RPL5
and RPL11, from the nucleolus increases p53 levels through an interaction
with the MDM2 ubiquitin ligase, resulting in increased levels of p53
36,37
.
Decreased expression of RPS19 or RPS14 causes selective activation of the
p53 pathway in erythroid progenitors, further establishing the sensitivity of
hematopoietic precursors to disruptions in ribosome biogenesis
38
. Thus, a
common mechanism underlying bone marrow failure states appears to be the
up-regulation of p53 through alterations in ribosome assembly.
Increased p53 stability also occurs when rRNA synthesis is decreased,
either through down-regulation of the catalytic subunit of RNA Polymerase I
39
or through knock-out of TIF-1A, a protein required for rRNA transcription
40
. The increase in p53 expression results in enhanced apoptosis as well as in
disruption of nucleolar architecture. The chromatin status of rRNA genes
maintains the appropriate ratio of expression among genes that are actively
transcribed and those that are not, thereby regulating cellular proliferation 12.
The nucleolar remodeling complex (NoRC), an ATP-dependent chromatin
remodeling complex that regulates rRNA gene silencing, interacts both with
DNA methyltransferase 1 (DNMT1) and with the histone deacetylase
HDAC1
26,41
. Our data raise the possibility that alterations in rRNA
12
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expression may underlie the up-regulation of p53 and apoptosis of bone
marrow progenitor cells in MDS patients. Further characterization of the
epigenetic events regulating nucleolar rRNA gene expression, including both
the extent of DNA methylation and the nature of histone modifications, may
help to further our understanding of BMF syndromes.
Reversal of DNA methylation by DNMT inhibitors has constituted a
major rationale for treating MDS patients with these drugs. Although
clinically significant responses occur in MDS patients treated with both 5AC
and DAC 3,4,42,43 and although both drugs cause global DNA hypomethylation
31,44-46
, specific methylation changes have not been identified that predict the
clinical response
2,7-9
. In addition, most patients ultimately develop resistance
to 5AC or DAC 8,9. Our in vitro data with cell lines suggest that the response
to hypomethylating agents could involve an increase in rRNA transcription,
resulting in decreased apoptosis. This supposition is supported by recent data
demonstrating that upregulation of rRNA expression increases ribosome
biogenesis and ubiquitination of p53, thereby reducing p53 levels 39. It will be
of interest to obtain clinical data on the response of rRNA expression in
CD34+ cells of patients receiving hypomethylating agents in relation to the
durability of their response to this intervention.
13
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Acknowledgements: This study was supported by a Leukemia and
Lymphoma Society SCOR and translational grant award to B.S. Mitchell.
Authorship Contributions:
Planning experiment: A.R., P.L.G and B.S.M.
Executing experiment: A.R., K.S. and S.P.
Analyzing experiment: A.R., B.B.T and B.S.M.
Writing Manuscript: A.R., B.B.T and B.S.M.
Disclosure of Conflicts of Interest: The authors declare no competing
financial interests.
Corresponding Author:
Beverly S. Mitchell M.D.
George E Becker Professor of Medicine
Stanford Cancer Institute,
LLSC Research Building (SIM1),
265 Campus Drive, Room G2167,
Stanford, CA 94305-5458.
Phone: 650-736-7716
Fax: 650-736-0607
E-mail: [email protected]
14
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Figure Legends
Figure 1. Pre-rRNA expression in CD34+ cells from normal and MDS
bone marrow.
A. Levels of pre-rRNA expression were determined in CD34+ cells from 5
normal individuals and 6 patients with MDS in a single representative
experiment. GAPDH was used as the internal control and samples were run
in triplicates represented by circles in controls and squares in patients. B.
Cumulative data showing levels of pre-rRNA in CD34+ cells from 10 normal
control and 22 MDS samples. GAPDH was used as the internal control. The
ends of the whiskers represent minimum and maximum values while 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 (blue line) was amplified and the extent of
methylation at each of 23/29 CpG sites (-195 to +52 bp) was determined by
pyrosequencing using primers P1 to P3. 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.
19
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Figure 3. rRNA promoter methylation by bisulfite sequencing
DNA derived from CD34+ normal and MDS samples were treated with
bisulfite, PCR amplified, cloned and sequenced. Each row represents an
individual clone. The open squares represent unmethylated CpGs and closed
squares represent methylated CpGs. Grey 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. The average percent DNA methylation across the 23CpGs of the rDNA
promoter is higher (p-value < 0.0001) and B. the pre-rRNA expression level
is lower (p-value < 0.01) in 13 MDS samples compared to 8 controls. The
ends of the whiskers represent minimum and maximum values while the bar
indicates the median value (50th percentile). Significance was determined
using the Mann-Whitney test.
Figure 5. rRNA expression correlates with the extent of rDNA promoter
methylation.
A. As MDS samples have higher average percent DNA methylation and
lower rRNA expression, among all data these variables are inversely
correlated (p-value < 0.01). B. Pearson correlation estimates between prerRNA expression and rDNA promoter methylation at 23CpGs in 8 control
20
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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 0.1 μM
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 triplicates. D-F The cells were treated with DMSO or
0.1 μM 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.
21
A
Pre-rRNA/GAPDH mRNA
1
2
3
4
CD34+ Normal Ct
B
Pre-rRNA/GAPDH mRNA
Figure 1
5
M1 M2 M3 M4 M5 M6
CD34+ MDS Samples
Upstream Control Element
Core Promoter
A2
A3
CD34+ Normal Controls
A1
+1
CD34+ MDS Samples
Figure 2
100%
0% CH3
CD34+ Normal Controls
Figure 3
1
2
3
CD34+ MDS Samples
1
2
3
Unmethylated
CpG
Methylated
CpG
4
NA
A
Pre-rRNA/GAPDH mRNA
Figure 4
B
Figure 5
A
B
Expression vs. Average Methylation
By Disease Status
Correlation Between rDNA Promoter Methylation
And rRNA Expression: Estimates with 95% CI
Figure 6
D
% Methylation
A
120
THP1 Control
100
THP1 DAC
80
60
40
20
0
CpGs 7-23
E
120
% Methylation
B
F
MM6 Control
MM6 DAC
80
60
40
20
0
120
% Methylation
C
100
100
80
60
40
20
0
ML2 Control
ML2 DAC
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Prepublished online October 15, 2012;
doi:10.1182/blood-2012-04-423111
Reduced ribosomal RNA expression and increased ribosomal DNA
promoter methylation in CD34+ cells of patients with myelodysplastic
syndromes
Aparna Raval, Kunju J. Sridhar, Shripa Patel, Brit B. Turnbull, Peter L. Greenberg and Beverly S. Mitchell
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