Aberrant promotor methylation in MDS hematopoietic cells during in vitro lineage specific differentiation is differently associated with DNMT isoforms.
Aberrant promoter methylation may contribute to the hematopoietic disturbances in myelodysplastic syndromes (MDS). To explore a possible mechanism, we therefore analyzed expression of DNA methyltransferase (DNMT) subtypes kinetics and aberrant promoter methylation of key regulatory genes during MDS hematopoiesis. An in vitro model of MDS lineage-specific hematopoiesis was generated by culturing CD34+ cells from healthy donors (n=7) and MDS patients (low-risk: RA/n=6, RARS/n=3; high-risk: RAEB/n=4, RAEB-T/n=2) with EPO, TPO and GCSF. Promoter methylation analysis of key genes involved in the control of apoptosis (p73, survivin, DAPK), DNA-repair (hMLH1), differentiation (RARb, WT1) and cell cycle control (p14, p15, p16, CHK2) was performed by methylation specific PCR of bisulfite-treated genomic DNA. Expression of DNMT1, DNMT3a and DNMT3b was analyzed and correlated with gene promoter methylation for each lineage at different time points. DNMT expression (all isoforms) was increased during thrombopoiesis whereas elevated DNMT1 level were seen during erythropoiesis. Associations between aberrant promoter methylation and DNMT expression were found in high-risk MDS for all lineages and during erythropoiesis. Hypermethylation of p15, p16, p73, survivin, CHK2, RARb and DAPK were associated with elevated DNMT isoform expression. No general overexpression of DNMT subtype was detected during MDS hematopoiesis. However a negative association of DNMT3a and 3b expression with MDS disease risk (IPSS) could be observed. Our data indicate that all mammalian DNMT isoforms may be involved in the aberrantly methylated phenotype in MDS but seem also to be essential for the differentiation of normal hematopoietic stem cells. In particular elevated DNMT1 expression may in particular contribute to ineffective erythropoiesis in MDS.
Hopfer
Olaf
O
Department of Hematology, Oncology and Transfusion Medicine, Charité University Hospital, Campus Benjamin Franklin, 12203 Berlin, Germany.
Komor
Martina
M
Koehler
Ina Sabine
IS
Freitag
Claudia
C
Schulze
Matthias
M
Hoelzer
Dieter
D
Thiel
Eckhard
E
Hofmann
Wolf-Karsten
WK
D017209:Apoptosis / Q000235:genetics; D016022:Case-Control Studies; D002453:Cell Cycle / Q000235:genetics; D002454:Cell Differentiation; D002478:Cells, Cultured; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000032:analysis*; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D004260:DNA Repair / Q000235:genetics; D004920:Erythropoiesis; D006410:Hematopoiesis; D006412:Hematopoietic Stem Cells / Q000473:pathology*; D006801:Humans; D009190:Myelodysplastic Syndromes / Q000235:genetics* / Q000473:pathology; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms / Q000032:analysis; D000097584:DNA Methyltransferase 3B
D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D020033:Protein Isoforms; D000097584:DNA Methyltransferase 3B; C000717967:DNMT3A protein, human; C000619380:DNMT1 protein, human