Epigenetic Dysregulation of GATA1 but Not Downstream Notch Effectors is Involved in MDS Dyserythropoiesis.

Abstract

Myelodysplastic syndromes (MDS) are mainly characterized by dyserythropoiesis resulting in anemia. So far, this pathological hallmark is incompletely understood. Notch signalling has recently been linked to impaired erythropoiesis and megakaryopoiesis of CD34+ progenitor cells, however its role in MDS is unclear. On the other hand, in MDS demethylating therapy results in decreased transfusion dependency, indicating aberrant methylation of key erythroid genes. Therefore, we have analyzed Notch pathway elements and its association with the key erythroid factors GATA1 and BCLxl and examined the methylation of CpGs flanking cis-regulatory elements (including N-box suppressor binding site for HES1 and GATA box) of the GATA1 erythroid promoter in differentiating CD34+ cells selected from MDS patients. We have generated an in-vitro model of MDS lineage-specific hematopoietic differentiation by culturing CD34+ bone marrow 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. Cell harvest was at days 0, 4, 7 and 11. RNA-expression of GATA1, BCLxl, DLK1, Notch1, HES1 and HERP2 was measured by real time RT-PCR (qPCR). GPI was used as a housekeeping gene. DNA methylation at 7 CpGs of the GATA1 gene promoter was quantitatively analyzed by Pyrosequencing (Pyro Mark ID, Biotage, Uppsala, Sweden) of bisulfite treated genomic DNA at any specific time point. In normal erythropoietic cells, RNA expression of GATA1 and of BCLxl was steadily up regulated, particularly during late erythropoietic differentiation. In contrast, during MDS erythropoiesis a loss of typical up regulation of GATA1 (day 11: 2.08 vs. 0.11; p=0.001) and BCLxl (day 11: 7.46 vs. 0.16; p=0.0005) was observed. Notch ligand DLK1 showed increased expression during erythropoiesis particularly in high risk MDS as compared to normal controls (days 4–11: 0.38 vs. 0.03; p=0.02). Furthermore, expression of HES1 was increasing during the course of normal erythropoietic differentiation but not in lineage specific cells from MDS patients (day 11: 0.01 vs. 0.006; p=0.1). No hypomethylation of CpGs flanking repressor HES1 binding site within the 5′-GATA region was detected in MDS erythropoiesis. Interestingly, decremental GATA1 promotor methylation values were seen during normal erythropoiesis matching GATA1 RNA up regulation in contrast to MDS erythropoiesis (day 11: 26% vs. 58%; p=0.00004). Our data show that the critical erythropoietic transcription factor GATA1 as well as the antiapoptotic molecule BCLxl fail to be up regulated during MDS erythropoiesis. The higher residual 5′-GATA1 methylation values in MDS erythropoiesis but decremental loss thereof in normal erythropoiesis suggests a gene dose effect for GATA1 during erythropoiesis being finely tuned by CpG methylation. Its dysregulation may contribute to the ineffective erythropoiesis observed in MDS. However, a transcriptional activation of the Notch pathway leading to increased expression of the GATA1 repressor HES1 and a hypomethylation of its binding site could not be detected in MDS.