SUMMARY Hematopoietic stem cell differentiation involves the silencing of self-renewal genes and induction of a specific transcriptional program. Identification of multiple covalent cytosine modifications raises the question of how these derivatized bases influence stem cell commitment. Using a replicative primary human hematopoietic stem/progenitor cell differentiation system, we demonstrate dynamic changes of 5-hydroxymethylcytosine (5-hmC) during stem cell commitment and differentiation to the erythroid line-age. Genomic loci that maintain or gain 5-hmC density throughout erythroid differentiation contain binding sites for erythroid transcription factors and several factors not previously recognized as erythroid-specific factors. The functional importance of 5-hmC was demonstrated by impaired erythroid differentiation, with augmentation of myeloid potential, and disrupted 5-hmC patterning in leukemia patient-derived CD34+ stem/early progenitor cells with TET methylcytosine dioxygenase 2 (TET2) mutations. Thus, chemical conjugation and affinity purification of 5-hmC-enriched sequences followed by sequencing serve as resources for deciphering functional implications for gene expression during stem cell commitment and differentiation along a particular lineage.

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Background: Not much is known about epigenomic changes during the differentiation of human stem cells into mature enucleated red cells. Results: Methylome analysis during human erythropoiesis revealed that global hypomethylation occurs during this process and correlates with transcriptomic changes. Conclusion: Integrative analysis also allowed us to identify novel regulatory areas of the genome. Significance: Progressive functional hypomethylation during human erythroid differentiation changes the current paradigm.

Anemia is the predominant clinical manifestation of myelodysplastic syndromes (MDS). Loss or deletion of chromosome 7 is commonly seen in MDS and leads to a poor prognosis. However, the identity of functionally relevant, dysplasia-causing, genes on 7q remains unclear. Dedicator of cytokinesis 4 (DOCK4) is a GTPase exchange factor, and its gene maps to the commonly deleted 7q region. We demonstrate that DOCK4 is underexpressed in MDS bone marrow samples and that the reduced expression is associated with decreased overall survival in patients. We show that depletion of DOCK4 levels leads to erythroid cells with dysplastic morphology both in vivo and in vitro. We established a novel single-cell assay to quantify disrupted F-actin filament network in erythroblasts and demonstrate that reduced expression of DOCK4 leads to disruption of the actin filaments, resulting in erythroid dysplasia that phenocopies the red blood cell (RBC) defects seen in samples from MDS patients. Reexpression of DOCK4 in −7q MDS patient erythroblasts resulted in significant erythropoietic improvements. Mechanisms underlying F-actin disruption revealed that DOCK4 knockdown reduces ras-related C3 botulinum toxin substrate 1 (RAC1) GTPase activation, leading to increased phosphorylation of the actin-stabilizing protein ADDUCIN in MDS samples. These data identify DOCK4 as a putative 7q gene whose reduced expression can lead to erythroid dysplasia.yelodysplastic syndromes (MDS) are a group of clonal hematopoietic disorders that are characterized by cytopenias caused by ineffective hematopoiesis (1-3). Even though MDS may transform to acute leukemia in one-third of patients who have MDS, cytopenias drive morbidity for most patients (4). Most of the morbidity experienced by these patients is due to low red blood cell (RBC) counts; therefore, studies on the molecular pathogenesis of dysplastic erythropoiesis are critically needed. Cytogenetic studies have shown that stem and progenitor cells in MDS contain deletions in chromosomes 5, 7, 20, and others (5). Deletions of the chromosomal 7q region are seen in 10% of cases and are associated with significantly worse survival (6). These genomic deletions are usually large, and even though some candidate pathogenic genes have been postulated (7), it is not clear which of the deleted genes contribute to the pathogenesis of ineffective erythropoiesis and dysplasia in MDS.In a previous study (8), we had observed that numerous 7q genes, including dedicator of cytokinesis 4 (DOCK4), were deleted or epigenetically silenced in MDS, thus prompting an examination of its role in erythropoiesis in the present study. DOCK4 belongs to a large family of proteins (CED5/DOCK180/MYOBLAST CITY class) that is well conserved in mammals (9-11). They are large proteins (>200 kb) that act as signaling intermediates and provide docking sites for many other signaling molecules. One of the welldescribed functions of DOCK4 is its ability to activate GTPases, such as ras-related C3 botulinum toxin substrate 1 (RAC1) and RAP1, in man...

Even though the Ten-eleven translocation (TET) enzymes catalyze the generation of 5-hydroxymethylcytosines required for lineage commitment and subsequent differentiation of stem cells into erythroid cells, the mechanisms that link extracellular signals to TET activation and DNA hydroxymethylation are unknown. We demonstrate that hematopoietic cytokines phosphorylate TET2, leading to its activation in erythroid progenitors. Specifi cally , cytokine receptorassociated JAK2 phosphorylates TET2 at tyrosines 1939 and 1964. Phosphorylated TET2 interacts with the erythroid transcription factor KLF1, and this interaction with TET2 is increased upon exposure to erythropoietin. The activating JAK2 V617F mutation seen in myeloproliferative disease patient samples and in mouse models is associated with increased TET activity and cytosine hydroxymethylation as well as genome-wide loss of cytosine methylation. These epigenetic and functional changes are also associated with increased expression of several oncogenic transcripts. Thus, we demonstrate that JAK2-mediated TET2 phosphorylation provides a mechanistic link between extracellular signals and epigenetic changes during hematopoiesis. SIGNIFICANCE: Identifi cation of TET2 phosphorylation and activation by cytokine-stimulated JAK2 links extracellular signals to chromatin remodeling during hematopoietic differentiation. This provides potential avenues to regulate TET2 function in the context of myeloproliferative disorders and myelodysplastic syndromes associated with the JAK2 V617F-activating mutation.