Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

RNAseq of PRMT4KD in human cord blood derived CD34+ cells

ABSTRACT: Defining the role of epigenetic regulators in normal hematopoiesis has become critically important, as recurrent mutations or aberrant expression of these genes has been identified in both myeloid and lymphoid hematological malignancies. We have found that PRMT4, a type I arginine methyltransferase, whose function in normal and malignant hematopoiesis is unknown, is overexpressed in AML patient samples. In support of an oncogenic role for PRMT4, we find that its overexpression blocks the myeloid differentiation of human stem/progenitor cells (HSPCs) while its knockdown (KD) is sufficient to induce myeloid differentiation of HSPCs and multiple AML cell lines. Although classically thought of as a co-activator, we found that PRMT4 functions to repress the expression of miR-223 in HSPCs via the methylation of RUNX1, which triggers the assembly of a multi-protein repressor complex that includes DPF2. As part of a feedback loop, PRMT4 expression is repressed post-transcriptionally by miR-223 during the normal differentiation process. These data reveal an unidentified role of PRMT4 in myeloid differentiation and its unexpected repressive role in transcriptional regulation. Furthermore, depletion of PRMT4 results in the differentiation of myeloid leukemia cells in vitro and their decrease proliferation in vivo. Thus, targeting PRMT4 holds potential as a novel therapy for acute myelogenous leukemia. Purified human primary CD34+ cells were transduced with lentiviruses carrying PRMT4KD or scramble control shRNAs. Total RNA was extrated. RNAseq was performed to identify target genes that are regulated by PRMT4. Experiments were performed in triplicate.

ORGANISM(S): Homo sapiens

SUBMITTER: Bic MSKCC

PROVIDER: E-GEOD-46056 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

PRMT4 blocks myeloid differentiation by assembling a methyl-RUNX1-dependent repressor complex.

Vu Ly P LP Perna Fabiana F Wang Lan L Voza Francesca F Figueroa Maria E ME Tempst Paul P Erdjument-Bromage Hediye H Gao Rui R Chen Sisi S Paietta Elisabeth E Deblasio Tony T Melnick Ari A Liu Yan Y Zhao Xinyang X Nimer Stephen D SD

Cell reports 20131212 6

Defining the role of epigenetic regulators in hematopoiesis has become critically important, because recurrent mutations or aberrant expression of these genes has been identified in both myeloid and lymphoid hematological malignancies. We found that PRMT4, a type I arginine methyltransferase whose function in normal and malignant hematopoiesis is unknown, is overexpressed in acute myelogenous leukemia patient samples. Overexpression of PRMT4 blocks the myeloid differentiation of human stem/proge ...[more]

PMID: 24332853

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Project description:BCOR is a component of a variant Polycomb group repressive complex 1 (PRC1) complex. Recently, we and others reported recurrent somatic BCOR loss-of-function mutations in myelodysplastic syndrome and acute myelogenous leukaemia (AML). However the role of BCOR in normal hematopoiesis is largely unknown. Here, we explored the function of BCOR in myeloid cells using myeloid murine cell models with Bcor conditional loss-of-function or overexpression alleles. Bcor mutant bone marrow cells showed significantly higher proliferation and differentiation rates with reduced protein levels of RING1B, a ubiquitin ligase subunit of PRC1 family complexes. Global RNA expression profiling in murine cells and AML patient samples with BCOR loss-of-function mutation suggested that loss of BCOR expression is associated with proliferation and myeloid differentiation and decreased stem cell quiescence. Further, we used a MLL-AF9 murine model of AML and found that loss of Bcor increased serial replating efficiency, enhanced MLL-AF9 in blocking cell differentiation, and increased expression of Evi1 which is associated with leukemic transformation. Our results strongly suggest that BCOR plays an indispensable role in maintaining hematopoietic stem cell (HSC) quiescence by inhibiting myeloid stem cell proliferation and differentiation and offer a mechanistic explanation for how BCOR regulates gene expression such as Hox genes. We took advantage of a mouse conditional Bcor allele which has exons 9 and 10 been flanked by LoxP sites to allow their removal via expression of Cre recombinase. Excision of these exons results in a frame shift and a premature stop codon causing nonsense mediated decay and/or carboxy-terminal deletion of the BCOR protein. CD34+ cells were sorted from control or Bcor mutant cells cultured under myeloid stem/progenitor conditions (IL3, 6 and SCF). We used microarrays to detail the global programme of gene expression in Control and Bcor mutant CD34+ cells.

Project description:In vitro generation of mature neutrophils from human induced pluripotent stem cells (iPSCs) requires hematopoietic progenitor development followed by myeloid differentiation. The purpose of our studies was to extensively characterize this process, focusing on the critical window of development between hemogenic endothelium, hematopoietic stem/progenitor cells (HSPCs), and myeloid commitment, to identify associated regulators and markers that might enable the stem cell field to improve the efficiency and efficacy of iPSC hematopoiesis. We utilized a 4-stage differentiation protocol involving: embryoid body (EB) formation (Stage-1); EB culture with hematopoietic cytokines (Stage-2); HSPC expansion (Stage-3); and neutrophil maturation (Stage-4). CD34+CD45- putative hemogenic endothelial cells were observed in Stage-3 cultures, and expressed VEGFR-2/Flk-1/KDR and VE-cadherin endothelial markers, GATA-2, AML1/RUNX1, and SCL/TAL1 transcription factors, and endothelial/HSPC-associated microRNAs miR-24, miR-125a-3p, miR-126/126*, and miR-155. Upon further culture, CD34+CD45- cells generated CD34+CD45+ HSPCs that produced hematopoietic CFUs. Mid-Stage-3 CD34+CD45+ HSPCs exhibited increased expression of GATA-2, AML1/RUNX1, SCL/TAL1, C/EBPÎ±, and PU.1 transcription factors, but exhibited decreased expression of HSPC-associated microRNAs, and failed to engraft in immune-deficient mice. Mid-stage-3 CD34-CD45+ cells maintained PU.1 expression and exhibited increased expression of hematopoiesis-associated miR-142-3p/5p and a trend towards increased miR-223 expression, indicating myeloid commitment. By late Stage-4, increased CD15, CD16b, and C/EBPÎµ expression were observed, with 25-65% of cells exhibiting morphology and functions of mature neutrophils. These studies demonstrate that hematopoiesis and neutrophil differentiation from human iPSCs recapitulates many features of embryonic hematopoiesis and neutrophil production in marrow, but reveals unexpected molecular signatures that may serve as a guide for enhancing iPSC hematopoiesis. miRNA expression profiles were analyzed in iNC-01-3 and iNC-01-4 induced pluripotent stem cell lines at day 0 (undifferentiated iPSCs), day 18 of differentiation (embryoid bodies), and in 3 FACS-sorted cell populations at day 22 of differentiation (CD34+CD45- cells, CD34+CD45+ cells, and CD34-CD45+ cells). Comparative CT analysis was normalized to U6 snRNA and RNU48 control RNAs relative to expression in undifferentiated iPSCs. Data includes 2 files for each sample, one for the card A array and one for the card B array. This includes a total of 32 data files consisting of: 5 replicates for undifferentiated iPSCs (10 files), 2 replicates for EB (4 files), 3 replicates for CD34+CD45- (6 files), 3 replicates for CD34+CD45+ (6 files), and 3 replicates for CD34-CD45+ (6 files).

Dataset Information

RNAseq of PRMT4KD in human cord blood derived CD34+ cells

Publications

PRMT4 blocks myeloid differentiation by assembling a methyl-RUNX1-dependent repressor complex.

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