Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

The comparison of CpG methylation profiles between CD34+ and CD34- human umbilical cord blood cells

ABSTRACT: Evidence for the epigenetic regulation of hematopoietic stem cells (HSCs) is growing, but the genome-wide epigenetic signature of HSCs and its functional significance remain unclear. In this study, we used high-resolition (Agilent, CpG Island ChIP-on-chip) platforms for genome-wide comparison of CpG methylation in human CD34+ and CD34- cells. CD34+ and CD34- cells were purified from human umbilical cord blood. Genomic DNA obtained from CD34+ and CD34- cells was fragmented by either sonication or digestion with MseI. Recombinant methyl-binding domain (Genomictree, Daejeon, Korea) was incubated with sonicated genomic DNA in a binding reaction mixture. The methyl-enriched DNA fraction was purified using Qiaquick PCR purification kits (Qiagen, Hilden, Germany) and amplified using a whole genome amplification kit as recommended by Agilent. Amplified DNA products from CD34+ and CD34- cells were labeled with Cy3-dUTP and Cy5-dUTP, respectively, by random priming and hybridized onto Agilent human CpG island microarrays using conditions specified by the manufacturer (Agilent, Santa Clara, CA). After washing, the slides were scanned using an Agilent scanner and images were quantified using the Feature Extraction Software v. 9.3 (Agilent).

ORGANISM(S): Homo sapiens

SUBMITTER: Il-Hoan Oh

PROVIDER: E-GEOD-17833 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Undifferentiated hematopoietic cells are characterized by a genome-wide undermethylation dip around the transcription start site and a hierarchical epigenetic plasticity.

Chung Yun Shin YS Kim Hye Joung HJ Kim Tae-Min TM Hong Sung-Hyun SH Kwon Kyung-Rim KR An Sungwhan S Park Jung-Hoon JH Lee Suman S Oh Il-Hoan IH

Blood 20090914 24

Evidence for the epigenetic regulation of hematopoietic stem cells (HSCs) is growing, but the genome-wide epigenetic signature of HSCs and its functional significance remain unclear. In this study, from a genome-wide comparison of CpG methylation in human CD34(+) and CD34(-) cells, we identified a characteristic undermethylation dip around the transcription start site of promoters and an overmethylation of flanking regions in undifferentiated CD34(+) cells. This "bivalent-like" CpG methylation p ...[more]

PMID: 19752395

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Project description:We show the molecular and functional characterization of a novel population of lineage-negative CD34-negative (Lin- CD34-) hematopoietic stem cells (HSCs) from chronic myelogenous leukemia (CML) patients at diagnosis. Molecular caryotyping and quantitative analysis of BCR/ABL transcript demonstrated that about one third of CD34- was leukemic. CML CD34- cells showed kinetic quiescence and limited clonogenic capacity. However, stroma-dependent cultures and cytokines induced CD34 expression on some HSCs, cell cycling, acquisition of clonogenic activity and increased expression of BCR/ABL transcript. CML CD34- cells showed an engraftment rate in immunodeficient mice similar to that of CD34+ cells. Gene expression profiling revealed the down-regulation of cell cycle arrest genes together with genes involved in antigen presentation and processing, while the expression of angiogenic factors was strongly up-regulated when compared to normal counterparts. Flow cytometry analysis confirmed the significant down-regulation of HLA class I and II molecules in CML CD34-cells. Increasing doses of imatinib mesilate (IM) did not affect fusion transcript levels, BCR-ABL kinase activity and the clonogenic efficiency of CML CD34- cells as compared to leukemic CD34+cells. Thus, we identified in CML a novel CD34- leukemic stem cell subset with peculiar molecular and functional characteristics which may be a potential target for CML therapeutics. Leukemic cells were obtained from 12 chronic phase Ph+ CML patients at diagnosis and before treatment. Normal samples were leukapheresis products from 12 healthy stem cell donors receiving recombinant human granulocyte colony-stimulating factor (G-CSF; Lenograstim, Sanofi-Aventis, Milan, Italy). The protocol was approved by the ethical committee of the University Hospital and each patient/donor gave written informed consent. Hemopoietic stem/progenitor cell purification and phenotypic analyses were performed as previously described (Lemoli et al, Br J Haematol, 2003; Lemoli RM et al., Blood, 1997). Aliquots of sorted Lin-CD34-, Lin-CD34+ and Lin+CD34+ were reanalyzed by FacScan (Becton Dickinson, Franklin Lakes, NJ) to assess their purities. Total cellular RNA was extracted from 0.5x105 cells of each sample using RNeasy Micro kit (Qiagen, Valencia, CA) following the protocol supplied by the manufacturer. Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit, Agilent Technologies, Waldbrunn, Germany) were used to determine the concentration and purity/integrity of RNA samples using Agilent 2100 Bioanalyzer. RNAs originating from 12 normal donors or from 12 CML patients were pooled in order to obtain at least 2 mg per sample. One-cycle target labeling assays, as well as the Affymetrix Human HG-U95Av2 GeneChip arrays hybridization, staining, and scanning, were performed, using Affymetrix standard protocols (Affymetrix, Santa Clara,CA).

Dataset Information

The comparison of CpG methylation profiles between CD34+ and CD34- human umbilical cord blood cells

Publications

Undifferentiated hematopoietic cells are characterized by a genome-wide undermethylation dip around the transcription start site and a hierarchical epigenetic plasticity.

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