Unknown,Transcriptomics,Genomics,Proteomics

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Epigenetic and transcriptional control in hematopoietic development and lineage differentiation

ABSTRACT: Hematopoiesis is a well-established model system to study molecular mechanisms of lineage-specific differentiation. Key transcription factors (TFs), such as PU.1, NF-E2 and GATA1 are implicated in crucial aspects of distinct hematopoietic lineages. How TFs collaborate with histone modificatons and how they affect the chromatin status remains to be elucidated. Chromatin-Immunoprecipitation followed by next-generation sequencing (ChIP-seq) has been proven to be an excellent tool to study chromatin modifications genome-wide. In this study, ChIP-seq was used to investigate the H3K4me2 landscape at enhancers during hematopoietic differentiation, starting from the hematopoietic stem cell (HSC) up to the fully differentiated erythrocyte, megakaryocyte or granulocyte. Although cell morphology and gene expression profiles differ extensively in committed erythrocytes, megakaryocytes and granulocytes, the genomic landscape of H3K4me2 is surprisingly alike across the three cell types. Granulocytes, in particular, seem to display an ‘erythocyte-like’ chromatin pattern. Similar results were observed for another chromatin mark, H3K27ac. Unexpectedly, the common progenitors of erythrocytes and granulocytes do not display a similar chromatin pattern, excluding the idea that the H3K4me2 mark is placed early on during differentiation. These results also suggest that the chromatin state is probably not the determining factor for lineage differentiation, but that rather lineage specific TF binding is important. In agreement with this, mature megakaryocytes loose the H3K4me2 mark surrounding erythrocyte-specific genes. This might be explained because megakaryocytes and erythrocytes both express the lineage specific TFs GATA1 and NF-E2. Altogether, we show that committed granulocytes and erythrocytes display similar H3K4me2 and H3K27ac patterns, and that those marks alone cannot predict which genes will be expressed eventually. Genomic ChIP-Seq on key transcription factors and histone modification marks in hematopoiesis

ORGANISM(S): Mus musculus

SUBMITTER: Chongzhi Zang

PROVIDER: E-GEOD-42110 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Active enhancers are delineated de novo during hematopoiesis, with limited lineage fidelity among specified primary blood cells.

Luyten Annouck A Zang Chongzhi C Liu X Shirley XS Shivdasani Ramesh A RA

Genes & development 20140801 16

Tissues may adopt diverse strategies to establish specific transcriptional programs in daughter lineages. In intestinal crypts, enhancers for genes expressed in both major cell types appear broadly permissive in stem and specified progenitor cells. In blood, another self-renewing tissue, it is unclear when chromatin becomes permissive for transcription of genes expressed in distinct terminal lineages. Using chromatin immunoprecipitation (ChIP) combined with deep sequencing (ChIP-seq) to profile ...[more]

PMID: 25128499

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Project description:Background: Children with Down syndrome (DS) are predisposed to acute megakaryoblastic leukemia (AMKL, or AML M7) and a related myeloid disorder, referred to as transient myeloproliferative disorder (TMD), or transient leukemia (TL). Recently, acquired mutations in the erythroid/megakaryocytic lineage-specific transcription factor GATA-1 have been identified in megakaryoblasts from virtually all DS patients with AMKL (DS-AMKL), as well as in nearly all DS neonates with TMD. These mutations prevent synthesis of full-length GATA-1, but lead to synthesis of a truncated GATA-1 protein (GATA-1s) that lacks the N-terminal transactivation domain. To determine the in vivo requirement of GATA-1 N-terminal domain in hematopoiesis and to model DS megakaryocytic leukemia initiated by GATA-1 mutation, we generated a GATA-1 knockin allele with a truncation at this domain (GATA-1deltaN). ES cells bearing this mutant allele generated a unique type of large Thrombopoietin (Tpo)-dependent megakaryocytes-containing colonies, when differentiated in vitro. Fetal livers from mice carrying this mutant allele contained elevated numbers of CD41+ cells throughout embryonic development, especially during mid-gestation. In in vitro hematopoietic colony assays, mutant cells from yolk sacs and mid-gestation fetal livers gave rise to a large number of macroscopic hyperproliferative megakaryocytic colonies (CFU-MKs). Consistent with that, when mutant fetal liver progenitors were cultured in liquid medium in the presence of Tpo, the derived megakaryocytes displayed marked hyperproliferation and grew for longer period of time than WTs. Specific aims: To understand the molecular basis for the megakaryocytic hyperproliferation phenotype of the GATA-1deltaN mutants, we plan to profile gene expression patterns of fetal liver-derived primary megakaryocytes and megakaryocytic progenitors from the GATA-1deltaN mutants and compare them with WTs, as well as GATA-1deltaNeodeltaHS mutants (GATA-1megakaryocytes). Experimental design: E12.5 fetal livers were harvested from either GATA-1 WT and mutant embryos. Single cell suspensions were made from these livers and the cells were cultured in liquid medium in the presence of Tpo for 3 days. On day 3, primary megakaryocytes were enriched by a BSA gradient, and further enriched by anti-CD41 antibody and magnetic microbeads. Megakaryocytic progenitors were collected by FACS sorting for small cells in the same culture that express low level of CD41. Total RNAs were then prepared from these two populations and submitted for microarray profiling. Conclusions: When examining genes that are normally upregulated upon megakaryocyte differentiation (many of this class are megakaryocyte-specific genes), we found in GATA-1deltaN megakaryocytes, the majority of these genes are upregulated, although not to the full extent as seen in WTs. In contrast, this class of genes is largely not upregulated in GATA-1deltaNeodeltaHS megakaryocytes, which are blocked at an immature stage of differentiation. Genes that are downregulated during megakaryocyte differentiation include those that are normally repressed by GATA-1. Among genes that are not properly downregulated in GATA-1deltaN megakaryocytes, five transcription factors (Myc, Myb, GATA-2, PU.1, Ikaros) are of particular interest. Inadequate GATA-1s-mediated repression of transcription factors required for proliferation of hematopoietic progenitors (Myc, Myb and GATA-2) and for specifying lineage choices other than megakaryocyte/erythrocyte (PU.1 and Ikaros) may contribute to hyperproliferation of GATA-1deltaN megakaryocytes.

Project description:Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts, and can be preserved in vitro in a naive inner-cell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSK3b signalling (termed 2i/LIF conditions). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer, retaining a pre-inactivation X chromosome state, global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters.Upon withdrawal of 2i/LIF, naM-CM-/ve mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast. Although human ES cells share several molecular features with naive mouse ES cells, they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs). These include use of the proximal enhancer element to maintain OCT4 expression, pronounced tendency for X chromosome inactivation in most female human ES cells, increase in DNA methylation and prominent deposition of H3K27me3 and bivalency acquisition on lineage regulatory genes. The feasibility for establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in rodent ES cells remains to be defined. Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells, from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts. The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells, and distinct from conventional primed human pluripotent cells. This includes competence in the generation of cross-species chimaeric embryos that underwent organogenesis following microinjection of human naive iPS cells into mouse morulas. Collectively, our findings establish new avenues for regenerative medicine, patient-specific iPS cell disease modelling and the study of early human development in vitro and in vivo. Four chromatin marks H3K4me1, H3K4me3, H3K27ac and H3K27me3 were measured from 3 cell lines: C1 and WIBR3 (naM-CM-/ve and conventional/primed stem cells), and BGO1 (only naM-CM-/ve stem cells).

Dataset Information

Epigenetic and transcriptional control in hematopoietic development and lineage differentiation

Publications

Active enhancers are delineated de novo during hematopoiesis, with limited lineage fidelity among specified primary blood cells.

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