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Role of ZBP-89 in human globin gene regulation and erythroid differentiation.

ABSTRACT: The molecular mechanisms underlying erythroid-specific gene regulation remain incompletely understood. Closely spaced binding sites for GATA, NF-E2/maf, and CACCC interacting transcription factors play functionally important roles in globin and other erythroid-specific gene expression. We and others recently identified the CACCC-binding transcription factor ZBP-89 as a novel GATA-1 and NF-E2/mafK interacting partner. Here, we examined the role of ZBP-89 in human globin gene regulation and erythroid maturation using a primary CD34(+) cell ex vivo differentiation system. We show that ZBP-89 protein levels rise dramatically during human erythroid differentiation and that ZBP-89 occupies key cis-regulatory elements within the globin and other erythroid gene loci. ZBP-89 binding correlates strongly with RNA Pol II occupancy, active histone marks, and high-level gene expression. ZBP-89 physically associates with the histone acetyltransferases p300 and Gcn5/Trrap, and occupies common sites with Gcn5 within the human globin loci. Lentiviral short hairpin RNAs knockdown of ZBP-89 results in reduced Gcn5 occupancy, decreased acetylated histone 3 levels, lower globin and erythroid-specific gene expression, and impaired erythroid maturation. Addition of the histone deacetylase inhibitor valproic acid partially reverses the reduced globin gene expression. These findings reveal an activating role for ZBP-89 in human globin gene regulation and erythroid differentiation.

SUBMITTER: Woo AJ

PROVIDER: S-EPMC3186340 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Role of ZBP-89 in human globin gene regulation and erythroid differentiation.

Woo Andrew J AJ Kim Jonghwan J Xu Jian J Huang Hui H Cantor Alan B AB

Blood 20110809 13

The molecular mechanisms underlying erythroid-specific gene regulation remain incompletely understood. Closely spaced binding sites for GATA, NF-E2/maf, and CACCC interacting transcription factors play functionally important roles in globin and other erythroid-specific gene expression. We and others recently identified the CACCC-binding transcription factor ZBP-89 as a novel GATA-1 and NF-E2/mafK interacting partner. Here, we examined the role of ZBP-89 in human globin gene regulation and erythr ...[more]

PMID: 21828133

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Project description:BackgroundThe guanine nucleotide binding protein (G protein)-coupled receptors (GPCRs) regulate cell growth, proliferation and differentiation. G proteins are also implicated in erythroid differentiation, and some of them are expressed principally in hematopoietic cells. GPCRs-linked NO/cGMP and p38 MAPK signaling pathways already demonstrated potency for globin gene stimulation. By analyzing erythroid progenitors, derived from hematopoietic cells through in vitro ontogeny, our study intends to determine early markers and signaling pathways of globin gene regulation and their relation to GPCR expression.ResultsHuman hematopoietic CD34+ progenitors are isolated from fetal liver (FL), cord blood (CB), adult bone marrow (BM), peripheral blood (PB) and G-CSF stimulated mobilized PB (mPB), and then differentiated in vitro into erythroid progenitors. We find that growth capacity is most abundant in FL- and CB-derived erythroid cells. The erythroid progenitor cells are sorted as 100% CD71+, but we did not find statistical significance in the variations of CD34, CD36 and GlyA antigens and that confirms similarity in maturation of studied ontogenic periods. During ontogeny, beta-globin gene expression reaches maximum levels in cells of adult blood origin (176 fmol/μg), while gamma-globin gene expression is consistently up-regulated in CB-derived cells (60 fmol/μg). During gamma-globin induction by hydroxycarbamide, we identify stimulated GPCRs (PTGDR, PTGER1) and GPCRs-coupled genes known to be activated via the cAMP/PKA (ADIPOQ), MAPK pathway (JUN) and NO/cGMP (PRPF18) signaling pathways. During ontogeny, GPR45 and ARRDC1 genes have the most prominent expression in FL-derived erythroid progenitor cells, GNL3 and GRP65 genes in CB-derived cells (high gamma-globin gene expression), GPR110 and GNG10 in BM-derived cells, GPR89C and GPR172A in PB-derived cells, and GPR44 and GNAQ genes in mPB-derived cells (high beta-globin gene expression).ConclusionsThese results demonstrate the concomitant activity of GPCR-coupled genes and related signaling pathways during erythropoietic stimulation of globin genes. In accordance with previous reports, the stimulation of GPCRs supports the postulated connection between cAMP/PKA and NO/cGMP pathways in activation of γ-globin expression, via JUN and p38 MAPK signaling.

Project description:The aim of the present study has been to establish serum-free culture conditions for ex vivo expansion and differentiation of human CD34(+) cells into erythroid lineage and to study the chromatin structure, gene expression, and transcription factor recruitment at the alpha-globin locus in the developing erythron.A basal Iscove's modified Dulbecco's medium cell culture medium with 1% bovine serum albumin as a serum replacement and a combination of cytokines and growth factors was used for expansion and differentiation of the CD34(+) cells. Expression patterns of the alpha- and beta-like genes at various stages of erythropoiesis was studied by reverse transcriptase quantitative polymerase chain reaction analysis, profile of key erythroid transcription factors was investigated by Western blotting, and the chromatin structure and transcription factor recruitment at the alpha-globin locus was investigated by chromatin immunoprecipitation quantitative polymerase chain reaction analysis.Human CD34(+) cells in the serum-free medium undergo near synchronous erythroid differentiation to yield large amount of cells at different differentiation stages. We observe distinct patterns of the histone modifications and transcription factor binding at the alpha-globin locus during erythroid differentiation of CD34(+) cells. Nuclear factor erythroid-derived 2 (NF-E2) was present at upstream activator sites even before addition of erythropoietin (EPO), while bound GATA-1 was only detectable after EPO treatment. After 7 days of EPO treatment, H3K4Me2 modification uniformly increases throughout the alpha-globin locus. Acetylation at H3K9 and binding of Pol II, NF-E2, and GATA-1 were restricted to certain hypersensitive sites of the enhancer and theta gene, and were conspicuously low at the alpha-like globin promoters. Rearrangement of the insulator binding factor CTCF took place at and around the alpha-globin locus as CD34(+) cells differentiated into erythroid pathway.Our results indicate that remodeling of the upstream elements may be the primary event in activation of alpha-globin gene expression. Activation of alpha-globin genes upon EPO treatment involves initial binding of Pol II, downregulation of pre-existing factors like NF-E2, removal of CTCF from the locus, then rebinding of CTCF in an altered pattern, and concurrent or subsequent binding of transcription factors like GATA-1.

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Role of ZBP-89 in human globin gene regulation and erythroid differentiation.

Publications

Role of ZBP-89 in human globin gene regulation and erythroid differentiation.

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