Dynamics of alpha-globin locus chromatin structure and gene expression during erythroid differentiation of human CD34(+) cells in culture.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Mahajan
Milind C
MC
Department of Genetics, The Anlyan Center, Yale University School of Medicine, New Haven, CT 06510, USA.
Karmakar
Subhradip
S
Newburger
Peter E
PE
Krause
Diane S
DS
Weissman
Sherman M
SM
D018952:Antigens, CD34 / Q000032:analysis; D000076246:CCCTC-Binding Factor; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D042002:Chromatin Assembly and Disassembly / Q000502:physiology*; D004742:Enhancer Elements, Genetic / Q000235:genetics; D015672:Erythroid Precursor Cells / Q000187:drug effects* / Q000378:metabolism; D004920:Erythropoiesis / Q000235:genetics*; D004921:Erythropoietin / Q000494:pharmacology*; D050982:GATA1 Transcription Factor / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D006021:Glycophorins / Q000096:biosynthesis / Q000235:genetics; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology; D006657:Histones / Q000378:metabolism; D006801:Humans; D038101:Insulator Elements / Q000235:genetics; D050984:NF-E2 Transcription Factor, p45 Subunit / Q000378:metabolism; D011485:Protein Binding; D012319:RNA Polymerase II / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D055542:alpha-Globins / Q000235:genetics*
D018952:Antigens, CD34; D000076246:CCCTC-Binding Factor; C000619697:CTCF protein, human; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; C409972:GYPA protein, human; D006021:Glycophorins; D016298:Hematopoietic Cell Growth Factors; D006657:Histones; D050984:NF-E2 Transcription Factor, p45 Subunit; C494692:NFE2 protein, human; D012097:Repressor Proteins; D014157:Transcription Factors; D055542:alpha-Globins; D004921:Erythropoietin; D012319:RNA Polymerase II