title: Krüppel-like Factor 4 activates HBG gene expression in primary erythroid cells.
abstract: The SP1/Krüppel-like Factor (SP1/KLF) family of transcription factors plays a role in diverse cellular processes, including proliferation, differentiation and control of gene transcription. The discovery of KLF1 (EKLF), a key regulator of HBB (β-globin) gene expression, expanded our understanding of the role of KLFs in erythropoiesis. In this study, we investigated a mechanism of HBG (γ-globin) regulation by KLF4. siRNA-mediated gene silencing and enforced expression of KLF4 in K562 cells substantiated the ability of KLF4 to positively regulate endogenous HBG gene transcription. The physiological significance of this finding was confirmed in primary erythroid cells, where KLF4 knockdown at day 11 significantly attenuated HBG mRNA levels and enforced expression at day 28 stimulated the silenced HBG genes. In vitro binding characterization using the γ-CACCC and β-CACCC probes demonstrated KLF4 preferentially binds the endogenous γ-CACCC, while CREB binding protein (CREBBP) binding was not selective. Co-immunoprecipitation studies confirmed protein-protein interaction between KLF4 and CREBBP. Furthermore, sequential chromatin immunoprecipitation assays showed co-localization of both factors in the γ-CACCC region. Subsequent luciferase reporter studies demonstrated that KLF4 trans-activated HBG promoter activity and that CREBBP enforced expression resulted in gene repression. Our data supports a model of antagonistic interaction of KLF4/CREBBP trans-factors in HBG regulation.
authors.affiliation: Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, TX, USA.
authors.forename: Inderdeep S
authors.initials: IS
authors.lastname: Kalra
authors.forename: Md M
authors.initials: MM
authors.lastname: Alam
authors.forename: Pankaj K
authors.initials: PK
authors.lastname: Choudhary
authors.forename: Betty S
authors.initials: BS
authors.lastname: Pace
mesh_terms: D001667:Binding, Competitive; D050882:CREB-Binding Protein / Q000378:metabolism; D002478:Cells, Cultured; D015672:Erythroid Precursor Cells / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology*; D020868:Gene Silencing; D006801:Humans; D020014:K562 Cells; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D015533:Transcriptional Activation / Q000502:physiology; D014407:Tumor Cells, Cultured; D055544:beta-Globins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: C436492:KLF12 protein, human; C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D034741:RNA, Small Interfering; D055544:beta-Globins; D050882:CREB-Binding Protein; C494363:CREBBP protein, human