Transcription Factors KLF1 and KLF2 Positively Regulate Embryonic and Fetal β-Globin Genes through Direct Promoter Binding

Krüppel-like factors (KLFs) control cell differentiation and embryonic development. KLF1 (erythroid Krüppel-like factor) plays essential roles in embryonic and adult erythropoiesis. KLF2 is a positive regulator of the mouse and human embryonic β-globin genes. KLF1 and KLF2 have highly homologous zinc finger DNA-binding domains. They have overlapping roles in embryonic erythropoiesis, as demonstrated using single and double KO mouse models. Ablation of the KLF1 or KLF2 gene causes embryonic lethality, but double KO embryos are more anemic and die sooner than either single KO. In this work, a dual human β-globin locus transgenic and KLF knockout mouse model was used. The results demonstrate that the human ∈- (embryonic) and γ-globin (fetal) genes are positively regulated by KLF1 and KLF2 in embryos. Conditional KO mouse experiments indicate that the effect of KLF2 on embryonic globin gene regulation is at least partly erythroid cell-autonomous. KLF1 and KLF2 bind directly to the promoters of the human ∈- and γ-globin genes, the mouse embryonic Ey- and βh1-globin genes, and also to the β-globin locus control region, as demonstrated by ChIP assays with mouse embryonic blood cells. H3K9Ac and H3K4me3 marks indicate open chromatin and active transcription, respectively. These marks are diminished at the Ey-, βh1-, ∈- and γ-globin genes and locus control region in KLF1−/− embryos, correlating with reduced gene expression. Therefore, KLF1 and KLF2 positively regulate the embryonic and fetal β-globin genes through direct promoter binding. KLF1 is required for normal histone modifications in the β-globin locus in mouse embryos. Krüppel-like factors (KLFs) control cell differentiation and embryonic development. KLF1 (erythroid Krüppel-like factor) plays essential roles in embryonic and adult erythropoiesis. KLF2 is a positive regulator of the mouse and human embryonic β-globin genes. KLF1 and KLF2 have highly homologous zinc finger DNA-binding domains. They have overlapping roles in embryonic erythropoiesis, as demonstrated using single and double KO mouse models. Ablation of the KLF1 or KLF2 gene causes embryonic lethality, but double KO embryos are more anemic and die sooner than either single KO. In this work, a dual human β-globin locus transgenic and KLF knockout mouse model was used. The results demonstrate that the human ∈- (embryonic) and γ-globin (fetal) genes are positively regulated by KLF1 and KLF2 in embryos. Conditional KO mouse experiments indicate that the effect of KLF2 on embryonic globin gene regulation is at least partly erythroid cell-autonomous. KLF1 and KLF2 bind directly to the promoters of the human ∈- and γ-globin genes, the mouse embryonic Ey- and βh1-globin genes, and also to the β-globin locus control region, as demonstrated by ChIP assays with mouse embryonic blood cells. H3K9Ac and H3K4me3 marks indicate open chromatin and active transcription, respectively. These marks are diminished at the Ey-, βh1-, ∈- and γ-globin genes and locus control region in KLF1−/− embryos, correlating with reduced gene expression. Therefore, KLF1 and KLF2 positively regulate the embryonic and fetal β-globin genes through direct promoter binding. KLF1 is required for normal histone modifications in the β-globin locus in mouse embryos. IntroductionErythroid cells are one of the first differentiated cell types in embryos (1Baron M.H. Fraser S.T. Curr. Opin. Hematol. 2005; 12: 217-221Crossref PubMed Scopus (33) Google Scholar). There are two unique processes in development: primitive and definitive erythropoiesis. Primitive erythropoiesis initiates from the extraembryonic mesoderm of the yolk sac as early as embryonic day 7.5 (E7.5) 2The abbreviations used are: E7.5embryonic day 7.5∈human embryonic globin geneγhuman fetal globin geneβhuman adult globin geneCBPCREB-binding proteinPCAFp300/CBP-associated factorLCRlocus control regionHShypersensitive site(s)KLFKrüppel-like factorqRT-PCRquantitative RT-PCRGPAglycophorin AH3K9Achistone 3 lysine 9 acetylationH3K4me3histone 3 lysine 4 trimethylation. in mice (2Qiu C. Olivier E.N. Velho M. Bouhassira E.E. Blood. 2008; 111: 2400-2408Crossref PubMed Scopus (129) Google Scholar, 3McGrath K.E. Palis J. Exp. Hematol. 2005; 33: 1021-1028Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Definitive erythropoiesis is detected in the mouse fetal liver by E11.5 (4Brotherton T.W. Chui D.H. Gauldie J. Patterson M. Proc. Natl. Acad. Sci. U.S.A. 1979; 76: 2853-2857Crossref PubMed Scopus (122) Google Scholar). The human β-globins are encoded by four major genes, ∈ (embryonic), Gγ and Aγ (fetal), and β (adult), located on chromosome 11. The mouse β-globin locus contains four genes, two embryonic (Ey and βh1), and two adult (βmaj and βmin).The expression of the β-globin genes is jointly regulated by elements in the promoter regions and an upstream enhancer region, the locus control region (LCR). The human β-globin LCR, located 6–22 kb upstream of the ∈-globin gene, contains multiple erythroid-specific DNase I hypersensitive sites (HS) and plays a crucial role in maintaining β-globin gene expression (5Grosveld F. van Assendelft G.B. Greaves D.R. Kollias G. Cell. 1987; 51: 975-985Abstract Full Text PDF PubMed Scopus (1430) Google Scholar, 6Fraser P. Hurst J. Collis P. Grosveld F. Nucleic Acids Res. 1990; 18: 3503-3508Crossref PubMed Scopus (140) Google Scholar). Examples of regulatory elements within the promoters of all β-globin genes are TATA, CAAT, and CACCC (7Lloyd J.A. Lee R.F. Lingrel J.B. Nucleic Acids Res. 1989; 17: 4339-4352Crossref PubMed Scopus (18) Google Scholar).Krüppel-like factors (KLFs) are a family of transcription factors that bind GC-rich sequences such as CACCC elements. The KLFs bind DNA via three carboxyl-terminal Cys-2/His-2 zinc fingers (8Bieker J.J. J. Biol. Chem. 2001; 276: 34355-34358Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar). Seventeen mammalian proteins have been identified in this family and are designated KLF1 to KLF17. KLFs are implicated in many cellular functions, such as erythropoiesis, cell differentiation, proliferation, and tissue development (9McConnell B.B. Yang V.W. Physiol. Rev. 2010; 90: 1337-1381Crossref PubMed Scopus (655) Google Scholar). The human and mouse KLF proteins are highly conserved. For example, KLF1 is 73% similar in the two species (although 90% similar within the zinc finger domain), and KLF2 is 90% similar in mouse and man.KLF1, also known as erythroid Krüppel-like factor or EKLF, is expressed only in erythroid cells and plays essential roles in embryonic and adult β-globin gene expression (10Perkins A.C. Sharpe A.H. Orkin S.H. Nature. 1995; 375: 318-322Crossref PubMed Scopus (524) Google Scholar, 11Nuez B. Michalovich D. Bygrave A. Ploemacher R. Grosveld F. Nature. 1995; 375: 316-318Crossref PubMed Scopus (476) Google Scholar, 12Basu P. Lung T.K. Lemsaddek W. Sargent T.G. Williams Jr., D.C. Basu M. Redmond L.C. Lingrel J.B. Haar J.L. Lloyd J.A. Blood. 2007; 110: 3417-3425Crossref PubMed Scopus (48) Google Scholar). KLF1 is a master regulator of adult β-globin gene expression (10Perkins A.C. Sharpe A.H. Orkin S.H. Nature. 1995; 375: 318-322Crossref PubMed Scopus (524) Google Scholar, 11Nuez B. Michalovich D. Bygrave A. Ploemacher R. Grosveld F. Nature. 1995; 375: 316-318Crossref PubMed Scopus (476) Google Scholar). Semiquantitative ChIP assays revealed that HA-tagged KLF1 binds to the promoters of the embryonic β-like globin genes (Ey and βh1), to HS1, HS2, HS3, and HS5 in mouse primitive erythroid cells, and to the promoter of the mouse adult βmaj-globin gene in primitive and definitive cells (13Zhou D. Pawlik K.M. Ren J. Sun C.W. Townes T.M. J. Biol. Chem. 2006; 281: 16052-16057Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). KLF1 interacts with CBP, p300, and PCAF, which have histone acetyltransferase activity (14Zhang W. Bieker J.J. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 9855-9860Crossref PubMed Scopus (326) Google Scholar). KLF1 is also implicated in erythroid processes other than β-globin gene regulation, such as cell maturation and cell membrane integrity (15Hodge D. Coghill E. Keys J. Maguire T. Hartmann B. McDowall A. Weiss M. Grimmond S. Perkins A. Blood. 2006; 107: 3359-3370Crossref PubMed Scopus (169) Google Scholar, 16Pilon A.M. Arcasoy M.O. Dressman H.K. Vayda S.E. Maksimova Y.D. Sangerman J.I. Gallagher P.G. Bodine D.M. Mol. Cell. Biol. 2008; 28: 7394-7401Crossref PubMed Scopus (82) Google Scholar).KLF2, originally known as lung KLF or LKLF, has important roles in T-cell differentiation and blood vessel development (9McConnell B.B. Yang V.W. Physiol. Rev. 2010; 90: 1337-1381Crossref PubMed Scopus (655) Google Scholar). KLF2 is a positive regulator of the mouse and human embryonic β-globin genes (17Basu P. Morris P.E. Haar J.L. Wani M.A. Lingrel J.B. Gaensler K.M. Lloyd J.A. Blood. 2005; 106: 2566-2571Crossref PubMed Scopus (64) Google Scholar). KLF1 and KLF2 have high homology within their DNA-binding domains and reside close to each other on the same chromosome in human and mouse, suggesting that they originated from a gene duplication event (17Basu P. Morris P.E. Haar J.L. Wani M.A. Lingrel J.B. Gaensler K.M. Lloyd J.A. Blood. 2005; 106: 2566-2571Crossref PubMed Scopus (64) Google Scholar). KLF1 and KLF2 can partially functionally compensate for each other in regulating the mouse embryonic β-globin genes. When both KLF1 and KLF2 are simultaneously ablated in mice, the amounts of Ey- and βh1-globin mRNA are reduced more than in KLF1 or KLF2 single knockouts (12Basu P. Lung T.K. Lemsaddek W. Sargent T.G. Williams Jr., D.C. Basu M. Redmond L.C. Lingrel J.B. Haar J.L. Lloyd J.A. Blood. 2007; 110: 3417-3425Crossref PubMed Scopus (48) Google Scholar). Like KLF1, KLF2 recruits proteins with histone acetyltransferase activity such as CBP, p300 and PCAF (18SenBanerjee S. Lin Z. Atkins G.B. Greif D.M. Rao R.M. Kumar A. Feinberg M.W. Chen Z. Simon D.I. Luscinskas F.W. Michel T.M. Gimbrone Jr., M.A. García-Cardeña G. Jain M.K. J. Exp. PubMed Scopus Google and KLF2 regulate the mouse embryonic β-globin genes. In this to KLF1 and KLF2 also control the human embryonic and fetal β-globin genes. to genes in with was to the roles of KLF1 and KLF2 in globin gene In this work, was that KLF1 and KLF2 mRNA are expressed in similar amounts in mouse primitive erythroid cells. The expression of KLF1 but KLF2 mRNA is in definitive erythroid cells. mice that the human β-globin locus K.M. M. Proc. Natl. Acad. Sci. U.S.A. 90: PubMed Scopus (126) Google Scholar, J. Grosveld F. PubMed Scopus Google in with gene used to that KLF1 and KLF2 positively regulate human ∈- and γ-globin gene expression in the KLF2 has an erythroid cell role in embryonic globin gene regulation, also have In ChIP KLF1 and KLF2 bind to the promoters of the mouse embryonic Ey- and βh1-globin and the human ∈- and γ-globin genes in mouse primitive erythroid cells. KLF1, but is required to the normal histone in the mouse and human β-globin in mouse primitive erythroid cells. both KLF1 and KLF2 directly globin gene regulation through the same DNA their of to was originally that KLF1 is required only for adult β-globin gene expression KLF1 KO mice die the embryonic to adult β-globin gene (10Perkins A.C. Sharpe A.H. Orkin S.H. Nature. 1995; 375: 318-322Crossref PubMed Scopus (524) Google Scholar, 11Nuez B. Michalovich D. Bygrave A. Ploemacher R. Grosveld F. Nature. 1995; 375: 316-318Crossref PubMed Scopus (476) Google Scholar). more mRNA in KLF1 KO mouse embryos that KLF1 mouse Ey- and βh1-globin gene expression in primitive erythroid cells (12Basu P. Lung T.K. Lemsaddek W. Sargent T.G. Williams Jr., D.C. Basu M. Redmond L.C. Lingrel J.B. Haar J.L. Lloyd J.A. Blood. 2007; 110: 3417-3425Crossref PubMed Scopus (48) Google Scholar). KLF2 also has a role in mouse Ey- and βh1-globin gene regulation (17Basu P. Morris P.E. Haar J.L. Wani M.A. Lingrel J.B. Gaensler K.M. Lloyd J.A. Blood. 2005; 106: 2566-2571Crossref PubMed Scopus (64) Google Scholar). in transgenic mouse KLF2 the human ∈- and γ-globin genes in the yolk sac (17Basu P. Morris P.E. Haar J.L. Wani M.A. Lingrel J.B. Gaensler K.M. Lloyd J.A. Blood. 2005; 106: 2566-2571Crossref PubMed Scopus (64) Google Scholar). that KLF1 is also required for normal expression of the human embryonic ∈- and fetal γ-globin genes mouse primitive is that KLF2 has an erythroid cell role in mouse embryonic βh1-globin gene as directly the globin genes. KLF1 and KLF2 the promoters of the mouse Ey- and βh1-globin and human ∈- and γ-globin genes, their direct roles in globin gene The ChIP the first that KLF1 binds to the β-globin promoters primitive erythropoiesis. KLF1 at the promoter of the βmaj-globin gene, as was in assays using a KLF1 in primitive erythroid cells (13Zhou D. Pawlik K.M. Ren J. Sun C.W. Townes T.M. J. Biol. Chem. 2006; 281: 16052-16057Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). KLF2 directly binds to the of of cell in cells J. J. P. S. Biol. 2008; PubMed Scopus Google Scholar). that KLF2 is directly to the promoters of the and human embryonic and fetal β-globin genes in primitive erythroid that KLF1 directly as a positive regulator of the human ∈- and γ-globin and mouse Ey- and βh1-globin genes embryonic erythropoiesis. is in to that has an role for KLF1 in γ-globin gene regulation adult erythropoiesis. in the human KLF1 gene have been with of fetal C. D. C. B. A. P. T. G. A. J. G. J. 2010; Full Text Full Text PDF PubMed Scopus Google Scholar, M. K.E. M. Bieker J.J. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: PubMed Scopus Google Scholar, J. P. M. G. P. M. van W. R. van W. Z. J. M. Grosveld M. S. 2010; PubMed Scopus Google Scholar, S. P. A. A. R. PubMed Scopus Google Scholar). was detected in with amounts of C. D. C. B. A. P. T. G. A. J. G. J. 2010; Full Text Full Text PDF PubMed Scopus Google Scholar). in the in the mouse, which has an in βh1-globin expression in the fetal liver and adult M. K.E. M. Bieker J.J. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: PubMed Scopus Google Scholar). is in a family with of fetal The the KLF1 zinc fingers and DNA J. P. M. G. P. M. van W. R. van W. Z. J. M. Grosveld M. S. 2010; PubMed Scopus Google Scholar). an in a family an in the zinc fingers S. P. A. A. R. PubMed Scopus Google Scholar). with the and a have of fetal S. P. A. A. R. PubMed Scopus Google Scholar). The for the effect of KLF1 on γ-globin gene regulation in the adult is via of J. P. M. G. P. M. van W. R. van W. Z. J. M. Grosveld M. S. 2010; PubMed Scopus Google Scholar, D. Sun C.W. Pawlik K.M. Townes T.M. 2010; PubMed Scopus Google Scholar). KLF1 can positively or γ-globin gene regulation, on the erythroid cell expression of KLF1 and KLF2 in primitive and definitive erythroid cells and a for the at the two The of KLF1 to KLF2 mRNA as erythroid cells from the primitive to the definitive KLF1 as a of differentiation genes and cells erythroid differentiation J. Fraser S.T. Z. M.H. Blood. 2010; PubMed Scopus Google Scholar). is that KLF1 is also in the from primitive to definitive erythropoiesis. is that KLF2 erythroid cells embryonic globin gene the role of and KLF2 have a high of homology in their zinc finger domains and can partially compensate for each other in embryonic erythroid cells, they regulate genes (12Basu P. Lung T.K. Lemsaddek W. Sargent T.G. Williams Jr., D.C. Basu M. Redmond L.C. Lingrel J.B. Haar J.L. Lloyd J.A. Blood. 2007; 110: 3417-3425Crossref PubMed Scopus (48) Google Scholar). family of transcription factors is and for example, can compensate or partially for each other erythropoiesis Williams A.M. Orkin S.H. Blood. PubMed Scopus Google Scholar, S. R. T. J. S. M. Blood. PubMed Google Scholar). Primitive erythroid cells are in KO embryos. KO embryos a in the of primitive erythroid both and are primitive erythroid cells are detected Williams A.M. Orkin S.H. Blood. PubMed Scopus Google Scholar). When expressed the control of the regulatory can the KO S. M. M. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar). can transcription factor family for example, positively KLF1 in primitive erythroid cells (12Basu P. Lung T.K. Lemsaddek W. Sargent T.G. Williams Jr., D.C. Basu M. Redmond L.C. Lingrel J.B. Haar J.L. Lloyd J.A. Blood. 2007; 110: 3417-3425Crossref PubMed Scopus (48) Google Scholar). is by KLF1 and by M. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). KLF1 also can by directly to promoter Keys J. M. Perkins A.C. M. Mol. Cell. Biol. 2007; PubMed Scopus Google Scholar). in definitive erythroid cells and G. Orkin S.H. PubMed Scopus Google Scholar, J.A. S. J. Weiss Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar, Blood. 2005; 106: PubMed Scopus Google indicate that at and H3K4me3 are at the globin genes, mouse and and human ∈ and have that H3K9Ac and H3K4me3 marks with each other in the human β-globin locus adult erythropoiesis C. J.A. G. P. F. R. M. M. Mol. Cell. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). the first in embryonic erythroid cells that H3K9Ac is only with the globin genes. In in a of mouse embryonic erythropoiesis using an that both H3K9Ac and and was at both the embryonic and adult β-globin genes G. M. Biol. PubMed Scopus Google Scholar). In the human β-globin and are detected within the LCR, and only at the active β-globin genes G. M. Biol. PubMed Scopus Google Scholar, W. P. G. J. Mol. Biol. PubMed Scopus Google Scholar). In a human cell that the fetal but the adult β-globin gene, and H3K4me3 are at the γ-globin gene A. A. Mol. Cell. Biol. 2007; PubMed Scopus Google Scholar). H3K4me3 is only in the fetal and adult β-globin genes in human and mouse tissue cell A. A. Mol. Cell. Biol. 2007; PubMed Scopus Google Scholar, G. C. R. J. S. M. M.A. Palis J. M. Blood. PubMed Scopus Google Scholar). results in embryonic erythroid cells in that H3K4me3 is at the mouse adult βmaj-globin gene at to binds to proteins such as CBP, p300, and PCAF (14Zhang W. Bieker J.J. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 9855-9860Crossref PubMed Scopus (326) Google Scholar, S. Lin Z. Atkins G.B. Greif D.M. Rao R.M. Kumar A. Feinberg M.W. Chen Z. Simon D.I. Luscinskas F.W. Michel T.M. Gimbrone Jr., M.A. García-Cardeña G. Jain M.K. J. Exp. PubMed Scopus Google Scholar, Nucleic Acids Res. PubMed Scopus Google Scholar). indicate for the first that H3K9Ac and H3K4me3 are reduced at the mouse Ey- and βh1-globin and human ∈- and γ-globin genes in primitive erythroid cells factors have been implicated in histone The of with histone and H3K4me3 at the β-globin locus J.A. Bieker J.J. Proc. Natl. Acad. Sci. U.S.A. 2005; PubMed Scopus Google Scholar). and H3K4me3 are reduced in the adult β-globin gene by the of KLF1 in fetal liver cells S. J. E. J. 2006; PubMed Scopus Google Scholar). is to and effect in is known reduced transcription of the of the transcription factor to and H3K4me3 or is a the of KLF1 and the of histone marks in model the is that KLF1 and KLF2 bind to the same CACCC elements in the and promoters in the β-globin locus and gene expression in the model is by the that KLF1 and KLF2 have similar DNA that is that they can and that they are to in similar amounts in embryonic blood cells. the effect of KLF1 on globin gene regulation to than that of on in the mouse KO models. this a with results histone marks in KLF1−/− and embryonic blood cells. In KLF1 with histone marks that are with transcription, KLF2 to histone modifications in the β-globin both KLF1 and KLF2 positively regulate globin gene transcription through direct to CACCC elements in the Therefore, are overlapping roles for KLF1 and KLF2 in embryonic blood cells. IntroductionErythroid cells are one of the first differentiated cell types in embryos (1Baron M.H. Fraser S.T. Curr. Opin. Hematol. 2005; 12: 217-221Crossref PubMed Scopus (33) Google Scholar). There are two unique processes in development: primitive and definitive erythropoiesis. Primitive erythropoiesis initiates from the extraembryonic mesoderm of the yolk sac as early as embryonic day 7.5 (E7.5) 2The abbreviations used are: E7.5embryonic day 7.5∈human embryonic globin geneγhuman fetal globin geneβhuman adult globin geneCBPCREB-binding proteinPCAFp300/CBP-associated factorLCRlocus control regionHShypersensitive site(s)KLFKrüppel-like factorqRT-PCRquantitative RT-PCRGPAglycophorin AH3K9Achistone 3 lysine 9 acetylationH3K4me3histone 3 lysine 4 trimethylation. in mice (2Qiu C. Olivier E.N. Velho M. Bouhassira E.E. Blood. 2008; 111: 2400-2408Crossref PubMed Scopus (129) Google Scholar, 3McGrath K.E. Palis J. Exp. Hematol. 2005; 33: 1021-1028Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Definitive erythropoiesis is detected in the mouse fetal liver by E11.5 (4Brotherton T.W. Chui D.H. Gauldie J. Patterson M. Proc. Natl. Acad. Sci. U.S.A. 1979; 76: 2853-2857Crossref PubMed Scopus (122) Google Scholar). The human β-globins are encoded by four major genes, ∈ (embryonic), Gγ and Aγ (fetal), and β (adult), located on chromosome 11. The mouse β-globin locus contains four genes, two embryonic (Ey and βh1), and two adult (βmaj and βmin).The expression of the β-globin genes is jointly regulated by elements in the promoter regions and an upstream enhancer region, the locus control region (LCR). The human β-globin LCR, located 6–22 kb upstream of the ∈-globin gene, contains multiple erythroid-specific DNase I hypersensitive sites (HS) and plays a crucial role in maintaining β-globin gene expression (5Grosveld F. van Assendelft G.B. Greaves D.R. Kollias G. Cell. 1987; 51: 975-985Abstract Full Text PDF PubMed Scopus (1430) Google Scholar, 6Fraser P. Hurst J. Collis P. Grosveld F. Nucleic Acids Res. 1990; 18: 3503-3508Crossref PubMed Scopus (140) Google Scholar). Examples of regulatory elements within the promoters of all β-globin genes are TATA, CAAT, and CACCC (7Lloyd J.A. Lee R.F. Lingrel J.B. Nucleic Acids Res. 1989; 17: 4339-4352Crossref PubMed Scopus (18) Google Scholar).Krüppel-like factors (KLFs) are a family of transcription factors that bind GC-rich sequences such as CACCC elements. The KLFs bind DNA via three carboxyl-terminal Cys-2/His-2 zinc fingers (8Bieker J.J. J. Biol. Chem. 2001; 276: 34355-34358Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar). Seventeen mammalian proteins have been identified in this family and are designated KLF1 to KLF17. KLFs are implicated in many cellular functions, such as erythropoiesis, cell differentiation, proliferation, and tissue development (9McConnell B.B. Yang V.W. Physiol. Rev. 2010; 90: 1337-1381Crossref PubMed Scopus (655) Google Scholar). The human and mouse KLF proteins are highly conserved. For example, KLF1 is 73% similar in the two species (although 90% similar within the zinc finger domain), and KLF2 is 90% similar in mouse and man.KLF1, also known as erythroid Krüppel-like factor or EKLF, is expressed only in erythroid cells and plays essential roles in embryonic and adult β-globin gene expression (10Perkins A.C. Sharpe A.H. Orkin S.H. Nature. 1995; 375: 318-322Crossref PubMed Scopus (524) Google Scholar, 11Nuez B. Michalovich D. Bygrave A. Ploemacher R. Grosveld F. Nature. 1995; 375: 316-318Crossref PubMed Scopus (476) Google Scholar, 12Basu P. Lung T.K. Lemsaddek W. Sargent T.G. Williams Jr., D.C. Basu M. Redmond L.C. Lingrel J.B. Haar J.L. Lloyd J.A. Blood. 2007; 110: 3417-3425Crossref PubMed Scopus (48) Google Scholar). KLF1 is a master regulator of adult β-globin gene expression (10Perkins A.C. Sharpe A.H. Orkin S.H. Nature. 1995; 375: 318-322Crossref PubMed Scopus (524) Google Scholar, 11Nuez B. Michalovich D. Bygrave A. Ploemacher R. Grosveld F. Nature. 1995; 375: 316-318Crossref PubMed Scopus (476) Google Scholar). Semiquantitative ChIP assays revealed that HA-tagged KLF1 binds to the promoters of the embryonic β-like globin genes (Ey and βh1), to HS1, HS2, HS3, and HS5 in mouse primitive erythroid cells, and to the promoter of the mouse adult βmaj-globin gene in primitive and definitive cells (13Zhou D. Pawlik K.M. Ren J. Sun C.W. Townes T.M. J. Biol. Chem. 2006; 281: 16052-16057Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). KLF1 interacts with CBP, p300, and PCAF, which have histone acetyltransferase activity (14Zhang W. Bieker J.J. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 9855-9860Crossref PubMed Scopus (326) Google Scholar). KLF1 is also implicated in erythroid processes other than β-globin gene regulation, such as cell maturation and cell membrane integrity (15Hodge D. Coghill E. Keys J. Maguire T. Hartmann B. McDowall A. Weiss M. Grimmond S. Perkins A. Blood. 2006; 107: 3359-3370Crossref PubMed Scopus (169) Google Scholar, 16Pilon A.M. Arcasoy M.O. Dressman H.K. Vayda S.E. Maksimova Y.D. Sangerman J.I. Gallagher P.G. Bodine D.M. Mol. Cell. Biol. 2008; 28: 7394-7401Crossref PubMed Scopus (82) Google Scholar).KLF2, originally known as lung KLF or LKLF, has important roles in T-cell differentiation and blood vessel development (9McConnell B.B. Yang V.W. Physiol. Rev. 2010; 90: 1337-1381Crossref PubMed Scopus (655) Google Scholar). KLF2 is a positive regulator of the mouse and human embryonic β-globin genes (17Basu P. Morris P.E. Haar J.L. Wani M.A. Lingrel J.B. Gaensler K.M. Lloyd J.A. Blood. 2005; 106: 2566-2571Crossref PubMed Scopus (64) Google Scholar). KLF1 and KLF2 have high homology within their DNA-binding domains and reside close to each other on the same chromosome in human and mouse, suggesting that they originated from a gene duplication event (17Basu P. Morris P.E. Haar J.L. Wani M.A. Lingrel J.B. Gaensler K.M. Lloyd J.A. Blood. 2005; 106: 2566-2571Crossref PubMed Scopus (64) Google Scholar). KLF1 and KLF2 can partially functionally compensate for each other in regulating the mouse embryonic β-globin genes. When both KLF1 and KLF2 are simultaneously ablated in mice, the amounts of Ey- and βh1-globin mRNA are reduced more than in KLF1 or KLF2 single knockouts (12Basu P. Lung T.K. Lemsaddek W. Sargent T.G. Williams Jr., D.C. Basu M. Redmond L.C. Lingrel J.B. Haar J.L. Lloyd J.A. Blood. 2007; 110: 3417-3425Crossref PubMed Scopus (48) Google Scholar). Like KLF1, KLF2 recruits proteins with histone acetyltransferase activity such as CBP, p300 and PCAF (18SenBanerjee S. Lin Z. Atkins G.B. Greif D.M. Rao R.M. Kumar A. Feinberg M.W. Chen Z. Simon D.I. Luscinskas F.W. Michel T.M. Gimbrone Jr., M.A. García-Cardeña G. Jain M.K. J. Exp. PubMed Scopus Google and KLF2 regulate the mouse embryonic β-globin genes. In this to KLF1 and KLF2 also control the human embryonic and fetal β-globin genes. to genes in with was to the roles of KLF1 and KLF2 in globin gene In this work, was that KLF1 and KLF2 mRNA are expressed in similar amounts in mouse primitive erythroid cells. The expression of KLF1 but KLF2 mRNA is in definitive erythroid cells. mice that the human β-globin locus K.M. M. Proc. Natl. Acad. Sci. U.S.A. 90: PubMed Scopus (126) Google Scholar, J. Grosveld F. PubMed Scopus Google in with gene used to that KLF1 and KLF2 positively regulate human ∈- and γ-globin gene expression in the KLF2 has an erythroid cell role in embryonic globin gene regulation, also have In ChIP KLF1 and KLF2 bind to the promoters of the mouse embryonic Ey- and βh1-globin and the human ∈- and γ-globin genes in mouse primitive erythroid cells. KLF1, but is required to the normal histone in the mouse and human β-globin in mouse primitive erythroid cells. both KLF1 and KLF2 directly globin gene regulation through the same DNA their of to