Differential Binding of Erythroid Krupple-like Factor to Embryonic/Fetal Globin Gene Promoters during Development

Abstract

The competition model for β-like globin gene switching during development predicts that differential binding of transcription factors to globin gene promoters and/or proximal enhancers regulate the competitive interactions of globin gene family members with the powerful locus control region (LCR). Direct interactions of individual genes with the LCR are essential for high level expression in erythroid cells. In this paper, we have demonstrated, by chromatin immunoprecipitation, that erythroid-Krupple-like factor (EKLF) binds to embryonic/fetal globin gene promoters in primitive (but not in definitive) erythroid cells. EKLF binds strongly to adult globin gene promoters and to LCR sequences HS4, HS3, HS2, and HS1 in both primitive and definitive erythroid cells. Trimethylation of histone H3K4 and H3K27 at the embryonic/fetal and adult globin gene promoters is equivalent in definitive cells; therefore, the differential binding of EKLF to these promoters does not appear to result from changes in chromatin configuration. Interestingly, the level of EKLF in definitive cells is 3-fold higher than the level in primitive cells. These results suggest that temporal-specific changes in EKLF abundance result in differential binding of this essential erythroid transcription factor to embryonic/fetal globin gene promoters during development and that these changes in EKLF binding specificity mediate the competitive interactions of globin gene family members with the LCR. The competition model for β-like globin gene switching during development predicts that differential binding of transcription factors to globin gene promoters and/or proximal enhancers regulate the competitive interactions of globin gene family members with the powerful locus control region (LCR). Direct interactions of individual genes with the LCR are essential for high level expression in erythroid cells. In this paper, we have demonstrated, by chromatin immunoprecipitation, that erythroid-Krupple-like factor (EKLF) binds to embryonic/fetal globin gene promoters in primitive (but not in definitive) erythroid cells. EKLF binds strongly to adult globin gene promoters and to LCR sequences HS4, HS3, HS2, and HS1 in both primitive and definitive erythroid cells. Trimethylation of histone H3K4 and H3K27 at the embryonic/fetal and adult globin gene promoters is equivalent in definitive cells; therefore, the differential binding of EKLF to these promoters does not appear to result from changes in chromatin configuration. Interestingly, the level of EKLF in definitive cells is 3-fold higher than the level in primitive cells. These results suggest that temporal-specific changes in EKLF abundance result in differential binding of this essential erythroid transcription factor to embryonic/fetal globin gene promoters during development and that these changes in EKLF binding specificity mediate the competitive interactions of globin gene family members with the LCR. The genes encoding β-like subunits of mammalian hemoglobins are expressed in a precise tissue- and developmental stage-specific pattern (1Stamatoyannopoulos G. Niehuis A.W. Majerus P. Varmus H. The Molecular Basis of Blood Diseases. W. B. Saunders Company, Philadelphia1994Google Scholar). Although erythroid-specific expression of human ε-, γ-, and β-globin genes in the yolk sac, fetal liver, and bone marrow has been known for decades, the precise molecular mechanisms that direct this exquisite regulation are still unknown. The ε-, γ-, and β-globin genes are linked on chromosome 11 in the order in which they are expressed during development, and this gene family is positioned downstream of a potent 22-kb enhancer/chromatin remodeling sequence termed the locus control region (LCR) 2The abbreviations used are: LCR, locus control region; EKLF, erythroid Krupple-like factor; ChIP, chromatin immunoprecipitation; HA, hemagglutinin; WT, wild-type; GFP, green fluorescent protein; dpc, days postcoitus; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TAP, tandem affinity purification. (2Behringer R.R. Ryan T.M. Palmiter R.D. Brinster R.L. Townes T.M. Genes Dev. 1990; 4: 380-389Crossref PubMed Scopus (192) Google Scholar, 3Engel J.D. Trends Genet. 1993; 9: 304-309Abstract Full Text PDF PubMed Scopus (97) Google Scholar, 4Forrester W.C. Takegawa S. Papayannopoulou T. Stamatoyannopoulos G. Groudine M. Nucleic Acids Res. 1987; 15: 10159-10177Crossref PubMed Scopus (294) Google Scholar, 5Grosveld F. van Assendelft G.B. Greaves D.R. Kollias G. Cell. 1987; 51: 975-985Abstract Full Text PDF PubMed Scopus (1439) Google Scholar, 6Orkin S.H. Cell. 1990; 63: 665-672Abstract Full Text PDF PubMed Scopus (323) Google Scholar, 7Townes T.M. Behringer R.R. Trends Genet. 1990; 6: 219-223Abstract Full Text PDF PubMed Scopus (137) Google Scholar). In 1990, two groups (2Behringer R.R. Ryan T.M. Palmiter R.D. Brinster R.L. Townes T.M. Genes Dev. 1990; 4: 380-389Crossref PubMed Scopus (192) Google Scholar, 7Townes T.M. Behringer R.R. Trends Genet. 1990; 6: 219-223Abstract Full Text PDF PubMed Scopus (137) Google Scholar, 8Enver T. Raich N. Ebens A.J. Papayannopoulou T. Costantini F. Stamatoyannopoulos G. Nature. 1990; 344: 309-313Crossref PubMed Scopus (238) Google Scholar) proposed that developmental control of human ε-, γ-, and β-globin gene expression is achieved by the competition of individual globin gene family members for interaction with the LCR. This “competition model” was proposed to explain the unexpected discoveries that 1) human β-globin gene developmental specificity was lost in transgenic mice when the gene was linked directly to the LCR (LCRβ) and 2) human β-globin gene developmental specificity was restored by including a human γ-globin gene in the transgene construct (LCRγ-β) (7Townes T.M. Behringer R.R. Trends Genet. 1990; 6: 219-223Abstract Full Text PDF PubMed Scopus (137) Google Scholar, 8Enver T. Raich N. Ebens A.J. Papayannopoulou T. Costantini F. Stamatoyannopoulos G. Nature. 1990; 344: 309-313Crossref PubMed Scopus (238) Google Scholar). These results strongly suggested that human γ-globin gene sequences are essential for correct human β-globin gene developmental control. Later experiments demonstrated that disruption of the γ-globin promoter CACCC box results in the loss of β-globin gene developmental specificity in transgenic mice (9Ryan T.M. Sun C.W. Ren J. Townes T.M. Nucleic Acids Res. 2000; 28: 2736-2740Crossref PubMed Google Scholar, 10Sargent T.G. Lloyd J.A. J. Biol. Chem. 2001; 276: 41817-41824Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Therefore, the γ-globin gene CACCC box appears to be essential for correct globin gene switching. The competition model proposes that erythroid-specific yolk sac, fetal liver, and bone marrow transcription factors (or stage-specific combinations of transcription factors) bind to globin gene promoters, which all contain CACCC boxes, and provide individual genes with a competitive advantage for interaction with the LCR at the appropriate developmental stage. Developmental stage-specific interactions between the LCR and embryonic/fetal and adult β-globin genes have recently been demonstrated in vivo by elegant RNA tagging and recovery of associated proteins (11Carter D. Chakalova L. Osborne C.S. Dai Y.F. Fraser P. Nat. Genet. 2002; 32: 623-626Crossref PubMed Scopus (511) Google Scholar) and chromosome conformation capture experiments (12Palstra R.J. Tolhuis B. Splinter E. Nijmeijer R. Grosveld F. de Laat W. Nat. Genet. 2003; 35: 190-194Crossref PubMed Scopus (438) Google Scholar). EKLF, GATA-1, and FOG-1 are essential for LCR interactions with adult β-globin genes in definitive erythroid cells (13Drissen R. Palstra R.J. Gillemans N. Splinter E. Grosveld F. Philipsen S. de Laat W. Genes Dev. 2004; 18: 2485-2490Crossref PubMed Scopus (308) Google Scholar, 14Vakoc C.R. Letting D.L. Gheldof N. Sawado T. Bender M.A. Groudine M. Weiss M.J. Dekker J. Blobel G.A. Mol. Cell. 2005; 17: 453-462Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar); however, the transcription factors that promote LCR interactions with embryonic/fetal globin genes in primitive erythroid cells are unknown. In this paper, we have demonstrated, by chromatin immunoprecipitation (ChIP), that EKLF binds to embryonic/fetal globin gene promoters in primitive (but not definitive) erythroid cells. Second, we have found that EKLF binds strongly to adult globin gene promoters in both primitive and definitive cells. Finally, we have demonstrated that EKLF concentrations increase ∼3-fold from primitive to definitive erythroid cells. We propose that this temporal-specific change in EKLF abundance is responsible for differential binding of EKLF to the embryonic/fetal globin gene promoters and, therefore, is critical for globin gene switching. Previously published functional data are consistent with this model. Underexpression of EKLF in heterozygous EKLF knock-out mice results in delayed switching (15Perkins A.C. Gaensler K.M. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12267-12271Crossref PubMed Scopus (130) Google Scholar, 16Wijgerde M. Gribnau J. Trimborn T. Nuez B. Philipsen S. Grosveld F. Fraser P. Genes Dev. 1996; 10: 2894-2902Crossref PubMed Scopus (183) Google Scholar), and overexpression of EKLF in transgenic mice results in premature globin gene switching (17Tewari R. Gillemans N. Wijgerde M. Nuez B. von Lindern M. Grosveld F. Philipsen S. EMBO J. 1998; 17: 2334-2341Crossref PubMed Scopus (71) Google Scholar). Knock-in Mouse Lines and Antibodies—Generation of homozygous hemagglutinin/erythroid Krupple-like factor/tandem affinity purification (HA/EKLF/TAP) knock-in mice was reported previously (18Zhou D. Ren J.X. Ryan T.M. Higgins N.P. Townes T.M. Nucleic Acids Res. 2004; 32: e128Crossref PubMed Scopus (31) Google Scholar). The mice used for ChIP analysis in Figs. 1 and 2 were also homozygous for the HbbS globin haplotype. The ΔLCR mice used for ChIP analysis in Fig. 4 were produced by removing HS1–4 in an 18-kb deletion on the HbbD background. 3K. M. Pawlik and T. M. Townes, unpublished data. ΔLCR mice were bred with HA/EKLF/TAP knock-in mice, and the offspring were mated to generate ΔLCR mutant mice (ΔLCR (D)/WT (S)) and wild-type mice (WT (D)/WT (S)); the ΔLCR and WT mice were also homozygous for HA/EKLF/TAP. Normal rabbit IgG (catalog number sc-2027), anti-HA antibody (catalog number sc-7392), and anti-GFP antibody (catalog number sc-9996) were obtained from Santa Cruz Biotechnology. Anti-trimethyl-histone H3 K4-(07-473) and anti-trimethyl-histone H3 K27 (07-449, 05-851) were purchased from Upstate Biotechnology. Anti-trimethyl-histone H3 K9 (catalog number ab8898) was obtained from Abcam.FIGURE 2ChIP analysis of histone H3 trimethylation at the εy2- and βS-globin gene promoters in definitive erythroid cells. Chromatin obtained from a 14.5-dpc fetal liver was immunoprecipitated with control rabbit IgG (lanes 1 and 6), anti-trimethyl-histone H3K9 (lanes 2 and 7), anti-trimethyl-histone H3K4 (lanes 3 and 8), and two anti-trimethyl-histone H3K27 antibodies (lanes4, 5, 9, and 10). Histone H3 trimethylation at the myoD1 gene was analyzed as a control. The results demonstrate that the εy2 promoter contains chromatin marks that are consistent with an open domain in definitive erythroid cells; therefore, the promoter appears to be accessible to EKLF.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4ChIP analysis of EKLF binding to β-globin promoter in the absence of the LCR. Ter119+ erythroid cells were purified from the bone marrow of either ΔLCR (A and B, lanes 1 and 2) or wild-type (A and B, lanes 3 and 4) mice. Chromatin DNA was prepared and precipitated with anti-HA or -GFP antibodies and analyzed by PCR. A, control experiment. Shown is allele-specific analysis of EKLF binding to HS3 in ΔLCR (D)/WT (S) mice by ChIP. As expected, only HS3 from the wild-type HbbS allele was enriched by anti-HA antibody in ΔLCR (D)/WT (S) mice (compare lane 2 with lane 4). B, allele-specific analysis of EKLF binding to the promoter-proximal region of the βmaj- and βS-globin genes in ΔLCR (D)/WT (S) mice. The results demonstrate that EKLF binds strongly to the βmaj gene promoter in the absence of the LCR (compare lanes 2 and 4).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Reverse Transcription-PCR of Yolk Sac and Fetal Liver RNA—Total RNA was purified from a 10.5-dpc yolk sac and a 14.5-dpc fetal liver using and with using as was to the abundance of εy2 and The εy2 and sequences are in the Chromatin were as previously Townes T.M. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar), for the of the from binding the anti-HA and were with purified and DNA in for at 1 at Ter119+ and cells were purified on as by the and were obtained from and were in 1 of globin gene 1 of the myoD1 gene of and of The of 4 at by of for for and for of the were the of allele-specific were with The were on a and with a Molecular The of β-globin sequences to myoD1 in ChIP experiments was as with with with with The DNA sequences for all of the ChIP are in the data. for HbbD and HbbS allele-specific analysis was as previously T. J. Bender M.A. Groudine M. Genes Dev. 2003; 17: PubMed Scopus Google Scholar). were produced from Ter119+ cells purified from a 10.5-dpc yolk sac and from 14.5-dpc fetal liver cells. were on and to a antibody Santa Cruz was used to EKLF and antibody was used to GAPDH, which as a control. were by and HA/EKLF/TAP were to EKLF to εy2 and in (but has been EKLF and Mol. Cell. Biol. 1993; PubMed Scopus Google Scholar) this erythroid-specific in still in of the in which EKLF globin gene expression in has been the of and EKLF antibodies that be used in ChIP experiments to EKLF binding to LCR and β-like globin genes in primitive and definitive erythroid cells. this we the EKLF gene with a and of the gene in cells and produced mice that are homozygous for the allele (18Zhou D. Ren J.X. Ryan T.M. Higgins N.P. Townes T.M. Nucleic Acids Res. 2004; 32: e128Crossref PubMed Scopus (31) Google Scholar). These mice were not and globin gene switching during development was with wild-type Therefore, the HA/EKLF/TAP knock-in mice be used to the developmental pattern of EKLF binding to globin locus sequences by ChIP experiments with the and anti-HA Fig. the β-globin locus and the of used for of immunoprecipitated chromatin ChIP results from bone fetal liver, and yolk sac cells of homozygous HA/EKLF/TAP mice. Fig. is a control and that of globin sequences was in anti-HA with chromatin from bone marrow cells of HA/EKLF/TAP mice. The data in Fig. demonstrate a of HS4, HS3, HS2, and promoter sequences in chromatin from Ter119+ bone marrow cells of HA/EKLF/TAP mice. These results strongly suggest that EKLF binds to these sequences in definitive erythroid cells in Interestingly, the data also demonstrate that EKLF does not bind to or to the εy2 and genes in definitive erythroid cells. Finally, the specificity of the was demonstrated by the absence of for sequences between and HS3 in These data are to the results recently reported by H. J.A. Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google Scholar) on EKLF binding to β-globin locus in cells M.J. Orkin S.H. Mol. Cell. Biol. 17: PubMed Scopus Google Scholar). demonstrate EKLF binding to HS3, HS2, and to the gene promoter in this definitive erythroid Interestingly, EKLF binding to was not in cells. EKLF binding to in Ter119+ bone marrow cells (but not in in the analyzed or in the antibodies for ChIP. As expected, the EKLF binding pattern that was in Ter119+ bone marrow cells was also in 14.5-dpc fetal liver cells EKLF strongly to HS4, HS3, HS2, and promoter sequences in chromatin from HA/EKLF/TAP and binding was at or we ChIP analysis of EKLF binding in 10.5-dpc yolk sac cells from HA/EKLF/TAP mice. The results of this are in Fig. As in definitive EKLF strongly to HS4, HS3, HS2, and we that EKLF to the promoters of and genes in these primitive erythroid cells. These results are for we not EKLF binding to the adult β-globin genes in primitive erythroid cells. The adult genes are expressed at in primitive cells and of globin gene switching have that EKLF is not to adult globin gene promoters at this stage. ChIP results demonstrate that EKLF binds to the βS-globin gene promoter in primitive cells and that the is equivalent to binding in definitive cells. we were to that EKLF to εy2- and gene promoters in primitive erythroid cells. Although and in experiments have demonstrated that the EKLF gene is expressed in yolk sac erythroid cells D. Townes T.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, K.M. Dev. 1996; PubMed Scopus Google Scholar), of globin gene switching not EKLF binding to εy2- and gene promoters at this developmental εy2- and genes are expressed at wild-type in EKLF knock-out mice B. D. A. R. Grosveld F. Nature. PubMed Scopus Google Scholar, A.C. Orkin S.H. Nature. PubMed Scopus Google Scholar). The knock-out result suggested that EKLF is not in expression of the embryonic/fetal globin the ChIP results demonstrate that EKLF binds to εy2 and promoters in yolk sac cells and suggest that EKLF an in globin gene expression at this developmental stage. In EKLF knock-out mice, proteins εy2 and in wild-type these proteins not a in globin gene are for the of EKLF binding to εy2 and promoters in definitive cells. is that bind to these promoters in definitive cells and EKLF to the CACCC have been and of binding in embryonic/fetal globin gene promoters result in of these genes in definitive cells A. J.D. A. Mol. Cell. Biol. 2005; PubMed Scopus Google Scholar, A. S. Stamatoyannopoulos G. EMBO J. 18: PubMed Scopus Google Scholar, Grosveld F. J. J.D. Genes Dev. 2000; PubMed Scopus Google Scholar). of the genes encoding these have not demonstrated of embryonic/fetal globin genes in definitive erythroid and ChIP experiments have not demonstrated the binding of these factors to embryonic/fetal globin gene promoters in of Histone H3 Trimethylation at εy2 and in an Chromatin for the of EKLF binding to εy2 and CACCC in definitive cells is that a chromatin is on these this we analyzed the εy2 and promoter for histone H3K27 which marks chromatin A. A. R. D. R. Genes Dev. 2004; 18: PubMed Scopus Google Scholar). antibodies to H3K27 were Fig. 2 that H3K27 trimethylation is not enriched on the εy2 promoter (lanes 4 and with the promoter (lanes and in 14.5-dpc fetal liver cells. The myoD1 control that H3K27 trimethylation is enriched on this which is in all erythroid cells. In histone H3K4 which marks chromatin H. R. L. H. P. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, M. F. Genes Dev. 2001; 15: PubMed Scopus Google Scholar, J.A. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar, S. H. P. D. Genes Dev. 2002; PubMed Scopus Google Scholar), is equivalent on εy2 and promoters lanes 3 and Finally, histone H3K9 which marks globin genes C.R. Blobel G.A. Mol. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar), is enriched on not on genes lanes 2 and in 14.5-dpc fetal liver cells. This is a control that in histone be in embryonic/fetal and adult globin genes in definitive erythroid cells. The data on and trimethylation suggest that the differential binding of EKLF to embryonic/fetal globin gene promoters during development does not result from changes in chromatin configuration. EKLF in during for the differential binding of EKLF to globin gene promoters in primitive and definitive cells is that the abundance of EKLF changes during in the of transcription factors have on gene regulation and R. D. P. H. Nat. 2003; 4: PubMed Scopus Google Scholar, H. 2000; PubMed Scopus Google Scholar, M. R. L. L. G. Orkin S.H. J. 2003; PubMed Scopus Google Scholar, E. H. T. 2000; PubMed Google Scholar, N. F. T. Genes Dev. PubMed Scopus Google Scholar, J. A. M. Dev. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, F. S. U. 2005; PubMed Scopus Google Scholar, M.A. Orkin S.H. EMBO J. PubMed Scopus Google Scholar, H. Orkin S.H. J. 2002; PubMed Scopus Google Scholar, P. G. J. A. N. Sun Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), and in the changes in transcription factor result in a change of Orkin S.H. 2002; PubMed Scopus Google Scholar). Therefore, we EKLF in primitive and definitive erythroid cells from homozygous HA/EKLF/TAP mice with anti-HA antibody Interestingly, we found that EKLF increase ∼3-fold in definitive with primitive erythroid Fig. 1 for This result is consistent with the 3-fold increase in EKLF that we previously in with erythroid cells D. Townes T.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We that the 3-fold increase in EKLF in with erythroid cells results in a of EKLF binding and that the binds to adult β-globin gene Therefore, adult globin gene family members are in definitive erythroid cells. EKLF to the EKLF binds to the LCR and to promoters of adult β-globin genes in erythroid cells at all developmental we EKLF binding to the β-globin gene promoter the LCR. We of the LCR in cells and produced mice that were heterozygous for this 18-kb ΔLCR were bred with HA/EKLF/TAP mice to that were heterozygous for the ΔLCR allele and homozygous for HA/EKLF/TAP. the binding of EKLF at WT and ΔLCR we used mice both HbbS (S) and HbbD and allele-specific ChIP as by Sawado T. J. Bender M.A. Groudine M. Genes Dev. 2003; 17: PubMed Scopus Google Scholar). HS3 from the HbbS allele is than HS3 from the HbbD As demonstrated in the control experiments in Fig. both HS3 were precipitated by anti-HA antibody in wild-type wild-type HbbD allele and wild-type HbbS and only HS3 from the HbbS allele was enriched in ΔLCR ΔLCR HbbD allele and wild-type HbbS allele-specific ChIP analysis of adult β-globin gene promoters was we found that EKLF was to the adult gene promoter of the ΔLCR allele as strongly as to the βS-globin gene promoter of the WT allele lanes 2 and 4). These results demonstrate that EKLF binding to the adult β-globin gene promoter does not the LCR. data GATA-1, and binding to β-globin gene promoters in the absence of the LCR in vivo suggest that the LCR does not these proteins to globin gene promoters C.R. Letting D.L. Gheldof N. Sawado T. Bender M.A. Groudine M. Weiss M.J. Dekker J. Blobel G.A. Mol. Cell. 2005; 17: 453-462Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, T. J. Bender M.A. Groudine M. Genes Dev. 2003; 17: PubMed Scopus Google Scholar). EKLF is essential for gene interactions (13Drissen R. Palstra R.J. Gillemans N. Splinter E. Grosveld F. Philipsen S. de Laat W. Genes Dev. 2004; 18: 2485-2490Crossref PubMed Scopus (308) Google Scholar), and EKLF binding in HS3 and are essential for LCR in primitive and definitive erythroid cells in A. and T. M. Townes, unpublished data. Therefore, EKLF binding to both LCR and globin gene promoter sequences in the yolk sac, fetal liver, and bone marrow appears to be critical for correct globin gene regulation during The demonstrate that EKLF binds to εy2 and promoters in primitive erythroid cells. The of these data is that EKLF εy2 and gene expression at this developmental stage. EKLF has two powerful transcription K.M. Dev. 1996; PubMed Scopus Google Scholar, D. Townes T.M. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar), and the binding of EKLF to the εy2 and promoters strongly a functional for this transcription factor in primitive erythroid cells. Although εy2- and genes are expressed at high in EKLF knock-out mice, CACCC proteins be responsible for this Interestingly, of yolk sac EKLF, also and as embryonic/fetal globin gene CACCC Sun and T. M. Townes, unpublished data. Therefore, and bind to embryonic/fetal globin gene CACCC in the absence of EKLF and or γ-globin gene model for globin gene switching that is consistent with the EKLF binding pattern is in Fig. EKLF in primitive and definitive erythroid and these direct globin gene switching. In primitive the EKLF 5, and binds to and adult β-globin gene CACCC however, εy2 and genes are these genes are proximal to the LCR. for the expression of εy2- and genes in primitive cells is that an EKLF binds to the adult globin gene promoters at this Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar, Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). In definitive a EKLF 5, and is as a result of the 3-fold increase in EKLF This binds to not to globin gene CACCC therefore, the adult genes are expressed at this developmental stage. This model does not an for transcription as P. M.A. Gaensler K.M. Lloyd J.A. 2005; PubMed Scopus Google Scholar), in globin gene switching the essential of be for correct globin gene switching. of the γ-globin gene CACCC box is to a change in developmental specificity high level expression of adult β-globin genes in primitive erythroid cells (9Ryan T.M. Sun C.W. Ren J. Townes T.M. Nucleic Acids Res. 2000; 28: 2736-2740Crossref PubMed Google Scholar, 10Sargent T.G. Lloyd J.A. J. Biol. Chem. 2001; 276: 41817-41824Abstract Full Text Full Text PDF PubMed Scopus (11) Google therefore, differential binding of EKLF to embryonic/fetal and adult globin gene CACCC appears to be critical for globin gene switching. Previously published functional data are consistent with this model. Underexpression of EKLF in heterozygous EKLF knock-out mice results in delayed switching (15Perkins A.C. Gaensler K.M. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12267-12271Crossref PubMed Scopus (130) Google Scholar, 16Wijgerde M. Gribnau J. Trimborn T. Nuez B. Philipsen S. Grosveld F. Fraser P. Genes Dev. 1996; 10: 2894-2902Crossref PubMed Scopus (183) Google Scholar), and overexpression of EKLF in transgenic mice results in premature globin gene switching (17Tewari R. Gillemans N. Wijgerde M. Nuez B. von Lindern M. Grosveld F. Philipsen S. EMBO J. 1998; 17: 2334-2341Crossref PubMed Scopus (71) Google Scholar). We a model for globin gene switching in which temporal-specific changes of EKLF abundance result in 1) a of EKLF 2) differential binding of EKLF to globin gene promoters, and developmental stage-specific globin gene of EKLF from primitive and definitive erythroid EKLF at these developmental and provide globin gene The results in this are consistent with the competition model for globin gene switching (7Townes T.M. Behringer R.R. Trends Genet. 1990; 6: 219-223Abstract Full Text PDF PubMed Scopus (137) Google Scholar) and suggest that developmental stage-specific changes in EKLF abundance mediate the competitive interactions of globin gene family members with the powerful locus control We and for Download with