T to C Substitution at –175 or –173 of the γ-Globin Promoter Affects GATA-1 and Oct-1 Binding in Vitro Differently but Can Independently Reproduce the Hereditary Persistence of Fetal Hemoglobin Phenotype in Transgenic Mice

Abstract

The T to C substitution at position –175 of the γ-globin gene has been identified in some individuals with non-deletion hereditary persistence of fetal hemoglobin (HPFH). In this study, the HPFH phenotype was reestablished in transgenic mice carrying the μ′LCRAγ–175ψβδβ construct, which contained a 3.1-kb μ′LCR cassette linked to a 29-kb fragment from the Aγ-to β-globin gene with the natural chromosome arrangement but with the –175 mutation, which provided evidence for this single mutation as the cause of this form of HPFH. The HPFH phenotype was also reproduced in transgenic mice carrying the μ′LCRAγ–173ψβδβ construct, in which the –175 T to C Aγ gene was substituted with the –173 T to C Aγ gene. In vitro experiments proved that the –175 mutation significantly reduced binding of Oct-1 but not GATA-1, whereas the –173 mutation dramatically decreased binding of GATA-1 but not Oct-1. These results suggest that abrogation of either GATA-1 or Oct-1 binding to this promoter region may result in the HPFH phenotype. An in vivo footprinting assay revealed that either the –175 mutation or the –173 mutation significantly decreased overall protein binding to this promoter region in adult erythrocytes of transgenic mice. We hypothesize that a multiprotein complex containing GATA-1, Oct-1, and other protein factors may contribute to the formation of a repressive chromatin structure that silences γ-globin gene expression in normal adult erythrocytes. Both the –173 and –175 T to C substitutions may disrupt the complex assembly and result in the reactivation of the γ-globin gene in adult erythrocytes. The T to C substitution at position –175 of the γ-globin gene has been identified in some individuals with non-deletion hereditary persistence of fetal hemoglobin (HPFH). In this study, the HPFH phenotype was reestablished in transgenic mice carrying the μ′LCRAγ–175ψβδβ construct, which contained a 3.1-kb μ′LCR cassette linked to a 29-kb fragment from the Aγ-to β-globin gene with the natural chromosome arrangement but with the –175 mutation, which provided evidence for this single mutation as the cause of this form of HPFH. The HPFH phenotype was also reproduced in transgenic mice carrying the μ′LCRAγ–173ψβδβ construct, in which the –175 T to C Aγ gene was substituted with the –173 T to C Aγ gene. In vitro experiments proved that the –175 mutation significantly reduced binding of Oct-1 but not GATA-1, whereas the –173 mutation dramatically decreased binding of GATA-1 but not Oct-1. These results suggest that abrogation of either GATA-1 or Oct-1 binding to this promoter region may result in the HPFH phenotype. An in vivo footprinting assay revealed that either the –175 mutation or the –173 mutation significantly decreased overall protein binding to this promoter region in adult erythrocytes of transgenic mice. We hypothesize that a multiprotein complex containing GATA-1, Oct-1, and other protein factors may contribute to the formation of a repressive chromatin structure that silences γ-globin gene expression in normal adult erythrocytes. Both the –173 and –175 T to C substitutions may disrupt the complex assembly and result in the reactivation of the γ-globin gene in adult erythrocytes. The human fetal globin genes (Gγ and Aγ) are mainly expressed in fetal liver. The switch from fetal to adult (δ and β) globin gene expression occurs in the perinatal period concomitantly with the establishment of the bone marrow as the main site of erythropoiesis. In some individuals, however, this switching is incomplete, and γ-globin expression persists in adult erythrocytes. Such a condition, found in syndromes of hereditary persistence of fetal hemoglobin (HPFH) 1The abbreviations used are: HPFH, hereditary persistence of fetal hemoglobin; LCR, locus control region; MEL, mouse erythroleukemia; DMS, dimethylsulfate; EMSA, electrophoretic mobility shift assay; ChIP, chromatin immunoprecipitation; LM-PCR, ligation-mediated PCR. and (δβ)0-thalassemias, can result from deletions within the β-globin gene cluster (1Stamatoyannopoulos G. Grosveld F. Stamatoyannopoulos G. Majerus P. Peelmutter R. The Molecular Basis of Blood Diseases. 3rd ed. W. B. Saunders, Philadelphia2001: 135-182Google Scholar). However, in some syndromes known as non-deletion HPFH, point mutations in the γ-globin gene promoter seem to be responsible for the continuance of γ-globin expression (1Stamatoyannopoulos G. Grosveld F. Stamatoyannopoulos G. Majerus P. Peelmutter R. The Molecular Basis of Blood Diseases. 3rd ed. W. B. Saunders, Philadelphia2001: 135-182Google Scholar). It has been revealed that most of the point mutations lie in or near the trans-acting factor binding motifs within the promoter and that the mutations usually affect the binding of some trans-acting factors in vitro (2Forget B.G. Ann. N. Y. Acad. Sci. 1998; 850: 38-44Crossref PubMed Scopus (192) Google Scholar). Studies in transgenic mice carrying either –117 Aγ HPFH cosmid or –117 Aγ HPFH yeast artificial chromosome showed that transgenic expression could mimic the developmental expression program observed in HPFH, providing powerful proof that the single point mutation was the cause of the Greek form of HPFH (3Berry M. Grosveld F. Dillon N.A. Nature. 1992; 358: 449-502Crossref Scopus (111) Google Scholar, 4Peterson K.R. Li Q.L. Clegg C.H. Furukawa T. Navas P.A. Norton E.J. Kimbrough T.G. Stamatoyannopoulos G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5655-5659Crossref PubMed Scopus (103) Google Scholar). Berry et al. (3Berry M. Grosveld F. Dillon N.A. Nature. 1992; 358: 449-502Crossref Scopus (111) Google Scholar) speculated that this change of γ-globin expression during development could be correlated with the loss of GATA-1 binding to the γ-promoter region around –117 and deduced that GATA-1 might act as a negative regulator of the γ-globin gene in normal adults. However, earlier in vitro experiments on a black form of HPFH point mutation, a T to C substitution at position –175 of the γ-globin promoter, showed no significant difference in the GATA-1 affinity for the promoter. Nevertheless, this mutation dramatically decreased the affinity of the Oct-1 transcription factor, which binds to an area partially overlapping the GATA-1 binding site (5Gumucio D.L. Rood K.L. Gray T.A. Riordan M.F. Sartor C.I. Collins F.S. Mol. Cell. Biol. 1988; 8: 5310-5322Crossref PubMed Scopus (79) Google Scholar, 6Lloyd J.A. Lee R.F. Lingrel J.B. Nucleic Acids Res. 1989; 17: 4339-4352Crossref PubMed Scopus (18) Google Scholar, 7Martin D.I.K. Tsai S.F. Orkin S.H. Nature. 1989; 338: 435-438Crossref PubMed Scopus (182) Google Scholar, 8McDonagh K.T. Lin H.J. Lowrey C.H. Bodine D.M. Nienhuis A.W. J. Biol. Chem. 1991; 266: 11965-11974Abstract Full Text PDF PubMed Google Scholar, 9Starck B.J. Sarkar R. Romana M. Bhargava A. Scarpa A.L. Tanaka M. Chamberlain J.W. Weissman S.M. Blood. 1994; 84: 1656-1665Crossref PubMed Google Scholar). To test whether the –175 T to C substitution might be the cause of the black form of HPFH, we introduced two β-globin minilocus constructs into mice. The first was μ′LCRAγψβδβ, which contained a 3.1-kb μ′LCR cassette linked to a 29-kb fragment from the Aγ- to β-globin gene with the natural chromosome arrangement. The second was μ′LCRAγ–175ψβδβ, which was similar to μ′LCRAγψβδβ except for a T to C substitution at position –175 of the Aγ promoter. Normal developmental expression of γ- and β-globin genes was observed in transgenic mouse lines with μ′LCRAγψβδβ, whereas persistent expression of the γ-globin gene in adults of transgenic mouse lines with μ′LCRAγ–175ψβδβ was noted, suggesting that this single mutation is responsible for the HPFH phenotype. To further investigate the relationship between GATA-1 or Oct-1 binding and the developmental regulation of the γ-globin gene, we substituted the –175 T to C Aγ-globin gene with a –173 T to C Aγ-globin gene in the construct. The –173 T base has been reported to be involved in the conserved GATA-1 binding motif (5Gumucio D.L. Rood K.L. Gray T.A. Riordan M.F. Sartor C.I. Collins F.S. Mol. Cell. Biol. 1988; 8: 5310-5322Crossref PubMed Scopus (79) Google Scholar, 6Lloyd J.A. Lee R.F. Lingrel J.B. Nucleic Acids Res. 1989; 17: 4339-4352Crossref PubMed Scopus (18) Google Scholar, 7Martin D.I.K. Tsai S.F. Orkin S.H. Nature. 1989; 338: 435-438Crossref PubMed Scopus (182) Google Scholar, 8McDonagh K.T. Lin H.J. Lowrey C.H. Bodine D.M. Nienhuis A.W. J. Biol. Chem. 1991; 266: 11965-11974Abstract Full Text PDF PubMed Google Scholar, 9Starck B.J. Sarkar R. Romana M. Bhargava A. Scarpa A.L. Tanaka M. Chamberlain J.W. Weissman S.M. Blood. 1994; 84: 1656-1665Crossref PubMed Google Scholar). By electrophoretic mobility shift assays (EMSAs), we found that the –173 mutation decreased GATA-1 binding dramatically but reduced Oct-1 binding only slightly. Despite these differences in transcription factor binding ability, the HPFH phenotype was reestablished in mice carrying μ′LCRAγ–173ψβδβ. These results suggested that the inability of either the GATA-1 or Oct-1 to bind this promoter region could result in the HPFH phenotype. We then tested the protein binding status within this promoter region in adult erythrocytes of mice carrying normal or mutant γ-globin genes by chromatin immunoprecipitation (ChIP) and in vivo footprint analysis. The results show that either the –175 or the –173 mutation significantly decreased protein binding to both the GATA-1 and Oct-1 binding sites in adult erythrocytes of transgenic mice. Taken together, these results suggest that the GATA-1 and Oct-1 might act together as part of a repressor complex that silences γ-globin gene expression in normal adult erythrocytes and that abrogation of binding by either disrupted the repressor complex and resulted in HPFH. Cosmid Constructs—The containing a 3.1-kb cassette of the of is a from The cosmid T. R. T. F. Stamatoyannopoulos G. Nature. PubMed Scopus Google Scholar) is a from G. The 3.1-kb μ′LCR was as a fragment from and into that contained a fragment with Aγ-globin gene and which contained the 3.1-kb the promoter, and a part of the Aγ-globin gene, was from and the fragment of cosmid cosmid μ′LCRAγψβδβ An T to C substitution at position –175 was introduced into the fragment of by and then into the The mutation was by the fragment of was linked to the 3.1-kb μ′LCR cassette and then as an fragment into the μ′LCRAγψβδβ, μ′LCRAγ–175ψβδβ to μ′LCRAγ–173ψβδβ for with and the point from the cosmid constructs and by 1991; Scholar). by in was with to a of and then on the at at for an was into the of the and of from with of and and the an the was and containing only the identified and The with the of with The was for at and but of the at the to a J.A. A. PubMed Scopus Google Scholar). An was used to by with a The with to a of of and into the of and then to B. R. F. the Scholar). mice identified by and of mice with to transgenic The of the constructs by the γ- and the β-globin gene and the μ′LCR as the by the of from the transgenic lines with that of human by analysis. mouse transgenic with to The the was observed was from the of mouse on from fetal on and and from the of as as adult B.J. Sarkar R. Romana M. Bhargava A. Scarpa A.L. Tanaka M. Chamberlain J.W. Weissman S.M. Blood. 1994; 84: 1656-1665Crossref PubMed Google Scholar). was with to the and globin by assays J.W. Stamatoyannopoulos G. Sci. J. Google Scholar). containing the promoter and a fragment of of the globin genes used as by a fragment of the human Aγ-globin gene into and with a fragment of from of the human Aγ by a fragment of the human β-globin in and with a fragment of from of the human by a to the of mouse gene, into and with a fragment of from of the mouse and by a mouse gene fragment into and with a fragment of The with by in vitro transcription of the with of was at with of by with and for at the assay The on and The of the mouse and and human as (5Gumucio D.L. Rood K.L. Gray T.A. Riordan M.F. Sartor C.I. Collins F.S. Mol. Cell. Biol. 1988; 8: 5310-5322Crossref PubMed Scopus (79) Google Scholar). In some to by the of to the for and by the of to the for The and as (5Gumucio D.L. Rood K.L. Gray T.A. Riordan M.F. Sartor C.I. Collins F.S. Mol. Cell. Biol. 1988; 8: 5310-5322Crossref PubMed Scopus (79) Google Scholar). The to of the γ-globin promoter with the normal or the –175 or –173 mutation was used as The with and –173 mutation and –175 mutation The as (5Gumucio D.L. Rood K.L. Gray T.A. Riordan M.F. Sartor C.I. Collins F.S. Mol. Cell. Biol. 1988; 8: 5310-5322Crossref PubMed Scopus (79) Google Scholar). contained and of was to of was and the was at for the of binding at for The of for GATA-1 and Oct-1 binding and was at in The then and by To differences in binding the of by an with and and as by et al. S.H. J. Nucleic Acids Res. PubMed Google with the marrow of the transgenic mice by to a of and at for with and in with on for the to to between and on The was to a and with and An of of protein was to the and at for The at for The was into of and to two whereas no was to the and then the at of the chromatin was the and with for at An of the was also by with for the human γ-globin promoter region of was to of human γ-globin promoter by at for by at for was at for for for for for for for and for of the of the with the and was at and the was in of the and The first a at and the at for The with and was by with at The of a was with of the was for the The first was with at for and the second was with for at for and the at for The of the at Normal of in with transgenic lines with of the μ′LCRAγψβδβ The of human Aγ- and β-globin as as mouse and by gene expression in transgenic mouse lines carrying of in to in fetal of in to in in a In the Aγ-globin was of mouse on significantly to in and to in adult result expression of the Aγ-globin gene during the fetal and The human β-globin gene was at the but in the fetal to and in the adult The of developmental expression of human γ- and β-globin genes in was similar to that in These results that transgenic mice carrying μ′LCRAγψβδβ can mimic the normal developmental expression of both the human Aγ- and β-globin is similar to that from a on transgenic mice carrying a that was the as μ′LCRAγψβδβ except for the of a cassette T. R. T. F. Stamatoyannopoulos G. Nature. PubMed Scopus Google Scholar). The HPFH in transgenic lines with of the μ′LCRAγ–175ψβδβ The expression of the human and globin during mouse development is in and gene expression in transgenic mouse lines carrying of in to in fetal of in to in in a The human from in only a with the Aγ-globin which from to in the fetal Aγ-globin gene expression in the adult was between and of the expression between the of γ-globin gene transcription in adult as with that in the fetal for mice carrying μ′LCRAγ–175ψβδβ significantly from the in mice carrying These results that the Aγ-globin gene with the –175 mutation during the adult and that the point mutation was responsible for the HPFH phenotype. The –175 and –173 GATA-1 and Oct-1 in region from to of the Aγ-globin gene promoter two GATA-1 binding motifs and Oct-1 binding motif of the normal fragment to of the γ-globin gene promoter and –173 mutant from and and as as both adult and and adult globin used for similar observed between the two lines not mobility and the normal fragment with the with the of the with the normal the of by the –173 mutation decreased by and the of by the mutation decreased by The assay that resulted from Oct-1 binding and that resulted from GATA-1 binding result that the –173 mutation dramatically decreased the binding affinity of GATA-1 to the mutant promoter but only reduced Oct-1 of with the –175 mutation been in (5Gumucio D.L. Rood K.L. Gray T.A. Riordan M.F. Sartor C.I. Collins F.S. Mol. Cell. Biol. 1988; 8: 5310-5322Crossref PubMed Scopus (79) Google Scholar, 6Lloyd J.A. Lee R.F. Lingrel J.B. Nucleic Acids Res. 1989; 17: 4339-4352Crossref PubMed Scopus (18) Google Scholar, 7Martin D.I.K. Tsai S.F. Orkin S.H. Nature. 1989; 338: 435-438Crossref PubMed Scopus (182) Google Scholar, 8McDonagh K.T. Lin H.J. Lowrey C.H. Bodine D.M. Nienhuis A.W. J. Biol. Chem. 1991; 266: 11965-11974Abstract Full Text PDF PubMed Google Scholar, 9Starck B.J. Sarkar R. Romana M. Bhargava A. Scarpa A.L. Tanaka M. Chamberlain J.W. Weissman S.M. Blood. 1994; 84: 1656-1665Crossref PubMed Google Scholar). results showed that with the normal the binding affinity of GATA-1 to the –175 mutation promoter decreased by whereas the binding affinity of Oct-1 decreased by is with the results of most of the the –175 mutation decreased GATA-1 binding but reduced Oct-1 binding which was the of the of the –173 The HPFH in further investigate whether –173 T to C substitution of the Aγ gene could the HPFH phenotype in we the μ′LCRAγ–173ψβδβ. transgenic lines carrying the and developmental expression of the globin genes was The human between and in whereas the Aγ-globin expression in the fetal of the transgenic was between and The Aγ-globin gene expression in the adult was between and of the expression Despite expression between the results showed that the Aγ-globin gene with the –173 mutation in the adult of mouse The –173 mutation resulted in the HPFH phenotype in transgenic similar to the of the Aγ-globin gene the –175 mutation gene expression in transgenic mouse lines carrying of in to in fetal of in to in in a of γ-globin gene expression in transgenic lines carrying of expression as by of human to in fetal on or are with in adult bone The results from lines carrying μ′LCRAγ–173ψβδβ are at the from lines carrying μ′LCRAγ–175ψβδβ are in the and from two lines carrying μ′LCRAγψβδβ are at the Both the –175 and –173 to the Aγ in marrow from a transgenic mouse carrying μ′LCRAγψβδβ a transgenic carrying μ′LCRAγ–175ψβδβ and a transgenic carrying μ′LCRAγ–173ψβδβ chromosome from the the with the or an of the the γ-promoter region was in of the with the and but not in the not in vitro experiments identified a GATA-1 binding site and an Oct-1 binding site within the a GATA-1 site within the –117 and two GATA-1 sites and an Oct-1 site within the –175 region of γ-promoter Google Scholar). The to chromatin was to the of the with the to the binding sites in to the binding sites within the –175 of the chromatin and revealed the of protein binding The results showed that both of the at the and of γ-globin promoter in adult bone marrow from transgenic mice carrying the construct, suggesting that bind to this promoter region of the these in vivo footprinting experiments could not which to this region of the chromatin in adult erythrocytes of the transgenic mice with the of GATA-1 and Oct-1 binding to this region by in vitro experiments suggested that the binding GATA-1 and Oct-1. The also revealed loss or significant of on the and in from transgenic mice carrying either μ′LCRAγ–175ψβδβ or suggesting that both the –175 mutation and the –173 mutation resulted in significantly decreased protein binding to the region of the in vitro experiments been to the of –175 HPFH. motifs for the protein GATA-1 are known to be near the –175 site within the γ-globin gene promoter. An Oct-1 binding motif is found between the GATA-1 sites and the GATA-1 The –175 HPFH mutation the GATA-1 site but also the base in the Oct-1 site is that the –175 mutation significantly Oct-1 the affinity of GATA-1 for the –175 region has been reported to be by the mutation or by (5Gumucio D.L. Rood K.L. Gray T.A. Riordan M.F. Sartor C.I. Collins F.S. Mol. Cell. Biol. 1988; 8: 5310-5322Crossref PubMed Scopus (79) Google Scholar, 7Martin D.I.K. Tsai S.F. Orkin S.H. Nature. 1989; 338: 435-438Crossref PubMed Scopus (182) Google Scholar, R. N. S. A. B. S. Nucleic Acids Res. 1988; PubMed Scopus Google Scholar). suggested that of the –175 mutation promoter was not to decreased Oct-1 binding but was on GATA-1 binding D.I.K. Tsai S.F. Orkin S.H. Nature. 1989; 338: 435-438Crossref PubMed Scopus (182) Google Scholar, 8McDonagh K.T. Lin H.J. Lowrey C.H. Bodine D.M. Nienhuis A.W. J. Biol. Chem. 1991; 266: 11965-11974Abstract Full Text PDF PubMed Google Scholar). et al. J. M. M. A. 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Biol. 1988; 8: PubMed Scopus Google Scholar, G. A. G. M. J. 1988; PubMed Scopus Google Scholar, Blood. PubMed Google Scholar). These that in vitro experiments could not the in vivo the –117 mutation reproduced the HPFH phenotype in transgenic mice (3Berry M. Grosveld F. Dillon N.A. Nature. 1992; 358: 449-502Crossref Scopus (111) Google Scholar, 4Peterson K.R. Li Q.L. Clegg C.H. Furukawa T. Navas P.A. Norton E.J. Kimbrough T.G. Stamatoyannopoulos G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5655-5659Crossref PubMed Scopus (103) Google Scholar) and the –175 mutation GATA-1 binding from the –117 mutation, is to test whether the –175 mutation of γ-globin promoter also the HPFH phenotype. In a study, transgenic mouse lines carrying the with or the –175 mutation Stamatoyannopoulos G. J. PubMed Scopus (18) Google Scholar). a of γ-globin expression was observed in the adult carrying with the –175 mutation, only a of γ-globin expression with the was observed the adult transgenic mice expressed of γ-globin The loss of developmental control of γ-globin gene expression in was to the of a between and of the Aγ promoter G. B. J.W. Li Mol. Cell. Biol. PubMed Google the of the region of the Aγ gene to a in Aγ gene N. Grosveld F. Nature. 1991; PubMed Scopus Google or from the of β-globin gene for with the T. R. T. F. Stamatoyannopoulos G. Nature. PubMed Scopus Google Scholar). To these we constructs containing a 3.1-kb cassette that the of the by a 29-kb fragment from the Aγ-to β-globin gene with normal chromosome similar cosmid construct, containing a cassette by the 29-kb has been to developmental control of the globin genes in transgenic mice T. R. T. F. Stamatoyannopoulos G. Nature. PubMed Scopus Google Scholar). In study, human and globin of mice carrying or two of the The γ-globin expression normal developmental control in mice carrying μ′LCRAγψβδβ, whereas continuance of γ-globin expression was observed in adult mice carrying These results provided proof that the –175 T to C substitution this form of non-deletion HPFH. on the that the –117 mutation reproduced the HPFH phenotype in transgenic mice as as dramatically GATA-1 Berry et al. (3Berry M. Grosveld F. Dillon N.A. Nature. 1992; 358: 449-502Crossref Scopus (111) Google Scholar) GATA-1 might act as a negative regulator of the γ-globin gene in normal adults (3Berry M. Grosveld F. Dillon N.A. Nature. 1992; 358: 449-502Crossref Scopus (111) Google Scholar). However, an binding of and and a decreased binding of in the region also been observed S.H. J. Nucleic Acids Res. PubMed Google Scholar, T.A. F. Blood. 1989; PubMed Google Scholar, A. Berry M. S. A. N. S. Grosveld F. Dillon N. J. PubMed Scopus Google Scholar, R. Mol. Cell. Biol. PubMed Google to the of GATA-1 in the developmental regulation of γ-globin gene of the HPFH phenotype in transgenic mice by the –175 mutation at that the decreased GATA-1 binding is not for expression of γ-globin in adults in this form of HPFH. However, be that GATA-1 binding in of the promoter. To further investigate the relationship between factor binding and developmental regulation of γ-globin we introduced a mutation at position to the –175 mutation, the –173 mutation decreased Oct-1 binding but reduced GATA-1 binding in Nevertheless, we introduced the μ′LCRAγ–173ψβδβ into the HPFH phenotype was also the –175 mutation decreased Oct-1 affinity for the promoter is to that this change is responsible for of the mutant in adults. In Oct-1 has been found to an in Mol. Cell. Biol. PubMed Scopus Google Scholar). The of Oct-1 as a repressor has been in of the gene, transcription factor gene, factor gene, and gene Mol. Cell. Biol. PubMed Scopus Google Scholar, M. M. F. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, N. R. Blood. 1998; 92: PubMed Google Scholar, K.L. R. J. PubMed Scopus Google Scholar). a of the region of human gene revealed that the transcription factor, Oct-1, an in the of the gene binding to an PubMed Scopus Google Scholar). Oct-1 also as a repressor for γ-globin regulation in adults However, the that the –173 mutation that only decreased the Oct-1 affinity for the promoter region resulted in the HPFH phenotype in transgenic mice reduced the that the change in Oct-1 binding γ-globin expression of the –175 HPFH. both the –175 and the –173 mutation, which binding of GATA-1 and Oct-1 to the promoter region in vitro resulted in HPFH we that the expression of γ-globin gene in adults is not to the decreased affinity of GATA-1 or Oct-1 to the promoter is to that Oct-1 and GATA-1 binding are both for of the γ-globin gene in adult erythrocytes. we the protein binding status within this promoter region in adult erythrocytes of transgenic mice carrying normal or mutant γ-globin the chromosome with or and to for the γ-promoter the in from bone marrow of transgenic mice. that the assay could not of GATA-1 and Oct-1 binding to the –175 region of the promoter. in vitro experiments identified a GATA-1 binding site and an Oct-1 site within the region and a GATA-1 site within the –117 region in to the two GATA-1 sites and the Oct-1 site within the –175 region of the γ-promoter Google the of chromatin was to the of the with the to the –175 region the a footprint assay was The result showed that both the at and of the γ-globin promoter in adult bone marrow from mice carrying μ′LCRAγψβδβ construct, suggesting that bind to this promoter region of the in vitro experiments that the –175 mutation and the –173 mutation Oct-1 and GATA-1 binding in vivo footprint revealed that both –175 and –173 mutation significantly decreased overall protein binding to the region of the in vivo footprint could not which to this region of the chromatin in adult erythrocytes of mice with μ′LCRAγψβδβ, we could that GATA-1 and Oct-1 the in vitro experiments proved binding of the two transcription factors to this a complex containing GATA-1, Oct-1, and other protein factors binds to this promoter region and to the formation of a repressive chromatin γ-globin gene expression in adult erythrocytes. Both the –175 and –173 mutations may the formation of a in the expression of the γ-globin gene in adult erythrocytes. experiments are to and the and of this complex in adult erythrocytes. We and for of the and G. Stamatoyannopoulos and Li for the cosmid and