Purification and Identification of Proteins That Bind to the Hereditary Persistence of Fetal Hemoglobin –198 Mutation in the γ-Globin Gene Promoter

Abstract

Expression of the ␥-globin gene is silenced in adult humans. However, certain point mutations in the ␥-globin gene promoter are capable of maintaining expression of this gene during adult erythropoiesis, a condition called non-deletion hereditary persistence of fetal hemoglobin (HPFH). Among these, the British form of HPFH carrying a T 3 C point mutation at position ؊198 of the A ␥-globin gene promoter results in 4 -10% fetal hemoglobin in heterozygotes. In this study, we used nuclear extracts from murine erythroleukemia cells to purify a protein complex that binds the HPFH ؊198 ␥-globin gene promoter. Members of this protein complex were identified by mass spectrometry and include DNMT1, the transcriptional coactivator p52, the protein SNEV, and RAP74 (the largest subunit of the general transcription factor IIF). Sp1, which was previously considered responsible for HPFH ؊198 ␥-globin gene activation, was not identified. The potential role of these proteins in the reactivation and/or maintenance of ␥-globin gene expression in the adult transcriptional environment is discussed.The human ␤-globin gene cluster consists of five functional globin genes (⑀, G ␥, A ␥, ␦, and ␤) arranged in the locus according to the order of their expression during development. Genetic and biochemical evidence indicates that the expression of these genes during development depends on interactions between the individual globin gene promoters and the locus control region located 6 -22 kb upstream of the ⑀-globin gene (1). Two developmental expression switches occur in the ␤-globin locus: one from the embryonic ⑀ gene to the fetal ␥ genes and later from the fetal ␥ genes to adult ␦ and ␤ genes. Adult individuals express very low levels of the ␥-globin genes (usually ϳ0.5% of the total hemoglobin). However, single point mutations occurring in either the G ␥-or A ␥-globin gene promoter result in continued expression of the ␥ gene in the adult, a condition termed non-deletion hereditary persistence of fetal hemoglobin (HPFH) 3 (reviewed in Refs. 1 and 2). Structural studies have shown that non-deletion HPFH point mutations are clustered in three regions of the ␥ gene promoters centered around positions Ϫ200, Ϫ175, and Ϫ115 relative to the transcriptional start site (2). The Ϫ200 region is a highly GC-rich region known to be the target for five different but closely spaced point mutations affecting the G ␥ promoter at position Ϫ202 (C 3 G) and the A ␥ promoter at position Ϫ202 (C 3 T), Ϫ198 (T 3 C), Ϫ196 (C 3 T), or Ϫ195 (C 3 G), respectively (1, 2). Two hypotheses have been proposed to explain the increased ␥-globin gene expression in adults carrying non-deletion HPFH mutations. The first proposes that these point mutations decrease the binding of a transcriptional repressor or a complex that is involved in the silencing of ␥-globin expression in the adult. Alternatively, these mutations may create binding sites that enhance the binding for a transcriptional activator or complex, thus increasing ␥ gene expression in the adult. Early in vit...

Correlations with Point Mutations and Severity of Hemolitic Anemias: The Example of Hereditary Persistence of Fetal Hemoglobin with Sickle Cell Anemia and Beta Thalassemia

“…This event allows the CACCC box interaction with the LCR, increasing the production of mRNA for  globin production. Besides SP1, Olave and colleagues identified in the same mutation (-198 T-C) another activator protein complex formed by DNMT1, CDC5-like protein, RAP74, SNEV and P52, that can bind to the promoter region of the  A globin gene, also reactivating the expression of this gene (Olave et al 2007). …”

“…The results showed that a protein complex binds to this region and is able to increase the HbF production. The authors suggested that DNMT1 protein, one member of this putative complex, acts as a chromatin remodeling protein opening the chromatin and triggering the  globin expression (Olave et al 2007). In another interesting study developed by Andrade et al, the pattern of gene expression was evaluated in reticulocytes of an individual with the deletional form of PHHF.…”

Fetal hemoglobin chemical inducers for treatment of hemoglobinopathies

“…The −200 region is a highly GC-rich region known to be the target of five different types of point mutations affecting either the G γ promoter at position −202 and the A γ promoter at positions −202, −198, −196, and −195. Biochemical studies have characterized the nuclear proteins able to bind to the HPFH −198 γ-globin gene promoter and include a protein complex formed by DNMT1, the transcriptional co-activator p52, the protein SNEV, and RAP74 (a subunit of the transcription factor IIF) [21].…”

High Levels of Human γ-Globin are Expressed in Adult Mice Carrying a Transgene of the Brazilian Type of Hereditary Persistence of Fetal Hemoglobin (Aγ −195)

“…The human b-globin gene cluster consists of five functional globin genes (e, A g, G g, d and b) arranged in the locus according to the order of their expression during development (1,2). The switch from fetal (HBG) to adult (HBD and HBB) globin gene expression occurs at birth, leading to the gradual replacement of Hb F (a 2 g 2 ) with Hb A (a 2 b 2 ) and a small amount of Hb A 2 (a 2 d 2 ).…”

“…Alternatively, these mutations may create binding sites that enhance the binding for a transcriptional activator or complex, thus increasing g gene expression in the adult. Early in vitro studies focused on characterizing the effects of these mutations on the binding of different DNA-binding proteins (1).…”