Abstract
Expression of the gamma-globin gene is silenced in adult humans. However, certain point mutations in the gamma-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-->C point mutation at position -198 of the Agamma-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 gamma-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 gamma-globin gene activation, was not identified. The potential role of these proteins in the reactivation and/or maintenance of gamma-globin gene expression in the adult transcriptional environment is discussed.
Highlights
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
Because the CACCC box is indispensable for ␥ gene expression in the adult, these results suggested that the hereditary persistence of fetal hemoglobin (HPFH) Ϫ198 mutation creates a new element that is able to substitute for the function of the CACCC box in adults
We have identified a set of proteins (DNMT1, CDC5-like protein, RAP74, SNEV, the coactivator p52, and a protein of unknown function) that are capable of binding to the HPFH Ϫ198 (T 3 C) mutation present in the A␥-globin gene promoter
Summary
The pooled Source Q protein fraction was supplemented with 5 mM MgCl2, 0.05% Nonidet P-40, and 3 g/ml oligo(dI-dC), bringing the buffer conditions similar to those used in gel mobility shift assays This mixture was incubated on ice for 15 min and centrifuged at 14,000 rpm at 4 °C for 10 min, and the supernatant was used as the input material for the tandem oligonucleotide affinity chromatography step. To visualize the eluted proteins from the tandem oligonucleotide affinity chromatography step, 150 –250-l aliquots of each of the column fractions were precipitated overnight at Ϫ20 °C with 4 volumes of cold acetone in the presence of 10 g of human recombinant insulin (Sigma) as a carrier This mixture was centrifuged at 14,000 rpm for 15 min at 4 °C, and the pelleted proteins were washed twice with 1 ml of cold acetone, dried at 37 °C, and suspended in 20 l of suspension buffer (50 mM Tris-HCl (pH 8.0), 1% SDS, and 5% glycerol). Online data mining was done with the Matrix Science Mascot tandem mass spectrometry (MS/MS) ion search algorithm (www.matrixscience.com) using both the mouse and NCBI non-redundant protein data bases
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