GATA-1 and Oct-1 are required for expression of the human alpha-hemoglobin-stabilizing protein gene.
Alpha-hemoglobin-stabilizing protein (AHSP) is an erythroid protein that binds and stabilizes alpha-hemoglobin during normal erythropoiesis and in pathological states of alpha-hemoglobin excess. AHSP has been proposed as a candidate gene in some Heinz body hemolytic anemias and as a modifier gene in the beta-thalassemia syndromes. To gain additional insight into the molecular mechanisms controlling the erythroid-specific expression of the AHSP gene and provide the necessary tools for further genetic studies of these disorders, we have initiated identification and characterization of the regulatory elements controlling the human AHSP gene. We mapped the 5'-end of the AHSP erythroid cDNA and cloned the 5'-flanking genomic DNA containing the putative AHSP gene promoter. In vitro studies using transfection of promoter/reporter plasmids in human tissue culture cell lines, DNase I footprinting analyses and gel mobility shift assays, identified an AHSP gene erythroid promoter with functionally important binding sites for GATA-1- and Oct-1-related proteins. In transgenic mice, a reporter gene directed by a minimal human AHSP promoter was expressed in bone marrow, spleen, and reticulocytes, but not in nonerythroid tissues. In vivo studies using chromatin immunoprecipitation assays demonstrated hyperacetylation of the promoter region and occupancy by GATA-1. The AHSP promoter is an excellent candidate region for mutations associated with decreased AHSP gene expression.
Gallagher
Patrick G
PG
Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06520-8021, USA. patrick.gallagher@yale.edu
Liem
Robert I
RI
Wong
Ellice
E
Weiss
Mitchell J
MJ
Bodine
David M
DM
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001798:Blood Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004920:Erythropoiesis / Q000235:genetics; D050982:GATA1 Transcription Factor / Q000378:metabolism*; D015870:Gene Expression; D005914:Globins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D018832:Molecular Chaperones / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D050811:Octamer Transcription Factor-1 / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
C468768:AHSP protein, human; D001798:Blood Proteins; D018076:DNA, Complementary; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; D018832:Molecular Chaperones; D050811:Octamer Transcription Factor-1; C494165:POU2F1 protein, human; D012333:RNA, Messenger; D011994:Recombinant Proteins; D005914:Globins