Genomic organisation and regulation of murine alpha haemoglobin stabilising protein by erythroid Kruppel-like factor.
Alpha haemoglobin stabilising protein (AHSP) binds free alpha-globin chains and plays an important role in the protection of red cells, particularly during beta-thalassaemia. Murine ASHP was discovered as a GATA-1 target gene and human AHSP is directly regulated by GATA-1. More recently, AHSP was rediscovered as a highly erythroid Kruppel-like factor (EKLF) -dependent transcript. We have determined the organisation of the murine AHSP gene and compared it to orthologs. There are two CACC box elements in the proximal promoter. The proximal element is absolutely conserved, but does not bind EKLF as it is not a canonical binding site. In rodents, the distal element contains a 3 bp insertion that disrupts the typical EKLF binding consensus region. Nevertheless, EKLF binds this atypical site by gel mobility shift assay, specifically occupies the AHSP promoter in vivo in a chromatin immunoprecipitation assay, and transactivates AHSP through this CACC site in promoter-reporter assays. Our results suggest EKLF can occupy CACC elements in vivo that are not predictable from the consensus binding site inferred from structural studies. We also propose that absence of AHSP in EKLF-null red cells exacerbates the toxicity of free alpha-globin chains, which exist because of the defect in beta-globin gene activation.
Keys
Janelle R
JR
Institute for Molecular Bioscience, University of Queensland, St Lucia, Qld, Australia.
Tallack
Michael R
MR
Hodge
Denise J
DJ
Cridland
Simon O
SO
David
Rakesh
R
Perkins
Andrew C
AC
D000818:Animals; D001483:Base Sequence; D001798:Blood Proteins / Q000235:genetics*; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D016384:Consensus Sequence; D017931:DNA Primers / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D004912:Erythrocytes / Q000378:metabolism*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D016678:Genome; D005914:Globins / Q000378:metabolism; D051741:Kruppel-Like Transcription Factors / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D018832:Molecular Chaperones / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D015533:Transcriptional Activation
C511177:Ahsp protein, mouse; D001798:Blood Proteins; D017931:DNA Primers; D051741:Kruppel-Like Transcription Factors; D018832:Molecular Chaperones; C080774:erythroid Kruppel-like factor; D005914:Globins