Genomic organisation and regulation of murine alpha haemoglobin stabilising protein by erythroid Kruppel‐like factor

Abstract

SummaryAlpha haemoglobin stabilising protein (AHSP) binds free a-globin chains and plays an important role in the protection of red cells, particularly during b-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 a-globin chains, which exist because of the defect in b-globin gene activation.

A global role for KLF1 in erythropoiesis revealed by ChIP-seq in primary erythroid cells

“…We expected KLF1 ChIP-seq peaks would be found at the proximal promoters of genes within the beta-globin gene cluster (e.g., Hbb-b1) (Im et al 2005;Keys et al 2007) and within the beta-globin LCR (Im et al 2005;Shyu et al 2006). Indeed, we observed KLF1 binding at the Hbb-b1 promoter and at HS1, HS2, HS4, and HS3 (weakly) of the beta-globin LCR (as defined in Hardison et al 1997) (Fig.…”

“…3A). Previous studies have also described KLF1 as a critical regulator of the gene encoding the alpha-globin chaperone protein Ahsp (Pilon et al 2006;Keys et al 2007). Unfortunately the Ahsp gene is missing from the mm8 and mm9 genome assemblies, preventing its inclusion in the comprehensive list of KLF1 ChIPseq target genes reported in this study.…”

“…ChIP assays were performed as previously described (Hodge et al 2006) using a rabbit polyclonal antibody raised against the N terminus of KLF1 (Perkins et al 1995;Keys et al 2007) in Klf3 À/À or wild-type (WT) fetal livers collected from E14.5 mouse embryos. At this stage of development the fetal liver is entirely erythroid and composed of cells at various stages of maturation from proerythroblasts to late enucleating normoblasts.…”

“…Direct transcriptional target genes of KLF1 are few to date, but loss of some are likely to explain these phenotypes, at least in part. These KLF1 target genes include Hbb-b1 (beta major globin), Ahsp, Epb4.9 (also known as Dematin), Klf3 (also known as Bklf ), E2f2, and Cdkn2c (also known as p18 INK4c ) (Miller and Bieker 1993;Nuez et al 1995;Perkins et al 1995;Drissen et al 2005;Hodge et al 2006;Pilon et al 2006Pilon et al , 2008Funnell et al 2007;Keys et al 2007;Tallack et al 2007Tallack et al , 2009.…”

Distinct modes of gene regulation by a cell-specific transcriptional activator

“…Array data and direct analysis have shown that the AHSP promoter is another EKLF-dependent transcriptional target that is highly expressed in erythroid cells (22,37,52). However, its promoter contains neither an INI nor a DPE.…”