Alterations in Expression and Chromatin Configuration of the Alpha Hemoglobin-Stabilizing Protein Gene in Erythroid Krüppel-Like Factor-Deficient Mice

Abstract

Erythroid Krüppel-like factor (EKLF) is an erythroid zinc finger protein identified by its interaction with a CACCC sequence in the ␤-globin promoter, where it establishes local chromatin structure permitting ␤-globin gene transcription. We sought to identify other EKLF target genes and determine the chromatin status of these genes in the presence and absence of EKLF. We identified alpha hemoglobin-stabilizing protein (AHSP) by subtractive hybridization and demonstrated a 95 to 99.9% reduction in AHSP mRNA and the absence of AHSP in EKLF-deficient cells. Chromatin at the AHSP promoter from EKLF-deficient cells lacked a DNase I hypersensitive site and exhibited histone hypoacetylation across the locus compared to hyperacetylation of wild-type chromatin. Wild-type chromatin demonstrated a peak of EKLF binding over a promoter region CACCC box that differs from the EKLF consensus by a nucleotide. In mobility shift assays, the AHSP promoter CACCC site bound EKLF in a manner comparable to the ␤-globin promoter CACCC site, indicating a broader recognition sequence for the EKLF consensus binding site. The AHSP promoter was transactivated by EKLF in K562 cells, which lack EKLF. These results support the hypothesis that EKLF acts as a transcription factor and a chromatin modulator for the AHSP and ␤-globin genes and indicate that EKLF may play similar roles for other erythroid genes.

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

“…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.…”

“…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.…”

Non-random subcellular distribution of variant EKLF in erythroid cells

“…Recent studies more directly show that there are other targets, including some in the primitive cell, that are also negatively affected by the absence of EKLF [17][18][19][20]. In the case of Band 4.9 and AHSP expression, this effect is as dramatic in the primitive cell as for β-globin in the definitive cell [17].…”

“…EKLF protein can interact with coactivators such as p300/CBP histone acetyltransferases as well as chromatin remodelers such as SWI/SNF to maximally transactivate the β-globin gene [15,16]. EKLF also activates protein-stabilizing, heme biosynthetic pathway, and red cell membrane protein genes in both primitive and definitive cells [17][18][19][20]. Although EKLF predominantly serves as a transcriptional activator, protein-protein interactions between EKLF and corepressors, such as mSin3A and HDAC1 [21], can result in the stage-specific repression of EKLF target genes [22].…”

Detection and characterization of silencers and enhancer-blockers in the greater CFTR locus

“…The CT-rich motif was quite simple, yet captured variability at individual positions in the silencer sequences. An example of a simple, yet potent, functional motif includes CCNCNCCCN, bound by KLF1 (erythroid Krüppel-like factor, formerly known as EKLF) (Pilon et al 2006). The Weeder analysis identified a 12-bp CT-rich motif in the same data set.…”