Multiple Defects in Erythroid Gene Expression in Erythroid Krupple-Like Factor (EKLF) Target Genes in EKLF-Deficient Mice.

Abstract

EKLF is the founding member of the KLF family of transcription factors. EKLF-deficient (−/−) mice die at d14-15 of gestation of severe anemia, attributed to decreased expression of the β-globin gene. Morphology of fetal-liver derived circulating erythroid cells in EKLF −/− mice does not mimic that seen in thalassemia, and mice expressing high levels of γ-globin bred onto an EKLF −/− background do not correct the anemia or rescue the −/− mice. These data suggest additional red cell defects are present in EKLF −/− mice contributing to the anemia. To address this hypothesis, we performed subtractive hybridization and microarray analyses with RNA isolated from d13.5 fetal livers of wild type (WT,+/+) and EKLF-deficient mice. In subtractive hybridization, WT (1X) was the tester population and EKLF −/− (30X) was the driver. >200 differentially expressed clones were sequenced. 122 clones were β-globin; 21 alpha hemoglobin stabilizing protein (AHSP); and the remainder were RBC membrane proteins including band 3 and β-spectrin. Microarray analyses were performed with Affymetrix GeneChip Mouse Genome 430 2.0 arrays; 3 independent EKLF +/+ and −/− RNA samples were analyzed. AHSP had the most significantly decreased expression in −/− samples, reduced to 5% of WT (p<0.0001). Other significantly down regulated genes, in addition to those identified by subtractive hybridization, included pyruvate kinase and ankyrin. Reduced expression in EKLF −/− RNA was confirmed using ribonuclease protection assay and/or real-time RT-PCR. AHSP mRNA was decreased by 75–90%; band 3 and β-spectrin were decreased by ~40%; ankyrin by 45%, and pyruvate kinase 15%. Flow cytometry of +/+ and −/ − fetal liver cells using TER119 revealed a TER119Hi population of cells absent in −/ − fetal liver cells, suggesting a block in differentiation to more mature erythroid progenitors. To ensure that the potential EKLF target genes were expressed in the TER119Lo population and were not identified because they were present only in the TER119Hi, the expression of each selected gene was analyzed in TER119Lo and TER119Hi cells by real time PCR. Target gene/α-globin ratios indicated the selected genes were expressed at levels >2 fold higher in TER119Lo than TER119Hi cells. Studies of the AHSP locus identified a strong DNaseI hypersensitive site (HS) in WT fetal liver nuclei between 5′ of the AHSP promoter, that was absent in −/ − chromatin. Chromatin immunoprecipitation analysis of WT fetal liver chromatin spanning 3.5kb of the AHSP locus using 13 primer pairs (~300bp intervals from >1kb 5′ and 3′) demonstrated 2 regions of hyperacetylation of histones H3 and H4. The 5′ region corresponded to the DNaseI HS, and the other region maps 3′ of the AHSP polyA signal. Histones H3 and H4 were also acetylated in the interval between these hyperacetylated regions, while the chromatin upstream and downstream of these regions were hypoacetylated. In chromatin from −/ − fetal liver cells, all sites were hypoacetylated, correlating with the severe reduction in AHSP gene expression. These results support the hypothesis that the anemia in EKLF −/ − mice is due to both decreased expression of the β-globin gene and other erythroid genes including those involved in membrane integrity, stabilization of α-globin protein, and glycolysis leading to defects in erythrocyte structure, function, and metabolism. They also suggest that EKLF may act as a transcription factor and a chromatin modulator for genes other than β-globin.