PgmNr M286: Aberrant DNA binding by mutant (E339D) KLF1 induces upregulation of embryonic β-globin in adult mice.

Authors:
D. M. Nebor ¹ ; R. R. Robledo ¹ ; A. C. Perkins ² ; K. Gillinder ² ; J. H. Graber ¹ ; D. Gatti ¹ ; V. Philip ¹ ; J. J. Bieker ³

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Institutes
1) The Jackson Laboratory, Bar Harbor, ME; 2) University of Queensland, Woolloongabba, Australia; 3) Mount Sinai School of Medicine, New York, NY.

Abstract:

The KLF1 transcription factor regulates nearly all aspects of erythroid differentiation including heme synthesis and globin regulation. Previously we showed that the inbred mouse model Nan (neonatal anemia) carries a missense mutation (E339D) in the second zinc finger of KLF1 that causes severe lifelong anemia in heterozygotes accompanied by a striking failure of hemoglobin switching. Embryonic Hbb-bh1 globin expression is significantly upregulated in Nan E14.5 fetal liver (FL) and in adult spleen. In humans elevation of fetal hemoglobin (HbF) ameliorates the severity of sickle cell disease and b-thalassemia. To examine the mechanisms of Hbb-bh1 regulation in adult Nan, we analyzed the global erythroid transcriptome by RNA-seq in wild type (WT) and Nan E14.5 FLs. Only 18% of upregulated and 52% of downregulated genes in Nan overlap known KLF1 target genes. Moreover, just 3% of upregulated Nan genes overlap genes normally activated by KLF1. We also analyzed the transcriptome of sorted erythroid precursors (pro, basophilic, polychromatophilic and orthochromatic erythroblasts) in adult spleens. Together the RNA-seq data show that extensive ectopic gene expression occurs in Nan. To analyze the DNA binding of KLF1^Nan vs. WT, we performed ChIP-seq in K1-ER and K1-NanER cell lines. Results show aberrant DNA binding, both absent and ectopic, by KLF1^Nan. To identify potential genetic modifiers of Hbb-bh1 expression, we performed expression QTL analysis of Hbb-bh1 in two Nan F2 intercrosses and in an outbred high resolution mapping population, the diversity outcross (DO). Statistical analyses reveal multiple significant and suggestive Hbb-bh1 eQTL. Among the genes present in these eQTL confidence intervals, those showing abnormal expression in Nan with dysregulated DNA binding by KLF1^Nan represent strong candidate genes for future functional anaylses as novel regulators of Hbb-bh1 expression. A better understanding of the genetic regulation of the switch of hemoglobin is key to the identification of new drug targets for the treatment of sickle cell disease and β-thalassemia.