EKLF/KLF1 Controls Cell Cycle Entry via Direct Regulation of E2f2

Andrew C. Perkins,Janelle R. Keys,Patrick O. Humbert,Michael R. Tallack

doi:10.1074/jbc.m109.006346

Andrew C. Perkins, Janelle R. Keys + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m109.006346

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Abstract

Differentiation of erythroid cells requires precise control over the cell cycle to regulate the balance between cell proliferation and differentiation. The zinc finger transcription factor, erythroid Krüppel-like factor (EKLF/KLF1), is essential for proper erythroid cell differentiation and regulates many erythroid genes. Here we show that loss of EKLF leads to aberrant entry into S-phase of the cell cycle during both primitive and definitive erythropoiesis. This cell cycle defect was associated with a significant reduction in the expression levels of E2f2 and E2f4, key factors necessary for the induction of S-phase gene expression and erythropoiesis. We found and validated novel intronic enhancers in both the E2f2 and E2f4 genes, which contain conserved CACC, GATA, and E-BOX elements. The E2f2 enhancer was occupied by EKLF in vivo. Furthermore, we were able to partially restore cell cycle dynamics in EKLF(-/-) fetal liver upon additional genetic depletion of Rb, establishing a genetic causal link between reduced E2f2 and the EKLF cell cycle defect. Finally, we propose direct regulation of the E2f2 enhancer is a generic mechanism by which many KLFs regulate proliferation and differentiation.

Highlights

Erythroid Kruppel-like factor (EKLF/KLF1),5 the founding member of the Kruppel-like factor (KLF) family of C2H2 zinc finger transcription factors, is essential for erythropoiesis [1,2,3]
We show partial rescue of the cell cycle phenotype in EKLFϪ/Ϫ mice is achieved by depletion of the E2F-binding protein Rb, providing further evidence for a genetic link between EKLF and the E2F-Rb G1/S checkpoint
There was a significant reduction in cells with S-phase DNA content during primitive (E10.5 peripheral blood, Fig. 1A) and definitive (E14.5 fetal liver, Fig. 1B) erythropoiesis in EKLFϪ/Ϫ (Ek) embryos when compared with wild-type littermates (WT)

Summary

EXPERIMENTAL PROCEDURES

Mouse Studies—Rbϩ/Ϫ EKLFϩ/Ϫ mice were obtained by mating EKLFϩ/Ϫ mice [3] and Rb ϩ/Ϫ mice [12]. Chromatin Immunoprecipitation (ChIP)—The erythroid cell line K1zf-ER was created by immortalization of EKLFϪ/Ϫ erythroid progenitors with J2 retrovirus as previously described [27] followed by infection with murine stem cell virus expressing the zinc finger DNA binding region (amino acids 273–376) of murine EKLF as an ERTM fusion. These cells or E14.5 fetal liver erythroid cells were used to determine in vivo chromatin occupancy of EKLF. Reverse oligonucleotides were annealed in excess after labeling of the forward strand with T4 polynucleotide kinase (Promega) and [␥-32P]ATP (PerkinElmer Life Sciences)

RESULTS

DISCUSSION

The KLF Family as Cell Cycle