Abstract

Krüppel-like factors (KLF) are a group of 17 transcription factors with highly conserved DNA-binding domains that contain three C-terminal C2H2-type zinc fingers and a variable N-terminal domain responsible for recruiting cofactors 1. KLFs participate in diverse roles in stem cell renewal, early patterning, organogenesis and tissue homeostasis. Krüppel-like factor 1 (KLF1) is an erythroid-specific KLF responsible for coordinating many aspects of terminal erythroid differentiation 2. It functions as a transcriptional activator by recruiting cofactors such as p300 and chromatin modifiers such as Brg1 via N-terminal transactivation domains 3. Krüppel-like factor 3 (KLF3) acts as a transcriptional repressor via recruitment of C-terminal binding proteins 4. In erythropoiesis, KLF1 directly activates KLF3 via an erythroid-specific promoter 5. Some KLF1 target genes are upregulated in Klf3-/- fetal liver suggesting possible competition between the two factors for promoter/enhancer occupancy. We generated three independent clones of the erythroid cell line, J2E, by retroviral transduction of a tamoxifen-inducible version of Klf3 (Klf3-ERTM) as previously described 6. Using next-generation sequencing of newly synthesised RNA (4sU-labeling), we show KLF3 induction leads to immediate repression of a set of ~580 genes; a subset of these (54) are also directly induced by KLF1 in K1-ER cells, suggesting antagonistic regulation. Indeed, ChIP-seq revealed KLF1 and KLF3 bind many of the same regulatory sites within the erythroid cell genome. KLF3 also binds an independent set of promoters which are not bound by KLF1, suggesting it also plays a KLF1-independent role in maintenance of gene repression. By de novo motif discovery we confirm KLF3 binds preferably to a extended CACCC motif, R-CCM-CRC-CCN, so the DNA-binding specificity in vivo is indistinguishable from the KLF1 binding specificity 7, and is independent of co-operating DNA-binding proteins or cofactors. Using Q-PCR of KLF1 ChIPed DNA in J2E-Klf3ER cells, we show that overexpression of KLF3 directly displaces KLF1 from many key target sites such as the E2f2 enhancer and this leads to down regulation of gene expression. This is the first proof that KLF1 and KLF3 directly compete for key promoters and enhancers which drive erythroid cell proliferation and differentiation. We propose KLF3 acts to 'fine-tune' transcription in erythropoiesis by repressing genes activated by KLF1 and that this negative feedback system is necessary for precise control over the generation of erythrocytes. It also works independently of KLF1 perhaps via competition for binding with other KLF/SP factors.

References:

1. van Vliet J, Crofts LA, Quinlan KG, Czolij R, Perkins AC, Crossley M. Human KLF17 is a new member of the Sp/KLF family of transcription factors. Genomics. 2006;87(4):474-482.

2. Tallack MR, Magor GW, Dartigues B, et al. Novel roles for KLF1 in erythropoiesis revealed by mRNA-seq. Genome Res. 2012.

3. Perkins A, Xu X, Higgs DR, et al. "Kruppeling" erythropoiesis: an unexpected broad spectrum of human red blood cell disorders due to KLF1 variants unveiled by genomic sequencing. Blood. 2016.

4. Dewi V, Kwok A, Lee S, et al. Phosphorylation of Kruppel-like factor 3 (KLF3/BKLF) and C-terminal binding protein 2 (CtBP2) by homeodomain-interacting protein kinase 2 (HIPK2) modulates KLF3 DNA binding and activity. J Biol Chem. 2015;290(13):8591-8605.

5. Funnell AP, Maloney CA, Thompson LJ, et al. Erythroid Kruppel-like factor directly activates the basic Kruppel-like factor gene in erythroid cells. Mol Cell Biol. 2007;27(7):2777-2790.

6. Coghill E, Eccleston S, Fox V, et al. Erythroid Kruppel-like factor (EKLF) coordinates erythroid cell proliferation and hemoglobinization in cell lines derived from EKLF null mice. Blood. 2001;97(6):1861-1868.

7. Tallack MR, Whitington T, Yuen WS, et al. A global role for KLF1 in erythropoiesis revealed by ChIP-seq in primary erythroid cells. Genome Res. 2010;20(8):1052-1063.

Disclosures

Perkins:Novartis Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria.

Author notes

*

Asterisk with author names denotes non-ASH members.