E-GEOD-44748 - Genome-wide binding profiles of KLF3 and KLF3 mutants in MEF cells
Released on 17 October 2013, last updated on 3 May 2014
Transcription factors are often regarded as being comprised of a DNA-binding domain and a functional domain. The two domains are considered separable and autonomous, with the DNA-binding domain directing the factor to its target genes and the functional domain imparting transcriptional regulation. We have examined a typical Zinc Finger (ZF) transcription factor from the Krüppel-like factor (KLF) family, KLF3. This factor has an N-terminal repression domain that binds the co-repressor C-terminal binding protein (CtBP), and a DNA-binding domain composed of three classical (ZFs) at its C-terminus. We established a system to compare the genomic occupancy profile of wildtype KLF3 with two mutants affecting the N-terminal functional domain: a mutant unable to contact its cofactor CtBP and a mutant lacking the entire N-terminal domain, but retaining the ZFs intact. We used chromatin immunoprecipitation followed by sequencing (ChIP-seq) to assess binding across the genome in murine embryonic fibroblasts. Our results define the in vivo recognition site for KLF3 and the two mutants as a typical CACCC-like element. Unexpectedly, we observe that mutations in the N-terminal functional domain severely affect DNA binding. In general, both mutations reduce binding but there are also instances where binding is retained or even increased. These results provide a clear demonstration that the correct localization of transcription factors to their target genes is not solely dependent on their DNA-contact domains. This informs our understanding of how transcription factors operate and is of relevance to the design of artificial ZF proteins. ChIP-seq was performed on the three samples, KLF3, ΔDL and DBD in duplicate (biological replicates). Input samples were used as controls.
Jon Burdach <email@example.com>, Alister P Funnell, Crisbel M Artuz, Lit Y Tan, Mak K Sin, Merlin Crossley, Richard C Pearson
Regions outside the DNA-binding domain are critical for proper in vivo specificity of an archetypal zinc finger transcription factor. Burdach J, Funnell AP, Mak KS, Artuz CM, Wienert B, Lim WF, Tan LY, Pearson RC, Crossley M. , Europe PMC 24106088