Insights into in vivo transcription factor targeting through studies of the archetypal zinc finger protein KLF3.

Background: Transcription factors (TFs) are major drivers of gene regulatory programs and underpin cellular processes including differentiation and development. TFs are often regarded as being minimally 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 examined an archetypal zinc finger (ZF) transcription factor, Krüppel-like factor 3 (KLF3) with an N-terminal domain that binds the co-repressor CtBP, and a DNA-binding domain comprised of three ZFs at its C-terminus. Methods: A system was established to compare the genomic occupancy profile of wild-type KLF3 with two mutants affecting the N-terminal functional domain: a mutant unable to contact the cofactor CtBP and a mutant lacking the entire N-terminal domain, but retaining the ZFs intact. Chromatin immunoprecipitation followed by high-throuput sequencing (CHIP-seq) was used to assess binding across the genome in murine embryonic fibroblasts. Results: The ChIP-seq experiments revealed that KLF3 occupancy is strongly enriched at gene promoters and that KLF3 peak height is related to degree of nucleosome depletion. De novo motif discovery showed that the KLF3 DNA-consensus motif was highly similar to known motifs for KLF1 and KLF4. Unexpectedly, we observed that mutations to the N-terminal domain of KLF3 dramatically altered KLF3 occupancy. Deletion of the N-terminal domain or disruption of the CtBP recruitment motif generally reduced binding, but also enabled retention or even an increase in binding at certain loci. Conclusion: These results provide a clear demonstration that the correct localisation 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 p