Regions outside the DNA-binding domain are critical for proper in vivo specificity of an archetypal zinc finger transcription factor

Abstract

Transcription factors (TFs) are often regarded as being composed of a DNA-binding domain (DBD) and a functional domain. The two domains are considered separable and autonomous, with the DBD directing the factor to its target genes and the functional domain imparting transcriptional regulation. We examined an archetypal zinc finger (ZF) TF, Krüppel-like factor 3 with an N-terminal domain that binds the corepressor CtBP and a DBD composed of three ZFs at its C-terminus. We established a system to compare the genomic occupancy profile of wild-type Krüppel-like factor 3 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 sequencing was used to assess binding across the genome in murine embryonic fibroblasts. Unexpectedly, we observe that mutations in the N-terminal domain generally reduced binding, but there were also instances where binding was retained or even increased. These results provide a clear demonstration that the correct localization of TFs to their target genes is not solely dependent on their DNA-contact domains. This informs our understanding of how TFs operate and is of relevance to the design of artificial ZF proteins.

KLF1 directly activates expression of the novel fetal globin repressor ZBTB7A/LRF in erythroid cells

“…20 Briefly, promoter regions were defined as being 210 to 11 kb from the RefSeq TSS, intronic regions were those lying between exons, and intergenic regions made up the remainder of the genome. Peaks that fell into coding exons or 59 and 39 untranslated region exons or close to the transcription termination sites (2100 bp to 11 kb) were labeled as other.…”

Pluripotent stem cells reveal erythroid-specific activities of the GATA1 N-terminus

“…These regions of the N-finger do not interact with DNA directly, but rather are believed to promote GATA1 chromatin occupancy by recruiting essential cofactors, including FOG1 and the TAL1/LMO2/LDB1 complex (35,38). Similarly, loss of the Kruppel-like factor 3 transcription N-terminus, which also does not bind DNA directly, alters chromatin occupancy selectively at a subset of in vivo target genes in mouse embryonic fibroblasts (54). Taken together, our current findings suggest that interactions between currently unidentified cofactors and the GATA1 N-terminus facilitate chromatin occupancy specifically at erythroid genes.…”

Editing the genome to introduce a beneficial naturally occurring mutation associated with increased fetal globin

“…ChIP was performed using B5 Â 10 7 cells per experiment 37 . Cells were crosslinked with 1% formaldehyde (Sigma-Aldrich) for 10 min at RT and reaction was quenched with glycine at a final concentration of 125 mM.…”