Analysis of an artificial zinc finger epigenetic modulator: widespread binding but limited regulation

Abstract

Artificial transcription factors (ATFs) and genomic nucleases based on a DNA binding platform consisting of multiple zinc finger domains are currently being developed for clinical applications. However, no genome-wide investigations into their binding specificity have been performed. We have created six-finger ATFs to target two different 18 nt regions of the human SOX2 promoter; each ATF is constructed such that it contains or lacks a super KRAB domain (SKD) that interacts with a complex containing repressive histone methyltransferases. ChIP-seq analysis of the effector-free ATFs in MCF7 breast cancer cells identified thousands of binding sites, mostly in promoter regions; the addition of an SKD domain increased the number of binding sites ∼5-fold, with a majority of the new sites located outside of promoters. De novo motif analyses suggest that the lack of binding specificity is due to subsets of the finger domains being used for genomic interactions. Although the ATFs display widespread binding, few genes showed expression differences; genes repressed by the ATF-SKD have stronger binding sites and are more enriched for a 12 nt motif. Interestingly, epigenetic analyses indicate that the transcriptional repression caused by the ATF-SKD is not due to changes in active histone modifications.

Prolonged re-expression of the hypermethylated geneEPB41L3using artificial transcription factors and epigenetic drugs

“…The functional effects we observed, however, may also be partly caused by the aspecific binding of the EPB41L3-targeting ATFs, as also observed for other ZFPs. 45 Such aspecific binding may result in modulation of off-target genes, although certain ZFATFs might regulate gene expression with single gene resolution. 25,46 A possible explanation for such observed gene specificity may be that most of the off-target promoters have an open chromatin structure which is easily accessible by the ATFs; however, the relative impact of ATFs on actively transcribed genes is small.…”

ZFX acts as a transcriptional activator in multiple types of human tumors by binding downstream from transcription start sites at the majority of CpG island promoters

“…It is possible that multiple sets of zinc fingers in ZFX may recognize a repeating unit of a short motif. Alternatively, it is possible that ZFX utilizes only a subset of its fingers to bind DNA; we have previously described this situation for two artificial six-finger ZNFs (Grimmer et al 2014). To further analyze the ZFX binding preferences, we focused on the AGGCCTAG motif, which is found in >90% of the ZFX peaks in each cell line.…”

Designing Epigenome Editors: Considerations of Biochemical and Locus Specificities

“…For example, it remains unclear how fusions to different DBDs might influence off-target binding. Previous studies suggest that the addition of a KRAB domain to a ZF can greatly increase the ZF’s off-target interactions [70]. This is in contrast to other studies that did not find evidence that addition of the KRAB domain to dCas9 increased off-target binding [78, 79].…”

“…For example, CMEs may have domains that interact with DNA or other proteins located at off-target loci [70]. This sequestering effect can be mitigated by fusing only the core catalytic domains of CMEs to DBDs, or by mutating residues in CMEs to reduce its intrinsic interactions with other binding partners.…”