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

Matthew R. Grimmer 1 2 Sabine Stolzenburg 0 5 Ethan Ford 4 Ryan Lister 4 Pilar Blancafort 0 3 5 Peggy J. Farnham 2 0 Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill , Chapel Hill, NC 27599, USA 1 Integrated Genetics and Genomics, University of California-Davis , Davis, CA 95616, USA 2 Norris Comprehensive Cancer Center, University of Southern California , Los Angeles, CA 90089, USA 3 Cancer Epigenetics Group, Harry Perkins Institute of Medical Research , Nedlands, WA 6008, Australia 4 Australian Research Council Centre of Excellence in Plant Energy Biology, The University of Western Australia , Perth, WA 6009, Australia 5 School of Anatomy, Physiology and Human Biology , M309, The University of Western Australia , Crawley, WA 6009, Australia - 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 sixfinger 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 5fold, 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. Dysregulation of normal epigenetic control of DNA methylation and histone modifications at gene promoters and enhancers is a hallmark of cancer (1). These regulatory layers are thought to cooperatively affect nucleosome positioning, chromatin structure and subsequent transcription factor accessibility to the genome (2). Epigenetic drugs targeting DNA methylation (3) and histone modifications (4 6) have shown promise in restoring some normal epigenetic features, but because they affect epigenome-regulating proteins, they are not sequence-specific. Just as modern small-molecule drugs can minimize classic chemotherapeutic side effects by specifically targeting disease-related proteins (7), artificial transcription factors (ATFs) may soon allow precise regulation of disease-related genetic loci. ATFs are proteins that can be engineered to bind and modulate the expression of specific genes through epigenetic effector domains (8,9). Epigenetic activation domains can be used to demethylate DNA (TET), recruit transcriptional machinery (VP64) or remove active histone marks (LSD). Conversely, epigenetic repression domains directly methylate DNA (DNMT3a) or recruit repressive histone modifiers (KRAB, SKD) (10). Genomic nucleases targeted by C2H2 zinc finger (ZNF) DNA binding domains were the first to be developed (11) and proteins composed of ZNF DNA binding domains fused to a nuclease have been employed in clinical trials (NCT00842634, NCT01044654 and NCT01252641). Endogenous ZNF proteins, which served as a model for ATF development, typically bind CG-rich regions, underwent a drastic expansion in vertebrates, and are the largest family of transcription factors in humans (12). ZNF DNA binding domains are comprised of tandem arrays of several ZNFs, each specifically interacting with 3 nucleotides (nt) of DNA in an antiparallel orientation. Observations of endogenous ZNF binding specificities and in vitro refinements lead to the development of the ZNF code for targeting DNA sequences of choice (1318). ATFs typically contain six C2H2 domains intended to target a unique 18 nt *To whom correspondence should be addressed. Tel: 323 442 8015. Email: Correspondence may also be addressed to Pilar Blancafort. Tel: + 61 8 6151 0990. Email: genomic sequence. The majority of endogenous ZNFs also contain an amino-terminal effector domain, facilitating interactions with epigenetic modifiers; the most common ZNF effector domain in humans is the Kru ppel-associated box (KRAB) domain (12). For example, the KRAB domain of ZNF274 recruits a complex, comprised of KAP1 and SETDB1, enriching the repressive histone modification, H3K9me3, at ZNF274 binding sites (19,20). Similarly, the addition of epigenetic effector domains to artificial ZNFs, creating ZNF toggle switches, allows modulation of the local chromatin structure at the intended target site. Unlike designer genomic nucleases or RNAi-based approaches, toggle switches can also upregulate silenced gene expression. For example, KRAB and VP64 effector domains were able to downregulate or upregulate the EGP2 promoter, respectively (21). A handful of genes have been successfully targeted in cancer cells and in tumors using ZNF toggle switches. The silenced tumor suppressor, Maspin, was upregulated using a VP64 activation domain (2224) and the TET DNA demethylase was able to re-activate the ICAM-1 promoter (25). A ZNF-DNMT3a fusion was used to force methylation and downregulate expression at the promoters of the Maspin gene and the oncogene SOX2 (26). Stolzenburg et al. also targeted the SOX2 promoter employing a super KRAB domain (SKD) to recruit repressive histone complexes (27). Effects from toggle switches alone have been fairly modest and no pattern has emerged to predict the efficacy of repression or activation. However, treatment of cells with broadly acting epigenetic drugs and toggle switches can have synergistic effects on target gene regulation (28). Recent studies of Cas9-based ATF toggle switches suggest that simultaneous epigenetic targeting of several neighboring sites may also have synergistic effects on gene regulation (29). This synergism suggests off-target effects of a single ATF may be minimal, but using multiple ATFs clearly requires monitoring genome-wide ATF binding patterns in vivo. Recent ChIP-seq studies of Transcription Activator-Like Effector (TALE)-based (30) and Cas9-based (31) ATFs have been informative, but to date, no genome-wide binding analysis of a ZNF-based ATF has been reported. In this study, we examine the genome-wide binding patterns of two previously reported ZNF effectors, 598-SKD and 552-SKD, which target the SOX2 promoter (26,27). As 598-SKD was more effective in downregulating SOX2 expression (27), we also profiled effects of this ATF on histone modifications and genome-wide expression. We found that ZNF-based ATFs bind very broa (...truncated)