Artificial Zinc Finger DNA Binding Domains: Versatile Tools for Genome Engineering and Modulation of Gene Expression

Abstract

Genome editing and alteration of gene expression by synthetic DNA binding activities gained a lot of momentum over the last decade. This is due to the development of new DNA binding molecules with enhanced binding specificity. The most commonly used DNA binding modules are zinc fingers (ZFs), TALE-domains, and the RNA component of the CRISPR/Cas9 system. These binding modules are fused or linked to either nucleases that cut the DNA and induce DNA repair processes, or to protein domains that activate or repress transcription of genes close to the targeted site in the genome. This review focuses on the structure, design, and applications of ZF DNA binding domains (ZFDBDs). ZFDBDs are relatively small and have been shown to penetrate the cell membrane without additional tags suggesting that they could be delivered to cells without a DNA or RNA intermediate. Advanced algorithms that are based on extensive knowledge of the mode of ZF/DNA interactions are used to design the amino acid composition of ZFDBDs so that they bind to unique sites in the genome. Off-target binding has been a concern for all synthetic DNA binding molecules. Thus, increasing the specificity and affinity of ZFDBDs will have a significant impact on their use in analytical or therapeutic settings.

Zinc-finger proteins in health and disease

“…This new class of proteins was able to bind specific sequences of DNA. 1 , 2 The zinc-finger structure (extensively reviewed in refs 3–7 ) is maintained by the zinc ion, which coordinates cysteine and histidine in different combinations. In classical C2H2 zinc-finger proteins, two cysteines in one chain and two histidines in other one are coordinated by a zinc ion.…”

Analysis of the role of the Hippo pathway in cancer

“…Transcription factors serve a central role in regulating gene expression and mediating a set of biological processes, including differentiation, development, metabolism, apoptosis, autologous phagocytosis and the maintenance of stem cells [73]. According to different DNA binding patterns, transcription factors are mainly divided into classical zinc fingers, homeodomains and a basic helix-loop-helix.…”

Nucleosome distortion as a possible mechanism of transcription activation domain function

“…Additional domains responsible for oligomerization, cellular localization, degradation, and other functions vary greatly between activators. DNA-binding domains are well characterized both by specificity of promoter sequence recognition and by distinct structural features [ 5 – 7 ]; ADs, however, despite being discovered more than three decades ago, remain largely enigmatic [ 3 , 8 ]. Although the broadly accepted mechanism of ADs’ action involves direct physical recruitment of a wide variety of enzymatic activities and protein complexes associated with chromatin remodeling and transcription initiation, the order of recruitment events, structural determinants, and specific amino acid sequences involved in recruitment events are often not clear and/or are contradictory.…”

“…The growing number of human diseases associated with the malfunction of gene expression argues for the development of new drugs affecting specific transcriptional activators or functionally substituting them [ 9 – 11 ]. Technical capabilities for small-molecule artificial activators have been demonstrated by a chemical synthesis of both functional DBDs [ 7 , 9 , 12 ] and ADs [ 13 , 14 ]. Nevertheless, the development of artificial activators with specific transcription activation potential is greatly hampered by deficiencies in our understanding of mechanisms of ADs’ function, which have been documented by different researchers over the past two decades [ 3 , 8 , 15 , 16 ].…”