Classical Zinc Finger Research Articles

Neural Zinc Finger Factor/Myelin Transcription Factor Proteins: Metal Binding, Fold, and Function.

Zinc finger (ZF) proteins make up a large family of metalloproteins that contain discrete domains with amino acid ligands (cysteine and histidine) that serve to coordinate zinc in a tetrahedral geometry. Upon zinc coordination, the domains adopt three-dimensional structure. The most well-studied ZFs are the "classical" ZFs, which use a Cys2His2 motif to bind zinc and adopt an antiparallel β sheet/α helical fold. In addition to the classical ZF class, at least 13 other ZF classes, collectively termed nonclassical ZFs, have been identified. These other classes are distinguished by the combination and order of the cysteine and histidine ligands within each domain, the spacing between each ligand (i.e., number and type of amino acid), and the structural architecture that the domain adopts in the presence of zinc. One class of nonclassical ZFs, the neural zinc finger/myelin transcription factor (NZF/MyT) class, contains ZF domains with a Cys2His2Cys ligand set, adopts a fold that consists of a series of loops in the presence of zinc, and functions as transcription factors by binding to and regulating genes that are critical for the development of the central nervous system. This Current Topic focuses on our understanding of the NZF/MyT class of nonclassical ZFs and presents current hypotheses regarding this class' unique mechanism of metal-mediated folding and function.

Myocardial ischemic preconditioning upregulated protein 1(Mipu1):zinc finger protein 667 - a multifunctional KRAB/C2H2 zinc finger protein.

Myocardial ischemic preconditioning upregulated protein 1 (Mipu1) is a newly discovered upregulated gene produced in rats during the myocardial ischemic preconditioning process. Mipu1 cDNA contains a 1824-base pair open reading frame and encodes a 608 amino acid protein with an N-terminal Krüppel-associated box (KRAB) domain and classical zinc finger C2H2 motifs in the C-terminus. Mipu1 protein is located in the cell nucleus. Recent studies found that Mipu1 has a protective effect on the ischemia-reperfusion injury of heart, brain, and other organs. As a nuclear factor, Mipu1 may perform its protective function through directly transcribing and repressing the expression of proapoptotic genes to repress cell apoptosis. In addition, Mipu1 also plays an important role in regulating the gene expression of downstream inflammatory mediators by inhibiting the activation of activator protein-1 and serum response element.

Is HNF4A a candidate to study zinc finger protein slug?

Protein-Protein Interactions (PPI) play a crucial role in deciphering function besides identifying candidates. While the experimental analysis is often time consuming involving number of experiments like pulldown assays, they are not necessarily limiting the ability to detect novel protein interactors. In this work, we discuss the role and putative interactors of SNAI2, a slug protein which is involved in the development of cancer progression. The protein interactions have been deciphered by domain pair exclusion method which gives confidence to already precluded interaction pairs. Additionally, conservation patterns of the slug protein have also been analysed by estimating site-specific evolutionary rates at structural level. Based upon the computational analysis, we consider HNF4A could be a putative candidate to study zinc finger protein slug. We believe, this candidate study augmented with structural conservation will definitely provide novel insights into the design and discovery of new interactions for zinc finger class of proteins besides providing possible clues for discovery of various cancer types associated with this class of proteins.

Macrophage-inducible C-type lectin Mincle-expressing dendritic cells contribute to control of splenic Mycobacterium bovis BCG infection in mice.

The macrophage-inducible C-type lectin Mincle has recently been identified to be a pattern recognition receptor sensing mycobacterial infection via recognition of the mycobacterial cell wall component trehalose-6',6-dimycolate (TDM). However, its role in systemic mycobacterial infections has not been examined so far. Mincle-knockout (KO) mice were infected intravenously with Mycobacterium bovis BCG to mimic the systemic spread of mycobacteria under defined experimental conditions. After intravenous infection with M. bovis BCG, Mincle-KO mice responded with significantly higher numbers of mycobacterial CFU in spleen and liver, while reduced granuloma formation was observed only in the spleen. At the same time, reduced Th1 cytokine production and decreased numbers of gamma interferon-producing T cells were observed in the spleens of Mincle-KO mice relative to the numbers in the spleens of wild-type (WT) mice. The effect of adoptive transfer of defined WT leukocyte subsets generated from bone marrow cells of zDC(+/DTR) mice (which bear the human diphtheria toxin receptor [DTR] under the control of the classical dendritic cell-specific zinc finger transcription factor zDC) to specifically deplete Mincle-expressing classical dendritic cells (cDCs) but not macrophages after diphtheria toxin application on the numbers of splenic and hepatic CFU and T cell subsets was then determined. Adoptive transfer experiments revealed that Mincle-expressing splenic cDCs rather than Mincle-expressing macrophages contributed to the reconstitution of attenuated splenic antimycobacterial immune responses in Mincle-KO mice after intravenous challenge with BCG. Collectively, we show that expression of Mincle, particularly by cDCs, contributes to the control of splenic M. bovis BCG infection in mice.

Structural metal sites in nonclassical zinc finger proteins involved in transcriptional and translational regulation.

Zinc finger (ZF) proteins are a large family of metalloproteins that utilize zinc for structural purposes. Zinc coordinates to a combination of cysteine thiol and histidine imidazole residues within the ZF polypeptide sequence resulting in a folded and functional protein. Initially, a single class of ZFs were identified. These ZFs, now referred to as the "classical" ZFs, utilize a Cys2His2 (CCHH) ligand set to bind zinc. Upon Zn coordination, the classical ZFs fold into a structure made up of an α helix and an antiparallel β sheet. When folded, classical ZFs recognize and bind to specific DNA targets and function as transcription factors. With the advent of genome sequencing and proteomics, many additional classes of ZFs were identified based upon their primary amino acid sequences. At least 13 additional classes of ZFs are known, and collectively these "nonclassical" ZFs differ in the ligand set involved in Zn(II) coordination, the organization of the ligands within the polypeptide sequence and the macromolecular targets. Some nonclassical ZFs are DNA binding "transcription factors", while others are involved in RNA regulation and protein recognition. Much less is known about these nonclassical ZFs with regards to the roles of metal coordination in fold and function. This Account focuses on our laboratory's efforts to characterize two families of "nonclassical" ZFs: the Cys3His (or CCCH) ZF family and the Cys2His2Cys (or CCHHC) ZF family. Our work on the CCCH ZF family has focused on the protein Tristetraprolin (TTP), which is a key protein in regulating inflammation. TTP contains two CCCH domains that were proposed to be ZFs based upon their sequence. We have shown that while this protein can coordinate Zn(II) at the CCCH sites, it can also coordinate Fe(II) and Fe(III). Moreover, the zinc and iron bound forms of TTP are equally adept at discriminating between RNA targets, which we have demonstrated via a fluorescence anisotropy based approach. Thus, CCCH type ZFs appear to be promiscuous with respect to metal preference and a role for iron coordination in CCCH ZF function is proposed. The CCHHC family of ZFs is a small family of nonclassical ZFs that are essential for the development of the central nervous system. There are three ZFs in this family: neural zinc finger factor-1 (NZF-1), myelin transcription factor-1 (MyT1), and suppressor of tumorgenicity 18 (ST18). All three proteins contain multiple clusters of "CCHHC" domains, which are all predicted to be Zn binding domains. We have focused on a tandem-CCHHC domain construct of NZF-1, which recognizes β-RARE DNA, and we have identified key residues required for DNA recognition. Unlike classical ZFs, for which a few conserved residues are required for DNA recognition, the CCHHC class of ZFs utilize a few nonconserved residues to drive DNA recognition leading us to propose a new paradigm for ZF/DNA binding.

KLF15 regulates slow myosin heavy chain expression through NFATc1 in C2C12 myotubes

A comprehensive understanding of genetic and environmental factors that control skeletal muscle fiber type specification and transformation is essential not only in sports science, but also in myopathy and metabolic disorders. Krüppel-like factors (KLFs) are a subfamily of the zinc-finger class of transcription factors, which are involved in the development, homeostasis, and pathology of cardiovascular systems. Compared to cardiac and smooth muscles, the role of KLFs in skeletal muscle is much less understood. In this study, the endogenous expression of KLF15 was analyzed in differentiating C2C12 muscle cells and mouse skeletal muscle. Our data indicated that Klf15 was upregulated during myogenic differentiation and higher levels of Klf15 mRNA were detected in mouse slow, oxidative soleus muscle (SL) compared to that in fast, glycolytic tibialis anterior muscle (TA), indicating that KLF15 may play a role in myogenesis or myofiber typing. Additional studies revealed that KLF15 regulated the expression of MHC-β/slow rather than muscle cell differentiation. Gene silencing, overexpression, and luciferase reporter assay showed that KLF15 regulated MHC-β/slow by binding to Nfatc1 promoter, inducing its activity, therefore mediating calcineurin/NFAT signaling. Our study contributed to the current knowledge on KLFs in skeletal muscle, and it indicated a need for further intensive studies on the redundant and divergent functions of KLFs.

An Experimentally Tested Scenario for the Structural Evolution of Eukaryotic Cys2His2 Zinc Fingers from Eubacterial Ros Homologs

The exact evolutionary origin of the zinc finger (ZF) domain is unknown, as it is still not clear from which organisms it was first derived. However, the unique features of the ZF domains have made it very easy for evolution to tinker with them in a number of different manners, including their combination, variation of their number by unequal crossing-over or tandem duplication and tuning of their affinity for specific DNA sequence motifs through point substitutions. Classical Cys2His2 ZF domains as structurally autonomous motifs arranged in multiple copies are known only in eukaryotes. Nonetheless, a single prokaryotic Cys2His2 ZF domain has been identified in the transcriptional regulator Ros from Agrobacterium tumefaciens and recently characterized. The present work focuses on the evolution of the classical ZF domains with the goal of trying to determine whether eukaryotic ZFs have evolved from the prokaryotic Ros-like proteins. Our results, based on computational and experimental data, indicate that a single insertion of three amino acids in the short loop that separates the β-sheet from the α-helix of the Ros protein is sufficient to induce a structural transition from a Ros like to an eukaryotic-ZF like structure. This observation provides evidence for a structurally plausible and parsimonious scenario of fold evolution, giving a structural basis to the hypothesis of a horizontal gene transfer (HGT) from bacteria to eukaryotes.

New Insights into DNA Recognition by Zinc Fingers Revealed by Structural Analysis of the Oncoprotein ZNF217

Classical zinc fingers (ZFs) are one of the most abundant and best characterized DNA-binding domains. Typically, tandem arrays of three or more ZFs bind DNA target sequences with high affinity and specificity, and the mode of DNA recognition is sufficiently well understood that tailor-made ZF-based DNA-binding proteins can be engineered. We have shown previously that a two-zinc finger unit found in the transcriptional coregulator ZNF217 recognizes DNA but with an affinity and specificity that is lower than other ZF arrays. To investigate the basis for these differences, we determined the structure of a ZNF217-DNA complex. We show that although the overall position of the ZFs on the DNA closely resembles that observed for other ZFs, the side-chain interaction pattern differs substantially from the canonical model. The structure also reveals the presence of two methyl-π interactions, each featuring a tyrosine contacting a thymine methyl group. To our knowledge, interactions of this type have not previously been described in classical ZF-DNA complexes. Finally, we investigated the sequence specificity of this two-ZF unit and discuss how ZNF217 might discriminate its target DNA sites in the cell.

Genetic and epigenetic mutations affect the DNA binding capability of human ZFP57 in transient neonatal diabetes type 1

In the mouse, ZFP57 contains three classical Cys2His2 zinc finger domains (ZF) and recognizes the methylated TGCmetCGC target sequence using the first and the second ZFs. In this study, we demonstrate that the human ZFP57 (hZFP57) containing six Cys2His2 ZFs, binds the same methylated sequence through the third and the fourth ZFs, and identify the aminoacids critical for DNA interaction. In addition, we present evidences indicating that hZFP57 mutations and hypomethylation of the TNDM1 ICR both associated with Transient Neonatal Diabetes Mellitus type 1 result in loss of hZFP57 binding to the TNDM1 locus, likely causing PLAGL1 activation.

Solution Structure of an Archaeal DNA Binding Protein with an Eukaryotic Zinc Finger Fold

While the basal transcription machinery in archaea is eukaryal-like, transcription factors in archaea and their viruses are usually related to bacterial transcription factors. Nevertheless, some of these organisms show predicted classical zinc fingers motifs of the C2H2 type, which are almost exclusively found in proteins of eukaryotes and most often associated with transcription regulators. In this work, we focused on the protein AFV1p06 from the hyperthermophilic archaeal virus AFV1. The sequence of the protein consists of the classical eukaryotic C2H2 motif with the fourth histidine coordinating zinc missing, as well as of N- and C-terminal extensions. We showed that the protein AFV1p06 binds zinc and solved its solution structure by NMR. AFV1p06 displays a zinc finger fold with a novel structure extension and disordered N- and C-termini. Structure calculations show that a glutamic acid residue that coordinates zinc replaces the fourth histidine of the C2H2 motif. Electromobility gel shift assays indicate that the protein binds to DNA with different affinities depending on the DNA sequence. AFV1p06 is the first experimentally characterised archaeal zinc finger protein with a DNA binding activity. The AFV1p06 protein family has homologues in diverse viruses of hyperthermophilic archaea. A phylogenetic analysis points out a common origin of archaeal and eukaryotic C2H2 zinc fingers.

Nucleophilic reactivity of Zinc-bound thiolates: subtle interplay between coordination set and conformational flexibility

During the past decades it has been established that zinc-bound cysteines in proteins can react with various electrophiles to perform physiological functions such as alkyl transfer reactions or oxidative stress sensing. Electrophiles targeting especially vulnerable structural zinc fingers have also been proposed as therapeutic agents against cancers or HIV. However, the nucleophilic reactivity of zinc fingers remains poorly understood. In this article, we investigate the nucleophilic reaction of zinc finger model peptides with H2O2 in order to get deeper insight into the factors governing the reactivity of zinc-bound cysteines of zinc finger sites. We use a set of nine peptides belonging to two different peptide families with (Cys)4, (Cys)3(His) and (Cys)2(His)2 coordination sets. One family is derived from the consensus peptide of classical ββα zinc fingers and the other one derived from the zinc finger site of the oxidative stress sensor Hsp33 that adopts a loosened zinc ribbon fold. The coordination properties and the structural behaviors of the new members of the latter family were carefully characterized. The rate constants of the reaction of the nine zinc finger models with H2O2 were measured at various temperatures to determine the activation parameters. In all cases, the reaction is characterized by a small enthalpy of activation, which shows that the nucleophilic reaction of zinc-bound cysteines is easy, and large unfavorable negative entropy of activation. Neutral Zn(Cys)2(His)2 cores are intrinsically less reactive than negatively charged Zn(Cys)4 and Zn(Cys)3(His). Interestingly, we observe that the more flexible zinc finger sites are the more reactive. Indeed the entropies of activation are strongly linked to the conformational behavior of the peptide in solution. This work reveals important factors that govern the reactivity of zinc-bound cysteines in these two structural classes of zinc fingers and can be used to identify reactive zinc fingers in proteins.

Effect of <i>MIG1</i> Gene Deletion on Glucose Repression in Baker’s Yeast

Mig1p, a zinc finger class of DNA-binding protein, plays a critical role in glucose repression for maltose utilization in Baker’s yeast. Maltose is the hydrolyzate of starch, which is the main component of dough. In this study, the recombinant yeast strains with MIG1 gene deletion were constructed, and the maltose metabolism of the parental and mutant strains in batch cultivations were investigated. Our results show that the degree of glucose repression of mutants △MIG1α and △MIG1a are reduced by 49.88% and 41.59% respectively compared to their parental strains, suggesting that MIG1 deletion can partially relieve glucose repression of maltose metabolism.

The Multi-zinc Finger Protein ZNF217 Contacts DNA through a Two-finger Domain

Classical C2H2 zinc finger proteins are among the most abundant transcription factors found in eukaryotes, and the mechanisms through which they recognize their target genes have been extensively investigated. In general, a tandem array of three fingers separated by characteristic TGERP links is required for sequence-specific DNA recognition. Nevertheless, a significant number of zinc finger proteins do not contain a hallmark three-finger array of this type, raising the question of whether and how they contact DNA. We have examined the multi-finger protein ZNF217, which contains eight classical zinc fingers. ZNF217 is implicated as an oncogene and in repressing the E-cadherin gene. We show that two of its zinc fingers, 6 and 7, can mediate contacts with DNA. We examine its putative recognition site in the E-cadherin promoter and demonstrate that this is a suboptimal site. NMR analysis and mutagenesis is used to define the DNA binding surface of ZNF217, and we examine the specificity of the DNA binding activity using fluorescence anisotropy titrations. Finally, sequence analysis reveals that a variety of multi-finger proteins also contain two-finger units, and our data support the idea that these may constitute a distinct subclass of DNA recognition motif.

Genome-wide mapping of ZNF652 promoter binding sites in breast cancer cells

A significant proportion of transcription factors encoded by the human genome are classical C(2) H(2) zinc finger proteins that regulate gene expression by directly interacting with their cognate DNA binding motifs. We previously showed that one such C(2) H(2) zinc finger DNA binding protein, ZNF652 (zinc finger protein 652), specifically and functionally interacts with CBFA2T3 to repress transcription of genes involved in breast oncogenesis. To identify potential targets by which ZNF652 exerts its putative tumour suppressive function, its promoter-specific cistrome was mapped by ChIP-chip. De novo motif scanning of the ZNF652 binding sites identified a novel ZNF652 recognition motif that closely resembles the previously characterised in vitro binding site, being a 10 nucleotide core of that 13 nucleotide sequence. Genes with ZNF652 binding sites function in diverse cellular pathways, and many are involved in cancer development and progression. Characterisation of the in vivo ZNF652 DNA binding motif and identification of potential ZNF652 target genes are key steps towards elucidating the function(s) of this transcription factor in the normal and malignant breast cell.

Novel human BTB/POZ domain-containing zinc finger protein ZBTB1 inhibits transcriptional activities of CRE

BTB/POZ protein family plays a key role in many biological processes by regulating the transcriptional activities of some downstream genes. Here, we characterized the member of C(2)H(2) type zinc finger gene, Zinc finger and BTB domain containing 1 (ZBTB1). The complete sequence of ZBTB1 cDNA contains a 2142bp open reading frame (ORF) and encodes a 713 amino acid protein with an N-terminal BTB/POZ domain that is similar to the same domain of other known transcription regulators and eight classical zinc finger C(2)H(2) motifs in the C-terminus. Subcellular localization analysis demonstrated that ZBTB1 was localized to the nucleus, forming dot-like structures. Transcriptional activity assays showed that ZBTB1 was a transcription repressor and overexpression of ZBTB1 in the COS7 cells reduced the transcriptional activities of cAMP response element (CRE). Further studies showed that the BTB domain and ZNF motifs of ZBTB1 may both be involved in this suppression. These results suggest that ZBTB1 protein may act as a transcription repressor in the activation of CREB and cAMP-mediated signal transduction pathways to mediate cellular functions.

Classical Cys2His2Zinc Finger Peptides Are Rapidly Oxidized by Either H2O2or O2Irrespective of Metal Coordination

ZIF268, a member of the classical zinc finger protein family, contains three Cys(2)His(2) zinc binding domains that together recognize the DNA sequence 5'-AGCGTGGGCGT-3'. These domains can be fused to an endonuclease to make a chimeric protein to target and cleave specific DNA sequences. A peptide corresponding to these domains, named ZIF268-3D, has been prepared to determine if the zinc finger domain itself can promote DNA cleavage when a redox active metal ion, Fe(II), is coordinated. The UV-vis absorption spectrum of Fe(II)-ZIF268-3D is indicative of Fe(II) coordination. Using fluorescence anisotropy, we demonstrate that Fe(II)-ZIF268-3D binds selectively to its target DNA in the same manner as Zn(II)-ZIF268-3D. In the presence of added oxidant, H(2)O(2) or O(2), DNA cleavage is not observed by Fe(II)-ZIF268-3D. Instead, the peptide itself is rapidly oxidized. Similarly, Zn(II)-ZIF268-3D and apo-ZIF268-3D are rapidly oxidized by H(2)O(2) or O(2), and we propose that ZIF268-3D is highly susceptible to oxidation.

Cysteine and histidine shuffling: mixing and matching cysteine and histidine residues in zinc finger proteins to afford different folds and function

Zinc finger proteins utilize zinc for structural purposes: zinc binds to a combination of cysteine and histidine ligands in a tetrahedral coordination geometry facilitating protein folding and function. While much is known about the classical zinc finger proteins, which utilize a Cys(2)His(2) ligand set to coordinate zinc and fold into an anti-parallel beta sheet/alpha helical fold, there are thirteen other families of 'non-classical' zinc finger proteins for which relationships between metal coordination and protein structure/function are less defined. This 'Perspective' article focuses on two classes of these non-classical zinc finger proteins: Cys(3)His type zinc finger proteins and Cys(2)His(2)Cys type zinc finger proteins. These proteins bind zinc in a tetrahedral geometry, like the classical zinc finger proteins, yet they adopt completely different folds and target different oligonucleotides. Our current understanding of the relationships between ligand set, metal ion, fold and function for these non-classical zinc fingers is discussed.

Genome wide identification of Dof transcription factor gene family in sorghum and its comparative phylogenetic analysis with rice and Arabidopsis

The Dof (DNA binding with One Finger) family represents a classic zinc-finger transcription factors involved with multifarious roles exclusively in plants. There exists great diversity in terms of number of Dof genes observed in different crops. In current study, a total of 28 putative Dof genes have been predicted in silico from the recently available whole genome shotgun sequence of Sorghum bicolor (L.) Moench (with assigned accession numbers TPA:BK006983-BK007006 and TPA:BK007079-BK007082). The predicted SbDof genes are distributed on nine out of ten chromosomes of sorghum and most of these genes lack introns based on canonical intron/exon structure. Phylogenetic analysis of 28 SbDof proteins resulted in four subgroups constituting six clusters. The comparative phylogenetic analysis of these Dof proteins along with 30 rice and 36 Arabidopsis Dof proteins revealed six major groups similar to what has been observed earlier for rice and Arabidopsis. Motif analysis revealed the presence of conserved 50-52 amino acids Dof domain uniformly distributed across all the 28 Dof proteins of sorghum. The in silico cis-regulatory elements analysis of these SbDof genes suggested its diverse functions associated with light responsiveness, endosperm specific gene expression, hormone responsiveness, meristem specific expression and stress responsiveness.

Coordination Properties of Zinc Finger Peptides Revisited: Ligand Competition Studies Reveal Higher Affinities for Zinc and Cobalt

Zinc fingers are ubiquitous small protein domains which have a Zn(Cys)(4-x)(His)(x) site. They possess great diversity in their structure and amino acid composition. Using a family of six peptides, it was possible to assess the influence of hydrophobic amino acids on the metal-peptide affinities and on the rates of metal association and dissociation. A model of a treble-clef zinc finger, a model of the zinc finger site of a redox-switch protein, and four variants of the classical ββα zinc finger were used. They differ in their coordination set, their sequence length, and their hydrophobic amino acid content. The speciation, metal binding constants, and structure of these peptides have been investigated as a function of pH. The zinc binding constants of peptides, which adopt a well-defined structure, were found to be around 10(15) at pH 7.0. The rates of zinc exchange between EDTA and the peptides were also assessed. We evidenced that the packing of hydrophobic amino acids into a well-defined hydrophobic core can have a drastic influence on both the binding constant and the kinetics of metal exchange. Notably, well-packed hydrophobic amino acids can increase the stability constant by 4 orders of magnitude. The half-life of zinc exchange was also seen to vary significantly depending on the sequence of the zinc finger. The possible causes for this behavior are discussed. This work will help in understanding the dynamics of zinc exchange in zinc-containing proteins.

The Discovery of Zinc Fingers and Their Applications in Gene Regulation and Genome Manipulation

An account is given of the discovery of the classical Cys(2)His(2) zinc finger, arising from the interpretation of biochemical studies on the interaction of the Xenopus protein transcription factor IIIA with 5S RNA, and of structural studies on its structure and its interaction with DNA. The finger is a self-contained domain stabilized by a zinc ion ligated to a pair of cysteines and a pair of histidines, and by an inner hydrophobic core. This discovery showed not only a new protein fold but also a novel principle of DNA recognition. Whereas other DNA binding proteins generally make use of the two-fold symmetry of the double helix, zinc fingers can be linked linearly in tandem to recognize nucleic acid sequences of varying lengths. This modular design offers a large number of combinatorial possibilities for the specific recognition of DNA (or RNA). It is therefore not surprising that the zinc finger is found widespread in nature, including 3% of the genes of the human genome. The zinc finger design is ideally suited for engineering proteins to target specific genes. In the first example of their application in 1994, a three-finger protein was constructed to block the expression of an oncogene transformed into a mouse cell line. In addition, a reporter gene was activated by targeting an inserted zinc finger promoter. Thus, by fusing zinc finger peptides to repression or activation domains, genes can be selectively switched off or on. It was also suggested that by combining zinc fingers with other effector domains, e.g., from nucleases or integrases, to form chimeric proteins, genomes could be manipulated or modified. Several applications of such engineered zinc finger proteins are described here, including some of therapeutic importance.