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Snail contributes to the epithelial–mesenchymal transition by suppressing E-cadherin in transcription processes. The Snail C2H2-type zinc-finger (ZF) domain functions both as a nuclear localization signal which binds to importin β directly and as a DNA-binding domain. Here, a 2.5 Å resolution structure of four ZF domains of Snail1 complexed with importin β is presented. The X-ray structure reveals that the four ZFs of Snail1 are required for tight binding to importin β in the nuclear import of Snail1. The shape of the ZFs in the X-ray structure is reminiscent of a round snail, where ZF1 represents the head, ZF2–ZF4 the shell, showing a novel interaction mode, and the five C-terminal residues the tail. Although there are many kinds of C2H2-type ZFs which have the same fold as Snail, nuclear import by direct recognition of importin β is observed in a limited number of C2H2-type ZF proteins such as Snail, Wt1, KLF1 and KLF8, which have the common feature of terminating in ZF domains with a short tail of amino acids.

C2H2 zinc fingers are found in several key transcriptional regulators in the immune system. However, these proteins usually contain more fingers than are needed for sequence-specific DNA binding, which suggests that different fingers regulate different genes and functions. Here we found that mice lacking finger 1 or finger 4 of Ikaros exhibited distinct subsets of the hematological defects of Ikaros-null mice. Most notably, the two fingers controlled different stages of lymphopoiesis, and finger 4 was selectively required for tumor suppression. The distinct defects support the hypothesis that only a small number of genes that are targets of Ikaros are critical for each of its biological functions. The subcategorization of functions and target genes by mutagenesis of individual zinc fingers will facilitate efforts to understand how zinc-finger transcription factors regulate development, immunity and disease.

Binding of transcription factors to DNA is a dynamic process allowing for spatial- and sequence-specificity. Many methods for determination of DNA-protein structures do not allow for identification of dynamics of the search process, but provide only a single snapshot of the most stable binding. In order to better understand the dynamics of DNA binding as a protein encounters its cognate site, we have created a computer-based DNA scanning array macro that sequentially inserts a high affinity DNA consensus binding site at all possible locations in a predicted protein-DNA interface. We show, using short molecular dynamic simulations at each location in the interface, that energy minimized states and decreased movement of evolutionary conserved amino acids can be readily observed and used to predict the consensus binding site. The macro was applied to SNAIL class C2H2 zinc finger family proteins. The analysis suggests that (1) SNAIL binds to the E-box in multiple states during its encounter with its cognate site; (2) several different amino acids contribute to the E-box binding in each state; (3) the linear array of zinc fingers contributes differentially to overall folding and base-pair recognition; and (4) each finger may be specialized for stability and sequence specificity. Moreover, the macromolecular movement observed using this dynamic approach may allow the NH2-terminal finger to bind without sequence specificity yet result in higher binding energy. This macro and overall approach could be applicable to many evolutionary conserved transcription factor families and should help to better elucidate the varied mechanisms used for DNA sequence-specific binding.

Miz-1 is a BTB/POZ transcription factor that contains 13C2H2 Zinc Finger domains (ZF). Miz-1 transactivates and represses the transcription of a myriad of genes involved in many aspects of the biology of the cell. The detailed molecular interactions through which Miz-1 controls transcription, including its specific DNA binding via its ZF domains, remain to be understood and documented. In our effort to shed light into the structural biology of Miz-1, we have undertaken the determination of the structure of all its ZF and the characterization of their interactions with cognate DNA. The structure of ZF 1 to 10 have already been solved and characterized. Here, we present the structure of the synthetic Miz-1 ZF13 determined by 2D (1)H-(1)H NMR.

Zfp206 (also named as Zscan10) belongs to the subfamily of C(2)H(2) zinc finger transcription factors, which is characterized by the N-terminal SCAN domain. The SCAN domain mediates self-association and association between the members of SCAN family transcription factors, but the structural basis and selectivity determinants for complex formation is unknown. Zfp206 is important for maintaining the pluripotency of embryonic stem cells presumably by combinatorial assembly of itself or other SCAN family members on enhancer regions. To gain insights into the folding topology and selectivity determinants for SCAN dimerization, we solved the 1.85 Å crystal structure of the SCAN domain of Zfp206. In vitro binding studies using a panel of 20 SCAN proteins indicate that the SCAN domain Zfp206 can selectively associate with other members of SCAN family transcription factors. Deletion mutations showed that the N-terminal helix 1 is critical for heterodimerization. Double mutations and multiple mutations based on the Zfp206SCAN-Zfp110SCAN model suggested that domain swapped topology is a possible preference for Zfp206SCAN-Zfp110SCAN heterodimer. Together, we demonstrate that the Zfp206SCAN constitutes a protein module that enables C(2)H(2) transcription factor dimerization in a highly selective manner using a domain-swapped interface architecture and identify novel partners for Zfp206 during embryonal development.

Zinc finger proteins (ZFPs) that contain multiple cysteine and/or histidine residues perform important roles in various cellular functions, including transcriptional regulation, cell proliferation, differentiation, and apoptosis. The Cys-Cys-His-His (C(2)H(2)) type of ZFPs are the well-defined members of this super family and are the largest and most complex proteins in eukaryotic genomes. In this study, we identified a novel C(2)H(2) type of zinc finger gene ZNFD from mice which has a 1,002 bp open reading frame and encodes a protein with 333 amino acid residues. The predicted 37.4 kDa protein contains a C(2)H(2) zinc finger domain. ZNFD gene is located on chromosome 18qD1. RT-PCR analysis revealed that the ZNFD gene was specifically expressed in mouse testis but not in other tissues. Subcellular localization analysis demonstrated that ZNFD was localized in the nucleus. Reporter gene assays showed that overexpression of ZNFD in the COS7 cells activates the transcriptional activities of heat shock element (HSE). Overall, these results suggest that ZNFD is a member of the zinc finger transcription factor family and it participates in the transcriptional regulation of HSE. Many heat shock proteins regulated by HSE are involved in testicular development. Therefore, our results suggest that ZNFD may probably participate in the development of mouse testis and function as a transcription activator in HSE-mediated gene expression and signaling pathways.

Regulation of endosomal trafficking by Rab GTPases depends on selective interactions with multivalent effectors, including EEA1 and Rabenosyn-5, which facilitate endosome tethering, sorting, and fusion. Both EEA1 and Rabenosyn-5 contain a distinctive N-terminal C 2 H 2 zinc finger that binds Rab5. How these C 2 H 2 zinc fingers recognize Rab GTPases remains unknown. Here, we report the crystal structure of Rab5A in complex with the EEA1 C 2 H 2 zinc finger. The binding interface involves all elements of the zinc finger as well as a short N-terminal extension but is restricted to the switch and interswitch regions of Rab5. High selectivity for Rab5 and, to a lesser extent Rab22, is observed in quantitative profiles of binding to Rab family GTPases. Although critical determinants are identified in both switch regions, Rab4-to-Rab5 conversion-of-specificity mutants reveal an essential requirement for additional substitutions in the proximal protein core that are predicted to indirectly influence recognition through affects on the structure and conformational stability of the switch regions.

It has been reported previously that the C2-L1Tc protein located in the Trypanosoma cruzi LINE (long interspersed nuclear element) L1Tc 3′ terminal end has NAC (nucleic acid chaperone) activity, an essential activity for retrotransposition of LINE-1. The C2-L1Tc protein contains two cysteine motifs of a C2H2 type, similar to those present in TFIIIA (transcription factor IIIA). The cysteine motifs are flanked by positively charged amino acid regions. The results of the present study show that the C2-L1Tc recombinant protein has at least a 16-fold higher affinity for single-stranded than for double-stranded nucleic acids, and that it exhibits a clear preference for RNA binding over DNA. The C2-L1Tc binding profile (to RNA and DNA) corresponds to a non-co-operative-binding model. The zinc fingers present in C2-L1Tc have a different binding affinity to nucleic acid molecules and also different NAC activity. The RRR and RRRKEK [NLS (nuclear localization sequence)] sequences, as well as the C2H2 zinc finger located immediately downstream of these basic stretches are the main motifs responsible for the strong affinity of C2-L1Tc to RNA. These domains also contribute to bind single- and double-stranded DNA and have a duplex-stabilizing effect. However, the peptide containing the zinc finger situated towards the C-terminal end of C2-L1Tc protein has a slight destabilization effect on a mismatched DNA duplex and shows a strong preference for single-stranded nucleic acids, such as C2-L1Tc. These results provide further insight into the essential properties of the C2-L1Tc protein as a NAC.

The C(2)H(2) zinc finger proteins form one of the largest families of transcriptional regulators in eukaryotes. We identified a Phytophthora sojae C(2)H(2) zinc finger (PsCZF1), that is highly conserved in sequenced oomycete pathogens. In transformants of P. sojae containing the PsCZF1 promoter fused to the beta-glucuronidase (GUS) reporter gene, GUS activity was highly induced in the P. sojae oospore stage and upregulated after infection. To elucidate the function of PsCZF1, its expression was silenced by introducing anti-sense constructs into P sojae. PsCZF1-silenced transformants did not exhibit altered cell size or morphology of sporangia and hyphae; however, hyphal growth rate was reduced by around 50% in the mutants. PsCZF1-deficient mutants were also impaired in production of oospores, swimming zoospores and germinating cysts, indicating that the gene is involved in various stages of the life cycle. Furthermore, we found that PsCZF1-deficient mutants lost virulence on host soybean cultivars. Our results suggest that this oomycete-specific C(2)H(2)-type zinc finger protein plays an important role in growth, development, and pathogenesis; therefore, PsCZF1 might be an attractive oomycete-specific target for chemical fungicide screening.

Motivation: Modeling and identifying the DNA-protein recognition code is one of the most challenging problems in computational biology. Several quantitative methods have been developed to model DNA-protein interactions with specific focus on the C2H2 zinc-finger proteins, the largest transcription factor family in eukaryotic genomes. In many cases, they performed well. But the overall the predictive accuracy of these methods is still limited. One of the major reasons is all these methods used weight matrix models to represent DNA-protein interactions, assuming all base-amino acid contacts contribute independently to the total free energy of binding. Results: We present a context-dependent model for DNA–zinc-finger protein interactions that allows us to identify inter-positional dependencies in the DNA recognition code for C2H2 zinc-finger proteins. The degree of non-independence was detected by comparing the linear perceptron model with the non-linear neural net (NN) model for their predictions of DNA–zinc-finger protein interactions. This dependency is supported by the complex base-amino acid contacts observed in DNA–zinc-finger interactions from structural analyses. Using extensive published qualitative and quantitative experimental data, we demonstrated that the context-dependent model developed in this study can significantly improves predictions of DNA binding profiles and free energies of binding for both individual zinc fingers and proteins with multiple zinc fingers when comparing to previous positional-independent models. This approach can be extended to other protein families with complex base-amino acid residue interactions that would help to further understand the transcriptional regulation in eukaryotic genomes. Availability:The software implemented as c programs and are available by request. http://ural.wustl.edu/softwares.html Contact: stormo@ural.wustl.edu

Q-type C2H2 zinc finger proteins (ZFPs) form a subfamily of transcription factors that contain a plant-specific QALGGH amino acid motif. A total of 47 expressed Q-type C2H2 zinc finger genes in bread wheat (Triticum aestivum) (designated TaZFP) were identified from the current databases. Protein sequence analysis for the presence of ERF-associated amphiphilic repressor (EAR) motif sequences from known transcriptional repressors revealed that 26% of the TaZFP subfamily members contain an EAR motif. Quantitative RT-PCR analysis of the mRNA distribution of 44 TaZFP genes in various organs revealed that 30 genes were predominantly expressed in the roots. The majority of the TaZFP genes showed significant changes in their mRNA levels during leaf development and aging. Expression of 37 TaZFP genes in the leaves and roots responded to drought stress at least in one organ with 74% of the drought-responsive TaZFP genes being down-regulated in the drought-stressed roots. In contrast, only 6 out of the 44 TaZFP genes showed expression changes in the leaves with sucrose treatment. Expression of 50% of the drought-responsive TaZFP genes in the leaves (16 genes analysed) did not respond to ABA treatment, indicating that some TaZFP genes are involved in ABA-independent signalling pathways. These results indicate that the Q-type TaZFP subfamily is likely to have an important role in wheat roots and is enriched with members that are potentially involved in regulating cellular activities during changes of the physiological status of plant cells, as it occurs during drought stress or leaf development/aging.

There are over 10,000 C2H2-type zinc finger (ZF) domains distributed among more than 1,000 ZF proteins in the human genome. These domains are frequently observed to be involved in sequence-specific DNA binding, and uncharacterized domains are typically assumed to facilitate DNA interactions. However, some ZFs also facilitate binding to proteins or RNA. Over 100 Cys2-His2 (C2H2) ZF-protein interactions have been described. We initially attempted a bioinformatics analysis to identify sequence features that would predict a DNA- or protein-binding function. These efforts were complicated by several issues, including uncertainties about the full functional capabilities of the ZFs. We therefore applied an unbiased approach to directly examine the potential for ZFs to facilitate DNA or protein interactions. The human OLF-1/EBF associated zinc finger (OAZ) protein was used as a model. The human O/E-1-associated zinc finger protein (hOAZ) contains 30 ZFs in 6 clusters, some of which have been previously indicated in DNA or protein interactions. DNA binding was assessed using a target site selection (CAST) assay, and protein binding was assessed using a yeast two-hybrid assay. We observed that clusters known to bind DNA could facilitate specific protein interactions, but clusters known to bind protein did not facilitate specific DNA interactions. Our primary conclusion is that DNA binding is a more restricted function of ZFs, and that their potential for mediating protein interactions is likely greater. These results suggest that the role of C2H2 ZF domains in protein interactions has probably been underestimated. The implication of these findings for the prediction of ZF function is discussed.

The zinc finger domain enables different proteins to interact with or bind DNA, RNA, or other proteins, and is present in the proteomes of many different organisms. Proteins containing zinc finger domain(s) were found to play important roles in eukaryotic cells regulating different signal transduction pathways and controlling processes, such as development and programmed cell death. There are many types of zinc finger proteins, classified according to the number and order of the Cys and His residues that bind the Zinc ion. Among these, the C2H2-type zinc finger proteins, with 176 members in Arabidopsis thaliana, constitute one of the largest families of transcriptional regulators in plants. They are mostly plant-specific and contain a conserved QALGGH sequence within their zinc finger domain. Recent studies revealed that C2H2 zinc finger proteins could function as key transcriptional repressors involved in the defense and acclimation response of plants to different environmental stress conditions. Here we highlight recent functional characterization studies of different C2H2 proteins in Arabidopsis, and suggest that many of these proteins function as part of a large regulatory network that senses and responds to different environmental stimuli.

Motivation: Mutual information can be used to explore covarying positions in biological sequences. In the past, it has been successfully used to infer RNA secondary structure conformations from multiple sequence alignments. In this study, we show that the same principles allow the discovery of transcription factor amino acids that are coevolving with nucleotides in their DNA-binding targets. Results: Given an alignment of transcription factor binding domains, and a separate alignment of their DNA target motifs, we demonstrate that mutually covarying base-amino acid positions may indicate possible protein–DNA contacts. Examples explored in this study include C2H2 zinc finger, homeodomain and bHLH DNA-binding motif families, where a number of known base-amino acid contacting positions are identified. Mutual information analyses may aid the prediction of base-amino acid contacting pairs for particular transcription factor families, thereby yielding structural insights from sequence information alone. Such inference of protein–DNA contacting positions may guide future experimental studies of DNA recognition. Contact:shaun.mahony@ccbb.pitt.edu or benos@pitt.edu

Caenorhabditis elegans is a powerful model for studying gene regulation, as it has a compact genome and a wealth of genomic tools. However, identification of regulatory elements has been limited, as DNA-binding motifs are known for only 71 of the estimated 763 sequence-specific transcription factors (TFs). To address this problem, we performed protein binding microarray experiments on representatives of canonical TF families in C. elegans, obtaining motifs for 129 TFs. Additionally, we predict motifs for many TFs that have DNA-binding domains similar to those already characterized, increasing coverage of binding specificities to 292 C. elegans TFs (∼40%). These data highlight the diversification of binding motifs for the nuclear hormone receptor and C2H2 zinc finger families and reveal unexpected diversity of motifs for T-box and DM families. Motif enrichment in promoters of functionally related genes is consistent with known biology and also identifies putative regulatory roles for unstudied TFs. DOI: http://dx.doi.org/10.7554/eLife.06967.001, eLife digest Many scientists use ‘model’ species—such as the fruit fly or a nematode worm called Caenorhabditis elegans—in their research because these organisms have useful features that make it easier to carry out many experiments. For example, C. elegans has a smaller genome compared to many other animals, which is useful for studying the roles of individual genes or stretches of DNA. Transcription factors are a type of protein that can bind to specific stretches of DNA and help to switch certain genes on or off. These ‘motifs’ may be close to the gene or further away in the genome, and therefore, must stand out amongst the rest of the DNA, like lights on a landing strip. However, the motifs for only 10% of the estimated 763 transcription factors in C. elegans have been identified so far. In this study, Narasimhan, Lambert, Yang et al. used a technique called a ‘protein binding microarray’ to identify the motifs for many more of the C. elegans transcription factors. These findings were then used to predict motifs for other transcription factors. Together, these methods increased the proportion of C. elegans transcription factors with known DNA-binding motifs from 10% to around 40%. Now that more DNA motifs have been identified, it is possible to look for similarities and differences between them. For example, Narasimhan, Lambert, Yang et al. found that transcription factors with similar sequences can bind to very varied motifs. On the other hand, some transcription factors that are very different are able to recognize very similar motifs. The experiments also indicate that motifs found very close to genes—in sequences known as ‘promoters’—may be able to interact with many proteins to influence the activity of genes. Narasimhan, Lambert, Yang et al.'s findings increase the number of C. elegans transcription factors with a motif, bringing the knowledge of these proteins more in line with the better-studied transcription factors of humans and fruit flies. The next challenge is to identify DNA motifs for the remaining 60% of transcription factors. DOI: http://dx.doi.org/10.7554/eLife.06967.002

The SCAN domain mediates interactions between members of a subfamily of zinc-finger transcription factors and is found in more than 60 C2H2 zinc finger genes in the human genome, including the tumor suppressor gene myeloid zinc finger 1 (MZF1). Glutathione-S-transferase pull-down assays showed that the MZF1 SCAN domain self-associates, and a Kd value of 600 nM was measured by intrinsic tryptophan fluorescence polarization. The MZF1 structure determined by NMR spectroscopy revealed a domain-swapped dimer. Each monomer consists of five alpha helices in two subdomains connected by the alpha2-alpha3 loop. Residues from helix 3 of each monomer compose the core of the dimer interface, while the alpha1-alpha2 loop and helix 2 pack against helices 3 and 5 from the opposing monomer. Comprehensive sequence analysis is coupled with the first high-resolution structure of a SCAN dimer to provide an initial view of the recognition elements that govern dimerization for this large family of transcription factors.

Zinc finger proteins play important roles in various cellular functions, including cell proliferation, differentiation, and apoptosis. Mitogen-activated protein kinase (MAPK) signal transduction pathways are one of the most common mechanisms in eukaryotic cell regulation. Many transcription factors are important targets of MAPKs. In this study, we identified a novel gene encoding a zinc finger protein named ZNF445. The ZNF445 mRNA consists of 9105 nucleotides and has a 1031-amino acid open reading frame. The predicted 119-kDa protein contains a leucine-rich region (LER or SCAN domain) at the N-terminus, followed by a well-conserved Krüppel-associated box (KRAB) domain. At the C-terminus of the protein, there are 14 C2H2 (Cys2-His2) zinc finger motifs. ZNF445 gene is mapped to chromosome 3p21.32. Northern blot analysis indicates that a 9.1 kb transcript specific for ZNF445 is expressed in uterus, thymus, small intestine, colon, pancreas, peripheral blood leukocyte, and especially at a higher level in the testis and skeletal muscle in human adult tissues. ZNF445 protein was located in the nucleus when overexpressed in cultured cells. Reporter gene assays showed that ZNF445 is a transcriptional repressor, and overexpression of ZNF445 in the HEK 293T cells activates the transcriptional activities of AP1 and SRE. Deletion studies showed that the SCAN domain of ZNF445 may be involved in this activation. Furthermore, we found that expression of ZNF445 can increase p42/44 MAPK, MEK and Raf-1 phosphorylation. These results clearly indicate that ZNF445 is a member of the zinc finger transcription factor family and may function in MAPK pathway through Raf-1/MEK/p42/44 MAPK signals.

A novel 114-kDa zinc finger protein, ZEC, has been found by cDNA cloning and characterized. ZEC was strongly expressed in the testis, liver and kidney, and also in embryonic stem cells. Epitope-tagged experiments indicated nuclear localization of ZEC. ZEC contained 18 C2H2 zinc fingers which were organized in two clusters. A ZEC binding DNA sequence, C/GA/TA/TGGTTGGTTGC, which we have designated the GT box, was identified by random oligonucleotide binding selection assay. The GT box did not contain binding sites for other previously characterized transcription factors and thus represented a potentially novel DNA target sequence. Electrophoretic mobility shift assay (EMSA) showed that both clusters of zinc fingers bound to the same DNA sequence. Site-directed mutagenesis revealed that the core sequence TTGGTT within the GT box was essential to ZEC binding, while DNA sequences outside of the core sequence enhanced this interaction. Furthermore, co-transfection assays demonstrated that ZEC could activate a reporter luciferase gene driven by this DNA sequence.

Zinc finger proteins play important roles in a variety of cellular functions, including cell growth, proliferation, apoptosis, and intracellular signal transduction, and the zinc finger-containing transcription factor has been implicated as a critical regulator of multiple cardiac-expressed genes as well as a regulator of inducible gene expression in response to hypertrophic stimulation. With the aim of identifying the genes involved in human heart development and diseases, we have isolated a novel LER-related zinc finger gene named ZNF394 from human heart cDNA library. ZNF394 gene has a predicted 561-amino acid open reading frame, encoding a 64kDa zinc finger protein. The N-terminus of ZNF394 protein has a leucine-rich region (LER or SCAN domain), followed by a well-conserved krüppel-associated box domain. The C-terminus of the protein contains 7 C2H2 zinc finger motifs in tandem arrays with the highly conserved space region of the H/C-link. ZNF394 gene is mapped to chromosome 7q11.21. Northern blot analysis indicates that a 2.18kb transcript specific for ZNF394 is specifically expressed in the heart, skeletal muscle, and brain in human adult tissues. ZNF394 protein is expressed in cell nucleus. Overexpression of ZNF394 in the cell inhibits the transcriptional activities of c-Jun and AP-1 reporters, suggesting that ZNF394 is a new transcriptional repressor in mitogen-activated protein kinase signaling pathways and may play an important role in cardiac development and/or cardiac function.

Flowers of the parthenocarpic knuckles mutant are conditionally male sterile and contain ectopic stamens and carpels that originate from placental tissue within developing gynoecia. The mutation was mapped to a 123 Kb interval on chromosome 5 using molecular markers. All aspects of the knuckles phenotype could be complemented by a genomic fragment from the region which contained the annotated MAC12.2 gene. A guanine to adenine transition within a predicted C2H2 zinc finger-encoding region of MAC12.2 causes the second of two critical zinc-binding cysteine residues to be replaced by a tyrosine. Transgenic plants in which translational fusions of the GUS reporter to KNUCKLES were driven by the presumptive KNUCKLES promoter indicate that the gene is expressed first in developing carpel primordia, and later in stamens and ovules of flower buds. In situ hybridization experiments showed a broader pattern of transcript localization, suggesting that post-transcriptional regulatory mechanisms may limit KNUCKLES protein accumulation and localization. Based on genetic evidence and the presence of a carboxy-terminal motif demonstrated by others to function as an active repression domain, we propose that KNUCKLES might function as a transcriptional repressor of cellular proliferation that regulates floral determinacy and relative size of basal pattern elements along the proximo-distal axis of the developing Arabidopsis gynoecium.

The transferase activities that add uridylyl residues to RNA have been reported in several unicellular and metazoan organisms. Thus far, the two terminal uridylyltransferases (TUTases) involved in uridine insertion/deletion mRNA editing in mitochondria of trypanosomes were the only known enzymes with confirmed UTP specificity. Here, we demonstrate that protein sequences of editing TUTases may be used to predict novel UTP-specific enzymes by data mining. The highest-scoring open reading frame from Trypanosoma brucei was expressed and recombinant protein purified. This enzyme catalyzes a processive UMP incorporation and is not localized to the mitochondria suggesting a non-editing biological function.

In this study, we first cloned nlz2, a second zebrafish member of the nlz-related zinc-finger gene family. nlz2 was expressed together with nlz1 in a broad posterior domain during gastrula stages as well as at the midbrain-hindbrain boundary and in the hindbrain caudal to rhombomere 4 during segmentation. nlz2 was also expressed in regions distinct from nlz1, notably in the forebrain, midbrain, and trunk. Misexpression of nlz2 in zebrafish embryos disrupted gene expression in the rostral hindbrain, similar to the effect of misexpressing nlz1. We next compared the nlz1 and nlz2 sequences to identify and characterize domains conserved within this family. We found a C-terminal domain required for nuclear localization and two conserved domains (the Sp motif and a putative C(2)H(2) zinc finger) required for nlz1 function. We also demonstrate that Nlz1 self-associated via its C terminus, interacted with Nlz2, and bound to histone deacetylases. Last, we found two forms of Nlz1 generated from alternative translation initiation sites in vivo. These forms have distinct activities, apparently depending on the function of the N-terminal Sp motif. Our data demonstrate that nlz2 functions similarly to nlz1 and define conserved domains essential for nuclear localization, self-association, and corepressor binding in this novel family of zinc-finger genes.

Present address: Department of Trait and Technology Development, Pioneer Hi-Bred International, Inc., 7250 NW 62nd Avenue, Johnston, IA 50131, USA. Present address: Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121, USA. INNER NO OUTER (INO) expression is limited to the abaxial cell layer of the incipient and developing outer integument in Arabidopsis ovules. Using deletion analysis of the previously defined INO promoter (P-INO), at least three distinct regions that contribute to the endogenous INO expression pattern were identified. One such positive element, designated POS9, which comprises at least three distinct subelements, was found to include sufficient information to duplicate the INO expression pattern when four or more copies were used in conjunction with a heterologous minimal promoter. While known regulators of INO, including INO, SUPERMAN, BELL1, and AINTEGUMENTA, did not detectably interact with POS9 in yeast one-hybrid assays, two groups of proteins that interact specifically with POS9 were identified in one-hybrid library screens. Members of one group include C2H2 zinc finger motifs. Members of the second group contain a novel, conserved DNA-binding region and were designated the BASIC PENTACYSTEINE (BPC) proteins on the basis of conserved features of this region. The BPC proteins are nuclear localized and specifically bind in vitro to GA dinucleotide repeats located within POS9. The widespread expression patterns of the BPCs and the large number of GA repeat potential target sequences in the Arabidopsis genome indicate that BPC proteins may affect expression of genes involved in a variety of plant processes.

We report a discovery that, of the 226 C2H2 zinc-finger (C2H2-ZNF) genes on human chromosome 19, 12 genes each have two open reading frames (ORFs) that are in different reading frames but that can be translated into the same type of C2H2-ZNF proteins. We came to this observation after using standard tools in an original manner. First, we found that the both ORFs of such a gene contained the same type of significant C2H2-ZNF domain with e-values of e–2 or better. Second, the both ORFs had a promoter, a transcription start site, a start codon, a Kozak pattern and a poly(A) site; hence, each of them can be viewed as a gene in terms of a gene's primary structure. Third, both the ORFs matched not only human C2H2-ZNF expressed sequence tags (ESTs) but also human C2H2-ZNF proteins with e-values of e–50 or better. This indicates that the both ORFs can be transcribed and translated into the same zinc-finger proteins. More importantly, we observed that the phenomenon—a DNA can be translated into the same type of proteins after a frameshift—also occurred in a set of 160 human C2H2-ZNF ESTs and in a set of nine cDNAs of human C2H2-ZNF proteins. This observation based on the two sets of wet-experimental data much strengthened our confidence on the discovery. Our discovery is useful in the deeper understanding of a gene's regulatory mechanism to maintain its function. Supplementary information: http://zfgene.i2r.a-star.edu.sg

Transcription factor IIIA from S. pombe exhibits a novel structural organization compared to its homologues in other species. TFIIIA from S. cerevisiae or vertebrates contains a total of nine C2H2 zinc-finger domains and a non-zinc finger region at its C terminus. In addition, the S. cerevisiae protein possesses an 81-amino acid spacer between zinc fingers eight and nine. In contrast, the S. pombe TFIIIA sequence includes ten potential zinc finger motifs, with a 53-amino acid spacer between fingers nine and ten. Zinc finger nine of the S. pombe protein deviates from the consensus for a C2H2 zinc finger, however, in that it does not include an appropriately positioned second Zn2+-coordinating histidine. We demonstrate here, through analysis of mutated forms of the protein, that the non-canonical ninth zinc finger is functional in both DNA binding and transcription. In addition, we have shown that the spacer preceding finger ten and finger ten itself are essential for the transcriptional function of S. pombe TFIIIA, but neither is required for wild-type 5 S rRNA gene-binding activity.

We have identified two novel human SCAN domain genes ZNF396 and ZNF397, which are clustered within the region of chromosome 18q12. Three isoforms of ZNF396 transcript, 1.5, 2.5 and 3.9-kb, are expressed highly in liver. Four isoforms of ZNF397 transcript, 1.7, 2.5, 7.0 and 9.0-kb, are expressed in a variety of tissues, with varying levels. The SCAN-(C2H2)X genes encode two distinct proteins due to a unique alternative splicing mechanism. Both ZNF396-fu (full zinc fingers) and ZNF397-fu consist of a SCAN domain in the N-terminal region and many consecutive C2H2 zinc finger repeats in the C-terminal region. ZNF396-nf (no zinc fingers) and ZNF397-nf encode 210 and 198 amino acids, respectively, containing the SCAN domain only. ZNF396-fu, ZNF396-nf, ZNF397-fu or ZNF397-nf can homo-associate, while ZNF396-fu hetero-associates with ZNF396-nf, and ZNF397-fu hetero-associates with ZNF397-nf. ZNF396-nf and ZNF397-nf polypeptides are expressed diffusely in the cells, while ZNF396-fu and ZNF397-fu polypeptides target specifically to the nuclei. ZNF396-fu, ZNF396-nf and ZNF397-nf can repress reporter gene transcription, with ZNF397-nf having the strongest repression activity. Deletion analysis revealed that ZNF397-fu is a transcriptional activator without its nine zinc finger repeats.

The C2H2 zinc finger is the most prevalent protein motif in the mammalian proteome. Two C2H2 fingers in Ikaros are dedicated to homotypic interactions between family members. We show here that these fingers comprise a bona fide dimerization domain. Dimerization is highly selective, however, as homologous domains from the TRPS-1 and Drosophila Hunchback proteins support homodimerization, but not heterodimerization with Ikaros. Ikaros-Hunchback selectivity is determined by 11 residues concentrated within the alpha-helical regions typically involved in base recognition. Preferential homodimerization of one chimeric protein predicts a parallel dimer interface and establishes the feasibility of creating novel dimer specificities. These results demonstrate that the C2H2 motif provides a versatile platform for both sequence-specific protein-nucleic acid interactions and highly specific dimerization.

Nearly one-third of Krupple-type C2H2 zinc finger proteins have a krupple-associated box (KRAB) domain, which may act as a transcriptional repressor. ZNF268, which was novelty isolated from early human embryo, is a typical krupple-type C2H2 zinc finger protein with a conserved KRAB domain. In this report, the KRAB domain of ZNF268 is identified to localize in the nucleus and has transcriptional repressor activity.

The development of teeth through epithelial-mesenchymal interactions is mediated on a molecular level by a network of secreted growth factors and responsive transcription factors. Although zinc finger transcription factors constitute by far the largest class of transcriptional regulators with an estimated number of approximately 1000 genes present in mammals [14], little is known about their role in the regulation of mineralized tissue formation. A fragment (Y150) of the novel C2H2 zinc finger transcription factor Krox-26 has initially been isolated from highly proliferative dental pulp tissue in rats [19]. The objective of this study was to clone the full-length cDNA sequence of the murine homologue and to determine its mRNA and protein expression pattern during mouse embryonic development. Mouse Krox-26 contains five C2H2 zinc finger repeats. Its expression was found to be most prominent in the developing craniofacial bones and dental organs. These results suggest Krox-26 as a potential regulator of gene transcription during the development of teeth and the craniofacial skeleton.

C2H2 type of zinc finger transcription factors (TFs) play crucial roles in plant stress response and hormone signal transduction. Hence considering its importance, genome-wide investigation and characterization of C2H2 zinc finger proteins were performed in Arabidopsis, rice and poplar but no such study was conducted in foxtail millet which is a C4 Panicoid model crop well known for its abiotic stress tolerance. The present study identified 124 C2H2-type zinc finger TFs in foxtail millet (SiC2H2) and physically mapped them onto the genome. The gene duplication analysis revealed that SiC2H2s primarily expanded in the genome through tandem duplication. The phylogenetic tree classified these TFs into five groups (I-V). Further, miRNAs targeting SiC2H2 transcripts in foxtail millet were identified. Heat map demonstrated differential and tissue-specific expression patterns of these SiC2H2 genes. Comparative physical mapping between foxtail millet SiC2H2 genes and its orthologs of sorghum, maize and rice revealed the evolutionary relationships of C2H2 type of zinc finger TFs. The duplication and divergence data provided novel insight into the evolutionary aspects of these TFs in foxtail millet and related grass species. Expression profiling of candidate SiC2H2 genes in response to salinity, dehydration and cold stress showed differential expression pattern of these genes at different time points of stresses.

Aim: To study the characteristics of a novel zinc finger protein designated as monocytic differentiation-associated zinc finger protein (MDZF) and its role in the differentiation of leukemia cells. Methods: The mRNA expression of MDZF in tissues and cells was analyzed by Northern blot and RT-PCR. Polyclonal antibodies against the N terminus of MDZF were used to analyze protein expression in hematopoietic cell lines and subcellular location of MDZF in promyelocytic NB4 cells. The NB4 cells treated with TPA or ATRA at different intervals were harvested and then the expression level of MDZF protein was determined by Western blot. Result: A full-length cDNA was successfully identified from a human monocyte-derived dendritic cell cDNA library which encodes 610 amino acids with eight C2H2 zinc finger motifs and one POZ domain. It was located on chromosome 3 according to the genome database. This novel zinc finger protein was designated as MDZF. One transcript isoform of MDZF was also cloned by RT-PCR. Northern blot showed that MDZF mRNA was restrictedly expressed in heart, skeleton muscle, kidney, liver, and placenta. MDZF protein was expressed in all hematopoietic cell lines examined. Immunoblotting and confocal analysis indicated a dominant cytoplasmic location of MDZF in NB4 cells. Furthermore, after NB4 cells were treated with TPA for 48 h and differentiated into monocytes, MDZF expression increased fivefold in the NB4 cells, but no effect was observed in NB4 cells treated with ATRA. Conclusion: A novel zinc finger protein MDZF was cloned. MDZF is upregulated in monocytic, but not granulocytic, differentiation of NB4 acute promyelocytic leukemia cells. MDZF may be a candidate regulator of monocytic differentiation.

C2H2 zinc finger proteins probably comprise the largest family of regulatory proteins in mammals. Most zinc fingers bind to a cognate DNA. In addition to DNA, many of the proteins also bind to RNA or protein, and some bind to RNA only. The binding properties depend on the amino acid sequence of the finger domains and of the linker between fingers, as well as on the higher-order structures and the number of fingers. C2H2 zinc finger proteins contain from 1 to more than 30 figures. Based on the number and the pattern of the fingers, most of the proteins can be classified into one of three groups: triple-C2H2, multiple-adjacent-C2H2, and separated-paired-C2H2 finger proteins. In contrast to proteins with triple-C2H2 fingers, proteins with multiple-adjacent-C2H2 fingers can bind multiple, different ligands. Proteins with a number of separated-paired fingers bind to the target by means of only a single pair.

We have identified a novel zinc finger gene, ZNF232 , mapped to human chromosome 17p12. The coding region of the gene is organized in three exons corresponding to a 417 amino acid long polypeptide containing a SCAN/LeR domain and five C 2 H 2 -type zinc fingers. ZNF232 is possibly a nuclear protein, as suggested by expression analysis of GFP/ZNF232 chimeric constructs. ZNF232 transcripts were detected in a wide collection of adult human tissues. The gene is possibly subjected to tissue-specific post-transcriptional regulation by means of alternative splicing.

Two novel and related C2H2 zinc finger proteins that are highly expressed in the brain, CTIP1 and CTIP2 (COUPTF-interacting proteins1 and 2, respectively), were isolated and shown to interact with all members of the chicken ovalbumin upstream promoter transcription factor (COUP-TF) subfamily of orphan nuclear receptors. The interaction of CTIP1 with ARP1 was studied in detail, and CTIP1 was found to harbor two independent ARP1 interaction domains, ID1 and ID2, whereas the putative AF-2 of ARP1 was required for interaction with CTIP1. CTIP1, which exhibited a punctate staining pattern within the nucleus of transfected cells, recruited cotransfected ARP1 to these foci and potentiated ARP1-mediated transcriptional repression of a reporter construct. However, transcriptional repression mediated by ARP1 acting through CTIP1 did not appear to involve recruitment of a trichostatin A-sensitive histone deacetylase(s) to the template, suggesting that this repression pathway may be distinct from that utilized by several other nuclear receptors.

The Drosophila melanogaster ovo locus codes for several tissue- and stage-specific proteins that all possess a common C-terminal array of four C(2)H(2)zinc fingers. Three fingers conform to the motif framework and are evolutionarily conserved; the fourth diverges considerably. The ovo genetic function affects germ cell viability, sex identity and oogenesis, while the overlapping svb function is a key selector for epidermal structures under the control of wnt and EGF receptor signaling. We isolated synthetic DNA oligomers bound by the OVO zinc finger array from a high complexity starting population and derived a statistically significant 9 bp long DNA consensus sequence, which is nearly identical to a consensus derived from several Drosophila genes known or suspected of being regulated by the ovo function in vivo. The DNA consensus recognized by Drosophila OVO protein is atypical for zinc finger proteins in that it does not conform to many of the 'rules' for the interaction of amino acid contact residues and DNA bases. Additionally, our results suggest that only three of the OVO zinc fingers contribute to DNA-binding specificity.

Zinc finger proteins are known to mediate various transcriptional control mechanisms and other cellular functions in human cells. Tumor necrosis factor alpha (TNFalpha) induces a variety of genes in human endothelial cells including A20, an antiapoptotic zinc finger protein. In order to identify other zinc finger protein genes induced by TNFalpha, we studied the gene expression profile of human umbilical vein endothelial cells (HUVECs) stimulated by TNFalpha by means of oligonucleotide microarrays. Among the 155 genes encoding zinc finger motif, the level of EST M88357 mRNA encoding a novel designated EZFIT (endothelial zinc finger protein induced by TNFalpha) was induced most profoundly (>19 fold). The EZFIT gene is located on the chromosome 19q13.4. Isolation of the full length cDNA coding sequence by PCR using primers architected from the genomic sequence revealed that EZFIT has 490 amino acids which contain 13 C2H2 zinc finger motifs. Among 24 human organs and cell types studied, EZFIT mRNA was found to be most highly expressed in the placenta followed by the brain, testis, pancreas, heart, small intestine, muscle, uterus, prostate and peripheral blood leukocytes. EZFIT mRNA was not detected in the liver, lung, colon, stomach, the salivary gland or the thyroid gland.

Kid1 encodes a transcriptional repressor implicated in the differentiation of renal epithelial cells. Here we report the characterisation of Kid3, a novel mouse gene related to Kid1. Kid3 encodes a C2H2 zinc finger protein with an N-terminal KRAB transcriptional repression domain. It maps to chromosome 11, adjacent to Kid1 and another related gene Kid2. Northern analysis shows that Kid3 is highly expressed in embryonic and adult brain, with lower levels in adult and embryonic (E16.5) kidney, gut, lung and heart. Expression of Kid3 in the kidney is developmentally regulated and suggests a role for Kid3 in the early stages of nephrogenesis.

The Drosophila gene buttonhead (btd) is required for the formation of the mandibular, the intercalary and the antennal head segments of the embryo. The btd protein (BTD) is functionally and structurally related to the human C2H2 zinc finger transcription factor Sp1. A second Sp1-like Drosophila gene, termed Drosophila Sp1 (D-Sp1), had been identified on the basis of a partial sequence showing that the gene encodes a characteristic zinc finger domain, composed of three finger motifs similar to both Sp1 and btd. D-Sp1 is located in the same cytological location as btd in chromosome band 9A on the X-chromosome. It had been proposed that D-Sp1 and btd are likely to act as a gene pair and function in a at least partially redundant manner. Here we report the molecular analysis of D-Sp1 and its expression pattern during embryonic and larval development. We show that D-Sp1 acts as a transcriptional regulator. Lack-of-function analysis combined with rescue and gain-of-function studies indicates that btd and D-Sp1 play essential and redundant roles for mechanosensory organ development. However, D-Sp1 lacks the specific features of BTD required for embryonic intercalary and antennal segment formation.

A novel cDNA clone was identified through yeast two-hybrid experiments. Following cross-examination between the cDNA clones, EST clones, and the cosmid clone, we could digitally assemble a new zinc finger transcription factor gene. This predicted gene has a cDNA size of about 1960 bp and is translated into a 487-amino-acid protein. According to database analysis, this gene contains three C2H2 zinc finger motifs and is highly related to human PLZF (promyelocytic leukemia zinc finger protein). The full-length coding region of the gene was isolated, and its sequences were confirmed by DNA sequencing. Interestingly, one splicing variant lacking exon III was also identified. Northern blot analysis revealed that this gene is mainly expressed in human testis. In conclusion, we have identified a new member of the PLZF zinc finger protein family, the testis zinc finger protein (TZFP), which is mainly expressed in testis tissue.

The Drosophila gene jim was identified by an enhancer trap line showing asymmetric dorso-ventral expression in the follicular epithelium. It gives rise to the jim-1 and jim-2 transcripts that contain distinct 5′-UTRs but encode the same nine C2H2 zinc finger protein. From stage 10A onward, jim-1 RNA is transcribed in squamous cells while jim-2 RNA is specific to all non-antero-dorsal columnar cells as the result of repression in antero-dorsal cells by the DER pathway.

During our search for the familial Mediterranean fever (FMF) gene, we identified by cDNA selection a 1.2 kb cDNA fragment representing a novel human gene that is expressed in a wide variety of tissues. This gene spans approx. 8.0 kb genomic DNA and has seven exons. Its 3' untranslated region contains a long tandem repeat that gives rise to a polymorphism with two alleles of approx. 1.1 kb and 1.0 kb, with the 1.1 kb allele in strong linkage disequilibrium with FMF in patients of different ethnic backgrounds. However, both genetic and mutational analyses have excluded this gene as the one responsible for FMF. The predicted 424 amino acid protein, designated ZNF213, contains three C2H2 zinc fingers, a Kruppel associated A box and a leucine rich motif (LeR domain/SCAN box), strongly suggestive of a transcription factor.

The study of zinc finger proteins has revealed their potential to act as oncogenes or tumor suppressors. Here we report the molecular, biochemical, and functional characterization of KS1 (KRAB/zinc finger suppressor protein 1), a novel, ubiquitously expressed zinc finger gene initially isolated from a rat pancreas library. KS1 contains 10 C2H2 zinc fingers, a KRAB-A/B motif, and an ID sequence that has been shown previously to participate in growth factor-regulated gene expression. Northern blot analysis using pancreatic cell lines demonstrates that KS1 mRNA is inducible by serum and epidermal growth factor, suggesting a role for this gene in cell growth regulation. Biochemical analysis reveals that KS1 is a nuclear protein containing two transcriptional repressor domains, R1 and R2. R1 corresponds to the KRAB-A motif, whereas R2 represents a novel sequence. Transformation assays using NIH3T3 cells demonstrate that KS1 suppresses transformation by the potent oncogenes Ha-ras, Galpha12, and Galpha13. Deletion of the R1/ KRAB-A domain does not modify the transformation suppressive activity of KS1, whereas deletion of R2 abolishes this function. Thus, KS1 is a novel growth factor-inducible zinc finger transcriptional repressor protein with the potential to protect against neoplastic transformation induced by several oncogenes.

Roaz, a rat C2H2 zinc finger protein, plays a role in the regulation of olfactory neuronal differentiation through its interaction with the Olf-1/EBF transcription factor family. An additional role for the Roaz/Olf-1/EBF heterodimeric protein is suggested by its ability to regulate gene activation at a distinct promoter lacking Olf-1/EBF-binding sites. Using an in vitro binding-site selection assay (Selex), we demonstrate that Roaz protein binds to novel inverted perfect or imperfect repeats of GCACCC separated by 2 bp. We show that Roaz is capable of binding to a canonical consensus recognition sequence with high affinity (Kd = 3 nM). Analysis of the structural requirement for protein dimerization and DNA binding by Roaz reveals the role of specific zinc finger motifs in the Roaz protein for homodimerization and heterodimerization with the Olf-1/EBF transcription factor. The DNA-binding domain of Roaz is mapped to the N-terminal 277 amino acids, containing the first seven zinc finger motifs, which confers weak monomeric binding to a single half site and a stronger dimeric binding to the inverted repeat in a binding-site-dependent manner. Full-length protein can form dimers on both the inverted repeat and direct repeat but not on a single half site. These findings support the role of the TFIIIA-type Zn fingers in both protein-protein interaction and protein-DNA interaction and suggest distinct functions for specific motifs in proteins with a large number of zinc finger structures.

The acetamidase-encoding amdS gene of Aspergillus nidulans has been shown to be controlled by multiple regulatory genes. A new gene, amdX, involved in amdS regulation was identified during the characterization of a translocation affecting amdS control. The amdX gene is predicted to encode a 1150-amino-acid polypeptide which contains two Cys-2-His-2 (C2H2) zinc finger DNA-binding motifs. Insertional inactivation of amdX and the phenotypes of transformants containing multiple copies of the amdX gene show that it is an activator of amdS expression. A fusion protein containing the AmdX zinc fingers was found to bind to sequences in the 5' region of amdS which overlap binding sites for the CreA and AmdA regulatory proteins. Evidence is presented for AmdX acting at these sites in vivo.

From a human pancreas cDNA library we isolated and characterized a novel zinc finger gene encoding a protein homologous to ZNF187, a serum response element-binding protein. The full-length cDNA contained an open reading frame of 1,686 nucleotides encoding a predicted 562-amino-acid peptide that included an ATP-GTP binding site and seven C2H2 zinc finger domains. The consensus sequence of the C2H2 domains (CX2CX3FX5LX2HX3H) is common in the SRE-binding region present in Drosophila Krüppel proteins. An alternatively spliced form of the transcript found in the cDNA library lacked both the ATP-GTP binding site and any C2H2 zinc finger domains. We localized this gene (ZNF188) to chromosome band 7q22.1→q22.3 by fluorescence in situ hybridization.

Myc-interacting zinc finger protein-1 (Miz-1) is a BTB/POZ transcription factor that activates the transcription of cytostatic genes, such as p15(INK4B) or p21(CIP1). The C-terminus of Miz-1 contains 13 consensus C2H2 zinc finger domains (ZF). ZFs 1-4 have been shown to interact with SMAD3/4, while the remaining ZFs are expected to bind the promoters of target genes. We have noted unusual features in ZF 5 and the linker between ZFs 5 and 6. Indeed, a glutamate is found instead of the conserved basic residue two positions before the second zinc-coordinating histidine on the ZF 5 helix, and the linker sequence is DTDKE in place of the classical TGEKP sequence. In a canonical ββα fold, such unusual primary structure elements should cause severe electrostatic repulsions. In this context, we have characterized the structure and the dynamics of a Miz-1 construct comprising ZFs 5-8 (Miz 5-8) by solution-state NMR. Whilst ZFs 5, 7 and 8 were shown to adopt the classical ββα fold for C2H2 ZFs, the number of long-range NOEs was insufficient to define a classical fold for ZF 6. We show by using (15)N-relaxation dispersion experiments that this lack of NOEs is due to the presence of extensive motions on the μs-ms timescale. Since this negatively charged region would have to be located near the phosphodiester backbone in a DNA complex, we propose that in addition to promoting conformational searches, it could serve as a hinge region to keep ZFs 1-4 away from DNA.

International audience; 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.

Many of the vertebrate zinc finger factors of the Kruppel type (C2H2 zinc fingers) contain in their N-terminus a conserved sequence referred to as the KRAB (Kruppel-associated box) domain that, when tethered to DNA, efficiently represses transcription. Using the yeast two-hybrid system, we have isolated an 835 amino acid RING finger (C3HC4 zinc finger) protein, TIF1 beta (also named KAP-1), that specifically interacts with the KRAB domain of the human zinc finger factor KOX1/ZNF10. TIF1 beta, TIF1 alpha, PML and efp belong to a characteristic subgroup of RING finger proteins that contain one or two other Cys/His-rich clusters (B boxes) and a putative coiled-coil in addition to the classical C3HC4 RING finger motif (RBCC configuration). Like TIF1 alpha, TIF1 beta also contains an additional Cys/His cluster (PHD finger) and a bromo-related domain. When tethered to DNA, TIF1 beta can repress transcription in transiently transfected mammalian cells both from promoter-proximal and remote (enhancer) positions, similarly to the KRAB domain itself. We propose that TIF1 beta is a mediator of the transcriptional repression exerted by the KRAB domain.

C2H2 zinc finger protein genes encode nucleic acid-binding proteins involved in the regulation of gene activity. AtZFP1 (Arabidopsis thaliana zinc finger protein 1) is one member of a small family of C2H2 zinc finger-encoding sequences previously characterized from Arabidopsis. The genomic sequence corresponding to the AtZFP1 cDNA has been determined. Molecular analysis demonstrates that AtZFP1 is a unique, intronless gene which encodes a 1100 nucleotides mRNA highly expressed in roots and stems. A construct in which 2.5 kb of AtZFP1 upstream sequences is linked to the beta-glucuronidase gene was introduced into Arabidopsis by Agrobacterium-mediated transformation of roots. Histochemical analysis of transgenic Arabidopsis carrying the AtZFP1 promoter: beta-glucuronidase fusion shows good correlation with RNA blot hybridization analysis. This transgenic line will be a useful tool for analyzing the regulation of AtZFP1 to further our understanding of its function.

Clone ZF388 was identified during the course of expressed sequence tag analysis of human adult testis cDNAs as being a member of the C2H2 zinc finger gene family. Northern blot analysis of a range of 16 different human tissues has shown that clone ZF388 detects a single 2.2-kb transcript and that the expression of this mRNA species is strictly specific to the testis. Sequence analysis of ZF388 and other clones isolated by rescreening a testis cDNA library has defined a transcript with an extensive N-terminal acidic domain containing 17% aspartic and glutamic acid residues and a C-terminal domain composed of five zinc finger motifs linked through the highly conserved sequence TGE-KPYE. This gene has been designated ZNF165. Analysis of somatic cell hybrids containing single human chromosomes shows that ZNF165 maps to chromosome 6. Fluorescence in situ hybridization of cDNA insert probes to male metaphase spreads shows that the ZNF165 transcript maps to 6p21 close to the human MHC region. Human 6p21 is homologous with the distal inversion of the mouse t-complex, which has been shown to contain a cluster of genes expressed specifically in the testis.