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C2H2 zinc finger proteins (ZFPs) play essential roles in leaf morphogenesis and floral development, as well as heat stress response and trichome formation, which activate or inhibit gene transcription mainly through interactions with nucleic acids, such as single-strand DNA, RNA binding or RNA/DNA bidirectional binding, and protein interaction. Single C2H2 ZFPs is the subfamily of ZFPs, but little of single C2H2 ZFP family is known in tomato. In this study, we identified 30 single ZFP genes in tomato using bioinformatics-based methods. Gene structures, phylogeny, conserved motifs, cis-element of promoter, chromosomal localization, gene duplication, and expression patterns of these single C2H2 ZFP genes were analyzed. Sequence analysis showed that most single C2H2 ZFP genes possessed only one exon, except for SlC1-liZFP1 and SlC1-liZFP2. These single C2H2 ZFP genes were asymmetrically distributed on 10 chromosomes, excluding 2 and 12 chromosomes. In addition, 24 of these genes were predicated to have experienced segmental duplication. Cis-element prediction indicated that many important elements were located in the putative promoter regions, like light and gibberellic acid (GA)-responsive elements. The expression profiles of these genes in different tissues and various hormones and stress treatment were further analyzed. Many genes were lowly expressed in all tissues, whereas some were specifically expressed in certain tissues, like SlC1-liZFP2 in young leaves, and SlC1-liZFP15 in fruits. Furthermore, these genes could also be induced by several hormones and stresses, including IAA, ETH, GA, cold, and drought. This study sets a good foundation for further characterizing the biological roles of single C2H2 ZFP genes in tomato.

The ZNF280D is a member of the C2H2 zinc finger family, which plays important roles in cell and organismal growth, secretion, and other biological processes. This study aimed to identify the alternative splicing of pig ZNF280D, and to predict the structure and function of the encoded proteins, as well as to study the spatio-temporal expression patterns of each transcript. RNA-seq and qPCR technology were used to identify the transcripts of pig ZNF280D. We found two transcripts, ZNF280D1 and ZNF280D2 (GenBank accession number: MF279143). ZNF280D1 is the common transcript, whereas ZNF280D2 lacked a large region at exon 8. In addition, ZNF280D2 had an additional C2H2 zinc finger domain, changing the protein from a tC2H2 to a maC2H2 zinc finger protein. The expression of two transcripts in various tissues of pig was differ significant. ZNF280D2 is the main subtype. The expression of ZNF280D2 in pig muscle was found to increase gradually with age. The ZNF280D1 was first decreased and then increased slightly. We successfully identified two transcripts of pig ZNF280D, and we suggested important functions for ZNF280D in digestive absorption and growth as well as in the development of skeletal muscles, the specific mechanisms for which would require further study.

Background C2H2 zinc finger proteins (C2H2 ZFPs) play vital roles in shaping many aspects of plant growth and adaptation to the environment. Plant genomes harbor hundreds of C2H2 ZFPs, which compose one of the most important and largest transcription factor families in higher plants. Although the C2H2 ZFP gene family has been reported in several plant species, it has not been described in the model leguminous species Medicago truncatula. Results In this study, we identified 218 C2H2 type ZFPs with 337 individual C2H2 motifs in M. truncatula. We showed that the high rate of local gene duplication has significantly contributed to the expansion of the C2H2 gene family in M. truncatula. The identified ZFPs exhibit high variation in motif arrangement and expression pattern, suggesting that the short C2H2 zinc finger motif has been adopted as a scaffold by numerous transcription factors with different functions to recognize cis-elements. By analyzing the public expression datasets and quantitative RT-PCR (qRT-PCR), we identified several C2H2 ZFPs that are specifically expressed in certain tissues, such as the nodule, seed, and flower. Conclusion Our genome-wide work revealed an expanded C2H2 ZFP gene family in an important legume M. truncatula, and provides new insights into the diversification and expansion of C2H2 ZFPs in higher plants.

C2H2 type of zinc finger transcription factors (C2H2-ZFP TFs) play crucial roles in plant developments and stress response. Regarding its importance, genome-wide study of C2H2-ZFs were performed in multiple important plant species, but any such investigation was not fulfilled in Triticum turgidum ssp. Durum (durum wheat) as an important nutritional crop. The present study identified 122 C2H2-ZFs in durum wheat and physically mapped them onto the genome. The phylogenetic analysis classified these TFs into six major groups. Genes structure and conserved motifs assay showed TtC2H2-ZF involvement in the important cellular functions. Comparative phylogeny between durum wheat TtC2H2-ZF genes and the orthologs in rice revealed the evolutionary relationships of C2H2-ZF proteins. The gene ontology and promoter cis-element analysis indicated that most of TtC2H2-ZF genes are involved in multiple molecular functions including metal ion-binding and various stimuli responses. Further, the miRNAs targeting TtC2H2-ZF transcripts, homology modeling and proteins interaction network were also demonstrated, suggesting the vital cellular functions of TtC2H2-ZFs during various circumstances. The expression heatmap demonstrated differential and tissue-specific expression patterns of these genes. Expression profiling of this gene family members in response to dehydration and heat stresses showed differential expression pattern of these genes at multiple time points of stresses. This study can prepare a comprehensive overview of the durum wheat C2H2-ZF gene family and may provide a new perspective on the evolution of them, which will form the basis for further investigation of the roles of this family members and future genetic engineering studies in crops.

The zinc fingers proteins (ZNF) are the largest family of DNA binding proteins and can act as transcriptional factors in eukaryotes. ZNF are implicated in activation in response to environmental stimulus by biometals such as Zn2+. Many of these proteins have the classical C2H2 zinc finger motifs (C2H2-ZNFm) of approximately 30 amino acids, where a Zn2+ ion is coordinated by two cysteine and two histidine residues. Trichomonas vaginalis is a protozoan parasite than responds to environmental changes including Zn2+. Until now has not been described any ZNF that could be involved in the regulation of genic expression of T. vaginalis. Here, we characterized in silico and experimentally an annoted ZNF (TvZNF1) from T. vaginalis and isolated the gene, tvznf1 encoding it. TvZNF1 have eight C2H2-ZNFm with residues that maybe involved in the structural stability of DNA binding motifs. In this work we confirmed the Zn2+ upregulation expression of tvznf1 gene. Recombinant TvZNF1 was able to bind to specific DNA sequences according to EMSA assay. Additionally, we demonstrated that recombinant TvZNF1 bind to MRE signature in vitro, which strongly suggests its role in transcriptional regulation, similar to the one observed for mammalian MTF-1. This result suggested a conserved mechanism of genic regulation mediated by ZNFs in T. vaginalis.

Background C2H2-zinc finger protein family is commonly found in the plant, and it is known as the key actors in the regulation of transcription and vital component of chromatin structure. A large number of the C2H2-zinc finger gene members have not been well characterized based on their functions and structure in cotton. However, in other plants, only a few C2H2-zinc finger genes have been studied. Results In this work, we performed a comprehensive analysis and identified 386, 196 and 195 C2H2-zinc finger genes in Gossypium hirsutum (upland cotton), Gossypium arboreum and Gossypium raimondii, respectively. Phylogenetic tree analysis of the C2H2-zinc finger proteins encoding the C2H2-zinc finger genes were classified into seven (7) subgroups. Moreover, the C2H2-zinc finger gene members were distributed in all cotton chromosomes though with asymmetrical distribution patterns. All the orthologous genes were detected between tetraploid and the diploid cotton, with 154 orthologous genes pair detected between upland cotton and Gossypium arboreum while 165 orthologous genes were found between upland cotton and Gossypium raimondii. Synonymous (Ks) and non-synonymous (Ka) nucleotide substitution rates (Ka/Ks) analysis indicated that the cotton C2H2-zinc finger genes were highly influenced mainly by negative selection, which maintained their protein levels after the duplication events. RNA-seq data and RT-qPCR validation of the RNA seq result revealed differential expression pattern of some the C2H2-zinc finger genes at different stages of cotton fiber development, an indication that the C2H2-zinc finger genes play an important role in initiating and regulating fiber development in cotton. Conclusions This study provides a strong foundation for future practical genome research on C2H2-zinc finger genes in upland cotton. The expression levels of C2H2-zinc finger genes family is a pointer of their involvement in various biochemical and physiological functions which are directly related to cotton fiber development during initiation and elongation stages. This work not only provides a basis for determining the nominal role of the C2H2-zinc finger genes in fiber development but also provide valuable information for characterization of potential candidate genes involved in regulation of cotton fiber development.

The large C2H2-Zinc Finger (C2H2-ZNF) gene family has rapidly expanded in primates through gene duplication. There is consequently considerable sequence homology between family members at both the nucleotide and amino acid level, allowing for coordinated regulation and shared functions. Here we show that multiple C2H2-ZNF mRNAs experience differential polyadenylation resulting in populations with short and long poly(A) tails. Furthermore, a significant proportion of C2H2-ZNF mRNAs are retained in the nucleus. Intriguingly, both short poly(A) tails and nuclear retention can be specified by the repeated elements that encode zinc finger motifs. These Zinc finger Coding Regions (ZCRs) appear to restrict polyadenylation of nascent RNAs and at the same time impede their export. However, the polyadenylation process is not necessary for nuclear retention of ZNF mRNAs. We propose that inefficient polyadenylation and export may allow C2H2-ZNF mRNAs to moonlight as non-coding RNAs or to be stored for later use.

Plant C2H2-type zinc finger proteins (ZFPs) play essential roles in developmental control and stress responses. The whole complement of ZFP genes has been identified in Arabidopsis and rice, while the genome-scale identification and functional analysis of maize ZFPs is not yet reported. Hence, we performed a comprehensive analysis, including gene structure, chromosome location, duplicated event, selective pressure, phylogeny, gene ontology annotation, and expression profiling under developmental stages and abiotic stresses. Phylogenetic analyses suggested that the ZmZFP gene family can be grouped into three classes (A, B, and C). The analysis of differential gene expression in different developmental stages and stress treatments (drought, salt, and cold) was conducted based on microarray and RNA-seq data. A total of 99.05 % (209 genes) of the total ZmZFP genes (211 genes) were detected in 60 different tissues in microarray data. Under drought stress, 13 differentially expressed genes were found in leaf, of which 7 and 6 genes were up-regulated and down-regulated, respectively. For salt stress, crown root (CR), primary root (PR) and seed root (SR) each had one significantly elevated gene, while 2, 1, and 7 genes were obviously down-regulated in CR, PR and SR, respectively. Additionally, 8 and 3 genes were significantly up-regulated and down-regulated, respectively, in the cold-tolerant line ETH-DH7. This study will lay the foundation for understanding the roles of ZFPs in maize growth and stress resistance, contributing to the molecular breeding of maize for food.

Cys2-His2 zinc finger (C2H2-ZF) proteins represent the largest class of putative human transcription factors. However, for most C2H2-ZF proteins it is unknown whether they even bind DNA or, if they do, to which sequences. Here, by combining data from a modified bacterial one-hybrid system with protein-binding microarray and chromatin immunoprecipitation analyses, we show that natural C2H2-ZFs encoded in the human genome bind DNA both in vitro and in vivo, and we infer the DNA recognition code using DNA-binding data for thousands of natural C2H2-ZF domains. In vivo binding data are generally consistent with our recognition code and indicate that C2H2-ZF proteins recognize more motifs than all other human transcription factors combined. We provide direct evidence that most KRAB-containing C2H2-ZF proteins bind specific endogenous retroelements (EREs), ranging from currently active to ancient families. The majority of C2H2-ZF proteins, including KRAB proteins, also show widespread binding to regulatory regions, indicating that the human genome contains an extensive and largely unstudied adaptive C2H2-ZF regulatory network that targets a diverse range of genes and pathways.

Q-type zinc finger proteins have been studied in several plant species and have been associated with response to stress. A whole genome analysis of Arabidopsis identified 176 putative C2H2 transcription factors (TF). Q-type C2H2 TFs containing the QALGGH motif and are a subset of these. In Arabidopsis, 18 have been described that contain two zinc finger motifs per protein, and several of these have been associated with a response to abiotic stress. The zinc fingers bind to the promoters of genes and negatively regulate transcription via the ethylene-responsive element-binding factor-associated amphiphilic repression (EAR) motif. Previously, we have identified StZFP2 from potato as induced upon infestation by insect pests. Interest in finding similar proteins in the newly annotated cabbage genome (Brassica oleracea var. capitata) led to the discovery of 25 proteins with 2-Q-type zinc finger domains. Examination of several transcriptome studies of B. oleracea submitted to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) confirms the expression of all of these genes in at least one tissue type. Identification of the nearest homologs of these proteins in Arabidopsis and comparison of the map positions of both Arabidopsis and B. oleracea genes supports both the synteny between the genetic maps of these related species and the triplication event in Brassica species hypothesized to occur prior to the split from the ancestral progenitor of Arabidopsis and B. oleracea.

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.

Al tolerance is one of the most important trait for worldwide crop production. Using microarrays, we previously identified a transcription factor belonging to the C2H2 zinc finger protein (ZFP) family associated with Al tolerance in wheat (Houde and Diallo, BMC Genomics 9:400, 2008). The current work aimed to identify specific members of the C2H2 ZFP family that are associated with Al tolerance. Wheat ESTs were used to assemble C2H2 ZFP family members that do not contain a classical EAR repressor domain. Specific primers were designed for qRT-PCR expression profiling of wheat root tips exposed to Al and H2O2. Two Al-tolerant and sensitive wheat cultivars including a pair of near-isogenic lines differing in Al tolerance were used. We reconstituted 16 wheat Q-type C2H2 ZFP. Expression profiling identified two transcripts (TaZFP2 and TaZFP3) that accumulate rapidly upon exposure to Al or in response to H2O2 in two tolerant wheat cultivars, including the tolerant near-isogenic line. The responsiveness of these transcripts to H2O2 suggests that they may represent the wheat orthologs of ZFP transcription factors ZAT7 and ZAT12 that were shown to improve ROS tolerance in Arabidopsis. Thus, they may play a crucial role in the improvement of oxidative stress tolerance in wheat.

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.

The Arabidopsis transcription factor (At1g26610) was shown to be down-regulated in male sterile lines compared to their maintainer line in Chinese cabbage (Brassica campestris ssp. chinensis) in our previous study. The BcMF20 gene, homologue of the At1g26610 gene was isolated from Chinese cabbage. It encodes a putative C2H2 zinc finger protein. The spatial and temporal expression patterns examined by qRT-PCR and in situ hybridization, indicated BcMF20 is specifically expressed in the developing pollen grains and the tapetum from the uninucleate pollen stage to mature pollen stage.

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.

In this paper, a modified particle swarm optimisation algorithm is proposed for protein sequence motif discovery. Protein sequences are represented as a chain of symbols and a protein sequence motif is a short sequence that exists in most of the protein sequence families. Protein sequence symbols are converted into numbers using a one to one amino acid translation table. The simulation uses EGF protein and C2H2 Zinc Finger protein families obtained from the PROSITE database. Simulation results show that the modified particle swarm optimisation algorithm is effective in obtaining global optimum sequence patterns, achieving 96.9 and 99.5 classification accuracy respectively in EGF and C2H2 Zinc Finger protein families. A better true positive hit result is achieved when compared to the motifs published in PROSITE database.

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 describe an Israeli Jewish Moroccan family presenting with autosomal dominant seborrhea-like dermatosis with psoriasiform elements, including enhanced keratinocyte proliferation, parakeratosis, follicular plugging, Pityrosporum ovale overgrowth and dermal CD4 lymphocyte infiltrate. We mapped the disease gene to a 0.5-cM region overlapping the PSORS2 locus (17q25) and identified a frameshift mutation in ZNF750, which encodes a putative C2H2 zinc finger protein. ZNF750 is normally expressed in keratinocytes but not in fibroblasts and is barely detectable in CD4 lymphocytes.

The crystal structure of the dimerization domain of the V(D)J recombination-activating protein, RAG1, was solved using zinc anomalous scattering. The structure reveals an unusual combination of multi-class zinc-binding motifs, including a zinc RING finger and a C2H2 zinc finger, that together from a single structural domain. The domain also contains a unique zinc binuclear cluster in place of a normally mononuclear zinc site in the RING finger. Together, four zinc ions help organize the entire domain, including the two helices that form the dimer interface.

In animal systems the C2H2 zinc finger protein (ZFP) genefamily is the largest group of regulatory proteins and its members havea wide and important role in growth and development. It is likely thatthis family of ZFP transcription factors will also be important inplants. We have used a PCR approach employing highly degenerateoligonucleotide primers to isolate several Arabidopsis genomic andcDNA clones encoding potential ZFPs. In addition we have used thesequence information from these clones to identify two ESTs as membersof this family. Five two-fingered and one three-fingered ZFPs have beenidentified. Outside of the zinc finger regions, there is considerablesequence diversity, including the sequence and length of the interfingerregion; the expression pattern of each gene is different. This is thefirst report of the isolation of a three-fingered plant C2H2ZFP gene and we discuss its possible evolutionary origin fromtwo-fingered ZFP genes.

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.

A possible function for microsatellite elements still remains elusive. Here we demonstrate that (TA)n microsatellites can be recognized in vivo in transient assays by a combination of naturally occurring C2H2 zinc fingers with high affinity that depends on specific protein-DNA contacts. This interaction results in an upregulation of the basal transcription level, when (TA)n elements are placed upstream of a minimal promoter. Upregulation depends on the length of the element. Our results suggest that specific interaction of transcription factors with microsatellite sequences may underlie effects on transcriptional regulation exerted by these sequences. Identification of such interactions would be a first step towards understanding the function and evolution of microsatellite elements.

Background Caenorhabditis elegans sarcomeres have been studied extensively utilizing both forward and reverse genetic techniques to provide insight into muscle development and the mechanisms behind muscle contraction. A previous genetic screen investigating early muscle development produced 13 independent mutant genes exhibiting a Pat (p aralyzed and a rrested elongation at the t wo-fold length of embryonic development) muscle phenotype. This study reports the identification and characterization of one of those genes, pat-9. Results Positional cloning, reverse genetics, and plasmid rescue experiments were used to identify the predicted C. elegans gene T27B1.2 (recently named ztf-19) as the pat-9 gene. Analysis of pat-9 showed it is expressed early in development and within body wall muscle lineages, consistent with a role in muscle development and producing a Pat phenotype. However, unlike most of the other known Pat gene family members, which encode structural components of muscle attachment sites, PAT-9 is an exclusively nuclear protein. Analysis of the predicted PAT-9 amino acid sequence identified one putative nuclear localization domain and three C2H2 zinc finger domains. Both immunocytochemistry and PAT-9::GFP fusion expression confirm that PAT-9 is primarily a nuclear protein and chromatin immunoprecipitation (ChIP) experiments showed that PAT-9 is present on certain gene promoters. Conclusions We have shown that the T27B1.2 gene is pat-9. Considering the Pat-9 mutant phenotype shows severely disrupted muscle attachment sites despite PAT-9 being a nuclear zinc finger protein and not a structural component of muscle attachment sites, we propose that PAT-9 likely functions in the regulation of gene expression for some necessary structural or regulatory component(s) of the muscle attachment sites.

Background Expansion of multi-C2H2 domain zinc finger (ZNF) genes, including the Krüppel-associated box (KRAB) subfamily, paralleled the evolution of tetrapodes, particularly in mammalian lineages. Advances in their cataloging and characterization suggest that the functions of the KRAB-ZNF gene family contributed to mammalian speciation. Results Here, we characterized the human 8q24.3 ZNF cluster on the genomic, the phylogenetic, the structural and the transcriptome level. Six (ZNF7, ZNF34, ZNF250, ZNF251, ZNF252, ZNF517) of the seven locus members contain exons encoding KRAB domains, one (ZNF16) does not. They form a paralog group in which the encoded KRAB and ZNF protein domains generally share more similarities with each other than with other members of the human ZNF superfamily. The closest relatives with respect to their DNA-binding domain were ZNF7 and ZNF251. The analysis of orthologs in therian mammalian species revealed strong conservation and purifying selection of the KRAB-A and zinc finger domains. These findings underscore structural/functional constraints during evolution. Gene losses in the murine lineage (ZNF16, ZNF34, ZNF252, ZNF517) and potential protein truncations in primates (ZNF252) illustrate ongoing speciation processes. Tissue expression profiling by quantitative real-time PCR showed similar but distinct patterns for all tested ZNF genes with the most prominent expression in fetal brain. Based on accompanying expression signatures in twenty-six other human tissues ZNF34 and ZNF250 revealed the closest expression profiles. Together, the 8q24.3 ZNF genes can be assigned to a cerebellum, a testis or a prostate/thyroid subgroup. These results are consistent with potential functions of the ZNF genes in morphogenesis and differentiation. Promoter regions of the seven 8q24.3 ZNF genes display common characteristics like missing TATA-box, CpG island-association and transcription factor binding site (TFBS) modules. Common TFBS modules partly explain the observed expression pattern similarities. Conclusions The ZNF genes at human 8q24.3 form a relatively old mammalian paralog group conserved in eutherian mammals for at least 130 million years. The members persisted after initial duplications by undergoing subfunctionalizations in their expression patterns and target site recognition. KRAB-ZNF mediated repression of transcription might have shaped organogenesis in mammalian ontogeny.

A novel zinc finger gene (ZNF182) was isolated from a human fetal cardiac cDNA library, using a consensus C2H2 zinc finger oligonucleotide probe. This gene was assigned to human chromosome 7p11.1-p12 by fluorescence in situ hybridization (FISH). Additional FISH signals were identified on both the long and the short arms of chromosome 19, suggesting the presence of homologous genes at these loci.

In this issue, Zhao and Stormo introduce a new method for deriving position weight matrices (PWMs) from Protein Binding microarrays (BEEML-PBM). Using this method, they challenge a central claim of our 2009 paper 1, by “[concluding] that the widespread phenomenon of secondary binding preference identified by Badis et al. is not supported by the data and is likely due to suboptimal estimation of the PWM.” BEEML-PBM is simple, elegant and corrects for a pronounced positional effect of TF binding in the PBM assay; however we do not agree with their overall conclusion and believe that it is based on incomplete and biased analysis of our data. Zhao and Stormo’s conclusions are based on comparing the performance of BEEML-PBM PWMs and our methods on held-out data. However, they over-estimate the performance of their PWMs and under-estimate the performance of our methods. First, their claims of suboptimality of our PWMs are based on results from only one of the three motif finders that we employed, Seed-and-Wobble (SnW). SnW was not developed to predict probe intensities and does not attempt to produce a summary PWM that optimizes performance over all probes in predicting probe intensities. Instead, it was developed for the purpose of summarizing the 8-mer data, seeding with the highest scoring 8-mer, in a compact way for use in visual depiction as sequence logos. In contrast, another of the methods we employed, RankMotif++, is designed to produce summary PWMs and we have previously reported2 that it, like BEEML-PBM, better predicts probe intensities than SnW. So we suspect its performance would be much more competitive. In fact, RankMotif++ is very similar to the BEEML base method3; it fits a PWM model using a regression-like procedure to optimally predict PBM intensity data. RankMotif++ differs from BEEML primarily in that regresses on a partial preference ordering of probes inferred from their PBM intensities rather than on their actual intensities themselves. We acknowledge that comparisons with RankMotif++ PWMs would have been difficult because, although the source code for RankMotif++ has been available for three years, the PWMs we learned for Badis et al. were until recently only available as sequence logos. However, we made the motifs available to Zhao and Stormo when we were notified of this oversight and before the final submission of their paper. The motifs are available here: http://the_brain.bwh.harvard.edu/suppl105/. Second, we note that Zhao and Stormo use a positional effect model when training their PWMs but do not allow the methods that they are comparing against the same opportunity to correct this bias during training. We propose that this correction is a major cause of BEEML-PBM’s success and that both the multiple PWM methods and the 8-mer affinity estimates we employed would greatly benefit from a similar correction, thus restoring our reported gain in performance. For example, the 8-mer median intensities used in Figure 2A are not corrected for positional biases and this leads to the counter-intuitive claim that for the 15–20 (of 41) data points that lie above the diagonal, BEEML-PBM PWMs capture more than 100% of the replicate reproducibility. A more appropriate comparison would either employ PWMs uncorrected for positional bias (as we did in our original paper) or to compare against similarly corrected 8-mer median intensities. Zhao and Stormo do neither and as such, we believe that their prediction accuracy estimates are inflated. Finally, we note that explaining 90% of the reproducible binding signal is not the same as explaining 100%, and proteins that we and others have confirmed have multiple binding modes do not satisfy Zhao and Stormo’s 90% cut -off. For example, we reported that Jundm2 (Jdp2) binds two half -sites with variable spacing between them; this is clearly observed in the top-scoring 8-mers1. This mode of binding is common among other bZIP proteins. Furthermore, Zhao and Stormo do not consider the PBM data for Bcl6b, a C2H2 zinc finger for which we obtained two very different PWMs; these are also clearly observed in the top-scoring 8-mers, and moreover enrichment for motif matches can be observed in associated ChIP-chip data1. In general, variable spacing in long C2H2 zinc finger array seems to be common; for example, ChIP-seq for REST also supports use of partial vs. full sites, and different spacings4. Single summary PWMs cannot capture these binding modes, and it is important to do so, as C2H2 zinc fingers are the most common domain in metazoa, and long arrays of these domains are common in human and mouse genomes. We agree that simple and accurate representation of TF sequence specificity on the basis of PBM data is an important problem. We ourselves have been working on extensions to our algorithms to capture the PBM positional and orientation effects (which we have previously reported5). We also recently conducted a DREAM competition in which the goal was to predict PBM probe intensities using a two-array framework and evaluation criteria similar to those employed in1 and Zhao et al. A manuscript describing these new data, the competition, the methods of ~20 groups, their evaluation, and a web site that allows benchmarking any method to the DREAM results is in preparation (M. Weirauch, G. Stolovitsky et al.). We have now obtained the BEEML-PBM code, and we look forward

A comparative study based on four fully sequenced eukaryotic genomes has revealed profound differences in the sets of transcription factors used by plants, fungi and animals.