C2H2 Zinc Finger Domain Research Articles

ZNF1 Encodes a Putative C2H2 Zinc-Finger Protein Essential for Appressorium Differentiation by the Rice Blast Fungus Magnaporthe oryzae.

The rice blast fungus Magnaporthe oryzae forms specialized infection structures called appressoria which are essential for gaining entry to plant tissue. Here, we report the identification of a novel nonpathogenic T-DNA-tagged mutant XF696 of M. oryzae with a single insertion in the promoter of ZNF1, which encodes a putative transcription factor (TF). Targeted gene deletion mutants of ZNF1 are nonpathogenic and unable to develop appressoria. However, Δznf1 mutants still respond to exogenous cyclic AMP on hydrophilic surfaces and can sense hydrophobic surfaces, initiating the differentiation of germ tubes. Interestingly, Δznf1 mutants also produce significantly more conidia compared with the isogenic wild-type strain. Quantitative reverse-transcription polymerase chain reaction analysis and green fluorescent protein fusion experiments revealed that expression of ZNF1 was highly induced during germination and appressorium development in M. oryzae and potentially regulated by the Pmk1 mitogen-activated protein kinase pathway. We observed that Δznf1 mutants are affected in mitosis and impaired in mobilization and degradation of lipid droplets and glycogen reserves during appressorium differentiation. Site-directed mutagenesis confirmed that three of the four C2H2 zinc-finger domains are essential for the function of Znf1. Taken together, we conclude that a C2H2 zinc-finger TF encoded by ZNF1 is essential for appressorium development by the rice blast fungus.

Tuberculate fruit gene Tu encodes a C2H2 zinc finger protein that is required for the warty fruit phenotype in cucumber (Cucumis sativus L.)

Cucumber fruits that have tubercules and spines (trichomes) are known to possess a warty (Wty) phenotype. In this study, the tuberculate fruit gene Tu was identified by map-based cloning, and was found to encode a transcription factor (TF) with a single C2 H2 zinc finger domain. Tu was identified in all 38 Wty lines examined, and was completely absent from all 56 non-warty (nWty) lines. Cucumber plants transgenic for Tu (TCP) revealed that Tu was required for the Wty fruit phenotype. Subcellular localization showed that the fusion protein GFP-Tu was localized mainly to the nucleus. Based on analyses of semi-quantitative and quantitative reverse transcription polymerase chain reaction (RT-PCR), and mRNA in situ hybridization, we found that Tu was expressed specifically in fruit spine cells during development of fruit tubercules. Moreover, cytokinin (CTK) content measurements and cytological observations in Wty and nWty fruits revealed that the Wty fruit phenotype correlated with high endogenous CTK concentrations. As a result of further analyses on the transcriptomic profile of the nWty fruit epidermis and TCP fruit warts, expression of CTK-associated genes, and hormone content in nWty fruit epidermis, Wty fruit warts and epidermis, and TCP fruit warts and epidermis, we found that Tu probably promoted CTK biosynthesis in fruit warts. Here we show that Tu could not be expressed in the glabrous and tubercule-free mutant line gl that contained Tu, this result that futher confirmed the epistatic effect of the trichome (spine) gene Gl over Tu. Taken together, these data led us to propose a genetic pathway for the Wty fruit trait that could guide future mechanistic studies.

The putative C2H2 transcription factor MtfA is a novel regulator of secondary metabolism and morphogenesis in Aspergillus nidulans.

Secondary metabolism in the model fungus Aspergillus nidulans is controlled by the conserved global regulator VeA, which also governs morphological differentiation. Among the secondary metabolites regulated by VeA is the mycotoxin sterigmatocystin (ST). The presence of VeA is necessary for the biosynthesis of this carcinogenic compound. We identified a revertant mutant able to synthesize ST intermediates in the absence of VeA. The point mutation occurred at the coding region of a gene encoding a novel putative C2H2 zinc finger domain transcription factor that we denominated mtfA. The A. nidulans mtfA gene product localizes at nuclei independently of the illumination regime. Deletion of the mtfA gene restores mycotoxin biosynthesis in the absence of veA, but drastically reduced mycotoxin production when mtfA gene expression was altered, by deletion or overexpression, in A. nidulans strains with a veA wild-type allele. Our study revealed that mtfA regulates ST production by affecting the expression of the specific ST gene cluster activator aflR. Importantly, mtfA is also a regulator of other secondary metabolism gene clusters, such as genes responsible for the synthesis of terrequinone and penicillin. As in the case of ST, deletion or overexpression of mtfA was also detrimental for the expression of terrequinone genes. Deletion of mtfA also decreased the expression of the genes in the penicillin gene cluster, reducing penicillin production. However, in this case, over-expression of mtfA enhanced the transcription of penicillin genes, increasing penicillin production more than 5 fold with respect to the control. Importantly, in addition to its effect on secondary metabolism, mtfA also affects asexual and sexual development in A. nidulans. Deletion of mtfA results in a reduction of conidiation and sexual stage. We found mtfA putative orthologs conserved in other fungal species.

Structural and dynamical characterization of the Miz-1 zinc fingers 5–8 by solution-state NMR

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.

Binding of Drosophila maternal Mamo protein to chromatin and specific DNA sequences

Alterations in chromatin structure dynamically occur during germline development in Drosophila and are essential for the production of functional gametes. We had previously reported that the maternal factor Mamo, which contains both a BTB/POZ domain and C2H2 zinc-finger domains and is enriched in primordial germ cells (PGCs), is required for the regulation of meiotic chromatin structure and the production of functional gametes. However, the molecular mechanisms by which Mamo regulates germline development remained unclear. To evaluate the molecular function of Mamo protein, we have investigated the binding of Mamo to chromatin and DNA sequences. Our data show that Mamo binds to chromatin and specific DNA sequences, particularly the polytene chromosomes of salivary gland cells. Overexpression of Mamo affected the organization of polytene chromosomes. Reduction in maternal Mamo levels impaired the formation of germline-specific chromatin structures in PGCs. Furthermore, we found that the zinc-finger domains of Mamo directly bind to specific DNA sequences. Our results suggest that Mamo plays a role in regulating chromatin structure in PGCs.

Isolation and characterization of differentially expressed genes in the mycelium and fruit body of Pleurotus ostreatus

The fruiting body of one of the most widely cultivated mushrooms, the oyster mushroom (Pleurotus ostreatus) is highly interesting, both commercially and scientifically. In the present study, we performed comparative proteomic profiling of P. ostreatus at two unique developmental stages; mycelium and fruit body, using two-dimensional gel electrophoresis (2-DE). Seven hundred fourteen (714) spots were detected and 29 spots (showing a high level of difference in their expressions) were identified by tandem mass spectrometry and basic local alignment search tool (BLAST) searching of an expressed sequence tag (EST) database of P. ostreatus. Among them, six proteins (putative fatty acid oxygenase, heat shock sks2, PriA homologue, Ap-1 like transcription factor YAP7, mung bean seed albumin, and C2H2 Zinc finger domain protein) and one protein (peroxisomal biogenesis factor 6) showed increased expression levels at the fruiting process and the mycelial stage, respectively. Through reverse transcriptase-polymerase chain reaction analysis, priA homologue and AP-1 like transcription factor yap7 showed gradually increased expression from mycelia to fruit body, whereas putative fatty acid oxygenase and heat shock protein sks2 were expressed only in the fruit body. These results provide useful information for future studies of mushroom development of P. ostreatus. Keywords : Developmental stage, mushroom fruiting, Pleurotus ostreatus , protein, two-dimensional gel electrophoresis African Journal of Biotechnology Vol. 12(24), pp. 3790-3796

Molecular cloning and expression analysis of Foxp3 from Nile tilapia

Foxp3 is a crucial transcription factor for the development and function of CD4+CD25+ regulatory T cells (Tr) which play a key role in preventing autoimmune diseases and maintaining maternal-fetal tolerance in mammals. However, the knowledge of Foxp3 and its regulation in teleosts is limited. In the present study, the Foxp3 cDNA was cloned from Nile tilapia and characterized. The full length cDNA of Nile tilapia (nt)Foxp3 contained a 5′UTR of 104bp, a 3′UTR of 239bp and an open reading frame of 1134bp. The putative ntFoxp3 contained a C2H2 zinc finger domain, a winged-helix/fork head domain and a leucine zipper-like domain with a consensus of V-x(6)-L-x(6)-L-x(6)-L, which are typical motifs of a Foxp3. The highest mRNA expression of ntFoxp3 was observed in the spleen, with moderate expression in the head kidney, intestine, kidney, liver and brain. Stimulation of peripheral blood mononuclear cells with PHA and LPS led to a significant increase of ntFoxp3 mRNA expression at 6 and 24h, respectively. In contrast, stimulation with PMA had no effect during the sampling period. After injecting 1×107cells of live Aeromonas hydrophila into adult female Nile tilapias, the mRNA expression of ntFoxp3 significantly increased in the gill and intestine at 6h, while unchanged in the spleen. In all the tissues examined, a clearly up-regulation of ntFoxp3 expression was detected at 24h after the second challenge. These results suggest that ntFoxp3 might be involved into lymphocyte activation and immune responses as reported in mammals. Meanwhile, the correlation between the expression profile of ntFoxp3 and serum estrogen (17-beta-estradiol, E2) concentration was investigated by real-time PCR and enzyme immunoassay. The results showed that E2 could regulate the expression of ntFoxp3 in the intestine and kidney but not in the spleen. This work will help future investigations into Tr cells and extend our understanding of interactions between sex hormones and immune related molecules in teleosts.

DLocalMotif: a discriminative approach for discovering local motifs in protein sequences

Local motifs are patterns of DNA or protein sequences that occur within a sequence interval relative to a biologically defined anchor or landmark. Current protein motif discovery methods do not adequately consider such constraints to identify biologically significant motifs that are only weakly over-represented but spatially confined. Using negatives, i.e. sequences known to not contain a local motif, can further increase the specificity of their discovery. This article introduces the method DLocalMotif that makes use of positional information and negative data for local motif discovery in protein sequences. DLocalMotif combines three scoring functions, measuring degrees of motif over-representation, entropy and spatial confinement, specifically designed to discriminatively exploit the availability of negative data. The method is shown to outperform current methods that use only a subset of these motif characteristics. We apply the method to several biological datasets. The analysis of peroxisomal targeting signals uncovers several novel motifs that occur immediately upstream of the dominant peroxisomal targeting signal-1 signal. The analysis of proline-tyrosine nuclear localization signals uncovers multiple novel motifs that overlap with C2H2 zinc finger domains. We also evaluate the method on classical nuclear localization signals and endoplasmic reticulum retention signals and find that DLocalMotif successfully recovers biologically relevant sequence properties. http://bioinf.scmb.uq.edu.au/dlocalmotif/

Molecular cloning, expression profile analysis and construction of adipose tissue specific expression vector of pig <I>Gli1</I> gene

The Hedgehog (Hh) signaling pathway inhibits fat accumulation, which is conserved in a wide variety of organisms from Drosophila to vertebrates, but few reports about its effect on pigs are available. In this study, pig Gli1 gene was cloned for the first time by rapid amplification of cDNA ends (RACE) and RT-PCR. Pig Gli1 expression profiles were then studied in different tissues and in different developmental stages of the adipose tissue of pigs using real-time PCR. Finally, the eukaryotic expression vector and the adipose tissue specific expression vector were constructed. The results showed that the full pig Gli1 cDNA length was 3 576 bp, the genomic sequence contained 10 715 bp with 12 exons, and 1 106 amino acids were encoded. Pig Gli1 was predicted as an unstable hydrophilic protein without a tans-membrane structure or a signal peptide. The C2H2 zinc finger domain and a nuclear localization sequence were found in pig Gli1. A homology analysis of the Gli1 amino acids and the genomic sequences among seven species showed that the identities were all greater than 80%, which indicates that Gli1 is highly conserved among different species. Tissue expression profile analysis showed that pig Gli1 was only expressed in the tone tissue of adult pigs. Analysis of the pig adipose tissue developmental process showed that Gli1 was detected in the adipose tissue of one-week-old pigs, but not in one-month-old and three-month-old pigs. Finally, a pig Gli1 eukaryotic expression vector was constructed and properly expressed with cell transfection. An adipose tissue specific expression vector was constructed for transgenic animal studies.

Human More Complex than Mouse at Cellular Level

The family of transcription factors with the C2H2 zinc finger domain is expanding in the evolution of vertebrates, reaching its highest numbers in the mammals. The question arises: whether an increased amount of these transcription factors is related to embryogenesis, nervous system, pathology or more of them are expressed in individual cells? Among mammals, the primates have a more complex anatomical structure than the rodents (e.g., brain). In this work, I show that a greater number of C2H2-ZFgenes are expressed in the human cells than in the mouse cells. The effect is especially pronounced for C2H2-ZF genes accompanied with the KRAB domain. The relative difference between the numbers of C2H2-ZF(-KRAB) genes in the human and mouse cellular transcriptomes even exceeds their difference in the genomes (i.e. a greater subset of existing in the genome genes is expressed in the human cellular transcriptomes compared to the mouse transcriptomes). The evolutionary turnover of C2H2-ZF(-KRAB) genes acts in the direction of the revealed phenomenon, i.e. gene duplication and loss enhances the difference in the relative number of C2H2-ZF(-KRAB) genes between human and mouse cellular transcriptomes. A higher amount of these genes is expressed in the brain and embryonic cells (compared with other tissues), whereas a lower amount - in the cancer cells. It is specifically the C2H2-ZF transcription factors whose repertoire is poorer in the cancer and richer in the brain (other transcription factors taken together do not show this trend). These facts suggest that increase of anatomical complexity is accompanied by a more complex intracellular regulation involving these transcription factors. Malignization is associated with simplification of this regulation. These results agree with the known fact that human cells are more resistant to oncogenic transformation than mouse cells. The list of C2H2-ZF genes whose suppression might be involved in malignization is provided.

Molecular phylogeny of OVOL genes illustrates a conserved C2H2 zinc finger domain coupled by hypervariable unstructured regions.

OVO-like proteins (OVOL) are members of the zinc finger protein family and serve as transcription factors to regulate gene expression in various differentiation processes. Recent studies have shown that OVOL genes are involved in epithelial development and differentiation in a wide variety of organisms; yet there is a lack of comprehensive studies that describe OVOL proteins from an evolutionary perspective. Using comparative genomic analysis, we traced three different OVOL genes (OVOL1-3) in vertebrates. One gene, OVOL3, was duplicated during a whole-genome-duplication event in fish, but only the copy (OVOL3b) was retained. From early-branching metazoa to humans, we found that a core domain, comprising a tetrad of C2H2 zinc fingers, is conserved. By domain comparison of the OVOL proteins, we found that they evolved in different metazoan lineages by attaching intrinsically-disordered (ID) segments of N/C-terminal extensions of 100 to 1000 amino acids to this conserved core. These ID regions originated independently across different animal lineages giving rise to different types of OVOL genes over the course of metazoan evolution. We illustrated the molecular evolution of metazoan OVOL genes over a period of 700 million years (MY). This study both extends our current understanding of the structure/function relationship of metazoan OVOL genes, and assembles a good platform for further characterization of OVOL genes from diverged organisms.

C. elegans PAT-9 is a nuclear zinc finger protein critical for the assembly of muscle attachments

BackgroundCaenorhabditis 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 (paralyzed and arrested elongation at the two-fold length of embryonic development) muscle phenotype. This study reports the identification and characterization of one of those genes, pat-9.ResultsPositional 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.ConclusionsWe 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.

Molecular cloning and expression analysis of a WRKY transcription factor in sugarcane

The WRKY proteins comprise a superfamily of transcription factors which are unique in plants. In the present study, a WRKY gene of sugarcane was isolated. This gene is 1 003 bp in full length, which includes 93 bp 5’ untranslated region (UTR) and 172 bp 3’ UTR. The open reading frame (ORF) of this gene is 738 bp which encodes 245 amino acids, with a typical WRKY DNA-binding domain at the 127 ~ 186 aa of the deduced amino acid sequences. The cysteine and histidine are also rich at the 153 ~ 184 aa, which is a C2H2 zinc finger domain of Class II and located at the C-terminal end of this deduced amino acid sequence; thus, this gene belongs to WRKYII. Furthermore, the prokaryotic expression vector of Sc-WRKY gene was successfully constructed and expressed in Escherichia coli (BL21 strain), and the molecular weight of the targeted product was about 25.7 kD. Real-time quantitative PCR analysis demonstrated that the Sc-WRKY gene was strongly induced by Ustilago scitaminea, salicylic acid (SA), NaCl and PEG, which suggests that this gene might play an important role in smut-resistant, drought-tolerant and salt-tolerant mechanism. Key words: Sugarcane (Saccharum officinarum), WRKY transcription factors, prokaryotic expression, real-time quantitative PCR.

Molecular Characterization and Expression Analysis of TaZFP15, a C2H2-Type Zinc Finger Transcription Factor Gene in Wheat (Triticum aestivum L.)

Based on sequencing of part clones in a root subtractive cDNA library, an expressed sequence tag (EST) sharing high similarity to a rice C2H2 zinc finger transcription factor (ZFP15) was obtained in wheat. Through bioinformatics approach, the wheat C2H2-type ZFP gene referred to TaZFP15 has been identified and characterized. As a full-length cDNA of 670 bp, TaZFP15 has an open reading frame of 408 bp and encodes a 135-aa polypeptide. TaZFP15 contains two C2H2 zinc finger domains and each one has a conserved motif QALGGH. The typical L-box, generally identified in the C2H2 type transcription factors, has also been found in TaZFP15. Phylogenetic analysis suggested that TaZFP15 shares high similarities with rice ZFP15 (GenBank accession no. AY286473), maize ZFP (GenBank accession no. NM_001159094) and a subset of other zinc-finger transcription factor genes in plant species. The expression of TaZFP15 was up-regulated by starved-Pi stress, showing a pattern to be gradually elevated along with the progression of the Pi-stress in a 23-h treatment regime. Similarly, the transcripts of TaZFP15 in roots were also induced by nitrogen deficiency, and abiotic stresses of drought and salinity. No responses of TaZFP15 were detected in roots to nutrition deficiencies of P, Zn, and Ca, and the external treatment of abscisic acid (ABA). TaZFP15 could be specifically amplified in genome A, B, and D, and without variability in the sequences, suggesting that TaZFP15 has multi-copies in the homologous hexaploid species. Transgenic analysis in tobacco revealed that up-regulation of TaZFP15 could significantly improve plant dry mass accumulation via increasing the plant phosphorus acquisition capacity under Pi-deficiency condition. The results suggested that TaZFP15 is involved in mediation of signal transductions of diverse external stresses.

Remote homologue identification of Drosophila GAGA factor in mouse.

GAGA factor (GAF) is involved in both gene activation and gene repression and plays a role in the modulation of chromatin structure. In Drosophila, Trithroax like (Trl) gene encodes the DNA binding protein called GAGA factor (GAF). Trl-GAF binds to GAGA sites through its C2H2 zinc finger domain and has an N-terminal BTB/POZ domain. Identification of Trl-GAF homologue in mouse helps in deeper understanding of the mechanism and function. Conventional alignment tools such as BLAST and FASTA cannot identify homologues in mouse genome as their sequence identity is below 30%. In the present study, various sequence and structure analyses were followed for the detection of remote homologues of Drosophila GAGA FACTOR in mouse to identify as Zbtb3. Through homology modeling and docking approach, the zinc finger region of mouse Zbtb3 showed conserved residues and favorable DNA binding sites with GAGA sites similar to that of Drosophila GAGA FACTOR.

The p97 ATPase associates with EEA1 to regulate the size of early endosomes

The AAA (ATPase-associated with various cellular activities) ATPase p97 acts on diverse substrate proteins to partake in various cellular processes such as membrane fusion and endoplasmic reticulum-associated degradation (ERAD). In membrane fusion, p97 is thought to function in analogy to the related ATPase NSF (N-ethylmaleimide-sensitive fusion protein), which promotes membrane fusion by disassembling a SNARE complex. In ERAD, p97 dislocates misfolded proteins from the ER membrane to facilitate their turnover by the proteasome. Here, we identify a novel function of p97 in endocytic trafficking by establishing the early endosomal autoantigen 1 (EEA1) as a new p97 substrate. We demonstrate that a fraction of p97 is localized to the early endosome membrane, where it binds EEA1 via the N-terminal C2H2 zinc finger domain. Inhibition of p97 either by siRNA or a pharmacological inhibitor results in clustering and enlargement of early endosomes, which is associated with an altered trafficking pattern for an endocytic cargo. Mechanistically, we show that p97 inhibition causes increased EEA1 self-association at the endosome membrane. We propose that p97 may regulate the size of early endosomes by governing the oligomeric state of EEA1.

A novel double-stranded RNA mycovirus from Fusarium graminearum: nucleic acid sequence and genomic structure

Ten Fusarium graminearum isolates from China were screened for dsRNA mycoviruses. Five dsRNAs (2.4 to 3.5 kbp) were purified from isolate China 9, cloned, and sequenced. BLAST analysis showed that the proteins encoded by dsRNA1 possess motifs that are conserved in RNA-dependent RNA polymerases, dsRNA2 resembles the hypothetical protein encoded by dsRNA3 of Magnaporthe oryzae chrysovirus 1, dsRNA4 shares no significant similarity to any published protein, and dsRNA5 has a C2H2 zinc finger domain. Tandem mass spectrophotometry, surface protein labeling of virus-like particles, SDS-PAGE, and protein BLAST results supports the notion that three of the virus segments code for structural proteins, of which dsRNA3 possibly codes for the capsid protein. Relative quantitative RT-PCR studies of the 5 dsRNAs suggested that the segments are encapsidated separately in unequal amounts. Genomic structure and phylogenic studies support the possibility that this virus may be a candidate for the type species of a novel genus in the family Chrysoviridae.

The C2H2 zinc finger transcription factors are likely targets for Ni(ii) toxicity

Ni(II) ions are able to hydrolyze Naa-(Ser/Thr) peptide bonds in Naa-(Ser/Thr)-Xaa-His-Zaa sequences. We found that various human transcription factors contain such nickel hydrolytic patterns within C2H2 zinc finger (ZF) domains. We demonstrated the hydrolysis on two peptide models, the 3rd ZF of the Sp1 transcription factor and the 1st ZF of the ZNF302 transcription factor. The experimentally studied reaction rates indicate that the hydrolysis reaction is likely to be an element of intracellular nickel toxicity.

ZNF274 Recruits the Histone Methyltransferase SETDB1 to the 3′ Ends of ZNF Genes

Only a small percentage of human transcription factors (e.g. those associated with a specific differentiation program) are expressed in a given cell type. Thus, cell fate is mainly determined by cell type-specific silencing of transcription factors that drive different cellular lineages. Several histone modifications have been associated with gene silencing, including H3K27me3 and H3K9me3. We have previously shown that genes for the two largest classes of mammalian transcription factors are marked by distinct histone modifications; homeobox genes are marked by H3K27me3 and zinc finger genes are marked by H3K9me3. Several histone methyltransferases (e.g. G9a and SETDB1) may be involved in mediating the H3K9me3 silencing mark. We have used ChIP-chip and ChIP-seq to demonstrate that SETDB1, but not G9a, is associated with regions of the genome enriched for H3K9me3. One current model is that SETDB1 is recruited to specific genomic locations via interaction with the corepressor TRIM28 (KAP1), which is in turn recruited to the genome via interaction with zinc finger transcription factors that contain a Kruppel-associated box (KRAB) domain. However, specific KRAB-ZNFs that recruit TRIM28 (KAP1) and SETDB1 to the genome have not been identified. We now show that ZNF274 (a KRAB-ZNF that contains 5 C2H2 zinc finger domains), can interact with KAP1 both in vivo and in vitro and, using ChIP-seq, we show that ZNF274 binding sites co-localize with SETDB1, KAP1, and H3K9me3 at the 3′ ends of zinc finger genes. Knockdown of ZNF274 with siRNAs reduced the levels of KAP1 and SETDB1 recruitment to the binding sites. These studies provide the first identification of a KRAB domain-containing ZNF that is involved in recruitment of the KAP1 and SETDB1 to specific regions of the human genome.

Human RING finger protein ZNF645 is a novel testis-specific E3 ubiquitin ligase

A large number of testis-specific genes are involved in the complex process of mammalian spermatogenesis. Identification of these genes and their roles is important for understanding the mechanisms underlying spermatogenesis. Here we report on a novel human RING finger protein, ZNF645, which contains a C3HC4 RING finger domain, a C2H2 zinc-finger domain, and a proline-rich region, indicating that it has a structure similar to that of the c-Cbl-like protein Hakai. ZNF645 was exclusively expressed in normal human testicular tissue. Immunohistochemical analysis confirmed that ZNF645 protein was present in spermatocytes, round and elongated spermatids, and Leydig cells. Immunofluorescence staining of mature sperms further showed that the ZNF645 protein was localized over the postacrosomal perinuclear theca region and the entire length of sperm tail. An in vitro ubiquitination assay indicated that the RING finger domain of the ZNF645 protein had E3 ubiquitin ligase activity. Therefore, we suggest that ZNF645 might act as an E3 ubiquitin-protein ligase and play a role in human sperm production and quality control.