An RNA-interference screen in Drosophila to identify ZAD-containing C2H2 zinc finger genes that function in female germ cells.
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ABSTRACT:
SUBMITTER: Shapiro-Kulnane L
PROVIDER: S-EPMC8022714 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:C2H2 zinc-finger proteins (ZFPs) constitute the largest family of nucleic acid binding factors in higher eukaryotes. In silico analysis identified a total of 326 putative ZFP genes in the Drosophila genome, corresponding to approximately 2.3% of the annotated genes. Approximately 29% of the Drosophila ZFPs are evolutionary conserved in humans and/or Caenorhabditis elegans. In addition, approximately 28% of the ZFPs contain an N-terminal zinc-finger-associated C4DM domain (ZAD) consisting of approximately 75 amino acid residues. The ZAD is restricted to ZFPs of dipteran and closely related insects. The evolutionary restriction, an expansion of ZAD-containing ZFP genes in the Drosophila genome and their clustering at few chromosomal sites are features reminiscent of vertebrate KRAB-ZFPs. ZADs are likely to represent protein-protein interaction domains. We propose that ZAD-containing ZFP genes participate in transcriptional regulation either directly or through site-specific modification and/or regulation of chromatin.
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Project description:H3K9me3-based gene silencing is a conserved strategy for securing cell fate, but the mechanisms controlling lineage-specific installation of this epigenetic mark remain unclear. In Drosophila, H3K9 methylation plays an essential role in securing female germ cell fate by silencing lineage inappropriate phf7 transcription. Thus, phf7 regulation in the female germline provides a powerful system to dissect the molecular mechanism underlying H3K9me3 deposition onto protein coding genes. Here we used genetic studies to identify the essential cis-regulatory elements, finding that the sequences required for H3K9me3 deposition are conserved across Drosophila species. Transposable elements are also silenced by an H3K9me3-mediated mechanism. But our finding that phf7 regulation does not require the dedicated piRNA pathway components, piwi, aub, rhino, panx, and nxf2, indicates that the mechanisms of H3K9me3 recruitment are distinct. Lastly, we discovered that an uncharacterized member of the zinc finger associated domain (ZAD) containing C2H2 zinc finger protein family, IDENTITY CRISIS (IDC; CG4936), is necessary for H3K9me3 deposition onto phf7. Loss of idc in germ cells interferes with phf7 transcriptional regulation and H3K9me3 deposition, resulting in ectopic PHF7 protein expression. IDC's role is likely to be direct, as it localizes to a conserved domain within the phf7 gene. Collectively, our findings support a model in which IDC guides sequence-specific establishment of an H3K9me3 mini domain, thereby preventing accidental female-to-male programming.
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Project description:C2H2 zinc finger (C2H2-ZNF) genes are one of the largest and most complex gene super-families in metazoan genomes, with hundreds of members in the human and mouse genome. The ongoing investigation of this huge gene family requires computational support to catalog genotype phenotype comparisons of C2H2-ZNF genes between related species and finally to extend the worldwide knowledge on the evolution of C2H2-ZNF genes in general. Here, we systematically collected all the C2H2-ZNF genes in the human and mouse genome and constructed a database named SysZNF to deposit available datasets related to these genes. In the database, each C2H2-ZNF gene entry consists of physical location, gene model (including different transcript forms), Affymetrix gene expression probes, protein domain structures, homologs (and synteny between human and mouse), PubMed references as well as links to relevant public databases. The clustered organization of the C2H2-ZNF genes is highlighted. The database can be searched using text strings or sequence information. The data are also available for batch download from the web site. Moreover, the graphical gene model/protein view system, sequence retrieval system and some other tools embedded in SysZNF facilitate the research on the C2H2 type ZNF genes under an integrative view. The database can be accessed from the URL http://epgd.biosino.org/SysZNF.
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Project description:BackgroundA recent study by Tadepally et al. describes the clustering of zinc finger (ZF) genes in the human genome and traces their evolutionary history among several placental mammals with complete or draft genome sequences. One of the main conclusions from the paper is that there is a dramatic rate of gene duplication and gene loss, including the surprising result that 118 human ZF genes are absent in chimpanzee. The authors also present evidence concerning the ancestral order in which the ZF-associated KRAB and SCAN domains were recruited to ZF proteins.ResultsBased on our analysis of two of the largest human ZF gene clusters, we find that nearly all of the human genes have plausible orthologs in chimpanzee. The one exception may be a result of the incomplete sequence coverage in the draft chimpanzee genome. The discrepancy in gene content analysis may result from the authors' dependence on the preliminary NCBI gene prediction set for chimpanzee, which appears to either fail to predict or to mispredict many chimpanzee ZF genes. Similar problems may affect the authors' interpretation of the more divergent dog, mouse, and rat ZF gene complements. In addition, we present evidence that the KRAB domain was recruited to ZF genes before the SCAN domain, rather than the reverse as the authors suggest. This discrepancy appears to result from the fact that the SCAN domain did indeed arise before the KRAB domain but is present only in non-ZF genes until a much later date.ConclusionWhen comparing gene content among species, especially when using draft genome assemblies, dependence on preliminary gene prediction sets can be seriously misleading. In such studies, genic sequences must be identified in a manner that is as independent as possible of prediction sets. In addition, we present evidence that provides a more parsimonious explanation for the large proportion of mammalian KRAB-ZF genes without a SCAN domain.
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Project description:C2H2 zinc finger proteins represent the largest and most enigmatic class of human transcription factors. Their C2H2-ZF arrays are highly variable, indicating that most will have unique DNA binding motifs. However, most of the binding motifs have not been directly determined. In addition, little is known about whether or how these proteins regulate transcription. Most of the ∼700 human C2H2-ZF proteins also contain at least one KRAB, SCAN, BTB, or SET domain, suggesting that they may have common interacting partners and/or effector functions. Here, we report a multifaceted functional analysis of 131 human C2H2-ZF proteins, encompassing DNA binding sites, interacting proteins, and transcriptional response to genetic perturbation. We confirm the expected diversity in DNA binding motifs and genomic binding sites, and provide motif models for 78 previously uncharacterized C2H2-ZF proteins, most of which are unique. Surprisingly, the diversity in protein-protein interactions is nearly as high as diversity in DNA binding motifs: Most C2H2-ZF proteins interact with a unique spectrum of co-activators and co-repressors. Thus, multiparameter diversification likely underlies the evolutionary success of this large class of human proteins.
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Project description:A ZAS gene encodes a large protein with two separate C2H2 zinc finger pairs that independently bind to specific DNA sequences, including the kappaB motif. Three paralogous mammalian genes, ZAS1, ZAS2, and ZAS3, and a related Drosophila gene, Schnurri, have been cloned and characterized. The ZAS genes encode transcriptional proteins that activate or repress the transcription of a variety of genes involved in growth, development, and metastasis. In addition, ZAS3 associates with a TNF receptor-associated factor to inhibit NF-kappaB- and JNK/ SAPK-mediated signaling of TNF-alpha. Genetic experiments show that ZAS3 deficiency leads to proliferation of cells and tumor formation in mice. The data suggest that ZAS3 is important in controlling cell growth, apoptosis, and inflammation. The potent vasoactive hormone endothelin and transcription factor AP2 gene families also each consist of three members. The ZAS, endothelin, and transcription factor AP2 genes form several linkage groups. Knowledge of the chromosomal locations of these genes provides valuable clues to the evolution of the vertebrate genome.
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Project description:Cessation of transcriptional activity is a hallmark of cell division. Many biochemical pathways have been shown and proposed over the past few decades to explain the silence of this phase. In particular, many individual transcription factors have been shown to be inactivated by phosphorylation. In this report, we show the simultaneous phosphorylation and mitotic redistribution of a whole class of modified transcription factors. C(2)H(2) zinc finger proteins (ZFPs) represent the largest group of gene expression regulators in the human genome. Despite their diversity, C(2)H(2) ZFPs display striking conservation of small linker peptides joining their adjacent zinc finger modules. These linkers are critical for DNA binding activity. It has been proposed that conserved phosphorylation of these linker peptides could be a common mechanism for the inactivation of the DNA binding activity of C(2)H(2) ZFPs, during mitosis. Using a novel antibody, raised against the phosphorylated form of the most conserved linker peptide sequence, we are able to visualize the massive and simultaneous mitotic phosphorylation of hundreds of these proteins. We show that this wave of phosphorylation is tightly synchronized, starting in mid-prophase right after DNA condensation and before the breakdown of the nuclear envelope. This global phosphorylation is completely reversed in telophase. In addition, the exclusion of the phospho-linker signal from condensed DNA clearly demonstrates a common mechanism for the mitotic inactivation of C(2)H(2) ZFPs.
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Project description:RNA interference systems depend on the synthesis of small RNA precursors whose sequences define the target spectrum of these silencing pathways. The Drosophila Heterochromatin Protein 1 (HP1) variant Rhino permits transcription of PIWI-interacting RNA (piRNA) precursors within transposon-rich heterochromatic loci in germline cells. Current models propose that Rhino's specific chromatin occupancy at piRNA source loci is determined by histone marks and maternally inherited piRNAs, but also imply the existence of other, undiscovered specificity cues. Here, we identify a member of the diverse family of zinc finger associated domain (ZAD)-C2H2 zinc finger proteins, Kipferl, as critical Rhino cofactor in ovaries. By binding to guanosine-rich DNA motifs and interacting with the Rhino chromodomain, Kipferl recruits Rhino to specific loci and stabilizes it on chromatin. In kipferl mutant flies, Rhino is lost from most of its target chromatin loci and instead accumulates on pericentromeric Satellite arrays, resulting in decreased levels of transposon targeting piRNAs and impaired fertility. Our findings reveal that DNA sequence, in addition to the H3K9me3 mark, determines the identity of piRNA source loci and provide insight into how Rhino might be caught in the crossfire of genetic conflicts.
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Project description:Myocardial ischemic preconditioning upregulated protein 1 (Mipu1) is a newly discovered upregulated gene produced in rats during the myocardial ischemic preconditioning process. Mipu1 cDNA contains a 1824-base pair open reading frame and encodes a 608 amino acid protein with an N-terminal Krüppel-associated box (KRAB) domain and classical zinc finger C2H2 motifs in the C-terminus. Mipu1 protein is located in the cell nucleus. Recent studies found that Mipu1 has a protective effect on the ischemia-reperfusion injury of heart, brain, and other organs. As a nuclear factor, Mipu1 may perform its protective function through directly transcribing and repressing the expression of proapoptotic genes to repress cell apoptosis. In addition, Mipu1 also plays an important role in regulating the gene expression of downstream inflammatory mediators by inhibiting the activation of activator protein-1 and serum response element.
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Project description:We have identified protein protein interactions of 118 C2H2 zinc finger proteins by AP-MS using GFP-tagged constructs in order to understand their role in transcription regulation.
2016-12-09 | PXD003431 | Pride