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1.
PLoS One ; 15(7): e0236943, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32735612

RESUMO

Halophyte Lobularia maritima LmSAP encodes an A20AN1 zinc-finger stress-associated protein which expression is up-regulated by abiotic stresses and heavy metals in transgenic tobacco. To deepen our understanding of LmSAP function, we isolated a 1,147 bp genomic fragment upstream of LmSAP coding sequence designated as PrLmSAP. In silico analyses of PrLmSAP revealed the presence of consensus CAAT and TATA boxes and cis-regulatory elements required for abiotic stress, phytohormones, pathogen, and wound responses, and also for tissue-specific expression. The PrLmSAP sequence was fused to the ß-glucuronidase (gusA) reporter gene and transferred to rice. Histochemical GUS staining showed a pattern of tissue-specific expression in transgenic rice, with staining observed in roots, coleoptiles, leaves, stems and floral organs but not in seeds or in the root elongation zone. Wounding strongly stimulated GUS accumulation in leaves and stems. Interestingly, we observed a high stimulation of the promoter activity when rice seedlings were exposed to NaCl, PEG, ABA, MeJA, GA, cold, and heavy metals (Al3+, Cd2+, Cu2+ and Zn2+). These results suggest that the LmSAP promoter can be a convenient tool for stress-inducible gene expression and is a potential candidate for crop genetic engineering.


Assuntos
Regulação da Expressão Gênica de Plantas/genética , Regiões Promotoras Genéticas , Plantas Tolerantes a Sal/genética , Estresse Fisiológico/genética , Dedos de Zinco/genética , Produtos Agrícolas/genética , Engenharia Genética , Glucuronidase/metabolismo , Metais Pesados/metabolismo , Especificidade de Órgãos , Oryza/genética , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Caules de Planta/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Tabaco/genética
2.
Nat Commun ; 11(1): 4136, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811816

RESUMO

During cellular reprogramming, the pioneer transcription factor GATA3 binds chromatin, and in a context-dependent manner directs local chromatin remodeling and enhancer formation. Here, we use high-resolution nucleosome mapping in human cells to explore the impact of the position of GATA motifs on the surface of nucleosomes on productive enhancer formation, finding productivity correlates with binding sites located near the nucleosomal dyad axis. Biochemical experiments with model nucleosomes demonstrate sufficiently stable transcription factor-nucleosome interaction to empower cryo-electron microscopy structure determination of the complex at 3.15 Å resolution. The GATA3 zinc fingers efficiently bind their target 5'-GAT-3' sequences in the nucleosome when they are located in solvent accessible, consecutive major grooves without significant changes in nucleosome structure. Analysis of genomic loci bound by GATA3 during reprogramming suggests a correlation of recognition motif sequence and spacing that may distinguish productivity of new enhancer formation.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Fator de Transcrição GATA3/química , Nucleossomos/química , Nucleossomos/genética , Motivos de Aminoácidos/genética , Sítios de Ligação , Sequenciamento de Cromatina por Imunoprecipitação , Microscopia Crioeletrônica , Elementos Facilitadores Genéticos , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Fator de Transcrição GATA3/ultraestrutura , Histonas/metabolismo , Humanos , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Ligação Proteica , Dedos de Zinco/genética
3.
Proc Natl Acad Sci U S A ; 117(29): 17151-17155, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32636268

RESUMO

Inherited bone marrow failure (BMF) syndromes are a heterogeneous group of diseases characterized by defective hematopoiesis and often predisposing to myelodysplastic syndrome (MDS) and acute myelogenous leukemia. We have studied a large family consisting of several affected individuals with hematologic abnormalities, including one family member who died of acute leukemia. By whole-exome sequencing, we identified a novel frameshift variant in the ubiquitously expressed transcription factor specificity protein 1 (SP1). This heterozygous variant (c.1995delA) truncates the canonical Sp1 molecule in the highly conserved C-terminal DNA-binding zinc finger domains. Transcriptomic analysis and gene promoter characterization in patients' blood revealed a hypermorphic effect of this Sp1 variant, triggering superactivation of Sp1-mediated transcription and driving significant up-regulation of Sp1 target genes. This familial genetic study indicates a central role for Sp1 in causing autosomal dominant transmission of BMF, thereby confirming its critical role in hematopoiesis in humans.


Assuntos
Transtornos da Insuficiência da Medula Óssea/genética , Mutação da Fase de Leitura/genética , Fator de Transcrição Sp1/genética , Transcrição Genética/genética , Feminino , Humanos , Masculino , Linhagem , Transcriptoma/genética , Regulação para Cima/genética , Dedos de Zinco/genética
4.
PLoS One ; 15(6): e0235317, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32598401

RESUMO

The Dof (DNA-binding one zinc finger) transcription factor family is a representative of plant-specific classes of transcription factors. In this study, we performed a genome-wide screening and characterization of the Dof gene family within two tetraploid species Gossypium barbadense, Gossypium hirsutum, and two diploid species Gossypium arboreum, Gossypium raimondii. 115, 116, 55 and 56 Dof genes were identified respectively and all of the genes contain a sequence encoding the Dof DNA-binding domain. Those genes were unevenly distributed across 13/26 chromosomes of the cotton. Genome comparison revealed that segmental duplication may have played crucial roles in the expansion of the cotton Dof gene family, and tandem duplication also played a minor role. Analysis of RNA-Seq data indicated that cotton Dof gene expression levels varied across different tissues and in response to different abiotic stress. Overall, our results could provide valuable information for better understanding the evolution of cotton Dof genes, and lays a foundation for future investigation in cotton.


Assuntos
Proteínas de Ligação a DNA/genética , Diploide , Genoma de Planta , Gossypium/genética , Proteínas de Plantas/genética , Tetraploidia , Dedos de Zinco/genética , Cromossomos de Plantas/genética , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Gossypium/crescimento & desenvolvimento , Gossypium/metabolismo , Família Multigênica , Filogenia , Proteínas de Plantas/metabolismo
5.
Nucleic Acids Res ; 48(11): 6382-6402, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32383734

RESUMO

The Cys2His2 zinc finger is the most common DNA-binding domain expanding in metazoans since the fungi human split. A proposed catalyst for this expansion is an arms race to silence transposable elements yet it remains poorly understood how this domain is able to evolve the required specificities. Likewise, models of its DNA binding specificity remain error prone due to a lack of understanding of how adjacent fingers influence each other's binding specificity. Here, we use a synthetic approach to exhaustively investigate binding geometry, one of the dominant influences on adjacent finger function. By screening over 28 billion protein-DNA interactions in various geometric contexts we find the plasticity of the most common natural geometry enables more functional amino acid combinations across all targets. Further, residues that define this geometry are enriched in genomes where zinc fingers are prevalent and specificity transitions would be limited in alternative geometries. Finally, these results demonstrate an exhaustive synthetic screen can produce an accurate model of domain function while providing mechanistic insight that may have assisted in the domains expansion.


Assuntos
Modelos Moleculares , Domínios Proteicos/fisiologia , Dedos de Zinco/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA/síntese química , DNA/genética , DNA/metabolismo , Aprendizado Profundo , Humanos , Ligação de Hidrogênio , Domínios Proteicos/genética , Reprodutibilidade dos Testes , Especificidade por Substrato/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Dedos de Zinco/genética
6.
Cell Mol Life Sci ; 77(20): 3991-4014, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32303790

RESUMO

The diverse eukaryotic proteins that contain zinc fingers participate in many aspects of nucleic acid metabolism, from DNA transcription to RNA degradation, post-transcriptional gene silencing, and small RNA biogenesis. These proteins can be classified into at least 30 types based on structure. In this review, we focus on the CCHC-type zinc fingers (ZCCHC), which contain an 18-residue domain with the CX2CX4HX4C sequence, where C is cysteine, H is histidine, and X is any amino acid. This motif, also named the "zinc knuckle", is characteristic of the retroviral Group Antigen protein and occurs alone or with other motifs. Many proteins containing zinc knuckles have been identified in eukaryotes, but only a few have been studied. Here, we review the available information on ZCCHC-containing factors from three evolutionarily distant eukaryotes-Saccharomyces cerevisiae, Arabidopsis thaliana, and Homo sapiens-representing fungi, plants, and metazoans, respectively. We performed systematic searches for proteins containing the CX2CX4HX4C sequence in organism-specific and generalist databases. Next, we analyzed the structural and functional information for all such proteins stored in UniProtKB. Excluding retrotransposon-encoded proteins and proteins harboring uncertain ZCCHC motifs, we found seven ZCCHC-containing proteins in yeast, 69 in Arabidopsis, and 34 in humans. ZCCHC-containing proteins mainly localize to the nucleus, but some are nuclear and cytoplasmic, or exclusively cytoplasmic, and one localizes to the chloroplast. Most of these factors participate in RNA metabolism, including transcriptional elongation, polyadenylation, translation, pre-messenger RNA splicing, RNA export, RNA degradation, microRNA and ribosomal RNA biogenesis, and post-transcriptional gene silencing. Several human ZCCHC-containing factors are derived from neofunctionalized retrotransposons and act as proto-oncogenes in diverse neoplastic processes. The conservation of ZCCHCs in orthologs of these three phylogenetically distant eukaryotes suggests that these domains have biologically relevant functions that are not well known at present.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas Fúngicas/genética , Saccharomyces cerevisiae/genética , Dedos de Zinco/genética , Animais , Proteínas de Ligação a DNA/genética , Estudo de Associação Genômica Ampla/métodos , Humanos
7.
Gene ; 745: 144651, 2020 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-32259633

RESUMO

Sexual differentiation and ovotestis development are closely associated with cortisol levels, the principal indicator of stress, via the glucocorticoid receptor (GR) in teleosts. Thus, GR is regarded as a mediator to expound the relationship between social stress and gonad development. In the present study, two gr genes (gr1 and gr2) were cloned and analyzed from a protandrous hermaphroditic teleost, the yellowtail clownfish (Amphiprion clarkii). GR1 was found to display a conserved nine-amino-acid insert, WRARQNTDG, between two zinc finger domains. The phylogenetic tree of GR showed that yellowtail clownfish GR1 and GR2 are clustered to teleost GR1 and teleost GR2 separately, and differ from tetrapod GR. The result of real-time PCR revealed that high-level gr1 was mainly distributed in the cerebellum, hypothalamus and heart. The gr2 gene was abundant in the pituitary and liver of females and nonbreeders, while gr2 was mainly detected in the medulla oblongata and middle kidney of males. Moreover, GRs can be expressed in cultured eukaryotic cells and functionally interact with dexamethasone (exogenous glucocorticoid), thereby triggering downstream signaling pathways of different potentials. GR1 and GR2 can be activated by 10 nM dexamethasone treatment in HEK-293T cells. Notably, real-time PCR analysis among three social status groups demonstrated that gr2 expression was the highest in the hypothalamus of nonbreeders, but gr1 was no difference. We speculate that social stress would increase the expression of gr2 gene expression in the hypothalamus to inhibit sexual development. These data provide evidence of social stress involving reproductive regulation, which may help to elucidate the underlying mechanism of sex differentiation and change.


Assuntos
Proteínas de Peixes/genética , Regulação da Expressão Gênica no Desenvolvimento , Perciformes/genética , Receptores de Glucocorticoides/genética , Diferenciação Sexual/genética , Animais , Dexametasona/farmacologia , Feminino , Proteínas de Peixes/metabolismo , Células HEK293 , Humanos , Masculino , Família Multigênica , Perciformes/metabolismo , Filogenia , Receptores de Glucocorticoides/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reprodução/genética , Transdução de Sinais/efeitos dos fármacos , Estresse Psicológico/genética , Dedos de Zinco/genética
8.
PLoS One ; 15(3): e0230177, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32182273

RESUMO

Jasmonates (JAs) are key phytohormones involved in regulation of plant growth and development, stress responses, and secondary metabolism. It has been reported that treatments with JAs could increase the contents of Amaryllidaceae alkaloids in Amaryllidaceae plants. Jasmonate ZIM (zinc-finger inflorescence meristem) domain (JAZ) proteins are key components in JA signal processes. However, JAZ proteins have not been characterized in genus Lycoris. In this study, we identified and cloned seven differentially expressed JAZ genes (namely LaJAZ1-LaJAZ7) from Lycoris aurea. Bioinformatic analyses revealed that these seven LaJAZ proteins contain the ZIM domain and JA-associated (Jas, also named CCT_2) motif. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed that these LaJAZ genes display different expression patterns in L. aurea tissues, and most of them are inducible when treated with methyl jasmonate (MeJA) treatment. Subcellular localization assay demonstrated that LaJAZ proteins are localized in the cell nucleus or cytoplasm. In addition, LaJAZ proteins could interact with each other to form homodimer and/or heterodimer. The findings in this study may facilitate further functional research of the LaJAZ genes, especially the potential regulatory mechanism of plant secondary metabolites including Amaryllidaceae alkaloids in L. aurea.


Assuntos
Regulação da Expressão Gênica de Plantas/genética , Lycoris/genética , Proteínas de Plantas/genética , Dedos de Zinco/genética , Acetatos/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Biologia Computacional/métodos , Ciclopentanos/farmacologia , Citoplasma/efeitos dos fármacos , Citoplasma/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Lycoris/efeitos dos fármacos , Oxilipinas/farmacologia , Reguladores de Crescimento de Planta/farmacologia , Domínios Proteicos/genética
9.
Nat Commun ; 11(1): 1478, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32198374

RESUMO

The Escherichia coli transcription-repair coupling factor Mfd displaces stalled RNA polymerase and delivers the stall site to the nucleotide excision repair factors UvrAB for damage detection. Whether this handoff from RNA polymerase to UvrA occurs via the Mfd-UvrA2-UvrB complex or alternate reaction intermediates in cells remains unclear. Here, we visualise Mfd in actively growing cells and determine the catalytic requirements for faithful recruitment of nucleotide excision repair proteins. We find that ATP hydrolysis by UvrA governs formation and disassembly of the Mfd-UvrA2 complex. Further, Mfd-UvrA2-UvrB complexes formed by UvrB mutants deficient in DNA loading and damage recognition are impaired in successful handoff. Our single-molecule dissection of interactions of Mfd with its partner proteins inside live cells shows that the dissociation of Mfd is tightly coupled to successful loading of UvrB, providing a mechanism via which loading of UvrB occurs in a strand-specific manner.


Assuntos
Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA/fisiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Imagem Individual de Molécula/métodos , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases , Proteínas de Bactérias , ATPases Bacterianas Próton-Translocadoras , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Complexos Multienzimáticos/metabolismo , Conformação Proteica , Dedos de Zinco/genética , Dedos de Zinco/fisiologia
10.
Nat Commun ; 11(1): 779, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034124

RESUMO

Engineering mammalian cells to carry out sophisticated and customizable genetic programs requires a toolkit of multiple orthogonal and well-characterized transcription factors (TFs). To address this need, we develop the COmposable Mammalian Elements of Transcription (COMET)-an ensemble of TFs and promoters that enable the design and tuning of gene expression to an extent not, to the best of our knowledge, previously possible. COMET currently comprises 44 activating and 12 inhibitory zinc-finger TFs and 83 cognate promoters, combined in a framework that readily accommodates new parts. This system can tune gene expression over three orders of magnitude, provides chemically inducible control of TF activity, and enables single-layer Boolean logic. We also develop a mathematical model that provides mechanistic insights into COMET performance characteristics. Altogether, COMET enables the design and construction of customizable genetic programs in mammalian cells.


Assuntos
Engenharia Genética/métodos , Mamíferos/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Animais , Linhagem Celular , Células HEK293 , Humanos , Plasmídeos/genética , Engenharia de Proteínas/métodos , Bibliotecas de Moléculas Pequenas/farmacologia , Transcrição Genética , Dedos de Zinco/genética
11.
Genet Test Mol Biomarkers ; 24(3): 145-149, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32105524

RESUMO

Introduction: The zinc finger homeobox 4 (ZFHX4) protein is a crucial molecular regulator of tumor-initiating stem cell-like functions. Objective: This study aimed to determine the role of ZFHX4 in the progression of ovarian serous cystadenocarcinoma (OSC). Methods: Differential gene expression ZFHX4 among low-stage (stages I and II), high-stage (stages III and IV), low-grade (grades I and II), and high-grade (grades III and IV) OSC patients was identified using four independent cohorts from the Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO). We compared ZFHX4 expression as a prognostic factor using Kaplan-Meier survival curves, multivariate analysis, the time-dependent area under the curve (AUC) of Uno's C-index, and the AUC of the receiver operating characteristics at 4 years post diagnosis. Results: ZFHX4 gene expression in high-stage tumors is significantly higher than in low-stage tumors (TCGA, p = 0.007; GSE9891, p = 0.001). A Kaplan-Meier analysis revealed that elevated expression of ZFHX4 was associated with a poor prognosis in OSC patients for all cohorts, regardless of stage and grade (TCGA, p = 1e-04; GSE9891, p = 0.0044; GSE13876, p = 0.00078; GSE26712, p = 0.039). Analysis of C-indices and the area under the receiver operating characteristic curve further supported this result (C-index: TCGA, 0.599; GSE9891, 0.642; GSE13876, 0.585; GSE26712, 0.597). Moreover, univariate and multivariate Cox hazards analyses confirmed the prognostic significance of ZFHX4 levels. Conclusion: Collectively, these findings suggest that ZFHX4 is a prognostic factor for OSC.


Assuntos
Cistadenocarcinoma Seroso/genética , Proteínas de Homeodomínio/genética , Neoplasias Ovarianas/metabolismo , Fatores de Transcrição/genética , Adulto , Idoso , Biomarcadores Tumorais/genética , Carcinoma Epitelial do Ovário/genética , China , Cistadenocarcinoma Seroso/metabolismo , Feminino , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Genes Homeobox/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Neoplasias Ovarianas/genética , Prognóstico , Curva ROC , Fatores de Transcrição/metabolismo , Transcriptoma/genética , Dedos de Zinco/genética
12.
Parasitol Res ; 119(2): 623-635, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31758298

RESUMO

Coccidiosis is caused by multiple species of the apicomplexan protozoa Eimeria. Among them, Eimeria tenella is frequently considered to be the most pathogenic. Zinc finger proteins (ZnFPs) are a type of protein containing zinc finger domains. In the present study, a putative Eimeria tenella AN1-like ZnFP (E. tenella AN1-like zinc finger domain-containing protein, putative partial mRNA, EtAN1-ZnFP) was cloned and characterized, and its immune protective effects were evaluated. The 798-bp ORF sequence of EtAN1-ZnFP that encoded a protein of approximately 27.0 kDa was obtained. The recombinant EtAN1-ZnFP protein (rEtAN1-ZnFP) was expressed in Escherichia coli. Western blot analysis showed that the recombinant protein was recognized by the anti-GST monoclonal antibody and anti-sporozoite protein rabbit serum. qPCR analysis revealed that EtAN1-ZnFP was highly expressed in unsporulated oocysts and sporozoites. Immunostaining with an anti-rEtAN1-ZnFP antibody indicated that EtAN1-ZnFP was uniformly distributed in the cytoplasm of sporozoites, except for the refractive body; furthermore, this protein was evenly distributed in the cytoplasm of immature schizonts but seldom distributed in mature schizonts. The results of the in vitro invasion inhibition assay indicated that the antibodies against rEtAN1-ZnFP efficiently reduced the ability of E. tenella sporozoites to invade host cells. Animal challenge experiments demonstrated that the chickens immunized with rEtAN1-ZnFP protein significantly decreased mean lesion scores and fecal oocyst output compared with challenged control group. The results suggest that EtAN1-ZnFP can induce partial immune protection against infection with E. tenella and could be an effective candidate for the development of new vaccines.


Assuntos
Galinhas , Eimeria tenella/genética , Doenças das Aves Domésticas/parasitologia , Proteínas de Protozoários/genética , Vacinas Protozoárias/genética , Dedos de Zinco/genética , Animais , Western Blotting , Clonagem Molecular , Coccidiose/parasitologia , Coccidiose/veterinária , Eimeria tenella/imunologia , Oocistos/metabolismo , Doenças das Aves Domésticas/imunologia , Proteínas de Protozoários/imunologia , Vacinas Protozoárias/imunologia , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Esporozoítos/imunologia
13.
Int J Mol Sci ; 20(23)2019 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-31783611

RESUMO

Retrotransposons can alter the regulation of genes both transcriptionally and post-transcriptionally, through mechanisms such as binding transcription factors and alternative splicing of transcripts. SINE-VNTR-Alu (SVA) retrotransposons are the most recently evolved class of retrotransposable elements, found solely in primates, including humans. SVAs are preferentially found at genic, high GC loci, and have been termed "mobile CpG islands". We hypothesise that the ability of SVAs to mobilise, and their non-random distribution across the genome, may result in differential regulation of certain pathways. We analysed SVA distribution patterns across the human reference genome and identified over-representation of SVAs at zinc finger gene clusters. Zinc finger proteins are able to bind to and repress SVA function through transcriptional and epigenetic mechanisms, and the interplay between SVAs and zinc fingers has been proposed as a major feature of genome evolution. We describe observations relating to the clustering patterns of both reference SVAs and polymorphic SVA insertions at zinc finger gene loci, suggesting that the evolution of this network may be ongoing in humans. Further, we propose a mechanism to direct future research and validation efforts, in which the interplay between zinc fingers and their epigenetic modulation of SVAs may regulate a network of zinc finger genes, with the potential for wider transcriptional consequences.


Assuntos
Elementos Alu/genética , Genoma Humano/genética , Repetições Minissatélites/genética , Retroelementos/genética , Epigênese Genética/genética , Evolução Molecular , Humanos , Transcrição Genética/genética , Dedos de Zinco/genética
14.
BMC Res Notes ; 12(1): 792, 2019 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-31801592

RESUMO

OBJECTIVE: Type C2H2 zinc fingers bind a variety of substrates, specific sequences in the double-stranded DNA counting among them. Engineering efforts led to the discovery of a set of general rules that enable obtaining zinc fingers modules that bind to almost any given sequence. The objective of this work was to determine an analogical set of rules for the binding of specific sequences in DNA-RNA hybrids using directed evolution of ZfQQR zinc finger. The target regions for evolution included the amino acid residues that directly interact with the substrate and linkers between the zinc finger modules. RESULTS: The directed evolution was performed using selection based on biopanning of phage-displayed libraries of randomized regions in the ZfQQR zinc finger. The applied strategy of randomization of the middle module of the zinc finger along with input library bias and materials used for biopanning hindered the selection of the modules with altered specificity. However, the directed evolution of the linker sequence between modules enabled selection of variants with improved selectivity towards DNA-RNA hybrids in the presence of double-stranded DNA in comparison to the original ZfQQR. This confirms the necessity of linker optimization between modules in zinc finger domains.


Assuntos
DNA/metabolismo , Evolução Molecular Direcionada , RNA/metabolismo , Dedos de Zinco/genética , Biblioteca Gênica , Ligação Proteica , Seleção Genética
15.
Stem Cell Res Ther ; 10(1): 380, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31842989

RESUMO

BACKGROUND: Spinal cord injury (SCI) results in glial scar formation and irreversible neuronal loss, which finally leads to functional impairments and long-term disability. Our previous studies have demonstrated that the ectopic expression of Zfp521 reprograms fibroblasts and astrocytes into induced neural stem cells (iNSCs). However, it remains unclear whether treatment with Zfp521 also affects endogenous astrocytes, thus promoting further functional recovery following SCI. METHODS: Rat astrocytes were transdifferentiated into neural stem cells in vitro by ZFP521 or Sox2. Then, ZFP521 was applied to the spinal cord injury site of a rat. Transduction, real-time PCR, immunohistofluorescence, and function assessments were performed at 6 weeks post-transduction to evaluate improvement and in vivo lineage reprogramming of astrocytes. RESULTS: Here, we show that Zfp521 is more efficient in reprogramming cultured astrocytes compared with Sox2. In the injured spinal cord of an adult rat, resident astrocytes can be reprogrammed into neurons through a progenitor stage by Zfp521. Importantly, this treatment improves the functional abilities of the rats as evaluated by the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale and further by calculation of its subscores. There was enhanced locomotor activity in the hind limbs, step length, toe spread, foot length, and paw area. In addition, motor evoked potential recordings demonstrated the functional integrity of the spinal cord. CONCLUSIONS: These results have indicated that the generation of iNSCs or neurons from endogenous astrocytes by in situ reprogramming might be a potential strategy for SCI repair.


Assuntos
Astrócitos/metabolismo , Regulação da Expressão Gênica/genética , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Traumatismos da Medula Espinal/genética , Fatores de Transcrição/genética , Dedos de Zinco/genética , Animais , Modelos Animais de Doenças , Masculino , Ratos , Ratos Sprague-Dawley
16.
Reprod Biol Endocrinol ; 17(1): 98, 2019 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-31759386

RESUMO

Ovol2, a mouse homolog of Drosophila ovo, was identified as a zinc finger transcription factor predominantly expressed in testis. However, the function of Ovol2 in postnatal male germ cell development remains enigmatic. Here, we firstly examined the mRNA and protein levels of Ovol2 in developing mouse testes by RT-qPCR and western blot and found that both mRNA and protein of Ovol2 are continually expressed in postnatal developing testes from postnatal day 0 (P0) testes to adult testes (P56) and exhibits its higher level at adult testis. Further testicular immuno-staining revealed that OVOL2 is highly expressed in the spermatogonia, spermatocytes and round spermatids. Interestingly, our conditional ovol2 knockout mouse model show that loss of ovol2 in embryonic germ cells does not affect fecundity in mice. Our data also show that Ovol1 may have compensated for the loss of Ovol2 functions in germ cells. Overall, our data indicate that ovol2 is dispensable for germ cell development and spermatogenesis.


Assuntos
Espermatogênese/genética , Testículo/metabolismo , Fatores de Transcrição/genética , Dedos de Zinco/genética , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fertilidade/genética , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos Knockout , Espermátides/citologia , Espermátides/metabolismo , Espermatócitos/citologia , Espermatócitos/metabolismo , Espermatogônias/citologia , Espermatogônias/metabolismo , Testículo/citologia , Testículo/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo
17.
PLoS One ; 14(11): e0224704, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31689304

RESUMO

SQUAMOSA promoter-binding protein-like (SPL), as plant specific transcription factors, is involved in many plant growth and development processes. However, there is less systematical study for SPL transcription factor in B. juncea (Cruciferae). Here, a total of 59 SPL genes classified into eight phylogenetic groups were identified in B. juncea, highly conserved within each ortholog were also found based on gene structure, conserved motif, as well as clustering level. In addition, clustering of SPL domain showed that two zinc finger-like structures and NLS segments were identified in almost of BjuSPLs. Analyzed of putative cis-elements for BjuSPLs demonstrated that SPL transcription factors were involved in adverse environmental changes, such as light, plant stresses and phytohormones response. Expression analysis showed that differentially expressed SPL genes were identified in flower and stem development of Cruciferae; such as BjuSPL3a-B, BjuSPL2b_B and BjuSPL2c_A were significantly expressed in flower; BjuSPL 3b_B and BjuSPL10a_A were significantly expressed in stem node (VP: vegetative period). Moreover, 28 of the 59 BjuSPLs were found involved in their posttranscriptional regulation targeted by miR156. We demonstrated that miR156 negatively regulated BjuSPL10a_A and BjuSPL3b_B to act for stem development in B. juncea.


Assuntos
Regulação da Expressão Gênica de Plantas , Família Multigênica/genética , Mostardeira/genética , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Genoma de Planta/genética , MicroRNAs/metabolismo , Mostardeira/crescimento & desenvolvimento , Mostardeira/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Dedos de Zinco/genética
18.
Mol Cancer ; 18(1): 133, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31481066

RESUMO

BACKGROUND: Circular RNAs (circRNAs) represent a subclass of regulatory RNAs that have been shown to have significant regulatory roles in cancer progression. However, the biological functions of circRNAs in bladder cancer (BCa) are largely unknown. METHODS: Cell invasion models were established, and invasion-related circRNAs were detected by qPCR. Using above method, circ-ZKSCAN1 was picked out for further study. Circ-ZKSCAN1 expression and survival analyses were performed through qPCR. The survival curves were generated by the Kaplan-Meier method, and the log-rank test was used to assess the significance. Cell proliferation, migration and invasion were examined to investigate the function of circ-ZKSCAN1. Tumorigenesis in nude mice was assessed to determine the effect of circ-ZKSCAN1 in bladder cancer. Biotin-coupled probe pull-down assays, FISH and luciferase reporter assays were conducted to confirm the relationship between circ-ZKSCAN1 and microRNA. RNA-seq revealed different molecular changes in downstream genes. RESULTS: Here, we found that circ-ZKSCAN1 was downregulated in BCa tissues and cell lines. Circ-ZKSCAN1 levels were associated with survival, tumor grade, pathological T stage and tumor recurrence. Overexpressed circ-ZKSCAN1 inhibits cell proliferation, migration, invasion and metastasis in vitro and in vivo. Mechanistically, we demonstrated that circ-ZKSCAN1 upregulated p21 expression by sponging miR-1178-3p, which suppressed the aggressive biological behaviors in bladder cancer. CONCLUSIONS: These results reveal that Circ-ZKSCAN1 acts as a tumor suppressor via a novel circ-ZKSCAN1/miR-1178-3p/p21 axis, which have the important role in the proliferation, migration and invasion ablitities of BCa cells and provide a novel perspective on circRNAs in BCa progression.


Assuntos
MicroRNAs/genética , RNA Circular , Neoplasias da Bexiga Urinária/genética , Dedos de Zinco/genética , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores Tumorais , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Interferência de RNA , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Bexiga Urinária/mortalidade
19.
Nucleic Acids Res ; 47(19): 10327-10339, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31504764

RESUMO

The essential splicing factor Cwc24 contains a zinc-finger (ZF) domain required for its function in splicing. Cwc24 binds over the 5' splice site after the spliceosome is activated, and its binding prior to Prp2-mediated spliceosome remodeling is important for proper interactions of U5 and U6 with the 5' splice site sequence and selection of the 5' splice site. Here, we show that Cwc24 transiently interacts with the 5' splice site in formation of the functional RNA catalytic core during spliceosome remodeling, and the ZF-motif is required for specific interaction of Cwc24 with the 5' splice site. Deletion of the ZF domain or mutation of the conserved ZF residues greatly weakened the association of Cwc24 with the spliceosome, and lowered the affinity and specificity of its interaction with the 5' splice site, resulting in atypical interactions of U5, U6 and Prp8 with the 5' splice site, and aberrant cleavage at the 5' splice site. Our results reveal a crucial role of the Cwc24 ZF-motif for defining 5' splice site selection in the first splicing step.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas com Motivo de Reconhecimento de RNA/genética , Fatores de Processamento de RNA/genética , Processamento de RNA/genética , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Ribonucleoproteína Nuclear Pequena U5/genética , Proteínas de Saccharomyces cerevisiae/genética , Spliceossomos/genética , Sequência de Bases/genética , Domínio Catalítico/genética , Humanos , Íntrons/genética , Mutação/genética , Sítios de Splice de RNA/genética , RNA Nuclear Pequeno/genética , Saccharomyces cerevisiae/genética , Dedos de Zinco/genética
20.
Nat Neurosci ; 22(9): 1413-1423, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31427770

RESUMO

Understanding the transcriptional changes that are engaged in stress resilience may reveal novel antidepressant targets. Here we use gene co-expression analysis of RNA-sequencing data from brains of resilient mice to identify a gene network that is unique to resilience. Zfp189, which encodes a previously unstudied zinc finger protein, is the highest-ranked key driver gene in the network, and overexpression of Zfp189 in prefrontal cortical neurons preferentially activates this network and promotes behavioral resilience. The transcription factor CREB is a predicted upstream regulator of this network and binds to the Zfp189 promoter. To probe CREB-Zfp189 interactions, we employ CRISPR-mediated locus-specific transcriptional reprogramming to direct CREB or G9a (a repressive histone methyltransferase) to the Zfp189 promoter in prefrontal cortex neurons. Induction of Zfp189 with site-specific CREB is pro-resilient, whereas suppressing Zfp189 expression with G9a increases susceptibility. These findings reveal an essential role for Zfp189 and CREB-Zfp189 interactions in mediating a central transcriptional network of resilience.


Assuntos
Adaptação Psicológica/fisiologia , Estresse Psicológico/genética , Dedos de Zinco/genética , Animais , Redes Reguladoras de Genes/genética , Camundongos , Camundongos Endogâmicos C57BL , Córtex Pré-Frontal/metabolismo , Transcrição Genética
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