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Easy Expression and Purification of Fluorescent N-Terminal BCL11B CCHC Zinc Finger Domain.

ABSTRACT: Zinc finger proteins play pivotal roles in health and disease and exert critical functions in various cellular processes. A majority of zinc finger proteins bind DNA and act as transcription factors. B-cell lymphoma/leukemia 11B (BCL11B) represents one member of the large family of zinc finger proteins. The N-terminal domain of BCL11B was shown to be crucial for BCL11B to exert its proper function by homodimerization. Here, we describe an easy and fast preparation protocol to yield the fluorescently tagged protein of the recombinant N-terminal BCL11B zinc finger domain (BCL11B_42-94) for in vitro studies. First, we expressed fluorescently tagged BCL11B_42-94 in E. coli and described the subsequent purification utilizing immobilized metal ion affinity chromatography to achieve very high yields of a purified fusion protein of 200 mg/L culture. We proceeded with characterizing the atypical zinc finger domain using circular dichroism and size exclusion chromatography. Validation of the functional fluorescent pair CyPet-/EYFP-BCL11B_42-94 was achieved with Förster resonance energy transfer. Our protocol can be utilized to study other zinc finger domains to expand the knowledge in this field.

SUBMITTER: Susemihl A

PROVIDER: S-EPMC8708588 | biostudies-literature |

REPOSITORIES: biostudies-literature

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The N-Terminal CCHC Zinc Finger Motif Mediates Homodimerization of Transcription Factor BCL11B.

Project description:The BCL11B gene encodes a Krüppel-like, sequence-specific zinc finger (ZF) transcription factor that acts as either a repressor or an activator, depending on its posttranslational modifications. The importance of BCL11B in numerous biological processes in multiple organs has been well established in mouse knockout models. The phenotype of the first de novo monoallelic germ line missense mutation in the BCL11B gene (encoding N441K) strongly implies that the mutant protein acts in a dominant-negative manner by neutralizing the unaffected protein through the formation of a nonfunctional dimer. Using a Förster resonance energy transfer-assisted fluorescence-activated cell sorting (FACS-FRET) assay and affinity purification followed by mass spectrometry (AP-MS), we show that the N-terminal CCHC zinc finger motif is necessary and sufficient for the formation of the BCL11B dimer. Mutation of the CCHC ZF in BCL11B abolishes its transcription-regulatory activity. In addition, unlike wild-type BCL11B, this mutant is incapable of inducing cell cycle arrest and protecting against DNA damage-driven apoptosis. Our results confirm the BCL11B dimerization hypothesis and prove its importance for BCL11B function. By mapping the relevant regions to the CCHC domain, we describe a previously unidentified mechanism of transcription factor homodimerization.

| S-EPMC5809685 | biostudies-literature

BCL11B_AP-MS - The N-terminal CCHC zinc finger motif mediates homodimerization of the transcription factor BCL11B

Project description:This study investigates whether the N-terminal CCHC zinc finger motif is necessary and sufficient for the formation of BCL11B dimers, using fluorescence microscopy and Förster resonance energy transfer-assisted fluorescence-activated cell sorting (FACSFRET). The role of the CCHC motif in homodimer assembly was further investigated using affinity purification followed by mass spectrometry (AP-MS). Functional studies investigated the role of dimerization in the induction of cell cycle arrest and protection against DNA damage-induced apoptosis.

2018-01-12 | PXD008606 | Pride

Target specificity of human immunodeficiency virus type 1 NCp7 requires an intact conformation of its CCHC N-terminal zinc finger.

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Genome-Wide Identification and Functional Characterization of CCHC-Type Zinc Finger Genes in Ustilaginoidea virens.

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| S-EPMC8619310 | biostudies-literature

Comprehensive Genome-Wide Identification, Characterization, and Expression Analysis of CCHC-Type Zinc Finger Gene Family in Wheat (Triticum aestivum L.).

Project description:The CCHC-type zinc finger proteins (CCHC-ZFPs) play versatile roles in plant growth, development and adaptation to the environment. However, little is known about functions of CCHC-ZFP gene family memebers in Triticum aestivum. In the present study, we identified a total of 50 TaCCHC-ZFP genes from the 21 wheat chromosomes, which were phylogenetically classified into eight groups based on their specific motifs and gene structures. The 43 segmentally duplicated TaCCHC-ZFP genes were retrieved, which formed 36 segmental duplication gene pairs. The collinearity analyses among wheat and other eight mono/dicots revealed that no gene pairs were found between wheat and the three dicots. The promoter analyses of the TaCCHC-ZFP genes showed that 636 environmental stress-responsive and phytohormone-responsive cis-elements. The gene ontology enrichment analysis indicated that all the TaCCHC-ZFP genes were annotated under nucleic acid binding and metal ion binding. A total of 91 MicroRNA (miRNA) binding sites were identified in 34 TaCCHC-ZFP genes according to the miRNA target analysis. Based on the public transcriptome data, the 38 TaCCHC-ZFP genes were identified as differentially expressed gene. The expression profiles of 15 TaCCHC-ZFP genes were verified by the quantitative real-time PCR assays, and the results showed that these genes were responsive to drought or heat treatments. Our work systematically investigated the gene structures, evolutionary features, and potential functions of TaCCHC-ZFP genes. It lays a foundation for further research and application of TaCCHC-ZFP genes in genetic improvement of T. aestivum.

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The N-terminal zinc finger domain of Tgf2 transposase contributes to DNA binding and to transposition activity.

Project description:Active Hobo/Activator/Tam3 (hAT) transposable elements are rarely found in vertebrates. Previously, goldfish Tgf2 was found to be an autonomously active vertebrate transposon that is efficient at gene-transfer in teleost fish. However, little is known about Tgf2 functional domains required for transposition. To explore this, we first predicted in silico a zinc finger domain in the N-terminus of full length Tgf2 transposase (L-Tgf2TPase). Two truncated recombinant Tgf2 transposases with deletions in the N-terminal zinc finger domain, S1- and S2-Tgf2TPase, were expressed in bacteria from goldfish cDNAs. Both truncated Tgf2TPases lost their DNA-binding ability in vitro, specifically at the ends of Tgf2 transposon than native L-Tgf2TPase. Consequently, S1- and S2-Tgf2TPases mediated gene transfer in the zebrafish genome in vivo at a significantly (p < 0.01) lower efficiency (21%-25%), in comparison with L-Tgf2TPase (56% efficiency). Compared to L-Tgf2TPase, truncated Tgf2TPases catalyzed imprecise excisions with partial deletion of TE ends and/or plasmid backbone insertion/deletion. The gene integration into the zebrafish genome mediated by truncated Tgf2TPases was imperfect, creating incomplete 8-bp target site duplications at the insertion sites. These results indicate that the zinc finger domain in Tgf2 transposase is involved in binding to Tgf2 terminal sequences, and loss of those domains has effects on TE transposition.

| S-EPMC4890040 | biostudies-literature

Expression, purification and DNA-binding properties of zinc finger domains of DOF proteins from Arabidopsis thaliana.

Project description:Introduction: DOF proteins are a family of plant-specific transcription factors with a conserved zinc finger (ZF) DNA-binding domain. Although several studies have demonstrated their specific DNA binding, quantitative affinity data is not available for the binding of DOF domains to their binding sites. Methods: ZF domains of DOF2.1, DOF3.4, and DOF5.8 from Arabidopsis thaliana were expressed and purified. Their DNA binding affinities were assessed using gel retardation assays and microscale thermophoresis with two different oligonucleotide probes containing one and two copies of recognition sequence AAAG. Results: DOF zinc finger domains (DOF-ZFs) were shown to form independently folded structures. Assessments using microscale thermophoresis demonstrated that DOF-ZFs interact more tightly (~ 100 fold) with double-motif probe than the single-motif probe. The overall Kd values for the DOF3.4-ZF and DOF5.8-ZF to the double-motif probe were ~2.3±1 and 2.5±1 µM, respectively. Conclusion: Studied DOF-ZF domains formed stable complexes with the double-motif probe. Although DOF3.4-ZF and DOF5.8-ZF do not dimerize with an appreciable affinity in the absence of DNA (judging from size-exclusion and multiangle laser light scattering data), it is possible that these ZFs form protein-protein contacts when bound to this oligonucleotide, consistent with previous reports that DOF proteins can homo- and hetero-dimerize.

| S-EPMC6128974 | biostudies-literature

The multi-zinc finger protein ZNF217 contacts DNA through a two-finger domain.

Project description:Classical C2H2 zinc finger proteins are among the most abundant transcription factors found in eukaryotes, and the mechanisms through which they recognize their target genes have been extensively investigated. In general, a tandem array of three fingers separated by characteristic TGERP links is required for sequence-specific DNA recognition. Nevertheless, a significant number of zinc finger proteins do not contain a hallmark three-finger array of this type, raising the question of whether and how they contact DNA. We have examined the multi-finger protein ZNF217, which contains eight classical zinc fingers. ZNF217 is implicated as an oncogene and in repressing the E-cadherin gene. We show that two of its zinc fingers, 6 and 7, can mediate contacts with DNA. We examine its putative recognition site in the E-cadherin promoter and demonstrate that this is a suboptimal site. NMR analysis and mutagenesis is used to define the DNA binding surface of ZNF217, and we examine the specificity of the DNA binding activity using fluorescence anisotropy titrations. Finally, sequence analysis reveals that a variety of multi-finger proteins also contain two-finger units, and our data support the idea that these may constitute a distinct subclass of DNA recognition motif.

| S-EPMC3207412 | biostudies-literature

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