znf143 Antibody

What is ZNF143 and what are its known biological functions?

ZNF143 (Zinc Finger Protein 143) is a 69 kDa member of the GLI C2H2-type zinc-finger protein family. The human ZNF143 protein is 638 amino acids in length and contains seven C2H2-type zinc fingers. It functions primarily as a transcriptional activator and is expressed in all tissues, with highest expression observed in ovarian tissue . Recent research has established that ZNF143 specifically activates nuclear-encoded mitochondrial genes, particularly those involved in mitochondrial ribosome function and oxidative phosphorylation complexes . The protein serves several key functions:

• Transcriptional activation of target genes

• Activation of selenocysteine tRNA (tRNAsec) genes

• Binding to SPH motifs in small nuclear RNA (snRNA) gene promoters

• Facilitating efficient U6 RNA polymerase III transcription through interaction with CHD8

Unlike previously thought, recent studies have demonstrated that ZNF143 does not function as a general chromatin looping factor .

How is ZNF143 antibody typically used in research applications?

ZNF143 antibodies are primarily used in immunological techniques to detect, visualize, and analyze ZNF143 protein expression and localization. Common research applications include:

• Immunocytochemistry/Immunofluorescence (ICC/IF): ZNF143 antibodies can be used to visualize nuclear localization of ZNF143 in fixed cells, as demonstrated in BG01V human embryonic stem cells .

• Chromatin Immunoprecipitation sequencing (ChIP-seq): ZNF143 antibodies are used to map genome-wide binding sites of ZNF143, identifying its target genes and regulatory elements .

• Western blotting: For detecting ZNF143 protein expression levels in cellular extracts and monitoring protein degradation in experimental systems .

• Protein interaction studies: To identify protein partners that associate with ZNF143 in transcriptional regulation complexes.

What critical validation steps should be performed before using a ZNF143 antibody?

Before utilizing a ZNF143 antibody in research applications, researchers should perform several essential validation steps:

• Antibody specificity assessment: Verify antibody specificity using cell lines with ZNF143 knockout or knockdown. Recent research revealed that a commonly used polyclonal antibody (Proteintech 16618-1-AP) cross-reacts with CTCF, leading to misattribution of functional roles to ZNF143 .

• Motif analysis of ChIP-seq data: When conducting ChIP-seq experiments, perform de novo motif analysis to confirm enrichment of known ZNF143 binding motifs (SBS1 and SBS2) in the identified peaks .

• Comparison with tagged ZNF143: When possible, compare results obtained with commercial antibodies to those using epitope-tagged ZNF143 (e.g., FLAG-tagged or HA-tagged) to confirm binding patterns .

• Western blot validation: Confirm the antibody detects a band of the expected molecular weight (approximately 69 kDa) without significant non-specific bands.

• Immunofluorescence controls: Include appropriate negative controls and validate nuclear localization pattern characteristic of ZNF143.

What are the optimal conditions for using ZNF143 antibody in immunofluorescence?

For optimal immunofluorescence results with ZNF143 antibody, follow these methodological guidelines:

• Fixation method: Use immersion fixation for adherent cells.

• Antibody concentration: A concentration of 10 μg/mL has been successfully used for ZNF143 detection in human embryonic stem cells .

• Incubation conditions: Incubate cells with primary antibody for 3 hours at room temperature for optimal binding .

• Detection system: Use appropriate species-specific fluorophore-conjugated secondary antibodies, such as NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody for mouse monoclonal anti-ZNF143 .

• Nuclear counterstaining: Include DAPI counterstaining to confirm nuclear localization of ZNF143 .

• Controls: Include negative controls (primary antibody omission, isotype controls) and positive controls (cell types known to express ZNF143) in each experiment.

How can researchers accurately distinguish between true ZNF143 binding sites and artifacts due to antibody cross-reactivity?

Recent research has revealed significant challenges in accurately identifying ZNF143 binding sites due to antibody cross-reactivity issues. To distinguish true binding sites from artifacts, researchers should implement the following strategies:

• Multiple antibody validation: Use multiple antibodies targeting different epitopes of ZNF143, or compare commercial antibody results with epitope-tagged ZNF143 ChIP-seq data .

• Motif enrichment analysis: True ZNF143 binding sites should show enrichment for known ZNF143 binding motifs (SBS1 and SBS2), while signals arising from cross-reactivity with CTCF will show CTCF motif enrichment .

• Comparison with CTCF ChIP-seq data: Compare ZNF143 ChIP-seq peaks with available CTCF ChIP-seq datasets to identify potential cross-reactivity. The Proteintech antibody was found to detect signal at CTCF binding sites where epitope-tagged ZNF143 showed no binding .

• Acute depletion experimental design: Utilize degradation tag (dTAG) systems to acutely deplete ZNF143 and assess which binding signals disappear (true ZNF143 sites) versus those that remain (likely cross-reactivity) .

• Signal intensity analysis: Cross-reactive signals at CTCF sites were observed to have lower intensity compared to true ZNF143 binding sites .

What are the most effective experimental systems to study ZNF143 function?

Based on recent advances in the field, the following experimental systems are particularly effective for studying ZNF143 function:

• Degradation tag (dTAG) systems: CRISPR-Cas9-mediated endogenous tagging of ZNF143 with the FKBP12 F36V domain allows for rapid, inducible degradation upon treatment with dTAG-V1 small molecule. This system enables the study of direct consequences of ZNF143 loss without confounding effects from long-term depletion .

• Mouse embryonic stem cells (mESCs): These cells provide an excellent model system for studying ZNF143 function in pluripotency and early development contexts .

• ChIP-seq with epitope tags: C-terminal tagging of ZNF143 with HA or FLAG epitopes allows specific detection without cross-reactivity issues observed with some commercial antibodies .

• Nascent transcript profiling: Techniques such as transient transcriptome sequencing (TT-seq) can be used to directly assess the immediate transcriptional consequences of ZNF143 depletion, helping identify direct target genes .

• In vitro embryo models: These models are valuable for studying the developmental consequences of ZNF143 loss .

How should researchers interpret discrepancies between ZNF143 ChIP-seq datasets in the literature?

The literature contains significant discrepancies between ZNF143 ChIP-seq datasets, particularly regarding the co-localization of ZNF143 with CTCF. Researchers should consider the following when interpreting these discrepancies:

• Antibody source evaluation: Carefully note the antibody used in each study. The Proteintech 16618-1-AP antibody has been demonstrated to cross-react with CTCF, resulting in false detection of ZNF143 at CTCF sites .

• Peak overlap analysis: When comparing datasets, calculate the proportion of ZNF143 peaks that overlap with CTCF peaks. Studies using the Proteintech antibody typically show >40% overlap with CTCF, compared to ~20% overlap with other antibodies .

• Motif enrichment comparison: Analyze motif enrichment in different datasets. True ZNF143 binding sites show strong enrichment for SBS1 and SBS2 motifs, while sites arising from cross-reactivity show CTCF motif enrichment .

• Data reconciliation strategies: When possible, focus on the consensus peaks identified by multiple antibodies and methodologies, particularly those showing the expected ZNF143 motif enrichment .

• Experimental validation: Consider performing targeted validation experiments for critical binding sites using alternative methods or tagged ZNF143 systems .

What is the current consensus on ZNF143's role in chromatin organization versus transcriptional activation?

Recent research has significantly clarified ZNF143's role, challenging previous assumptions. The current consensus based on the latest evidence indicates:

• Chromatin organization: Contrary to earlier reports, acute depletion of ZNF143 using degradation tag systems has demonstrated that ZNF143 does not function as a general chromatin looping factor. Both Hi-C and 4C-seq analyses following ZNF143 depletion showed no consistent changes in chromatin interactions .

• Misattribution due to antibody issues: The incorrect association of ZNF143 with chromatin looping likely stemmed from antibody cross-reactivity with CTCF, a well-established chromatin organization factor .

• Primary role in transcriptional activation: ZNF143 functions primarily as a transcriptional activator, specifically regulating nuclear-encoded mitochondrial genes. Acute ZNF143 depletion leads to immediate downregulation of these genes at both transcriptome and proteome levels .

• Mitochondrial function regulation: ZNF143 safeguards mitochondrial activity by regulating genes involved in mitochondrial ribosome function and oxidative phosphorylation complexes, which is essential for cellular proliferation and differentiation .

• Developmental importance: ZNF143 plays an essential role in organismal development, with its loss causing severe mitochondrial dysfunction .

What are common technical challenges when using ZNF143 antibody in ChIP-seq experiments?

Researchers working with ZNF143 antibodies in ChIP-seq applications should be aware of several common technical challenges:

• Antibody cross-reactivity: As extensively documented, some commercial antibodies (particularly Proteintech 16618-1-AP) cross-react with CTCF, leading to false positive binding sites. This cross-reactivity can be identified by motif enrichment analysis showing CTCF motifs instead of ZNF143 motifs (SBS1/SBS2) .

• Signal-to-noise ratio: ChIP-seq experiments may suffer from high background or low signal-to-noise ratios. Optimization of antibody concentration, chromatin fragmentation, and immunoprecipitation conditions is critical.

• Epitope accessibility: The conformation of ZNF143 when bound to chromatin may affect epitope accessibility. Testing multiple antibodies targeting different epitopes can help address this issue.

• Chromatin preparation: Inadequate chromatin preparation can impact ChIP-seq quality. For ZNF143, which binds primarily at promoters, ensure proper chromatin fragmentation and fixation conditions.

• Data analysis challenges: The overlap between ZNF143 and CTCF binding sites requires careful computational analysis to distinguish true binding sites. Researchers should incorporate motif analysis and comparison with multiple datasets to validate results .

How can researchers properly validate ZNF143 antibody specificity for their specific application?

To ensure ZNF143 antibody specificity for particular research applications, follow these validation approaches:

• CRISPR knockout or knockdown controls: Generate ZNF143 knockout or knockdown cell lines to serve as negative controls for antibody specificity testing. The acute degradation system using dTAG technology provides an excellent platform for this purpose .

• Peptide competition assays: Perform peptide competition assays using the immunizing peptide to confirm specific binding of the antibody to ZNF143.

• Recombinant protein controls: Use recombinant ZNF143 protein as a positive control in western blot or other applications to confirm antibody recognition of the target protein.

• Multiple antibody confirmation: Compare results obtained with multiple antibodies targeting different epitopes of ZNF143. Consistent signals across different antibodies increase confidence in specificity.

• Tagged protein comparison: When possible, compare results with epitope-tagged ZNF143 (HA-tag, FLAG-tag) detection to validate signals observed with commercial antibodies .

• Cross-reactivity assessment: Test for potential cross-reactivity with related zinc finger proteins or other nuclear factors, particularly CTCF, which has been shown to be detected by some ZNF143 antibodies .

How has our understanding of ZNF143 function evolved in recent years?

Recent research has led to significant revisions in our understanding of ZNF143 function:

• Redefining chromatin looping role: While ZNF143 was previously reported to cooperate with CTCF in establishing enhancer-promoter interactions, recent acute depletion studies have found no evidence for ZNF143 involvement in chromatin looping. This revision stems from the discovery of antibody cross-reactivity issues that led to misattribution of functions .

• Identification of true biological function: Current evidence establishes ZNF143 as a transcriptional activator specifically regulating nuclear-encoded mitochondrial genes, particularly those involved in mitochondrial ribosome function and oxidative phosphorylation .

• Developmental significance: ZNF143 has been identified as an essential regulator of organismal development through its role in safeguarding mitochondrial activity .

• Technical advancements: The development of acute protein degradation systems has enabled more precise examination of direct ZNF143 functions, distinguishing them from secondary effects observed in traditional knockout or knockdown approaches .

• Antibody specificity awareness: The field has gained critical awareness of antibody specificity issues, particularly cross-reactivity with CTCF, highlighting the importance of rigorous antibody validation in chromatin biology studies .

What are the implications of antibody cross-reactivity for interpreting historical ZNF143 research?

The discovery of antibody cross-reactivity has significant implications for interpreting previous ZNF143 research:

• Reevaluation of CTCF co-localization: Studies reporting extensive co-localization of ZNF143 with CTCF (>40% overlap) likely reflect antibody cross-reactivity rather than true biological co-occupancy. The actual co-localization is estimated to be approximately 20% .

• Reassessment of chromatin looping function: Claims about ZNF143's role in chromatin looping, particularly when based on data generated with the Proteintech antibody, should be critically reevaluated in light of cross-reactivity issues .

• Data reanalysis approaches: Historical ChIP-seq datasets generated with the Proteintech antibody can be reanalyzed using motif enrichment analysis to distinguish likely true ZNF143 binding sites (enriched for SBS1/SBS2 motifs) from potential CTCF cross-reactivity sites (enriched for CTCF motifs) .

• Experimental design considerations: Future studies should incorporate multiple antibody approaches, tagged protein systems, or genetic depletion controls to ensure accurate attribution of ZNF143 functions .

• Publication bias awareness: Researchers should be aware that publication records may disproportionately reflect findings based on cross-reactive antibodies, particularly those from large consortium projects that utilized the Proteintech antibody .

What are recommended protocols for ChIP-seq using ZNF143 antibodies?

Based on recent advances and technical considerations, the following protocol recommendations can help researchers conduct reliable ZNF143 ChIP-seq experiments:

• Antibody selection: Use either:

  • Well-validated commercial antibodies with confirmed specificity

  • Custom antibodies with demonstrated specificity

  • Epitope-tagged ZNF143 systems (HA-tag, FLAG-tag) which have shown higher specificity

• Controls to include:

  • Input chromatin control

  • IgG negative control

  • ZNF143 knockout/knockdown control to assess antibody specificity

  • CTCF ChIP-seq comparative analysis to identify potential cross-reactivity

• Validation approach:

  • Perform de novo motif analysis to confirm enrichment of ZNF143 binding motifs (SBS1/SBS2) in identified peaks

  • Compare peak sets with datasets generated using different antibodies

  • Conduct ChIP-qPCR validation of selected peaks representing different signal intensities

• Data analysis considerations:

  • Filter peaks based on motif presence (SBS1/SBS2)

  • Classify peaks based on overlap with CTCF sites and motif enrichment

  • Annotate peaks relative to genomic features, particularly promoters of nuclear-encoded mitochondrial genes

What experimental designs best capture the direct effects of ZNF143 on gene expression?

To accurately assess the direct effects of ZNF143 on gene expression, the following experimental designs are recommended:

• Acute depletion systems:

  • Utilize FKBP12 F36V degradation tag (dTAG) systems for rapid ZNF143 depletion

  • This approach enables examination of immediate transcriptional consequences before secondary effects emerge

• Nascent transcriptome profiling:

  • Employ transient transcriptome sequencing (TT-seq) to capture immediate transcriptional changes

  • This method is more sensitive to rapid changes than steady-state RNA measurements

• Time-course experiments:

  • Conduct time-resolved experiments (e.g., 2h, 6h, 24h post-depletion) to distinguish direct from indirect effects

  • Early timepoints (2-6h) reflect primary transcriptional responses

• Integration of binding and expression data:

  • Combine ZNF143 ChIP-seq with nascent transcriptome profiling

  • Direct target genes typically show both ZNF143 binding at promoters and rapid transcriptional changes upon ZNF143 depletion (>85% of downregulated genes at 2h have ZNF143 binding at their promoters)

• Mitochondrial function assessment:

  • Include measurements of mitochondrial function (e.g., oxygen consumption rate, ATP production)

  • These reflect the functional consequences of ZNF143-regulated gene expression changes

What are the most promising approaches for further elucidating ZNF143 function in mitochondrial regulation?

Based on recent discoveries about ZNF143's role in regulating nuclear-encoded mitochondrial genes, several promising research directions emerge:

• Tissue-specific regulation: Investigate tissue-specific functions of ZNF143 in mitochondrial homeostasis, particularly in tissues with high energy demands like cardiac and skeletal muscle.

• Stress response mechanisms: Examine how ZNF143 activity responds to mitochondrial stress conditions and its potential role in the mitochondrial unfolded protein response pathway.

• Interaction with other transcription factors: Explore how ZNF143 coordinates with other transcription factors known to regulate mitochondrial gene expression, such as NRF1, NRF2, and PGC-1α.

• Post-translational modifications: Investigate how post-translational modifications of ZNF143 might regulate its activity in response to cellular energy status and other signals.

• Therapeutic targeting: Explore the potential of modulating ZNF143 activity as a therapeutic approach for mitochondrial disorders or conditions with mitochondrial dysfunction components .

How might improvements in antibody technology address current limitations in ZNF143 research?

Advancing antibody technology could significantly enhance ZNF143 research by addressing current limitations:

• Monoclonal antibody development: Development of highly specific monoclonal antibodies targeting unique epitopes of ZNF143 not present in CTCF or other zinc finger proteins could eliminate cross-reactivity issues.

• Recombinant antibody fragments: Engineering of recombinant antibody fragments (e.g., single-chain variable fragments) with enhanced specificity for ZNF143-specific epitopes.

• Conformational epitope targeting: Developing antibodies that recognize ZNF143 in its native conformation when bound to chromatin could improve ChIP-seq sensitivity and specificity.

• Nanobody approach: Utilizing nanobodies (single-domain antibodies) which can access epitopes that conventional antibodies cannot might improve detection of ZNF143 in complex with chromatin.

• Multi-parameter ChIP technologies: Implementing CUT&Tag, CUT&RUN, or other advanced chromatin profiling methods that may be less susceptible to antibody cross-reactivity issues and provide higher resolution mapping of ZNF143 binding sites.

• Combinatorial epitope detection: Developing systems that require recognition of multiple ZNF143 epitopes simultaneously to generate signal, significantly reducing false positives from cross-reactivity .