ZNF800 Antibody

What is ZNF800 and what cellular functions does it regulate?

ZNF800 is a C2H2 zinc-finger transcription factor that functions primarily as a transcriptional repressor. It plays a critical role in intestinal epithelial cell differentiation, specifically as a master repressor of enteroendocrine cell (EEC) differentiation. ZNF800 is broadly expressed, including in the small intestine and colon epithelium. Knockout of ZNF800 results in increased EECs while reducing goblet and Paneth cell populations, indicating its essential role in balancing secretory lineage differentiation . At the molecular level, ZNF800 represses the endocrine transcription factor regulatory network by binding to gene loci involved in neural and endocrine-gland development pathways, particularly targeting transcription factors like INSM1, NEUROG3, and PAX4 .

How can I verify ZNF800 antibody specificity for immunological applications?

To verify ZNF800 antibody specificity:

• Positive controls: Use tissues with known ZNF800 expression such as small intestine and colon epithelium for immunostaining or Western blots .

• Negative controls: Employ ZNF800 knockout organoids or cells (as described in the research by Lin et al.) as negative controls .

• Peptide competition assay: Pre-incubate the antibody with purified ZNF800 peptide before immunostaining to demonstrate signal reduction.

• Multiple antibody validation: Compare staining patterns from antibodies targeting different epitopes of ZNF800.

• siRNA validation: Use siRNA knockdown of ZNF800 to show reduced antibody signal intensity.

For chromatin immunoprecipitation (ChIP) applications, validate using both anti-ZNF800 and anti-FLAG antibodies in parallel, similar to the approach taken in the study that identified 11,565 consensus ZNF800 binding peaks .

What are the recommended fixation and antigen retrieval methods for ZNF800 immunostaining?

For optimal ZNF800 immunostaining:

• Fixation: 4% paraformaldehyde (PFA) for 10-15 minutes for cells or 24 hours for tissues.

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes.

• Blocking: 5% normal serum (species different from antibody host) with 0.1% Triton X-100.

• Antibody incubation: Overnight at 4°C for primary antibody, followed by appropriate secondary antibody.

• Counterstaining: DAPI for nuclear visualization, similar to the approach used in dual RNA-FISH and protein immunofluorescent staining protocols .

For organoids specifically, fixation in 4% PFA followed by permeabilization with 0.3% Triton X-100 has been shown to be effective for ZNF800 detection while preserving the 3D structure.

What is the relationship between ZNF800 and its circular RNA form (circZNF800)?

ZNF800 gene produces both linear mRNA that encodes the ZNF800 protein and a circular RNA form known as circZNF800 (hsa_circ_0082096). While the ZNF800 protein functions as a transcriptional repressor regulating cell differentiation, circZNF800 has distinct functions:

• CircZNF800 is upregulated in cancer stem cell-enriched spheroids derived from colorectal cancer .

• It acts as a miRNA sponge, impeding expression of miR-140-3p, miR-382-5p, and miR-579-3p .

• By sequestering these miRNAs, circZNF800 promotes the expression of genes targeted by these miRNAs, including ALK/ACVR1C, FZD3, and WNT5A .

• CircZNF800 positively regulates intestinal stem cell markers, pluripotency factors, and cancer stem cell markers .

• It enhances cancer stem cell properties including cell proliferation, spheroid formation, and tumor growth in vivo .

This distinction highlights the complex regulatory networks involving different RNA species derived from the same gene locus.

How can I distinguish between ZNF800 protein and circZNF800 in experimental systems?

Distinguishing between ZNF800 protein and circZNF800 requires specific approaches:

For circZNF800-specific detection:

• Design divergent primers that amplify across the backsplice junction

• Treat RNA samples with RNase R to degrade linear RNAs while preserving circular RNAs

• For RNA-FISH, design probes targeting the backsplice junction sequence

• Validate using overexpression and knockdown controls as described in the literature

For ZNF800 protein-specific detection:

• Nuclear fractionation protocols can help enrich for the protein

• ChIP-seq can identify genomic binding sites unique to the protein function

What are the optimal experimental conditions for analyzing ZNF800 binding sites using ChIP-seq?

Based on successful ChIP-seq experiments identifying ZNF800 binding sites , the optimal conditions include:

• Crosslinking: 1% formaldehyde for 10 minutes at room temperature

• Sonication: Optimize to generate DNA fragments of 200-500 bp

• Antibody selection: Use both anti-ZNF800 and anti-FLAG antibodies (in FLAG-tagged ZNF800 systems) for validation

• Controls: Include IgG controls and input samples; consider using ZNF800 knockout samples as negative controls

• Sequencing depth: Aim for at least 20 million uniquely mapped reads per sample

• Peak calling: Use MACS2 with q-value < 0.01 as the significance threshold

• Analysis focus: Examine binding sites within ±5 kb of transcription start sites (TSS), as most ZNF800 binding was found in these regions

• Validation: Confirm key binding sites with ChIP-qPCR

In the published research, this approach successfully identified 11,565 consensus ZNF800 binding peaks in wild-type organoids and 7,085 peaks in rescued ZNF800-/- organoids, with significant overlap between the two datasets .

How can I effectively design CRISPR-based systems to manipulate ZNF800 or circZNF800 expression?

Different CRISPR approaches are needed for targeting ZNF800 protein versus circZNF800:

For ZNF800 protein knockout:

• Design sgRNAs targeting early exons of the ZNF800 gene using CRISPR-Cas9

• Verify knockout by Western blot and functional assays (e.g., changes in EEC differentiation)

• As a control, include rescue experiments using doxycycline-inducible ZNF800 expression systems

For circZNF800 manipulation:

• Use CRISPR-Cas13d system, which targets RNA rather than DNA

• Design crRNAs specifically targeting the backsplice junction of circZNF800

• Clone crRNAs into appropriate CRISPR-Cas13d vectors (e.g., Addgene #138148)

• Include scrambled sequence controls (crSC)

• Validate knockdown efficiency using qRT-PCR with divergent primers

• For overexpression studies, use in vitro synthesized and circularized circZNF800

The CRISPR-Cas13d approach has been successfully used both in vitro and in vivo for circZNF800 knockdown, demonstrating inhibition of tumor growth in xenografted mouse models .

What is the interplay between ZNF800 and other transcription factors in regulating cell differentiation?

ZNF800 interacts with multiple transcription factors in complex regulatory networks:

• ZNF800 and GFI1: Both function as repressors of EEC differentiation but operate independently. Double knockout of GFI1 and ZNF800 leads to further abrogation of goblet and Paneth cells and greater induction of EECs compared to single knockouts .

• ZNF800 and endocrine TF network: ZNF800 represses multiple endocrine-specific transcription factors:

  • Directly binds and represses INSM1, NEUROG3, and PAX4 loci

  • Regulates SOX4 and NEUROD2 expression

  • Indirectly affects NEUROD1 expression

• Regulatory dynamics:

  • Single-cell regulatory network inference and clustering (SCENIC) analysis identified 249 regulons with different activities between wild-type and ZNF800-/- organoids

  • Key regulons with higher activity in ZNF800-/- cells include SOX4, NEUROD2, and PAX4

  • Other TFs discovered in screens, such as TEF and NFIC, also show regulon activity in EEC lineages

This complex interplay suggests that ZNF800 functions as a master regulator at the top of a transcriptional hierarchy controlling secretory lineage differentiation in the intestinal epithelium.

What are the best approaches for studying ZNF800 function in organoid models?

Organoid models provide powerful systems for studying ZNF800 function:

For monitoring dynamic cellular processes:

• Use Fucci cell-cycle reporters to assess cell cycle progression

• Employ clonal formation efficiency assays to evaluate stem cell function

• Implement sequential doxycycline induction/withdrawal cycles to test the reversibility of phenotypes

How should I design experiments to study circZNF800 in cancer stem cell biology?

To study circZNF800 in cancer stem cell biology:

• Cell systems:

  • Use spheroidal culture to enrich for cancer stem cells

  • Establish paired adherent and spheroid cultures from the same cell lines for comparison

• Manipulation approaches:

  • Overexpression: Use in vitro synthesized and circularized circZNF800

  • Knockdown: CRISPR-Cas13d system targeting the backsplice junction

  • Include appropriate controls (GFP for overexpression, scrambled crRNA for knockdown)

• Functional assays:

  • Proliferation: EdU staining followed by flow cytometry

  • Stemness: Expression of CD44/CD133, Lgr5, and SOX9 by immunofluorescence

  • Self-renewal: Spheroid and colony formation assays

  • In vivo tumor growth: Xenograft models in nude mice

• Molecular mechanisms:

  • RNA pulldown assays to confirm circZNF800-miRNA interactions

  • Stem-loop qRT-PCR for miRNA quantification

  • Analysis of downstream targets (ALK/ACVR1C, FZD3, WNT5A) by qRT-PCR

• In vivo therapeutic targeting:

  • Injection of CRISPR-Cas13d-circZNF800 viral particles at tumor sites

  • Monitor tumor growth and cancer stem cell marker expression

What controls should be included when evaluating ZNF800 antibody performance in various applications?

Comprehensive controls for ZNF800 antibody validation:

Additional validation approaches:

• Peptide competition assays to demonstrate binding specificity

• Use of multiple antibodies targeting different epitopes of ZNF800

• Recombinant protein standards for quantitative applications

• Cross-validation with orthogonal methods (e.g., mass spectrometry, RNA-seq)

• Tissue microarrays for high-throughput screening of antibody performance across multiple samples

What is the optimal protocol for dual detection of ZNF800 protein and circZNF800 in tissue samples?

For simultaneous detection of ZNF800 protein and circZNF800:

• Sample preparation:

  • Fix tissue sections in 4% PFA

  • Perform heat-induced antigen retrieval in citrate buffer (pH 6.0)

  • Permeabilize with 0.3% Triton X-100

• RNA-FISH for circZNF800:

  • Synthesize Cy5-tagged circZNF800 antisense RNA probe targeting the backsplice junction

  • Prepare hybridization buffer containing the probe at 200 nM final concentration

  • Incubate overnight at appropriate temperature (typically 37°C)

  • Wash with saline sodium citrate (SSC) buffer

• Immunofluorescence for ZNF800 protein:

  • Block with 5% normal serum

  • Incubate with anti-ZNF800 primary antibody

  • Apply fluorescently-labeled secondary antibody (using a different fluorophore than the RNA-FISH probe)

• Counterstain and imaging:

  • Counterstain nuclei with DAPI (1:10,000 in PBS for 10 minutes)

  • Image using confocal microscopy (e.g., FV3000 Confocal Laser Scanning Microscope)

This protocol has been successfully adapted for dual detection of RNA and proteins such as Lgr5, Sox9, and Ki-67 in tissue samples , and can be modified for ZNF800 protein detection.

How can ZNF800 antibody be used to study enteroendocrine cell differentiation disorders?

ZNF800 antibody can be employed in several approaches to study enteroendocrine cell differentiation disorders:

• Diagnostic applications:

  • Immunohistochemical analysis of patient biopsies to assess ZNF800 expression levels and localization

  • Correlation of ZNF800 expression with enteroendocrine cell populations (CHGA+) in conditions like enteroendocrine cell hyperplasia or deficiency

• Mechanistic studies:

  • ChIP-seq to identify alterations in ZNF800 binding patterns in diseased tissues

  • Integrative analysis with transcriptomic data to identify disease-specific regulatory networks

  • Assessment of ZNF800 interaction with other transcription factors (NEUROG3, INSM1, PAX4) known to be involved in enteroendocrine disorders

• Therapeutic development:

  • High-throughput screening for compounds that modulate ZNF800 activity

  • Monitoring ZNF800 expression and localization as pharmacodynamic biomarkers in drug trials

  • Development of potential nucleic acid therapeutics targeting ZNF800 or its regulatory pathways

• Organoid disease modeling:

  • Generation of patient-derived organoids to study ZNF800 function in personalized models

  • CRISPR-based correction of ZNF800 mutations in patient-derived organoids to assess phenotypic rescue

What are the implications of targeting circZNF800 in cancer therapy development?

The research on circZNF800 reveals several promising therapeutic implications:

• Precision medicine approach:

  • CircZNF800 is overexpressed in late-stage colorectal cancer tissues

  • It promotes cancer stem cell properties and tumor growth

  • Targeting circZNF800 with CRISPR-Cas13d impedes tumor growth in xenograft models

• Combination therapy potential:

  • CircZNF800 impedes expression of miR-140-3p, miR-382-5p, and miR-579-3p

  • It promotes expression of ALK/ACVR1C, FZD3, and WNT5A

  • Combining circZNF800 inhibition with targeted therapies against these downstream pathways may enhance efficacy

• Cancer stem cell targeting:

  • CircZNF800 positively regulates intestinal stem cell, pluripotency, and cancer stem cell markers

  • Therapeutics targeting circZNF800 may help eliminate therapy-resistant cancer stem cell populations

  • This approach could potentially reduce tumor recurrence and metastasis

• Delivery mechanisms:

  • CRISPR-Cas13d-circZNF800 viral particles administered at CRC tumor sites successfully impeded tumor growth

  • Development of targeted nanoparticle delivery systems could enhance therapeutic specificity

What techniques can be used to investigate the protein-protein interactions of ZNF800?

To study ZNF800 protein-protein interactions:

For studying ZNF800's role in transcriptional complexes:

• Generate tagged ZNF800 constructs (FLAG, HA, or BioID) for efficient pulldown

• Perform cross-linking before immunoprecipitation to capture transient interactions

• Use size-exclusion chromatography to isolate native complexes

• Consider chromatin-focused approaches since ZNF800 functions as a transcriptional repressor

What are common issues with ZNF800 antibody applications and how can they be resolved?

When troubleshooting ZNF800 detection specifically:

• Consider its nuclear localization pattern when optimizing extraction methods

• For ChIP applications, note that most ZNF800 binding sites localize within ±5 kb of transcription start sites

• When studying both protein and circular RNA, implement the sequential protocol described for dual RNA-FISH and immunofluorescence