ZNF274 Antibody

What is ZNF274 and what cellular functions does it mediate?

ZNF274 is a KRAB-containing zinc finger protein that plays a crucial role in genome organization and gene silencing. It contains a distinctive SCAN domain positioned between two repressive KRAB motifs. Functionally, ZNF274 anchors target DNA sequences at the nucleolus and facilitates their compartmentalization through the SCAN domain . This protein is essential for sequestering lineage-specific gene clusters within nucleolus-associated domains (NADs), which results in their transcriptional silencing. When ZNF274 is ablated, these silenced regions undergo transcriptional activation, lose their repressive chromatin marks, and experience alterations in 3D genome architecture . This mechanism particularly affects protocadherin and KZFP-encoding genes, highlighting ZNF274's importance in regulating developmentally significant gene clusters.

Which applications are ZNF274 antibodies validated for?

ZNF274 antibodies have been validated for multiple experimental applications essential for molecular and cellular research. Commercial antibodies are typically validated for:

• Western Blot (WB): For detecting ZNF274 protein in cell or tissue lysates

• Immunohistochemistry (IHC): For visualizing ZNF274 expression patterns in tissue sections

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of ZNF274

• Chromatin Immunoprecipitation (ChIP): As demonstrated in research using ChIP-PCR assays to identify ZNF274 binding sites in regulatory regions

• Immunofluorescence microscopy: For subcellular localization studies showing ZNF274's accumulation in discrete nuclear dots and regions surrounding nucleoli

Each application requires specific optimization for antibody concentration, incubation time, and detection methods to ensure reliable results.

Which species show cross-reactivity with ZNF274 antibodies?

The reactivity profiles of ZNF274 antibodies vary depending on the specific product and immunogen used in their development. Based on available information:

This cross-reactivity information is vital when selecting an appropriate antibody for comparative studies across species. Researchers should verify reactivity when working with species not listed in the manufacturer's specifications through preliminary validation experiments.

What is the molecular basis of ZNF274's nuclear localization and function?

ZNF274's distinctive subcellular localization is critical to its function in genome organization. Confocal imaging of ZNF274 reveals its accumulation in discrete nuclear dots with frequent localization in compartments surrounding the nucleolus . This localization pattern is functionally significant as it facilitates the tethering of target DNA sequences to nucleolus-associated domains.

The protein contains multiple functional domains that contribute to its localization and activity:

• SCAN domain: Critical for homodimerization and nucleolar localization

• KRAB domains: Required for recruiting transcriptional repressor complexes

• Zinc finger domains: Mediate sequence-specific DNA binding

Deletion experiments have demonstrated that removing the KRAB domains results in more prominent nucleolar concentration, while deletion of the SCAN domain prevents nucleolar association entirely . The SCAN domain specifically enables ZNF274 to form homodimers, a property that appears essential for bringing bound loci into spatial proximity and establishing long-range chromosomal contacts.

How does ZNF274 contribute to 3D genome organization and nucleolus-associated domains?

ZNF274 plays a pivotal role in 3D genome architecture by facilitating the compartmentalization of specific genomic regions into nucleolus-associated domains (NADs). Approximately 44% of regions showing H3K9 hypomethylation upon ZNF274 knockout coincide with NAD-associated sequences . This suggests a preferential organization of repressive chromatin hubs near nucleoli.

Mechanistically, ZNF274:

• Binds to specific DNA sequences via its zinc finger domains

• Homodimerizes through the SCAN domain

• Accumulates in the perinucleolar environment

• Tethers bound genomic regions to the nucleolus

• Facilitates the formation of repressive chromatin compartments

Hi-C experiments reveal that contacts between ribosomal DNA (rDNA) and ZNF274-targeted DNA occur more frequently in wild-type cells compared to ZNF274 knockout cells . This provides strong evidence that ZNF274 directly links specific chromatin regions to the perinucleolar environment. DNA FISH combined with immunofluorescence for nucleolin has confirmed that ZNF274-regulated gene clusters frequently localize near nucleoli .

These findings illuminate how ZNF274 contributes to the spatial segregation of lineage-specific gene clusters, enabling selective expression and modulating cell differentiation programs during development.

What is the significance of ZNF274 in cancer biology and potential therapeutic implications?

Emerging evidence indicates that ZNF274 plays a significant role in cancer biology, particularly in colorectal cancer (CRC). Immunohistochemical analysis has revealed that ZNF274 expression is significantly higher in CRC tissues compared to adjacent non-tumor tissues . This overexpression correlates with poor clinical outcomes, as demonstrated by Kaplan-Meier survival analysis showing that CRC patients with elevated ZNF274 expression have shorter survival periods and worse prognoses .

The oncogenic properties of ZNF274 in CRC appear to be associated with:

• Promotion of cell proliferation, as evaluated by CCK-8 and colony formation assays

• Enhancement of cell migration, demonstrated through Transwell assays

• Transcriptional regulation of target genes such as MRPL40

• Involvement in IL-8-mediated signaling pathways

ZNF274 expression strongly correlates with adverse clinical parameters including:

• Larger tumor size

• Increased invasion

• Higher incidence of lymph node metastasis

• Advanced AJCC stage

These findings position ZNF274 as both a potential biomarker for prognostic assessment and a promising therapeutic target in CRC. Future research directions include exploring ZNF274 inhibition strategies and investigating its role in other cancer types.

How do researchers distinguish between ZNF274 isoforms in experimental settings?

Distinguishing between ZNF274 isoforms requires careful selection of antibodies and experimental approaches:

• Antibody Selection: Choose antibodies raised against specific regions that differ between isoforms:

  • N-terminal targeting antibodies such as those from Aviva Systems Biology

  • Antibodies raised against specific domains like the SCAN domain or KRAB domains

  • Antibodies targeting unique epitopes in different isoforms

• Western Blot Analysis: Western blotting can resolve different isoforms based on molecular weight. Researchers should:

  • Use high-resolution SDS-PAGE gels (8-10%) for optimal separation

  • Include positive controls expressing known isoforms

  • Implement longer running times to enhance band separation

  • Compare results with predicted molecular weights of each isoform

• PCR-Based Approaches: Design primers that specifically amplify different isoforms:

  • Exon-spanning primers can differentiate alternatively spliced transcripts

  • Quantitative RT-PCR with isoform-specific primers allows relative quantification

• Mass Spectrometry: For definitive isoform identification, immunoprecipitate ZNF274 and analyze by mass spectrometry to detect isoform-specific peptides.

These approaches can be combined to provide comprehensive isoform characterization in experimental systems.

What are the optimal immunohistochemistry protocols for ZNF274 detection in tissue samples?

For reliable ZNF274 detection in tissue samples, the following optimized protocol is recommended based on published methods:

• Tissue Preparation:

  • Fix tissues in 10% neutral buffered formalin

  • Embed in paraffin and section at 4-5 μm thickness

  • Mount on positively charged slides

• Antigen Retrieval:

  • Deparaffinize sections through xylene and graded alcohols

  • Block endogenous peroxidase activity with 3% H₂O₂ solution for 30 minutes

  • Perform heat-induced epitope retrieval using 10 mM citrate buffer (pH 6.0) for 20 minutes

• Antibody Incubation:

  • Block with normal serum from the species of secondary antibody for 30 minutes

  • Apply primary anti-ZNF274 antibody (recommended dilutions: 1:100-1:200) at 4°C overnight

  • Wash thoroughly with PBS (3 × 5 minutes)

  • Incubate with biotin-labeled secondary antibody for 30 minutes at room temperature

  • Wash with PBS (3 × 5 minutes)

• Detection and Visualization:

  • Apply streptavidin-peroxidase complex for 30 minutes

  • Develop with DAB (3,3'-diaminobenzidine) substrate

  • Counterstain with hematoxylin

  • Dehydrate, clear, and mount with neutral mounting medium

• Controls:

  • Include positive controls (tissues known to express ZNF274)

  • Include negative controls (omitting primary antibody)

  • Consider using ZNF274 knockout tissues as specificity controls

This protocol has been successfully employed to demonstrate ZNF274 overexpression in colorectal cancer tissues compared to adjacent normal tissues .

What are the critical considerations for ChIP-seq experiments using ZNF274 antibodies?

ChIP-seq experiments with ZNF274 antibodies require careful optimization and validation:

• Antibody Selection:

  • Choose ChIP-grade antibodies specifically validated for this application

  • Consider using multiple antibodies targeting different epitopes to cross-validate results

  • Verify antibody specificity using western blot or immunoprecipitation

• Chromatin Preparation:

  • Optimize crosslinking conditions (1% formaldehyde for 10 minutes is standard)

  • Ensure efficient sonication to generate 200-500 bp fragments

  • Verify fragmentation efficiency using agarose gel electrophoresis

• Immunoprecipitation Protocol:

  • Include appropriate controls (IgG control, input DNA)

  • Use sufficient antibody (typically 2-5 μg per ChIP reaction)

  • Extend incubation time (overnight at 4°C) to enhance binding

  • Implement stringent washing steps to reduce background

• Data Analysis Considerations:

  • Design primers for ChIP-qPCR validation of peaks

  • Compare ZNF274 binding sites with known sequence motifs

  • Integrate with other epigenetic marks, particularly H3K9me3

  • Analyze intersection with nucleolus-associated domains (NADs)

• Validation Strategies:

  • Confirm selected peaks by ChIP-qPCR

  • Validate functional relevance through gene expression analysis in ZNF274 knockout models

  • Correlate binding with chromatin states and three-dimensional genome organization

These considerations will help researchers generate high-quality ChIP-seq data for investigating ZNF274's genomic binding profile and regulatory functions.

How can researchers verify ZNF274 antibody specificity in their experimental systems?

Verifying antibody specificity is critical for reliable research outcomes. For ZNF274 antibodies, implement these validation strategies:

• Western Blot Analysis:

  • Compare wild-type versus ZNF274 knockout or knockdown samples

  • Verify band size matches the predicted molecular weight of ZNF274 (~65-70 kDa)

  • Examine cross-reactivity with other KRAB-ZFPs using recombinant proteins

• Peptide Competition Assay:

  • Pre-incubate antibody with the immunizing peptide

  • Perform parallel experiments with neutralized and non-neutralized antibody

  • Specific signals should be absent in the peptide-blocked condition

• Immunoprecipitation-Mass Spectrometry:

  • Perform IP with ZNF274 antibody followed by mass spectrometry

  • Verify that ZNF274 is the predominant protein identified

  • Assess co-immunoprecipitated proteins for known interactors (e.g., KAP1)

• Genetic Models:

  • Use CRISPR/Cas9-generated ZNF274 knockout cells

  • Compare antibody reactivity in wild-type versus knockout conditions

  • Rescue experiments with ZNF274 re-expression should restore signals

• Immunofluorescence Patterns:

  • ZNF274 should display characteristic nuclear dotted pattern with perinucleolar enrichment

  • Compare with published localization patterns

  • Co-staining with nucleolar markers (e.g., nucleolin) should show expected partial co-localization

A systematic application of these approaches will provide comprehensive validation of antibody specificity and reliability for ZNF274 research.

Why might Western blots with ZNF274 antibodies show multiple bands?

Multiple bands in ZNF274 Western blots can result from several factors:

• Isoform Expression:

  • ZNF274 has multiple isoforms due to alternative splicing

  • Different isoforms may show distinct molecular weights

• Post-translational Modifications:

  • Phosphorylation, SUMOylation, or ubiquitination can alter mobility

  • These modifications may be tissue or condition-specific

• Proteolytic Processing:

  • Sample preparation with insufficient protease inhibitors

  • Native proteolytic processing of ZNF274 in cells

• Cross-reactivity:

  • Antibody recognition of related KRAB-zinc finger proteins

  • Non-specific binding to unrelated proteins with similar epitopes

• Experimental Considerations:

  • Incomplete denaturation of protein complexes

  • Overloading protein samples causing band distortion

Troubleshooting approaches include:

• Comparing patterns across multiple cell lines

• Using phosphatase treatment to remove phosphorylation

• Testing alternative lysis buffers with varied detergent strengths

• Performing peptide competition assays to identify specific bands

• Using ZNF274 knockout samples as negative controls

What strategies can resolve low signal-to-background ratios in immunohistochemistry?

When facing high background or low signal in ZNF274 immunohistochemistry, consider these optimization strategies:

• Antibody Dilution Optimization:

  • Perform titration experiments (e.g., 1:50, 1:100, 1:200, 1:500)

  • Optimize both primary and secondary antibody concentrations

• Antigen Retrieval Enhancements:

  • Compare citrate buffer (pH 6.0) versus EDTA buffer (pH 9.0)

  • Test different retrieval times (10, 20, 30 minutes)

  • Compare microwave, pressure cooker, and water bath methods

• Blocking Improvements:

  • Extend blocking time (60 minutes instead of 30)

  • Try alternative blocking reagents (5% BSA, commercial blockers)

  • Add 0.1-0.3% Triton X-100 to reduce non-specific binding

• Washing Protocol Modifications:

  • Increase washing duration and number of washes

  • Add 0.05% Tween-20 to wash buffers

  • Use TBS instead of PBS if phosphate interferes

• Detection System Selection:

  • Compare polymer-based vs. avidin-biotin detection systems

  • Consider tyramide signal amplification for low-abundance targets

  • Reduce DAB incubation time to minimize background

• Tissue Preparation Considerations:

  • Control fixation time to prevent overfixation

  • Use freshly cut sections (within 1 week)

  • Reduce endogenous peroxidase blocking time if signal is weak

Systematically testing these variables will help optimize the signal-to-background ratio for ZNF274 immunohistochemistry.

How might ZNF274 research advance our understanding of 3D genome architecture in development and disease?

ZNF274 research offers promising avenues for understanding genome organization:

• Developmental Regulation:

  • Investigating how ZNF274 mediates developmental stage-specific gene silencing

  • Exploring ZNF274's role in establishing tissue-specific chromatin domains

  • Examining dynamic changes in ZNF274-mediated genome organization during differentiation

• Disease Mechanisms:

  • Expanding studies of ZNF274 dysregulation beyond colorectal cancer to other malignancies

  • Investigating potential roles in neurodevelopmental disorders given its regulation of protocadherin clusters

  • Exploring connections between ZNF274 dysfunction and chromatin organization diseases

• Technological Applications:

  • Developing ZNF274-based tools for targeted chromatin reorganization

  • Creating engineered ZNF274 variants to modulate gene expression in therapeutic contexts

  • Utilizing ZNF274's nucleolar targeting ability for novel gene silencing approaches

• Integration with Single-Cell Technologies:

  • Applying single-cell Hi-C with ZNF274 ChIP to reveal cell-to-cell variation in genome organization

  • Combining single-cell transcriptomics with ZNF274 binding data to correlate nuclear organization with gene expression heterogeneity

These research directions could significantly advance our understanding of how three-dimensional genome architecture influences cellular function in both normal development and pathological states.

What are emerging methods for studying ZNF274 binding dynamics and interactions?

Cutting-edge technologies are expanding our ability to study ZNF274:

• Live-Cell Imaging Approaches:

  • CRISPR-based tagging of endogenous ZNF274 with fluorescent proteins

  • Lattice light-sheet microscopy for high-resolution tracking of ZNF274 dynamics

  • FRAP (Fluorescence Recovery After Photobleaching) to assess binding kinetics

• Proximity Labeling Methods:

  • BioID or TurboID fusion with ZNF274 to identify proximal proteins in living cells

  • APEX2-based approaches for temporal control of proximity labeling

  • Combining proximity labeling with ChIP to identify proteins co-occupying ZNF274 binding sites

• Chromosome Conformation Technologies:

  • Micro-C for higher-resolution chromatin interaction mapping

  • PLAC-seq (Proximity Ligation-Assisted ChIP-seq) to identify ZNF274-mediated chromatin contacts

  • Hi-M for simultaneous visualization of multiple genomic loci with ZNF274 binding

• Functional Genomics Screens:

  • CRISPR interference/activation screens targeting ZNF274 binding sites

  • Domain-focused mutagenesis to dissect functional regions of ZNF274

  • Synthetic genetic interaction screens to identify pathways cooperating with ZNF274

These emerging technologies promise to reveal new insights into ZNF274's dynamic behavior, molecular interactions, and functional impact on genome organization and gene regulation.