Recombinant Mouse Zinc finger protein 76 (Znf76)

What is the basic structure and function of mouse ZNF76?

Mouse ZNF76 is a C2H2-type zinc finger protein that functions as a transcriptional repressor primarily through its interaction with TATA-binding protein (TBP). The protein has an estimated molecular weight of approximately 61.8 kDa and contains both N- and C-terminal domains that interact with TBP. The C-terminus contains a critical glutamic acid-rich domain that is essential for TBP interaction . ZNF76 belongs to the Krüppel C2H2-type zinc finger protein family, which is characterized by the zinc finger motif C-x-C-x-H-x-H .

From a functional perspective, ZNF76 prevents TBP from occupying promoter regions, such as the endogenous p21 promoter, thereby exerting an inhibitory effect on p53-mediated transactivation . This repressive activity can be modulated through post-translational modifications, particularly sumoylation at lysine 411 .

What are the recommended methods for detecting endogenous ZNF76 in mouse tissues?

For detecting endogenous ZNF76 in mouse tissues, Western blotting using specific antibodies is the most validated approach. Anti-ZNF76 antibodies reactive to mouse, human, and rat ZNF76 are commercially available . When performing Western blotting, a recommended dilution range of 1:500-1:1000 has been validated for detecting ZNF76 .

Methodology for optimal detection:

• Prepare tissue or cell lysates in PBS containing mammalian cell protease inhibitor cocktail

• Clarify lysates by centrifugation (20,000g at 4°C for 30 min)

• Determine protein concentration using BCA assay

• Run 20-40 μg of total protein on SDS-PAGE (recommended 10-12% gel)

• Transfer to PVDF membrane

• Block with 5% non-fat milk in TBST

• Incubate with anti-ZNF76 antibody at 1:500-1:1000 dilution

• Detect using appropriate secondary antibody and chemiluminescence

For immunohistochemistry or immunofluorescence approaches, additional optimization may be required as these applications have less validated protocols for ZNF76 detection.

What expression systems and purification strategies are most effective for producing recombinant mouse ZNF76?

Based on established protocols for similar zinc finger proteins, the following approaches are recommended for recombinant mouse ZNF76 production:

Expression Systems:

Purification Strategy:

• Add N-terminal His-tag or GST-tag to facilitate purification

• For His-tagged protein:

  • Lyse cells in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 10% glycerol, 1 mM PMSF

  • Include 10-20 μM ZnCl₂ in all buffers to maintain zinc finger structure

  • Purify using Ni-NTA affinity chromatography

  • Further purify by ion-exchange and size-exclusion chromatography

• For GST-tagged protein:

  • Use GST affinity purification followed by on-column cleavage

When expressing in mammalian systems like HEK293 cells, harvest and wash cells with PBS twice, then resuspend in ice-cold PBS containing protease inhibitor cocktail before lysis via freeze-thaw cycles .

How does ZNF76 mechanistically function as a transcriptional repressor?

ZNF76 acts as a transcriptional repressor through a well-characterized mechanism involving TBP interaction and promoter occupation prevention:

• TBP Binding: ZNF76 interacts with TBP through both its N and C termini, with the C-terminal glutamic acid-rich domain being particularly critical .

• Promoter Occupation: Chromatin immunoprecipitation experiments demonstrate that ZNF76 prevents TBP from occupying the endogenous p21 promoter .

• p53 Pathway Inhibition: ZNF76 exerts an inhibitory function on p53-mediated transactivation, affecting the expression of p53 target genes .

• Regulatory Mechanism: The repressive activity of ZNF76 is modulated by sumoylation at lysine 411 by PIAS1. Overexpression of PIAS1 and SUMO-1 abolishes the interaction between ZNF76 and TBP, partially relieving the repressive effect .

This mechanism follows the pattern observed in other zinc finger transcriptional repressors, where specific DNA binding through the zinc finger domains is coupled with protein-protein interactions that recruit or inhibit components of the transcriptional machinery.

What are the known protein interaction partners of ZNF76 and how do they affect its function?

ZNF76 participates in several protein-protein interactions that influence its function within transcriptional networks:

Confirmed Direct Interaction Partners:

• TBP (TATA-binding protein): Primary interaction partner, binds to both N and C termini of ZNF76. This interaction is critical for ZNF76's repressive function .

• PIAS1: Functions as a SUMO E3 ligase for ZNF76, mediating sumoylation at lysine 411 .

• p53 pathway components: While direct binding to p53 has not been conclusively demonstrated, ZNF76 functionally interferes with p53-mediated transactivation .

Potential Interaction Network Based on TF Atlas Data:
Analysis of transcription factor interaction atlases suggests that ZNF76, like other zinc finger proteins, may participate in combinatorial interactions with other transcription factors. These interaction networks are critical for directing tissue-specific gene expression, with approximately half of the measured interactions conserved between mouse and human .

The functional importance of these interactions is underscored by data showing that highly connected transcription factors (TF hubs) tend to be broadly expressed across tissues, while TFs with fewer interactions tend to be expressed in a tissue-specific pattern .

For identification of novel ZNF76 interaction partners, approaches such as co-immunoprecipitation followed by mass spectrometry or yeast two-hybrid screening are recommended.

How do post-translational modifications, particularly sumoylation, regulate ZNF76 activity?

Post-translational modifications significantly impact ZNF76 function, with sumoylation being the most well-characterized:

Sumoylation:

• Site: ZNF76 is sumoylated specifically at lysine 411, which is located within the minimal TBP-interacting region .

• Enzyme: PIAS1 functions as the SUMO E3 ligase for ZNF76 .

• Functional effect: Sumoylation modulates ZNF76's transcriptional repression activity by affecting its interaction with TBP. Overexpression of PIAS1 and SUMO-1 abolishes the interaction between ZNF76 and TBP, partially relieving the repressive effect of ZNF76 .

Methodology for studying ZNF76 sumoylation:

• Detection: Perform immunoprecipitation with anti-ZNF76 antibodies followed by Western blotting with anti-SUMO antibodies.

• Mutagenesis: Generate K411R mutants to abolish sumoylation and assess functional consequences.

• In vitro sumoylation: Reconstitute the sumoylation reaction using purified components (E1, E2, PIAS1, SUMO-1, and ZNF76).

• Functional assays: Compare wild-type and K411R mutant in reporter gene assays to assess the impact of sumoylation on transcriptional repression.

Other potential post-translational modifications that may regulate ZNF76 function include phosphorylation and acetylation, though these have not been as well characterized as sumoylation.

What methodologies are most effective for determining ZNF76 DNA-binding specificity?

Several complementary approaches can be employed to determine the DNA-binding specificity of ZNF76:

1. Chromatin Immunoprecipitation followed by Sequencing (ChIP-seq):

• Most definitive approach for identifying genomic binding sites in vivo

• Requires highly specific antibodies or epitope-tagged ZNF76 expression

• Analysis should include motif discovery to identify consensus binding sequences

2. Protein-Binding Microarrays (PBM):

• Allows unbiased assessment of binding to all possible DNA sequence variants

• Particularly useful for C2H2 zinc finger proteins like ZNF76

• Can identify both high-affinity primary sites and secondary binding preferences

3. Electrophoretic Mobility Shift Assay (EMSA):

• Useful for validating direct binding to specific DNA sequences

• Can determine relative binding affinities through competition assays

• Requires purified recombinant ZNF76 or relevant DNA-binding domains

4. SELEX (Systematic Evolution of Ligands by Exponential Enrichment):

• Highly effective for determining optimal binding sequences

• Can be coupled with high-throughput sequencing (SELEX-seq)

• Useful for defining extended binding motifs beyond core recognition sequences

5. In silico prediction based on zinc finger code:

• C2H2 zinc finger domains typically follow recognition rules where specific amino acids at positions -1, 3, and 6 of the α-helix recognize specific nucleotides

• This approach has limitations but can provide initial predictions to test experimentally

When analyzing results, it's important to consider that zinc finger proteins often show context-dependent binding, where the presence of cofactors or neighboring transcription factors can alter binding specificity in vivo compared to in vitro methods.

How has ZNF76 evolved across species and how does it relate to other zinc finger proteins?

ZNF76 belongs to the C2H2-type zinc finger protein family, one of the most abundant protein families, with 720 genes and 372 transcription factors in mammals . Evolutionary analysis reveals several key insights:

Conservation:

• ZNF76 is conserved across mammalian species, with orthologs identified in both human and mouse genomes

• The conservation of TF-TF interactions, including those potentially involving ZNF76, is estimated to be approximately 34-64% between human and mouse

Structure and Function Relationships:

• The C2H2 zinc finger motif (C-x-C-x-H-x-H) is highly conserved and represents one of the most common DNA-binding domains in eukaryotes

• ZNF76 contains multiple zinc finger domains, typical of the C2H2 class of zinc finger proteins

Evolutionary Context:
Unlike KRAB-zinc finger proteins, which underwent extensive expansion in response to transposable element activity , ZNF76 belongs to a more evolutionarily stable subset of zinc finger proteins that likely evolved to regulate specific genes rather than to suppress transposable elements.

Functional Conservation:
The transcriptional repressor function of ZNF76 through TBP interaction appears to be conserved, suggesting an important role in basic transcriptional regulation across species.

This evolutionary conservation makes ZNF76 an interesting candidate for comparative studies of transcriptional regulation across mammalian species, particularly in the context of developmentally regulated genes.

What is known about ZNF76 dysregulation in disease models, and does it have therapeutic potential?

While direct evidence for ZNF76 involvement in disease is limited, its role as a transcriptional repressor acting on the p53 pathway suggests potential implications for diseases involving dysregulated cell proliferation and apoptosis:

Potential Disease Associations:

• Cancer: As a repressor of p53-mediated transactivation , ZNF76 could potentially impact tumor suppression mechanisms. Dysregulation might contribute to altered p53 pathway activity in cancer cells.

• Developmental disorders: Given the importance of precise transcriptional regulation during development, mutations in ZNF76 could theoretically impact developmental processes.

Therapeutic Considerations:
The technology developed for synthetic zinc finger proteins could potentially be applied to ZNF76-related pathways:

• Engineered zinc finger proteins: Custom-designed zinc finger proteins could be developed to either mimic or counteract ZNF76 function at specific genomic loci.

• Small molecule modulators: Compounds targeting the ZNF76-TBP interaction or the sumoylation of ZNF76 could potentially modulate its activity.

• Gene editing approaches: CRISPR/Cas9-based approaches similar to those used for KRAB-ZFP clusters could be employed to study or modify ZNF76 function in disease models.

For researchers investigating ZNF76 in disease contexts, it is recommended to:

• Analyze ZNF76 expression levels in disease tissues compared to normal controls

• Examine genetic variants in ZNF76 for potential disease associations

• Consider the impact of ZNF76 on p53 pathway regulation in disease-relevant cell types

What cutting-edge approaches can be used to comprehensively identify and validate ZNF76 genomic targets?

To thoroughly identify and validate genomic targets of ZNF76, several advanced techniques can be employed:

1. CUT&RUN or CUT&Tag:

• Offers higher signal-to-noise ratio than conventional ChIP-seq

• Requires fewer cells and less sequencing depth

• Particularly valuable when working with limited biological samples

2. ChIP-exo or ChIP-nexus:

• Provides near base-pair resolution of protein-DNA binding sites

• Helps define the precise binding motifs for ZNF76

• Useful for distinguishing direct from indirect binding events

3. HiChIP or PLAC-seq:

• Combines chromatin immunoprecipitation with chromosome conformation capture

• Identifies long-range chromatin interactions mediated by ZNF76

• Helps determine how ZNF76 influences 3D genome organization

4. CRISPRi with ZNF76 binding sites:

• Use dCas9-KRAB to repress ZNF76 binding sites

• Assess transcriptional consequences of preventing ZNF76 binding

• Helps establish functional relevance of binding events

5. CRISPR activation/repression of ZNF76:

• Modulate ZNF76 expression using CRISPRa or CRISPRi

• Perform RNA-seq to identify genes responsive to ZNF76 levels

• Compare with ChIP-seq data to distinguish direct from indirect targets

6. Protein-centered Chromatin Conformation Capture (P3C):

• Identifies all genomic loci that interact with ZNF76-bound regions

• Helps understand the broader regulatory network affected by ZNF76

7. Single-cell approaches:

• scRNA-seq combined with ZNF76 perturbation to assess cell-type-specific effects

• scATAC-seq to examine chromatin accessibility changes upon ZNF76 modulation

These approaches should ideally be combined to build a comprehensive understanding of ZNF76 genomic targets and their functional relevance in different cellular contexts.

What mutagenesis strategies would be most informative for mapping functional domains of ZNF76?

A systematic mutagenesis approach would be highly valuable for elucidating the structure-function relationships of ZNF76:

Domain Deletion Strategy:

• N-terminal TBP-binding domain deletion: Remove the N-terminal region to assess its contribution to TBP binding and transcriptional repression.

• C-terminal glutamic acid-rich domain deletion: This domain is critical for TBP interaction , and its deletion would help quantify its contribution.

• Individual zinc finger deletions: Sequential deletion of each zinc finger domain to map their contributions to DNA binding specificity.

• SUMO-site mutation: K411R mutation to prevent sumoylation and assess its impact on protein-protein interactions and transcriptional repression .

Point Mutation Strategy:

• DNA-binding residues: Mutate key residues at positions -1, 3, and 6 within each zinc finger α-helix, which typically make specific contacts with DNA .

• Zinc-coordinating residues: Mutation of cysteine and histidine residues in the C2H2 motif to disrupt zinc binding and finger structure.

• TBP-interacting residues: Alanine scanning of the glutamic acid-rich domain to identify specific residues critical for TBP interaction.

• Conservation-guided mutagenesis: Target residues conserved between mouse and human ZNF76 to identify functionally important sites.

Chimeric Protein Strategy:

• Zinc finger swapping: Exchange individual zinc fingers with those from related proteins to alter DNA binding specificity.

• Domain swapping: Replace domains of ZNF76 with corresponding domains from other transcription factors to create functional chimeras.

For optimal functional assessment, each mutant should be tested in:

• Protein-protein interaction assays (co-IP with TBP)

• DNA binding assays (EMSA or similar)

• Transcriptional reporter assays

• Cellular localization studies