e4f1 Antibody

What is E4F1 and why is it significant in molecular biology research?

E4F1 (E4F transcription factor 1) is a multifunctional zinc finger protein initially identified as a cellular target of the adenoviral oncoprotein E1A. It functions primarily as a transcriptional regulator and an atypical E3 ubiquitin ligase. Its significance stems from its critical roles in multiple cellular processes including cell cycle control, DNA damage response (DDR), and metabolic regulation. E4F1 interacts with p53 and pRB tumor suppressor pathways, regulates cyclin A2 transcription, and controls a transcriptional program involved in pyruvate dehydrogenase (PDH) activity . Its functional diversity makes it an important target for researchers studying developmental biology, metabolism, and cancer pathways.

What types of E4F1 antibodies are available for research applications?

Several types of E4F1 antibodies are available with varying specifications:

Research applications include western blotting (WB), immunoprecipitation (IP), flow cytometry (FC), immunofluorescence (IF), and ELISA, with immunofluorescence being the most common application .

How does E4F1 protein structure influence antibody selection?

The human E4F1 canonical protein comprises 784 amino acid residues with a mass of approximately 83.5 kDa. Its structure includes multiple domains that serve different functions, including zinc finger motifs for DNA binding and regions involved in protein-protein interactions. When selecting antibodies, researchers should consider:

• The subcellular localization targeted – E4F1 is present in both nucleus and cytoplasm

• Post-translational modifications – E4F1 undergoes sumoylation, phosphorylation, and proteolytic cleavage which may affect epitope accessibility

• Research application – certain epitopes may be masked in specific applications (e.g., fixed versus native conditions)

• Protein isoforms – including p120E4F and p50E4F variants

For unbiased assessment of total E4F1 levels, antibodies targeting conserved regions are preferable, while phospho-specific antibodies may be required for studying its activation state.

What are the optimal protocols for using E4F1 antibodies in ChIP assays?

Chromatin immunoprecipitation (ChIP) using E4F1 antibodies requires careful optimization to identify E4F1 binding sites. Based on published protocols , a recommended approach includes:

• Sample preparation and crosslinking:

  • Use 3×10^7 cells or 100mg of tissue (e.g., gastrocnemius muscle)

  • Crosslink with 1% formaldehyde

  • Isolate nuclei, extract and sonicate chromatin (e.g., using Vibralcell bioblock)

• Immunoprecipitation:

  • Use affinity-purified rabbit anti-E4F1 polyclonal antibody

  • Pull down with Dynabeads coupled to protein G

  • Process input and immunoprecipitated DNA (decrosslinking, RNaseA treatment, proteinase K digestion)

  • Purify by phenol-chloroform-isoamylic-alcohol extraction/precipitation followed by column chromatography

• Analysis methodology:

  • For targeted analysis: qPCR with promoter-specific primers

  • For genome-wide analysis: deep-sequencing (Hi-SEq. 2000, Illumina) with mm9 mouse genome mapping

  • Bioinformatic analysis using combined approaches of two peak callers (Cisgenome and QESEQ) to define high-confidence binding sites

This methodology allows detection of E4F1 binding to promoters of genes involved in various pathways, including those regulating the pyruvate dehydrogenase complex .

How should antibody validation be performed for E4F1 detection in different experimental systems?

Comprehensive validation of E4F1 antibodies should include:

• Specificity validation:

  • Use E4f1-null cells as negative controls when available

  • Compare results from multiple antibodies targeting different epitopes

  • Perform siRNA-mediated depletion of E4F1 to confirm signal reduction

  • Include isotype controls (e.g., Mouse IgG1 [MOPC-21] for monoclonal antibodies)

• Application-specific validation:

  • Western blot: Confirm detection of appropriate molecular weight band (83.5 kDa)

  • Immunoprecipitation: Validate by immunoblotting of the immunoprecipitate

  • ChIP: Validate enrichment at known E4F1 target genes (e.g., Dlat, Dld, Brp44l/Mpc1, Slc25a19)

  • Immunofluorescence: Confirm expected subcellular localization (nuclear and cytoplasmic)

• Cross-species reactivity:

  • Test across multiple species if studying orthologous proteins

  • E4F1 gene orthologs have been reported in mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken

Proper validation ensures reliable results and minimizes potential artifacts in experimental findings.

How can E4F1 antibodies be effectively used to study DNA damage response mechanisms?

E4F1 plays crucial roles in DNA damage response (DDR), particularly in double-strand break (DSB) repair mechanisms. Advanced applications include:

• Laser microirradiation recruitment assays:

  • Use live-cell imaging with GFP-E4F1 fusion proteins to track E4F1 recruitment to DNA lesions

  • Analyze co-localization with γH2AX as a DNA damage marker

  • Quantify relocation dynamics to determine recruitment kinetics

• Proximity ligation assays (PLA):

  • Employ PLA to detect proximity between endogenous E4F1 and DDR proteins like ATR

  • Compare proximity signals under various conditions (e.g., baseline vs. after hydroxyurea treatment)

  • Quantify PLA signals to assess changes in protein-protein interactions

• Mechanistic studies:

  • Use E4F1 antibodies to investigate its PARP-1-dependent recruitment to DNA lesions

  • Study E4F1's interaction with BRG1/SMARCA4 (catalytic subunit of the SWI/SNF complex)

  • Analyze its role in γH2AX clearance, transcriptional silencing, and DNA-end resection

• Chromatin fraction analysis:

  • Isolate chromatin-bound fractions to assess E4F1 enrichment after DNA damage

  • Study its role in BRG1 recruitment to damaged chromatin

These approaches have revealed that E4F1 promotes ATR/CHK1 signaling and homologous recombination, contributing to genome integrity maintenance .

What are the key considerations when using E4F1 antibodies in tissue-specific knockout models?

When studying tissue-specific E4F1 knockout models, several methodological considerations are essential:

• Genotyping and knockout validation:

  • Verify Cre-mediated recombination efficiency by qPCR on genomic DNA from target tissues

  • Use primers specific for wild-type (E4f1^+^), floxed (E4f1^flox^), and null (E4f1^-^) alleles

  • Example primer sequences:

    • E4f1^+^ and E4f1^flox^: Fwd 5'-CCTTGAGCACGGAGGAGAGC-3', Rev 5'-GCCCTAGCCTGCTCTGCCATC-3'

    • E4f1^-^: Fwd 5'-CACTGCCTTGGAGGACTTTG-3', Rev 5'-CCTCTGTTCCACATACACTTCATTC-3'

• Antibody selection for tissue analysis:

  • Choose antibodies validated for the specific tissue type

  • Consider tissue-specific post-translational modifications

  • Validate antibody in tissue lysates from knockout models

• Experimental design for tissue-specific phenotypes:

  • In skeletal muscle-specific knockouts, examine PDH activity using appropriate assays

  • Assess physiological parameters relevant to E4F1 function (e.g., endurance tests, metabolic analyses)

  • Measure tissue-specific mRNA and protein levels of E4F1 target genes (e.g., Dlat, Brp44l, Slc25a19, Dld)

• Controls and comparisons:

  • Use heterozygous or tissue-specific Cre-expressing wild-type animals as controls

  • Compare multiple tissues (e.g., skeletal muscle vs. cardiac muscle) to assess tissue-specific effects

These approaches have successfully revealed the role of E4F1 in regulating PDH activity specifically in skeletal muscles but not in cardiac tissue .

How should researchers address discrepancies in E4F1 detection between different antibodies?

When faced with inconsistent results between different E4F1 antibodies, researchers should consider:

• Epitope accessibility issues:

  • Different epitopes may be masked by protein-protein interactions or post-translational modifications

  • Solution: Use antibodies targeting distinct epitopes (e.g., N-terminal vs. C-terminal) and compare results

  • For example, compare antibodies targeting residues 275-325 versus those targeting the C-terminus (aa 700+)

• Isoform-specific detection:

  • E4F1 exists in multiple forms (p120E4F, p50E4F)

  • Solution: Use isoform-specific antibodies or multiple antibodies to detect all relevant forms

  • Verify with recombinant protein standards of each isoform

• Application-specific optimization:

  • Different antibodies may perform optimally in specific applications

  • Solution: Test multiple antibodies for each application or use application-validated antibodies

  • For example, some antibodies may work better for immunofluorescence while others excel in western blotting

• Technical trouble-shooting matrix:

When publishing, researchers should report antibody clone/catalog numbers, validation methods, and experimental conditions to ensure reproducibility.

What analytical approaches are recommended for interpreting ChIP-seq data for E4F1 binding sites?

Analysis of E4F1 ChIP-seq data requires sophisticated computational approaches:

• Pre-processing and quality control:

  • Remove read pile-ups and filter out problematic genomic regions (Chromosomes Y and M)

  • Remove regions with high artifactual read density by analyzing inputs alone

  • Apply random sampling to standardize read counts (e.g., 10 million reads per sample)

• Peak calling strategy:

  • Use a combined approach with multiple peak callers (Cisgenome and QESEQ)

  • Cisgenome parameters: Seqpeak -b 50 -w 1 -e 150 -ts 1 -c 3 -maxgap 50 -minlen 100 -br 1 -bar 1 -dat 1 -bw 5 -lpois 1 -lpwin 10000 -lpcut 1e-5

  • QESEQ parameters: QESEQ -s 150 -v 1 -p 0.01 -c 20

• Motif discovery and validation:

  • Analyze peak sequences to identify E4F1 binding motifs

  • Validate motifs through in vitro binding assays with recombinant E4F1 protein

  • Confirm conservation of motifs across species

• Functional annotation:

  • Integrate ChIP-seq data with RNA-seq to identify direct transcriptional targets

  • Perform gene ontology analysis to identify enriched biological processes

  • In the case of E4F1, look specifically for enrichment of mitochondrial and metabolic genes

• Visualization and reporting:

  • Generate genome browser tracks (e.g., BAR files for visualization in IGB)

  • Create heatmaps showing E4F1 binding intensity at transcription start sites

  • Report peak distribution relative to genomic features (promoters, enhancers, etc.)

This approach has successfully identified E4F1-regulated genes involved in PDH activity and mitochondrial function .

How do E4F1 antibodies contribute to understanding metabolic regulation in disease models?

E4F1 antibodies have revealed critical insights into metabolic disorders through:

• PDH activity regulation:

  • E4F1 directly regulates genes encoding PDH components (Dlat, Dld) and transporters (Brp44l/Mpc1, Slc25a19)

  • In E4F1-deficient skeletal muscles, PDH activity is reduced by 80-90%

  • This leads to pyruvate accumulation, lactate production, and metabolic reprogramming

  • Research application: Use antibodies to monitor E4F1 binding to these gene promoters via ChIP-qPCR in disease models

• Adipocyte metabolism:

  • E4F1 expression is significantly higher in epididymal fat of obese (Ob/Ob) mice and in patients with high BMI

  • E4F1 regulates p53-associated metabolic functions in adipocytes

  • E4F1-deficient mice show resistance to diet-induced obesity

  • Research application: Use antibodies to study E4F1 expression patterns in various adipose tissues

• Clinical significance:

  • A homozygous nonsynonymous mutation in the E4F1 gene was identified in a patient with reduced PDH activity, muscular defects, and lactate acidemia

  • Mutations in E4F1-target genes (Dlat, Dld, Brp44l, Slc25a19) are associated with congenital metabolic disorders

  • Research application: Use antibodies to detect alterations in E4F1 levels or localization in patient samples

• Therapeutic potential:

  • E4F1 knockout animal models recapitulate symptoms of PDC-deficient patients

  • These models can be used for preclinical studies of therapeutic strategies

  • Research application: Use antibodies to monitor therapeutic effects on E4F1 and its downstream targets

This research has important implications for understanding and potentially treating metabolic disorders associated with PDH deficiency.

What are the emerging applications of E4F1 antibodies in cancer research?

E4F1 antibodies are revealing important connections between E4F1 and cancer pathways:

• DNA damage response and genomic stability:

  • E4F1 is rapidly recruited to DNA lesions in a PARP-1-dependent manner

  • It promotes γH2AX clearance, transcriptional silencing, and homologous recombination

  • E4F1 binds to BRG1/SMARCA4 and mediates its recruitment to DNA lesions

  • Research application: Use antibodies to study E4F1's role in DNA repair in cancer cells with defective DDR

• Oncogenic pathway interactions:

  • E4F1 interacts with p53 and pRB tumor suppressors to regulate cell cycle

  • It displays an atypical E3 ligase function that modifies but does not degrade p53

  • Research application: Use antibodies for co-immunoprecipitation to study these interactions in different cancer contexts

• Cancer genomic alterations:

  • A proportion of human breast cancers show amplification and overexpression of E4F1 or BRG1

  • These alterations are mutually exclusive with BRCA1/2 alterations

  • Research application: Use antibodies for immunohistochemistry to detect E4F1 overexpression in tumor samples

• Cell cycle regulation:

  • E4F1-depleted cells show G2 accumulation, reduced EdU incorporation, and delayed S phase progression

  • They exhibit increased phosphorylation of p53 at serine 15, indicating cellular stress

  • Research application: Use antibodies to monitor cell cycle-dependent expression and localization of E4F1

These applications highlight the importance of E4F1 in maintaining genome integrity and suggest it may serve as a potential biomarker or therapeutic target in certain cancers.