EP300 Antibody

What is EP300 and what cellular functions does it regulate?

EP300 (also known as p300, KAT3B, or E1A binding protein p300) is a transcriptional co-activator protein encoded by the EP300 gene located on the long arm of human chromosome 22 at position 13.2. It functions primarily as a histone acetyltransferase that regulates transcription via chromatin remodeling .

EP300 plays essential roles in:

• Cell growth and division regulation

• Cell differentiation

• Prevention of cancerous tumor growth

• Mediating cAMP-gene regulation by binding specifically to phosphorylated CREB protein

• Acting as a co-activator of HIF1A (hypoxia-inducible factor 1 alpha)

• Stimulating hypoxia-induced genes such as VEGF

At the molecular level, EP300 acetylates all four core histones in nucleosomes, providing epigenetic tags for transcriptional activation. Specifically, it mediates acetylation of histone H3 at 'Lys-122' (H3K122ac) and 'Lys-27' (H3K27ac), which are modifications that stimulate transcription by promoting nucleosome instability .

What are the primary applications of EP300 antibodies in research?

EP300 antibodies serve multiple critical research applications:

Primary Applications:

• Western Blotting (WB): For detecting EP300 protein expression levels in cell and tissue lysates

• Immunohistochemistry (IHC): For visualizing EP300 distribution in tissue sections

• Chromatin Immunoprecipitation (ChIP): For studying EP300 interactions with chromatin

• ELISA: For quantitative detection of EP300 in samples

Tissue and Cell Types:
EP300 antibodies have been validated for detection in various tissues and cell types including:

• Cervix carcinoma

• Erythroleukemia cells

• Leukemic T-cells

• Liver tissue

• Mouse intestine tissue

• NIH3T3 whole cell lysates

When selecting an EP300 antibody, researchers should consider the specific application and target species, as different antibodies demonstrate varied reactivity to human, mouse, and rat EP300 .

How can one reliably distinguish between EP300 and its paralog CBP using antibodies?

Background:
EP300 and CBP (CREB-binding protein) are paralogs with high sequence homology and overlapping yet distinct functions in many biological processes. Differentiating between these proteins is essential for understanding their specific roles.

Answer:
Distinguishing between EP300 and CBP requires careful antibody selection and validation:

• Epitope Selection: Choose antibodies raised against regions where EP300 and CBP sequences diverge. The N-terminal regions often show greater sequence dissimilarity and make better targets for specific antibody generation .

• Validation Methods:

  • Western Blot Size Discrimination: EP300 has an observed molecular weight of approximately 300 kDa, while CBP has a slightly different migration pattern that can be distinguished on high-resolution gels .

  • Immunoprecipitation-Western Blot: Perform IP with the target antibody followed by Western blot using another validated antibody to confirm specificity.

  • Knockout Controls: Use CRISPR-Cas9 generated EP300 or CBP knockout cells as negative controls to validate antibody specificity .

• Functional Discrimination:
  Research shows that EP300, but not CBP, physically interacts with transcription factors like TFAP2β and GATA3 in neuroblastoma cells. Immunoprecipitation of EP300 and CBP followed by Western blotting for these interacting partners can distinguish between the paralogs .

What are the optimal experimental conditions for EP300 ChIP-seq, and how does epitope tagging compare to antibody-based approaches?

Background:
Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a critical method for mapping EP300 genomic binding sites, but success depends heavily on antibody quality and experimental conditions.

Answer:
Research comparing traditional antibody-based ChIP-seq with biotin-based epitope tagging of EP300 (EP300fb) provides valuable insights:

• Antibody-Based ChIP-seq Limitations:

  • Lower sensitivity with typical peak detection yielding around 15,281 EP300-bound regions in ESCs

  • Moderate reproducibility between replicates (77.8% overlap)

  • Lower signal-to-noise ratio in tissue samples

• Epitope Tagging Advantages:

  • Much higher sensitivity with EP300fb bioChIP-seq detecting 48,963 EP300-bound regions

  • Superior reproducibility between replicates (93.6% overlap)

  • Stronger signal-to-noise ratio

  • 89.6% of regions detected by antibody methods were also found by bioChIP-seq

• Tissue-Specific Considerations:

  • In embryonic heart and forebrain tissues, EP300fb bioChIP-seq detected 9.5× and 3.0× more EP300 regions, respectively, compared to antibody-based methods

  • Higher reproducibility between biological duplicates (83% for heart, 93% for forebrain)

Researchers should consider employing epitope tagging approaches when possible, particularly for tissue samples or when studying low-abundance EP300 binding sites.

Why might EP300 antibodies show unexpected subcellular localization patterns, and how can specificity be validated?

Background:
Some researchers have observed unexpected cytoplasmic staining when using EP300 antibodies, raising questions about antibody specificity or biological relevance.

Answer:
When EP300 antibodies show unexpected localization patterns:

• Verify Expression Patterns in Literature:
  Research indicates that EP300 can be expressed in both nuclear and cytoplasmic compartments depending on cell type and condition. For example, cytoplasmic EP300 staining has been observed in cervix carcinoma and erythroleukemia cell lines .

• Validation Approaches:

  • Multiple Antibodies: Test at least two different antibodies recognizing different EP300 epitopes

  • Peptide Competition: Pre-incubate antibody with immunizing peptide to block specific binding

  • Genetic Knockdown/Knockout: Use siRNA, shRNA, or CRISPR to reduce or eliminate EP300 expression

  • Positive Controls: Include known EP300-expressing tissues (e.g., liver, leukemic T-cells)

• Protocol Optimization:

  • Optimize fixation conditions (formaldehyde vs. methanol)

  • Adjust antigen retrieval methods for IHC

  • Titrate antibody concentration

  • Include appropriate blocking reagents to reduce non-specific binding

• Biological Relevance Assessment:

  • Recent research suggests EP300 functions may extend beyond its well-characterized nuclear role

  • Consider co-localization studies with markers of specific subcellular compartments

How should researchers address contradictory results when using different EP300 antibodies in the same experimental system?

Background:
Contradictory results using different EP300 antibodies can create significant challenges in research interpretation and reproducibility.

Answer:
When faced with contradictory results:

• Antibody Characteristics Analysis:

  • Epitope Mapping: Different antibodies may target distinct EP300 domains that could be differentially accessible in various experimental conditions

  • Clonality Comparison: Compare monoclonal versus polyclonal antibody results, as polyclonals detect multiple epitopes while monoclonals target single epitopes

  • Host Species Effects: Different host species (rabbit vs. mouse) may introduce variability

• Technical Validation Approach:

  • Immunoprecipitation-Mass Spectrometry: Perform IP followed by mass spectrometry to confirm antibody specificity for EP300

  • Western Blot Analysis: Validate antibody specificity by Western blot with appropriate molecular weight controls

  • Epitope-Tagged Controls: Use cells expressing tagged EP300 (e.g., FLAG-EP300) to validate antibody recognition

• Experimental Design Refinement:

  • Report results using multiple EP300 antibodies

  • Include appropriate positive and negative controls

  • Consider the possibility that different antibodies may be detecting distinct EP300 conformations or post-translationally modified forms

How does EP300 overexpression or knockdown affect T cell metabolism and function in cancer models?

Background:
Understanding EP300's role in T cell function has important implications for cancer immunotherapy research.

Answer:
Recent research reveals EP300's critical role in T cell metabolism and anti-tumor function:

• EP300 Regulation of T Cell Glycolysis:

  • EP300 protein levels in CD8+ T cells decrease when cultured in cancer cell conditioned medium (CM)

  • Glucose supplementation restores EP300 levels

  • EP300 overexpression increases extracellular acidification rate (ECAR) of T cells

  • EP300 overexpression elevates glycolytic enzymes HK2 and PKM2 protein levels

• Effects on T Cell Function:

  • Proliferation: EP300 overexpression increases Ki67 expression and reduces apoptosis in T cells

  • Cytotoxic Function: EP300 overexpression elevates GZMB protein levels and increases IFN-γ+ and TNF-α+ T cell populations

  • Mechanism: EP300 promotes BPTF expression in CD8+ T cells via histone H3K27 acetylation, which enhances glycolytic activity

• Experimental Evidence:
  When BPTF was knocked down in CD8+ T cells overexpressing EP300:

  • ECAR decreased significantly

  • Glycolytic enzymes HK2 and PKM2 decreased

  • Apoptosis increased while proliferation (Ki67+ cells) decreased

  • GZMB levels and proportions of IFN-γ+ and TNF-α+ cells decreased

These findings suggest EP300 as a potential target for enhancing T cell function in cancer immunotherapy.

What are the latest approaches for selectively targeting EP300 versus CBP using PROTACs or other degraders?

Background:
Selective targeting of EP300 over its paralog CBP presents a significant challenge but offers potential therapeutic advantages in certain contexts.

Answer:
Recent advances in proteolysis-targeting chimeras (PROTACs) and other degraders provide promising approaches for selective EP300 targeting:

• PROTAC Design Strategies:

  • Selective JQAD1 PROTAC: Researchers have developed a novel PROTAC degrader called JQAD1 that displays strong selectivity for EP300 over CBP

  • Mechanism: This chimeric small molecule induces time-dependent loss of EP300, enhancer acetylation, and transcriptional output in neuroblastoma cells

  • Structural Basis: The selectivity exploits differences in protein-protein interactions, as EP300 (but not CBP) physically associates with transcription factors like TFAP2β and GATA3

• Structure-Based Approaches:

  • dCE-2 PROTAC Development: Another PROTAC called dCE-2 was designed based on an in-house-developed CBP/EP300 ligand with a K_D of 29/25 nM

  • Design Elements: This molecule incorporates a 3-methylcinnoline acetyl-lysine mimic identified by high-throughput docking, connected to a 10-atom aliphatic linker and thalidomide CRBN E3 ligand

  • Effectiveness: dCE-2 is active across multiple cell lines with DC₅₀ = 40 nM in LP1 cells after 16h treatment

• Key Considerations for PROTAC Development:

  • Despite modest K_D values toward CBP/EP300 bromodomains, degraders can be highly efficient due to their ability to switch between compact and extended conformations

  • Binary affinity should not be the only parameter in early PROTAC screening

  • Multidisciplinary approaches combining biological, biochemical, and computational techniques are essential for understanding PROTAC structure-activity relationships

These selective degraders provide valuable research tools for dissecting the distinctive roles of EP300 versus CBP in various biological contexts.

What are the optimal sample preparation protocols for EP300 detection in different experimental systems?

Background:
Successful EP300 detection requires careful consideration of sample preparation methods for different applications.

Answer:
Optimized protocols for key applications include:

• Western Blotting:

  • Lysis Buffer: Use RIPA buffer supplemented with deacetylase inhibitors (e.g., TSA, nicotinamide) and protease inhibitors

  • Protein Separation: Due to EP300's large size (300 kDa), use 6-8% SDS-PAGE gels or gradient gels (4-15%)

  • Transfer Conditions: Extend transfer time (overnight) or use specialized systems for high molecular weight proteins

  • Detection: Rabbit polyclonal antibodies like PB9178 show strong specific signals with minimal background

• Immunohistochemistry:

  • Fixation: 10% neutral-buffered formalin for 24-48 hours

  • Antigen Retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Blocking: 5-10% normal serum from the same species as the secondary antibody

  • Antibody Dilution: Typically 1:100-1:500 for primary antibodies like E-AB-16407

• Chromatin Immunoprecipitation (ChIP):

  • Cross-linking: 1% formaldehyde for 10 minutes at room temperature

  • Chromatin Shearing: Sonication to achieve fragments of 200-500 bp

  • Pre-clearing: With protein A/G beads to reduce background

  • Consideration: Biotin-tagging approaches (EP300fb) show superior results compared to traditional antibody-based ChIP

• Storage Conditions:

  • Store antibodies at -20°C or -80°C

  • Avoid repeated freeze-thaw cycles

  • For working solutions, store at 4°C with preservatives like sodium azide

How can researchers quantitatively assess EP300 binding to chromatin and transcriptional targets?

Background:
Accurate quantification of EP300 chromatin binding is essential for understanding its role in transcriptional regulation.

Answer:
Several quantitative approaches can be employed:

• ChIP-qPCR Analysis:

  • Principle: Combines ChIP with quantitative PCR to measure EP300 occupancy at specific genomic loci

  • Controls: Include input DNA, IgG controls, and positive control regions (known EP300 binding sites)

  • Normalization: Use spike-in exogenous DNA (e.g., E. coli DNA) as demonstrated in studies of TFAP2β knockout effects on H3K27ac

  • Analysis: Calculate percent input or fold enrichment over IgG control

• ChIP-seq with Spike-in Normalization:

  • Methodology: Add exogenous spike-in controls before immunoprecipitation

  • Application: This approach enabled detection of genome-wide loss of H3K27ac after TFAP2β knockout in neuroblastoma cells

  • Analysis: Normalize to spike-in control to allow quantitative comparisons between conditions

• CUT&RUN Sequencing:

  • Advantage: Higher signal-to-noise ratio than traditional ChIP-seq

  • Application: Used to demonstrate that loss of TFAP2β results in genome-wide loss of H3K27ac in neuroblastoma cells

  • Quantification: Include exogenous spike-in controls for normalization between samples

• Ternary Complex Formation Assays:

  • TR-FRET Method: Time-resolved fluorescence resonance energy transfer to measure formation of ternary complexes

  • FluoPPI System: Fluorescence-based protein-protein interaction assay used to monitor interactions between EP300 and other proteins in living cells

  • Cooperativity Assessment: Calculate cooperativity (α) values to determine interaction strength (e.g., α = 3.4 for dCE-2 interaction with CBP and CRBN)