EP300 Antibody

What is EP300 and why is it important in research?

EP300 (E1A binding protein p300, also known as p300 or KAT3B) is a histone acetyltransferase that regulates transcription via chromatin remodeling and is crucial for cell proliferation and differentiation. It functions by mediating cAMP-gene regulation through binding to phosphorylated CREB protein and acts as a co-activator of HIF1A, playing a role in stimulating hypoxia-induced genes like VEGF . EP300 is important in research because:

• It represents a critical epigenetic regulator with a calculated molecular weight of 264161 MW but is observed at approximately 300 kDa in experimental conditions

• Mutations in EP300 are associated with Rubinstein-Taybi syndrome and epithelial cancers

• It plays essential roles in the pathogenesis of non-Hodgkin B cell lymphoma and other cancer types

• It's involved in establishing histone H3 lysine 27 acetylation (H3K27ac), a key marker of active enhancers and promoters

What experimental applications are EP300 antibodies optimized for?

EP300 antibodies have been validated for multiple research applications, with different antibodies optimized for specific techniques:

The most extensively validated applications include Western blotting, immunohistochemistry, and ELISA, with ChIP-seq applications also reported in several research studies .

How can I validate the specificity of an EP300 antibody?

Validating EP300 antibody specificity requires multiple approaches:

• Knockdown/knockout validation: Generate EP300 knockdown cells using shRNAs targeting the 3'UTR region of EP300 mRNA as demonstrated in bladder cancer studies. This approach achieved efficient knockdown of endogenous EP300 at both mRNA and protein levels .

• Cross-reactivity testing: Verify that the antibody doesn't cross-react with other proteins, particularly the closely related paralog CBP. Antibodies like PB9178 have been validated for "no cross-reactivity with other proteins" .

• Immunoprecipitation followed by mass spectrometry: Perform co-IP with the EP300 antibody followed by mass spectrometry to confirm that the precipitated protein is indeed EP300 .

• Expected localization patterns: Confirm nuclear localization in immunofluorescence experiments, as EP300 functions primarily in the nucleus as a transcriptional co-activator .

• Molecular weight verification: Confirm detection at the expected molecular weight (~300 kDa) in Western blot applications .

What tissues and cell types express EP300?

EP300 shows varied expression across multiple tissues and cell types:

Researchers studying specific tissue types should note that EP300 expression can be detected in cytoplasm, as confirmed in customer inquiries about cervix carcinoma erythroleukemia tissue staining .

How can I distinguish between EP300 and its paralog CBP in experimental studies?

Distinguishing between EP300 and CBP requires careful experimental design:

• Selective antibodies: Use antibodies that have been validated for specificity to either EP300 or CBP with minimal cross-reactivity. For instance, PB9178 has been validated for EP300 specificity .

• Co-immunoprecipitation experiments: Studies have shown that EP300, but not CBP, physically interacts with specific transcription factors like TFAP2β and GATA3 in neuroblastoma cells. Immunoprecipitation of EP300 and CBP followed by Western blotting for these transcription factors can help distinguish their binding partners .

• ChIP-seq experimental design: When performing ChIP-seq experiments with EP300 and CBP antibodies, analyze overlapping and unique binding sites. Research has shown distinct binding patterns in neuroblastoma cells, with EP300 and CBP showing only partial overlap in genomic binding .

• Knockout/knockdown studies: CRISPR-Cas9 mediated knockout of either EP300 or CBP can reveal their differential dependencies in specific cell types. For example, most high-risk neuroblastoma cell lines require EP300 for cell growth while CBP is dispensable .

• Functional analysis: In neuroblastoma, EP300 controls enhancer acetylation by interacting with TFAP2β, while CBP has a limited role, demonstrating functional differences that can be exploited experimentally .

What are the optimal protocols and considerations for EP300 ChIP-seq experiments?

For successful EP300 ChIP-seq experiments:

• Antibody selection: Choose antibodies specifically validated for ChIP applications. Research has shown that EP300 antibody ChIP-seq typically yields fewer peaks compared to biotin-tagged EP300 approaches (bioChIP-seq) .

• Enhanced sensitivity approaches: Consider epitope-tagged approaches for improved sensitivity. Studies showed that EP300 fb bioChIP-seq identified 48,963 EP300-bound regions compared to only 15,281 for EP300 antibody ChIP-seq, with the epitope-tagged allele demonstrating superior sensitivity and specificity .

• Signal-to-noise optimization: Epitope-tagging approaches in mouse models (Ep300 fb/fb; Rosa26 BirA/BirA) showed high reproducibility (93.6% overlap between biological duplicates) compared to antibody-based approaches (77.8% overlap) .

• Tissue-specific considerations: In tissue-based experiments, antibody-based EP300 ChIP-seq yielded 9.5x or 3.0x fewer EP300 regions in heart and forebrain, respectively, compared to tagged approaches .

• Analysis considerations: When analyzing EP300 binding sites, perform motif enrichment analysis to identify transcription factor binding motifs preferentially associated with EP300 binding. Studies in neuroblastoma cells identified GATA3 and TFAP2β motifs enriched at EP300-bound sites .

How do EP300 mutations impact antibody recognition and functional studies?

EP300 mutations can significantly affect both antibody recognition and protein function:

• Structural impact: Mutations like R1627W in the HAT domain of EP300 can impact the protein's structure and function. Structural analysis showed this mutation affects a substrate-binding pocket, potentially disrupting polypeptide substrate binding .

• Epitope availability: Mutations near antibody epitopes can affect recognition. When working with mutated EP300, validate antibody binding to the specific mutant forms being studied .

• Functional consequences: Studies of bladder cancer-associated EP300 mutations (H1451L, D1485V, E1521Q, K1554N, R1627W, Q2295K) demonstrated differential effects on protein function. The R1627W mutation partially impaired EP300 HAT activity by interfering with substrate binding capacity .

• Experimental design for mutant studies: When studying EP300 mutations, establish knockdown cell lines for endogenous EP300 (using shRNAs against the 3'UTR) before introducing exogenous wild-type or mutant EP300. This approach minimizes interference from endogenous protein .

• Structural analysis tools: Use tools like the SWISS-MODEL online server for modeling EP300 mutations and predicting their impact on protein structure and function .

What role does EP300 play in T cell metabolism and cancer immunotherapy research?

EP300 has emerging importance in T cell function and cancer immunotherapy:

• Glycolytic regulation: EP300 restores glycolytic activity in T cells. When CD8+ T cells are cultured in cancer cell-conditioned medium, EP300 protein levels are substantially reduced but can be restored through glucose addition .

• Metabolic enhancement: Overexpression of EP300 in CD8+ T cells increases extracellular acidification rate (ECAR) and enhances expression of glycolytic enzymes HK2 and PKM2 .

• Anti-tumor function: EP300 overexpression in CD8+ T cells reduces apoptosis, increases proliferation marker Ki67, and enhances production of anti-tumor effectors like GZMB, IFN-γ, and TNF-α .

• Epigenetic mechanism: EP300 promotes expression of BPTF (a chromatin remodeling factor) through histone acetylation (H3K27ac) at the BPTF promoter, forming a mechanistic link between EP300 and T cell function .

• Experimental approach: Researchers can use lentivirus-carried overexpression plasmids (oe-EP300) to investigate EP300's role in immune cell function, as demonstrated in studies examining glucose metabolic disorders in CD8+ T cells .

What are emerging approaches for targeting EP300 in cancer research?

Novel approaches for targeting EP300 in cancer research include:

• Selective HAT inhibitors: A-485 is the most potent and specific HAT-inhibitory compound toward EP300/CBP developed to date, showing effectiveness in neuroblastoma cells .

• PROTAC degraders: Proteolysis-targeting chimeras (PROTACs) like "JQAD1" selectively target EP300 for degradation by forming a ternary complex between EP300, the PROTAC, and an E3 ubiquitin ligase, directing EP300 to proteasomal degradation .

• Structure-based design: Researchers have developed PROTACs like dCE-2 based on CBP/EP300 ligands that form ternary complexes with CBP and CRBN with high cooperativity (α = 3.4) .

• CRBN-dependent approaches: EP300 degraders like JQAD1 show CRBN-dependent activity, with effectiveness determined by cereblon expression across neuroblastoma cells .

• Experimental validation: These approaches can be validated by measuring loss of H3K27ac at enhancers and assessing transcriptional output changes in cancer cells .

How can I investigate EP300 interactions with specific transcription factors?

To study EP300 interactions with transcription factors:

• Co-immunoprecipitation: Immunoprecipitate EP300 followed by Western blotting for transcription factors of interest. In neuroblastoma, this approach demonstrated that EP300, but not CBP, physically interacts with TFAP2β and GATA3 .

• Reciprocal co-IP: Perform reciprocal co-IP by pulling down the transcription factor and probing for EP300, as demonstrated with TFAP2β in neuroblastoma cells .

• Motif enrichment analysis: Analyze the top EP300-bound peaks from ChIP-seq data for enriched transcription factor motifs. Studies in neuroblastoma identified GATA3 and TFAP2β motifs enriched under EP300-bound peaks .

• CRISPR-Cas9 knockout studies: Perform CRISPR-Cas9-based knockout of transcription factors to determine their role in controlling EP300 localization .

• H3K27ac co-IP followed by mass spectrometry: Immunoprecipitate H3K27ac from nuclear extracts followed by mass spectrometry to identify transcription factors associated with EP300-marked chromatin .

What methods are available for studying the histone acetyltransferase activity of EP300?

To investigate EP300's HAT activity:

• ChIP-qPCR assays: Use chromatin immunoprecipitation with qPCR to detect H3K27ac levels at specific gene promoters. This can reveal EP300's acetyltransferase activity at target genes .

• Western blot for histone modifications: Measure global changes in histone acetylation (especially H3K27ac) following EP300 manipulation or inhibition .

• HAT activity assays: In vitro histone acetyltransferase assays can measure the direct enzymatic activity of immunoprecipitated EP300 or recombinant EP300 proteins .

• Structural analysis of HAT domain mutations: Analyze how mutations in the EP300 HAT domain affect enzyme activity. For example, the R1627W mutation disrupts substrate binding capacity without completely eliminating HAT activity .

• Small molecule inhibitors: Use selective HAT inhibitors like A-485 to probe EP300's acetyltransferase function in cellular contexts .

• Target protein acetylation: Assess acetylation of non-histone EP300 targets such as ALX1, HDAC1, PRMT1, SIRT2, STAT3, or GLUL to understand the breadth of EP300's acetyltransferase activity .