YEATS4 Antibody

What is YEATS4 and what are its primary cellular functions?

YEATS4 (YEATS domain-containing protein 4) is a nuclear protein that contains a highly conserved YEATS domain, which has been identified as a novel reader of histone acetylation. In humans, the canonical YEATS4 protein has 227 amino acid residues with a molecular mass of approximately 26.5 kDa . YEATS4 is primarily localized in the nucleus and is expressed in multiple tissues including brain, heart, kidney, liver, lung, pancreas, placenta, and skeletal muscle .

The protein plays crucial roles in chromatin remodeling and cell cycle regulation. The YEATS domain is required for the recognition of acetylated histones and chromatin recruitment, while its coiled-coil domain and/or A box are involved in protein-protein interactions . YEATS4 is known to participate in repressing the p53 tumor suppressor pathway during normal cellular proliferation, suggesting its importance in regulating cell growth and proliferation . Additionally, YEATS4 has been implicated in epithelial-to-mesenchymal transition (EMT) in cancer progression, particularly through its interaction with ZEB1 promoter regions .

What are the common applications for YEATS4 antibodies in research?

YEATS4 antibodies are utilized in several key research applications, with Western blotting being the most widely employed technique. This application allows researchers to detect and quantify YEATS4 protein levels in various cell and tissue lysates, providing insights into its expression patterns across different experimental conditions .

Immunohistochemistry (IHC) is another important application, particularly for analyzing YEATS4 expression in tissue samples. IHC has been crucial in demonstrating that YEATS4 is more highly expressed in tumor tissues compared to adjacent normal tissues, as observed in bladder cancer and breast cancer studies .

Enzyme-linked immunosorbent assay (ELISA) is also employed for the quantitative detection of YEATS4 in research samples . For more specialized applications, YEATS4 antibodies can be used in chromatin immunoprecipitation (ChIP) experiments to investigate its association with specific genomic regions, particularly in the context of its role in histone modification recognition and transcriptional regulation .

What are the proper storage and handling conditions for YEATS4 antibodies?

The optimal storage conditions for YEATS4 antibodies typically involve maintaining them at -20°C for long-term preservation of antibody activity and stability . For short-term storage (typically up to a few weeks), refrigeration at 2-8°C is generally acceptable, though specific recommendations may vary by manufacturer .

When working with YEATS4 antibodies, it's advisable to aliquot the stock solution to minimize freeze-thaw cycles, which can degrade antibody quality over time. Most commercially available YEATS4 antibodies have a shelf life of approximately 12 months when stored properly according to manufacturer guidelines .

During experimental procedures, YEATS4 antibodies should be kept on ice when in use and returned to appropriate storage conditions promptly. For shipping purposes, YEATS4 antibodies are typically transported on blue ice to maintain their integrity . Specific handling recommendations may vary between different antibody preparations, so researchers should always consult the product-specific documentation for detailed instructions.

How can researchers validate the specificity of YEATS4 antibodies?

Validating YEATS4 antibody specificity is critical for ensuring experimental reliability. A comprehensive validation approach should include multiple complementary methods:

First, perform Western blot analysis using both positive and negative controls. A well-validated YEATS4 antibody should detect a band at approximately 26.5 kDa in cells expressing YEATS4 . One effective control strategy is to compare detection between non-transfected cells and cells transiently transfected with the YEATS4 gene, as demonstrated in validation studies where 293 cell lysates showed clear differential detection patterns .

Second, implement genetic approaches by comparing antibody reactivity in wild-type cells versus YEATS4 knockout cells. The search results describe CRISPR-Cas9 systems used to generate YEATS4 knockout cells, which provide excellent negative controls for antibody validation . The complete absence of signal in knockout cells strongly supports antibody specificity.

Third, use immunoprecipitation followed by mass spectrometry to confirm that the antibody is capturing the authentic YEATS4 protein. This approach can also identify potential cross-reactive proteins . Additionally, peptide competition assays, where pre-incubation of the antibody with the immunizing peptide blocks specific binding, provide further evidence of specificity.

Finally, compare results across multiple YEATS4 antibodies raised against different epitopes. Concordant results with antibodies recognizing distinct regions of YEATS4 substantially increases confidence in specificity.

What is the role of YEATS4 in cancer progression and how can antibodies help study this function?

YEATS4 plays significant roles in cancer progression through multiple mechanisms, and antibodies are essential tools for investigating these functions. Research has demonstrated that YEATS4 is amplified in various cancers, including glioblastomas and bladder cancer, and its elevated expression correlates with poor clinical outcomes .

In breast cancer, YEATS4 expression increases with disease progression and correlates with poor prognosis and distant metastasis. YEATS4 overexpression strengthens the malignant characteristics of breast cancer cells both in vitro and in vivo, particularly by inducing epithelial-to-mesenchymal transition (EMT) and enhancing metastatic capability .

Researchers can use YEATS4 antibodies in multiple experimental approaches to study these cancer-related functions:

• Immunohistochemistry to analyze YEATS4 expression patterns in patient tumor samples

• Western blotting to compare YEATS4 protein levels across different cancer cell lines

• Chromatin immunoprecipitation to investigate how YEATS4 regulates gene expression in cancer cells, particularly its interaction with the ZEB1 promoter region in breast cancer

• Proximity labeling assays to identify YEATS4-interacting proteins in cancer cells, providing insights into its regulatory networks

What are the known post-translational modifications of YEATS4 and how do they regulate its function?

YEATS4 undergoes several post-translational modifications that critically regulate its stability and function, with acetylation and ubiquitination being particularly important.

Acetylation: Research has identified that YEATS4 is acetylated at lysine residues K64, K65, and K69, mediated by the lysine acetyltransferase KAT8 . These acetylation events are crucial for stabilizing YEATS4 by preventing its degradation. Mass spectrometry analysis of purified SFB-YEATS4 proteins co-expressed with HA-KAT8 and treated with trichostatin A (TSA) and nicotinamide (NAM) has been instrumental in identifying these specific acetylation sites . The functional significance of these acetylation events is demonstrated by the finding that KAT8-mediated YEATS4 acetylation promotes DNA repair and tumor growth, and consequently results in cisplatin (DDP) resistance in bladder cancer .

Ubiquitination: YEATS4 is subject to ubiquitin-mediated proteasomal degradation, regulated by the E3 ubiquitin ligase HUWE1 . This was discovered through a Protein Stability Regulators Screening Assay (ProSRSA) combined with CRISPR-Cas9 screening targeting ubiquitin family genes. Importantly, KAT8-mediated acetylation of YEATS4 at K64, K65, and K69 blocks HUWE1 binding, thus preventing ubiquitination and subsequent degradation .

To study these modifications experimentally, researchers can use several approaches:

• Generate acetylation-mimetic or acetylation-deficient mutants (e.g., K→Q or K→R substitutions at positions 64, 65, and 69) to examine the functional consequences

• Employ site-specific acetylation antibodies to detect acetylated YEATS4

• Use proteasome inhibitors (e.g., bortezomib) and protein synthesis inhibitors (e.g., cycloheximide) in combination with Western blotting to assess YEATS4 protein stability

• Perform co-immunoprecipitation experiments to analyze interactions between YEATS4 and its regulators (KAT8, HUWE1) under various conditions

How does YEATS4 contribute to epigenetic regulation through histone interactions?

YEATS4 functions as an important epigenetic regulator through its interactions with acetylated histones and its participation in chromatin remodeling complexes. The YEATS domain of YEATS4 has been identified as a specialized "reader" of histone acetylation marks, allowing the protein to recognize specific modifications on histone tails and facilitate downstream chromatin-dependent processes .

In breast cancer, YEATS4 has been shown to mediate histone H3K27 acetylation (H3K27ac) at specific sites of the ZEB1 promoter, thereby regulating ZEB1 expression at the transcriptional level . This mechanism directly links YEATS4 to the regulation of epithelial-to-mesenchymal transition (EMT), as ZEB1 is a master regulator of this process. Experimental depletion of ZEB1 blocks YEATS4-induced EMT, migration, invasion, and metastasis, confirming the functional importance of this regulatory axis .

Researchers investigating YEATS4's epigenetic functions can employ the following experimental approaches:

• Chromatin immunoprecipitation (ChIP) assays to map YEATS4 binding sites across the genome and identify associated histone modifications

• ChIP-sequencing to generate genome-wide profiles of YEATS4 occupancy and correlate these with specific histone acetylation patterns

• RNA-sequencing following YEATS4 knockdown or overexpression to identify genes regulated by YEATS4-dependent epigenetic mechanisms

• Co-immunoprecipitation experiments to identify protein complexes containing YEATS4 and other chromatin regulators

• In vitro histone peptide binding assays to characterize the specificity of the YEATS domain for different histone modifications

What experimental approaches can be used to study YEATS4 protein-protein interactions?

Investigating YEATS4 protein-protein interactions is essential for understanding its biological functions and regulatory mechanisms. Several sophisticated experimental approaches can be employed:

Proximity Labeling Assay: This technique, as described in the research literature, is particularly powerful for identifying proteins that interact with YEATS4 in living cells . By expressing YEATS4 fused to a biotin ligase (TurboID), researchers can label proteins in close proximity to YEATS4. The procedure involves treating cells expressing YEATS4-V5-TurboID with biotin for 30 minutes, followed by cell lysis, sonication, and enrichment of biotinylated proteins using streptavidin beads. After washing with various buffers (1 M KCl, 0.1 M Na₂CO₃, 2 M urea), the captured proteins can be identified by mass spectrometry .

Co-immunoprecipitation (Co-IP): Traditional or tandem affinity purification Co-IP can be used to isolate YEATS4 complexes. The search results describe experiments using SFB-tagged YEATS4, which enables sequential purification through streptavidin beads followed by Flag antibody immunoprecipitation, improving specificity .

CRISPR-based Screening: The Protein Stability Regulators Screening Assay (ProSRSA) combined with CRISPR-Cas9 library screening has been successfully employed to identify proteins that regulate YEATS4 stability . This approach involves creating a cell line expressing a dual-reporter system (DsRed-P2A-EGFP-YEATS4) and using CRISPR to knockout ubiquitin family genes. Flow cytometry sorting based on EGFP/DsRed ratio allows identification of genes affecting YEATS4 protein levels.

Yeast Two-hybrid Screening: Although not specifically mentioned in the search results, this is a complementary approach to identify direct YEATS4 binding partners, particularly for mapping interaction domains.

Bimolecular Fluorescence Complementation (BiFC): This technique can visualize protein-protein interactions in living cells by fusing YEATS4 and potential interaction partners with complementary fragments of a fluorescent protein.

For all these approaches, appropriate controls are essential, including validation of interactions through reverse Co-IP, domain mapping to identify interaction regions, and functional studies to determine the biological significance of identified interactions.