KMT2D Antibody

What is KMT2D and why is it significant in cancer research?

KMT2D is the largest H3K4 methyltransferase in the COMPASS/Set1 family and represents one of the most frequently mutated genes across multiple cancer types. The enzymatic function of KMT2D depends on several conserved C-terminal domains, including a PHD domain, two FY-rich motifs (FYRC and FYRN), and a catalytic SET domain . It plays a crucial role as a bona fide tumor suppressor gene in follicular lymphoma and diffuse large B-cell lymphoma .

In recent studies, KMT2D mutations have been identified in over 20% of lung squamous cell carcinoma (LUSC) cases, where it functions as a key regulator of tumorigenesis . When KMT2D is deleted in experimental models, it can transform lung basal cell organoids to LUSC and promote overgrowth and squamous differentiation, consistent with early malignant transformation . Additionally, KMT2D loss increases activation of receptor tyrosine kinases (RTKs) EGFR and ERBB2, partly through reprogramming the chromatin landscape .

What applications are validated for KMT2D antibodies?

KMT2D antibodies have been validated for multiple experimental applications with specific dilution recommendations:

For IHC applications, it is recommended to perform antigen retrieval with TE buffer pH 9.0, with citrate buffer pH 6.0 as an alternative . Researchers should titrate the antibody in each testing system to obtain optimal results as outcomes can be sample-dependent .

How can researchers validate the specificity of KMT2D antibodies?

Antibody specificity can be validated through several approaches:

First, verify antibody reactivity using positive controls such as HEK-293 cells for Western blot applications, which show the expected 593 kDa molecular weight for the full-length KMT2D protein . Observe caution that both calculated and observed molecular weights of KMT2D are approximately 593 kDa, making it a challenging protein to resolve on standard gels .

Second, employ genetic approaches using KMT2D knockdown or knockout models. Evidence from the literature confirms the specificity of certain antibodies, as demonstrated in studies where KMT2D protein loss was verified by Western blot after CRISPR/Cas9-mediated gene targeting . Similarly, samples carrying biallelic truncating mutations that eliminate the epitope recognized by C-terminal targeted antibodies showed absence of full-length KMT2D protein expression .

Third, consider epitope location when selecting antibodies. For instance, antibodies targeting the C-terminal portion of KMT2D can identify full-length protein but may not detect truncated versions resulting from genetic alterations .

How do KMT2D mutations affect antibody reactivity and experimental design?

KMT2D undergoes two primary types of genomic alterations: truncation and missense mutations . These mutations can significantly impact antibody recognition and experimental outcomes.

For truncation mutations, C-terminal-directed antibodies will fail to detect the protein as the epitope is typically lost. Research has shown that in samples with biallelic truncating mutations, immunoblot analysis using C-terminal antibodies revealed complete absence of intact KMT2D protein . Interestingly, even when using antibodies directed against the N-terminal half of KMT2D, detection of truncated proteins remains challenging despite expression of the mutant cDNA .

For missense mutations, most maintain protein stability levels comparable to wild-type. In a systematic study testing 16 DLBCL-derived mutant alleles alongside three germline variants as controls, all but one (S5404F) produced similar amounts of both mRNA and protein as the wild-type allele, indicating that missense mutations generally do not affect KMT2D protein stability .

When designing experiments involving KMT2D detection in cancer samples, researchers should employ multiple antibodies targeting different epitopes and combine this approach with genetic sequencing to fully characterize KMT2D status. Additionally, functional assays measuring H3K4 methyltransferase activity should be considered, as mutations can impair enzymatic function without affecting protein expression .

What methodological approaches are recommended for studying KMT2D's role in epigenetic regulation?

To effectively study KMT2D's role in epigenetic regulation, a multi-faceted approach is recommended:

For ChIP applications, antibodies targeting KMT2D can identify genomic binding sites. Studies have shown that over 95% of KMT2D-bound chromatin in germinal center B cells is decorated by H3K4me1 (36% of peaks) and/or H3K4me3 (62% of peaks) . This finding underscores KMT2D's role as a non-redundant methyltransferase that controls the methylation state of numerous regions in mature B cell compartments.

For epigenomic profiling, researchers should combine KMT2D ChIP with histone modification mapping (particularly H3K4me1/me3) and assessment of chromatin accessibility. This integrated approach can reveal how KMT2D loss affects enhancer activation and gene expression programs, as demonstrated in studies where KMT2D deficiency led to diminished global H3K4 methylation in germinal-center B-cells and DLBCL cells .

For functional validation, genetic manipulation of KMT2D (through conditional deletion, CRISPR-mediated knockout, or expression of mutant forms) combined with transcriptomic and epigenomic profiling provides comprehensive insights. For instance, conditional deletion of Kmt2d early during B cell development resulted in increased germinal center B-cells and enhanced B cell proliferation in mice , demonstrating that timing of KMT2D loss can significantly impact phenotypic outcomes.

How does combined haploinsufficiency of KMT2D with other epigenetic regulators affect experimental outcomes?

Recent research indicates that KMT2D and CREBBP (another epigenetic regulator) are paradoxically co-mutated in lymphomas despite having similar enhancer regulatory functions . This suggests important mechanistic implications for researchers studying epigenetic regulators.

Studies demonstrate that combined haploinsufficiency of Crebbp and Kmt2d (referred to as C+K) accelerates lymphomagenesis . Specifically, this combined loss causes disruption of super-enhancers driving immune synapse genes, leading to reduction of CD8 cells in lymphomas—directly linking super-enhancer function to immune surveillance and potentially contributing to immunotherapy resistance .

When designing experiments investigating KMT2D function, researchers should consider potential cooperative effects with other epigenetic modifiers. Transcriptional analysis revealed that dual CREBBP and KMT2D haploinsufficiency results in cooperative repression of genes related to germinal center exit/immune synapse response and DNA repair, while simultaneously inducing biosynthetic programs normally restricted to centrocytes undergoing T cell help .

For experimental design, this suggests that single-gene manipulation studies may not fully capture the complexity of KMT2D function in disease contexts where multiple epigenetic regulators are altered. Integration of multi-omics approaches (including trajectory analysis of gene expression changes) becomes essential to elucidate the full spectrum of biological effects resulting from combinatorial epigenetic dysregulation .

How can KMT2D antibodies be utilized to investigate its role in lung cancer development?

KMT2D has emerged as a critical player in lung squamous cell carcinoma (LUSC), with mutations present in over 20% of cases . Researchers can employ KMT2D antibodies in multiple applications to investigate its role in lung cancer:

For immunohistochemical analysis, KMT2D antibodies can be used to assess protein expression in LUSC patient samples compared to normal lung tissues, as studies have shown significantly lower KMT2D expression in LUSC compared to normal lung tissue . The recommended dilution range of 1:50-1:500 for IHC applications allows for optimization depending on tissue type and fixation methods .

For mechanistic studies, Western blot analysis using KMT2D antibodies can verify knockout efficiency in experimental models, as demonstrated in studies utilizing CRISPR/Cas9 sgRNAs targeting Kmt2d in organoid models . This approach helps establish critical causative relationships between KMT2D loss and phenotypic changes in lung cancer progression.

For investigation of downstream effects, researchers can combine KMT2D expression analysis with assessment of receptor tyrosine kinase (RTK) activity, particularly EGFR and ERBB2, as KMT2D loss increases activation of these pathways . This multi-parametric approach helps identify therapeutic vulnerabilities in KMT2D-deficient tumors.

What considerations are important when using KMT2D antibodies to study B-cell development and lymphoma?

When investigating KMT2D in B-cell development and lymphoma, several important considerations should guide experimental design:

First, understand the expression pattern of KMT2D across B-cell developmental stages. Studies indicate KMT2D is expressed in all mature B cell compartments, including naïve, germinal center, and memory B cells . Co-immunofluorescence analysis using KMT2D and germinal center-specific markers like BCL6 in reactive human tonsils has confirmed KMT2D staining in nuclei across all mature B cell compartments, including germinal centers .

Second, consider the timing of KMT2D loss in experimental models. Research has demonstrated that conditional deletion of Kmt2d early during B cell development results in increased germinal center B-cells and enhanced B cell proliferation, while deletion after initiation of the germinal center reaction has different outcomes . This temporal aspect is critical when designing studies to recapitulate human disease pathogenesis.

Third, interpret results in the context of genetic cooperation. In BCL2-overexpressing mouse models that develop germinal center-derived lymphomas resembling human tumors, genetic ablation of Kmt2d leads to a further increase in tumor incidence . This indicates that KMT2D loss synergizes with other oncogenic events, which should be considered when analyzing human lymphoma samples with multiple genetic alterations.

How should researchers troubleshoot inconsistent KMT2D detection in different experimental systems?

Inconsistent detection of KMT2D across experimental systems can arise from several factors that require specific troubleshooting approaches:

For Western blot applications, KMT2D's large size (593 kDa) presents significant technical challenges . Researchers should employ low percentage (3-5%) SDS-PAGE gels or gradient gels optimized for high molecular weight proteins. Transfer efficiency should be monitored using appropriate size markers, and extended transfer times may be necessary. The recommended dilution range of 1:2000-1:16000 for WB applications allows flexibility for optimization based on specific sample types .

For detection of endogenous KMT2D in cell lines with potential mutations, consider the epitope location of your antibody. If using a C-terminal-directed antibody, samples with truncation mutations will not show signal despite expressing truncated protein . Using antibodies targeting different regions of KMT2D can help resolve such discrepancies.

For immunohistochemistry applications, antigen retrieval conditions are critical. The recommended protocol suggests TE buffer at pH 9.0, with citrate buffer pH 6.0 as an alternative . Systematic optimization of antigen retrieval methods, antibody concentration, and incubation conditions may be necessary for each tissue type.

When encountering negative results, validate antibody performance using known positive controls (HEK-293 cells for WB; human breast cancer, lymphoma, or colon cancer tissues for IHC) . Additionally, genetic confirmation through sequencing can help interpret negative immunostaining results, particularly in tumor samples where KMT2D mutations are common.

What strategies can help distinguish between wild-type and mutant KMT2D in research samples?

Distinguishing between wild-type and mutant KMT2D forms presents unique challenges that require integrated approaches:

For missense mutations, most mutant KMT2D proteins show similar expression levels to wild-type . In these cases, functional assays measuring methyltransferase activity provide more informative readouts than expression analysis alone. Researchers can assess H3K4 methylation status at KMT2D target loci as a surrogate for KMT2D function.

Genetic confirmation through DNA sequencing remains essential, particularly in primary tumor samples where multiple genetic alterations may coexist. This can be complemented with transcriptomic analysis to assess expression levels of KMT2D target genes, providing functional insights even when protein detection is challenging.

For complex samples containing mixed populations, consider single-cell approaches combining protein detection (through immunofluorescence or mass cytometry) with genetic analysis to correlate KMT2D protein status with mutational profile at the single-cell level.

How are KMT2D antibodies advancing our understanding of epigenetic dysregulation in cancer?

KMT2D antibodies have been instrumental in elucidating the epigenetic mechanisms underlying cancer development and progression. Through immunoblotting, immunohistochemistry, immunofluorescence, and chromatin immunoprecipitation approaches, these antibodies have helped establish KMT2D as a bona fide tumor suppressor in multiple cancer types, including follicular lymphoma, diffuse large B-cell lymphoma, and lung squamous cell carcinoma .

Key insights facilitated by KMT2D antibody-based research include the discovery that over 95% of KMT2D-bound chromatin in germinal center B cells is decorated by H3K4me1 and/or H3K4me3, establishing KMT2D as a non-redundant methyltransferase controlling the methylation state of numerous genomic regions in mature B cells . Furthermore, antibody-based approaches have demonstrated that KMT2D deletion can transform lung basal cell organoids to squamous cell carcinoma, highlighting its critical role in maintaining epithelial cell identity .

The integration of antibody-based detection methods with genetic, transcriptomic, and epigenomic analyses has revealed that KMT2D cooperates with other epigenetic regulators like CREBBP, and their combined haploinsufficiency accelerates lymphomagenesis through disruption of super-enhancers driving immune synapse genes . This mechanistic insight provides a potential explanation for immunotherapy resistance in certain cancers.