SETD1B Antibody

What is SETD1B and what are its primary biological functions?

SETD1B (SET Domain Containing 1B), also known as KMT2G, functions as a histone methyltransferase that catalyzes the transfer of methyl groups from S-adenosyl-L-methionine to the epsilon-amino group of lysine 4 on histone H3 (H3K4) through a non-processive mechanism . This enzyme is capable of generating all three methylation states (H3K4me1, H3K4me2, and H3K4me3) at active chromatin sites where transcription and DNA repair processes occur . SETD1B forms part of the chromatin remodeling machinery and plays an essential role in regulating the transcriptional programming of multipotent hematopoietic progenitor cells and lymphoid lineage specification during hematopoiesis . Recent studies have also identified SETD1B as a critical mediator of broad H3K4me3 formation, facilitating RNA Polymerase II recruitment to active genes .

How can researchers detect SETD1B expression in different tissue samples?

Researchers can detect SETD1B expression through several complementary methods:

• Immunohistochemistry (IHC): Validated antibodies such as the rabbit polyclonal SETD1B antibody (ab113984) can be used for IHC on formalin/PFA-fixed paraffin-embedded tissue sections . The recommended dilution is 1/1000 (1μg/ml) with DAB detection systems providing good visualization. Studies have successfully applied this approach to human ovarian carcinoma tissues .

• Quantitative RT-PCR: For mRNA expression analysis, researchers have successfully quantified SETD1B expression in sorted cell populations using specific primers . This approach has revealed that SETD1B is expressed across various hematopoietic lineages, with particularly high expression reported in CD11b+ Gr-1 high granulocytes in some studies .

• Immunoprecipitation (IP): SETD1B antibodies can be utilized for protein complex isolation, enabling the identification of interaction partners in chromatin remodeling complexes .

What experimental models are available for studying SETD1B function?

Several experimental models have been developed to study SETD1B function:

• Conditional knockout mouse models: Researchers have generated Setd1b-floxed mouse lines using CRISPR/Cas9 technology . These models can be crossed with specific Cre lines for tissue-specific deletion:

  • Rosa26-Cre-ERT2 for tamoxifen-inducible, ubiquitous deletion

  • Vav-Cre for hematopoietic-specific deletion

  • Stra8-GFP Cre for male germ cell-specific deletion

• Cellular models: CRISPR/Cas9-mediated knockout of SETD1B in lymphoma cell lines (such as OCI-LY19) has been used to study its role in apoptosis regulation and drug sensitivity .

• Transplantation models: HSC-enriched bone marrow from Setd1b conditional mice can be transplanted into irradiated wild-type recipients, followed by tamoxifen induction, enabling the study of SETD1B function specifically in the hematopoietic system .

How does SETD1B contribute to lymphoma pathogenesis and therapeutic resistance?

SETD1B functions as a tumor suppressor in lymphoma, with mutations and deletions occurring in 7% of follicular lymphomas (FL) and 16% of diffuse large B cell lymphomas (DLBCL) . Deficiency in SETD1B confers striking resistance to the BCL2 inhibitor Venetoclax and experimental MCL-1 inhibitors, indicating its critical role in apoptotic regulation .

Mechanistically, SETD1B is required for the expression of several proapoptotic BCL2 family proteins including BIM and BIK . When SETD1B is inactivated, these pro-apoptotic factors are downregulated, resulting in apoptosis resistance. Research has shown that SETD1B loss typically coincides with mutations in another histone methyltransferase, KMT2D, suggesting cooperative effects in lymphomagenesis .

Notably, SETD1B mutations demonstrate a hotspot at position 8, where frameshift insertions replace a histidine (H) with a proline (P) and cause premature termination (H8Pfs*30) . Most SETD1B lesions appear to be heterozygous or subclonal in human lymphomas, based on variant allele frequency analyses .

What is the relationship between SETD1B and other epigenetic regulators in disease contexts?

Several important relationships have been identified:

• SETD1B and KMT2D cooperation: SETD1B deficiency frequently co-occurs with KMT2D mutations in lymphomas . In vivo experiments have demonstrated powerful additive effects when both genes are inactivated, resulting in accelerated lymphomagenesis with over 95% disease penetrance at 100 days, compared to 60% with either gene knockout alone .

• SETD1B and TP53: Human genetic analyses suggest a mutually exclusive relationship between SETD1B mutations and TP53 mutations, particularly in follicular lymphoma. In human FL cohorts (n=216), TP53 mutations (occurring in 10% of samples) were found exclusively in SETD1B wild-type tumors . This suggests these alterations may represent alternative pathways to lymphomagenesis.

• SETD1B and KDM5 demethylases: Inhibition of KDM5 histone H3K4 demethylases can restore BIM and BIK expression and synergize with Venetoclax in SETD1B-deficient lymphomas, representing a potential therapeutic strategy .

What are the technical considerations for optimizing SETD1B antibody specificity in research applications?

When working with SETD1B antibodies, researchers should consider these technical aspects:

• Antibody validation: Comprehensive validation is critical due to potential cross-reactivity with other SET domain-containing proteins. Validation should include:

  • Western blot analysis comparing wild-type and SETD1B-deficient samples

  • Immunoprecipitation followed by mass spectrometry

  • ChIP-seq using multiple independent antibodies targeting different epitopes

• Epitope selection: The search results indicate that antibodies targeting synthetic peptides within human SETD1B aa 350-400 have demonstrated effectiveness for IHC-P and IP applications . This region appears to be sufficiently unique to avoid cross-reactivity.

• Application-specific optimization:

  • For IHC: Validated protocols suggest 1/1000 dilution (1μg/ml) with DAB detection systems

  • For IP: Optimize extraction conditions to preserve protein complexes

  • For ChIP: Crosslinking conditions and sonication parameters must be optimized for studying SETD1B binding to chromatin

How can researchers assess SETD1B-mediated histone methylation changes?

Researchers can employ several complementary approaches:

• ChIP-seq analysis: This technique allows genome-wide profiling of H3K4me1, H3K4me2, and H3K4me3 distribution. Studies have successfully used this approach to identify broad H3K4me3 domains regulated by SETD1B, particularly in spermatids .

• Immunofluorescence: Using specific antibodies against H3K4me1 and H3K4me3, researchers can visualize changes in histone methylation patterns at the cellular level. This approach revealed that in SETD1B cKO testes, H3K4me3 but not H3K4me1 signals were greatly reduced .

• Western blotting: This technique can be used to quantify global levels of H3K4 methylation states in wild-type versus SETD1B-deficient cells.

• CUT&RUN or CUT&Tag: These newer techniques offer advantages over traditional ChIP by providing higher signal-to-noise ratios and requiring fewer cells, making them valuable for studying SETD1B-mediated histone modifications in rare cell populations.

What protocols are recommended for studying SETD1B's role in apoptosis regulation?

Based on the search results, the following methodological approaches have been validated:

• Cell death assays: OCI-LY19 cells treated with Venetoclax (2.5 μM) or control DMSO for 6 hours can be stained with APC-Annexin V and 7-aminoactinomycin D for flow cytometry analysis using an LSR Fortessa Cell Analyzer .

• Gene expression analysis: Quantitative RT-PCR using TaqMan Gene Expression Assays for apoptotic genes such as BIK (Hs00154189_m1) with β-actin as a housekeeping gene for normalization has been successfully employed .

• CRISPR/Cas9-mediated gene editing: This approach can be used to generate SETD1B-deficient cell lines for functional studies. Two validated sgRNAs targeting SETD1B have been reported (sgSetd1b and sgSetd1b[sg2]) .

• Combinatorial drug treatments: Testing SETD1B-deficient cells with combinations of BCL2 inhibitors (e.g., Venetoclax) and KDM5 inhibitors to restore apoptotic sensitivity represents a validated approach .

What are the validated approaches for studying SETD1B in hematopoietic development?

Several experimental approaches have been validated for studying SETD1B's role in hematopoiesis:

• Conditional gene deletion: Using Rosa26-Cre-ERT2 or Vav-Cre systems to delete SETD1B in hematopoietic cells, followed by comprehensive phenotypic analyses .

• Flow cytometric analysis: Researchers can quantify hematopoietic stem and progenitor cell populations using established marker panels. Studies have demonstrated that SETD1B ablation disturbs homeostasis of hematopoietic stem and progenitor cells (HSPCs) .

• Competitive transplantation assays: These can assess the functional capacity of SETD1B-deficient HSCs compared to wild-type counterparts.

• Histopathological examination: Analysis of bone marrow, spleen, and liver tissues from SETD1B-deficient mice has revealed important insights, including increased presence of immature myeloid cells, dysplastic megakaryocytes, and altered ratios of different hematopoietic lineages .

How might SETD1B antibodies contribute to developing novel therapeutic strategies for lymphoma?

Current research suggests several promising therapeutic avenues:

• Biomarker development: SETD1B mutational status could serve as a biomarker for predicting resistance to BCL2 inhibitors like Venetoclax. Antibody-based detection methods could help stratify patients likely to respond to these therapies .

• Combination therapies: The finding that KDM5 histone demethylase inhibitors can restore sensitivity to Venetoclax in SETD1B-deficient lymphomas suggests a potential combination therapy approach . Antibody-based assays could be used to monitor restoration of pro-apoptotic protein expression.

• Drug screening platforms: SETD1B antibodies could be incorporated into high-throughput screening platforms to identify compounds that mimic SETD1B function or bypass its deficiency in regulating apoptotic pathways.

• Selective targeting of SETD1B-mutant cells: Identifying unique vulnerabilities in SETD1B-deficient cells could lead to synthetic lethal therapeutic approaches that specifically target cancer cells while sparing normal tissues.

What are the emerging roles of SETD1B in developmental processes?

Recent research has uncovered several developmental functions of SETD1B:

• Spermatogenesis: SETD1B is crucial for proper temporal gene expression during spermatogenesis, mediating broad H3K4me3 formation and facilitating RNA Polymerase II recruitment . SETD1B deficiency leads to aberrant gene expression timing, with genes typically expressed in specific stages becoming inappropriately activated in other stages .

• Intellectual development: Human genetic studies have linked SETD1B mutations to intellectual disability syndromes, suggesting important roles in neuronal development and function .

• Hematopoietic lineage specification: SETD1B plays essential roles in regulating the transcriptional programming of multipotent hematopoietic progenitor cells and lymphoid lineage specification .

What are common challenges in using SETD1B antibodies and how can they be addressed?

Based on the available research, researchers may encounter these challenges:

• Detection sensitivity: In some contexts, SETD1B expression might be relatively low, making detection challenging. Potential solutions include:

  • Using amplification systems (such as tyramide signal amplification) for IHC

  • Concentrating protein samples for Western blotting

  • Employing more sensitive PCR techniques when analyzing gene expression

• Background signal: Non-specific binding can complicate interpretation of results. Strategies to minimize this include:

  • Thorough blocking steps with appropriate blocking reagents

  • Including validated negative controls (SETD1B-deficient samples)

  • Using monoclonal antibodies with higher specificity for critical applications

• Epitope masking: In fixed tissues, formaldehyde crosslinking may mask SETD1B epitopes. Antigen retrieval methods, such as heat-induced epitope retrieval in citrate buffer (pH 6.0), have been successfully employed for IHC applications .

• Antibody validation: Given SETD1B's similarity to other SET domain proteins, rigorous validation is essential. The research community should establish standard validation protocols including knockout controls, peptide competition assays, and orthogonal detection methods.