MEF2B Antibody

What is MEF2B and how are antibodies used to study its role in lymphomagenesis?

MEF2B (Myocyte Enhancer Factor 2B) is a transcription factor with crucial roles in B-cell development and lymphomagenesis. MEF2B antibodies are essential tools for investigating this protein's function in normal and malignant contexts.

MEF2B has recurrent mutation hotspots at K4, Y69, and D83 in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma, suggesting its critical role in lymphoma development . When studying MEF2B in lymphoma contexts, researchers typically employ antibodies in multiple applications:

• Chromatin immunoprecipitation (ChIP): To identify genome-wide MEF2B binding sites

• Immunohistochemistry/Immunofluorescence: To examine expression patterns in tissue samples

• Western blotting: To quantify protein levels and detect post-translational modifications

• Co-immunoprecipitation: To identify protein-protein interactions

For optimal results, ChIP-certified antibodies are recommended when mapping genome-wide binding patterns . Research has demonstrated that MEF2B predominantly binds to GC-specific enhancer regions, with approximately 60% of MEF2B-bound regions overlapping with enhancers and super-enhancers in germinal center B cells .

What validation methods should be implemented when selecting MEF2B antibodies for research?

Selecting properly validated MEF2B antibodies is critical for reliable experimental results. According to established validation protocols, researchers should consider antibodies that have undergone:

• Standard validation:

  • Concordance with protein characterization data in UniProtKB/Swiss-Prot database

  • Validation scores typically categorized as Supported, Approved, or Uncertain

• Enhanced validation through at least one of the following:

  • siRNA knockdown: Measuring decrease in antibody staining upon target protein downregulation

  • Independent antibody validation: Comparing staining patterns of antibodies targeting different epitopes

  • Tagged GFP cell lines: Evaluating signal overlap between antibody staining and GFP-tagged protein

• Application-specific validation:

  • For immunohistochemistry: Validated staining patterns across 44 normal tissues

  • For immunocytochemistry: Validated protein localization in cell lines against external evidence

When selecting antibodies for specific applications, researchers should verify that validation has been performed in the intended application (IHC, ICC-IF, WB, ChIP, etc.) to ensure reliability .

How should researchers optimize ChIP-seq protocols when using MEF2B antibodies?

ChIP-seq with MEF2B antibodies requires specific optimization for successful genome-wide binding site mapping. Based on published protocols, researchers should:

• Antibody selection and validation:

  • Use ChIP-certified antibodies (e.g., anti-MEF2B polyclonal antibodies at 0.3 mg/ml concentration)

  • For tagged MEF2B studies, V5 antibodies have been successfully employed for ChIP-seq

• Binding reaction optimization:

  • Use conditions similar to: 150 mM NaCl, 10 mM HEPES (pH 7.6), 0.5 mM EDTA, 0.5 mM DTT, 0.05 mg/ml BSA, and 5% glycerol

  • For MEF2B DBD dimer studies, 20 nM protein concentration has proven effective

• Peak identification and analysis:

  • Use tools like ChIPseek for peak identification

  • Apply GREAT tool to identify genes potentially regulated by peak regions

  • Use rank product scores to prioritize candidate direct target genes based on fold change and proximity to peaks

• Controls:

  • Include input controls and negative controls (non-specific IgG)

  • For tagged MEF2B, include empty vector controls

Following this protocol has enabled researchers to identify approximately 20,000 MEF2B binding peaks, with 15% associated with promoter regions, 35% intragenic, and 50% intergenic .

How can researchers interpret MEF2B binding patterns in relation to transcriptional regulation?

Understanding MEF2B binding patterns requires comprehensive analysis of chromatin state and gene expression correlations:

• Integrating ChIP-seq with histone modifications:

  • MEF2B binding correlates with active regulatory elements marked by:

    • Promoters: H3K4me3+ and H3K27Ac+

    • Enhancers: H3K4me1+ and H3K27Ac+ (intergenic or intragenic)

  • Approximately 60% of MEF2B-bound regions overlap with enhancers and super-enhancers

• Correlating binding with gene expression:

  • To identify direct targets, integrate:

    • ChIP-seq data from human GC B cells

    • RNA-seq data from relevant cell populations (e.g., naïve, GC, memory B cells)

    • Gene expression data from knockout models

  • MEF2B predominantly functions as a transcriptional activator, with 89% of direct target genes showing increased expression

• Motif analysis at binding sites:

  • MEF2B binds to MEF2 DNA binding motifs

  • Co-enrichment for motifs of transcription factors involved in GC reaction (IRF, STAT, ETS family)

  • MEF2B uses base readout at its half-sites combined with shape readout at the center of its degenerate motif

This integrated approach has revealed that MEF2B directly regulates genes involved in cell cycle, DNA replication and repair, apoptosis, and germinal center B-cell confinement .

What methodologies are effective for studying MEF2B mutations using antibodies?

MEF2B mutations (particularly at K4, Y69, and D83) are recurrent in lymphomas, requiring specific methodologies for their study:

• Detection of mutant proteins:

  • Express tagged versions of wild-type and mutant MEF2B (e.g., FLAG-HA-MEF2B) in appropriate cell lines

  • Use tag-specific antibodies for immunoprecipitation and detection

  • Validate antibody recognition of mutant proteins vs. wild-type

• Comparative analysis of wild-type vs. mutant binding sites:

  • Perform parallel ChIP-seq with wild-type and mutant MEF2B

  • Compare genome-wide distribution patterns

  • Identify differential binding sites

• Functional analysis:

  • Use antibodies to monitor protein half-life after cycloheximide treatment

  • Quantify using densitometry of western blots

  • For K4E, Y69H, and D83V mutations, decreased capacity to promote gene expression has been observed in both HEK293A and DLBCL cells

• Interaction studies:

  • Use immunoprecipitation followed by mass spectrometry to identify differential protein interactions

  • Sequential IP (FLAG followed by HA) can be performed for double-tagged proteins

  • Analyze eluates by trypsin digestion and LC-MS/MS

These methodologies have revealed that MEF2B mutations decrease target gene activation and alter cell migration, providing insight into lymphoma development mechanisms .

What techniques are optimal for detecting MEF2B post-translational modifications?

MEF2B undergoes various post-translational modifications, particularly phosphorylation, which can be detected using specialized techniques:

• Phos-tag gel approach:

  • Effectively separates phosphorylated MEF2B species

  • Has been successfully applied to normal and malignant GC B cells

  • Allows visualization of multiple phosphorylation states

• Mass spectrometry for phosphosite mapping:

  • Immunoprecipitate MEF2B using sequential FLAG-HA purification

  • Perform trypsin digestion of purified protein

  • Analyze by nano-scale reverse-phase HPLC followed by electrospray ionization and LTQ Orbitrap Velos Pro ion-trap mass spectrometry

  • This approach has identified phosphorylation at T196 in both isoforms A and B, and isoform A-specific phosphorylation at S310, T313, and S324

• Antibody-based detection:

  • Western blotting with phospho-specific antibodies (when available)

  • For total MEF2B detection, antibody concentration of 0.3 mg/ml has been validated

  • Validate phospho-specific antibodies using lambda phosphatase treatment as a negative control

• Functional studies:

  • Combine detection of phosphorylation with functional assays to determine impact on:

    • DNA binding capability

    • Transcriptional activity

    • Protein stability and half-life

    • Protein-protein interactions

These approaches have helped characterize the complex regulatory mechanisms controlling MEF2B activity in normal and pathological contexts.

How can researchers analyze MEF2B-DNA binding mechanisms?

Studying MEF2B-DNA interactions requires specialized techniques to understand binding specificity and structural interactions:

• SELEX-seq approach:

  • Implement binding reactions with 200 nM SELEX-library DNA and 20 nM MEF2B DBD dimer

  • Conduct in buffer containing 150 mM NaCl, 10 mM HEPES (pH 7.6), 0.5 mM EDTA, 0.5 mM DTT, 0.05 mg/ml BSA, 5% glycerol

  • Use 8% polyacrylamide gel for EMSA

  • Isolate bound fragments, purify, and sequence

• Molecular dynamics (MD) simulations:

  • Start with co-crystal structure (e.g., PDB ID: 1TQE)

  • Solvate with explicit water molecules

  • Model with AMBER99-parmbsc1 force field

  • Equilibrate in NPT ensemble at 300K and 1 bar

  • Analyze trajectories by clustering and calculating interface residue distances

• Crystallographic analysis:

  • Has revealed that MEF2B uses base readout at half-sites combined with shape readout at the center of the motif

  • A-tract polarity dictates binding nuances

  • The MEF2D-MRE complex has been characterized at atomic resolution

• Binding motif analysis:

  • MEF2B binding sites are enriched for MEF2 DNA binding motifs

  • Also enriched for motifs of IRF, STAT, and ETS family transcription factors

  • DNA shape and flexibility contribute to binding specificity

These techniques have revealed that MEF2B binding specificity is determined by a combination of base-specific contacts and DNA shape recognition at the central AT-rich region .

What methods are effective for studying MEF2B's role in lymphoma development?

To investigate MEF2B's contribution to lymphomagenesis, researchers have employed several complementary approaches:

• Transgenic mouse models:

  • Conditional knockout models:

    • Generated by inserting loxP sites into intronic regions upstream of exon 2 and downstream of exon 9

    • Crossed with CD21-Cre or Cγ1-Cre mice to achieve B-cell specific deletion

  • Mutation knock-in models:

    • MEF2D-HNRNPUL1 (MH) knock-in mice develop progressive disease from impaired B-cell development to pre-leukemia

    • When combined with NRAS G12D, MEF2D fusions drive aggressive BCP-ALL

• Transcriptome and epigenome analysis:

  • RNA-seq and microarray to identify MEF2B target genes

  • ChIP-seq to map genome-wide binding sites

  • Integration of datasets to identify direct target genes

• Functional cellular assays:

  • Cell migration and chemotaxis assays

  • Cell viability and proliferation studies

  • Changes in expression of mesenchymal markers

• Therapeutic intervention studies:

  • Targeting MEF2B-driven transactivation (e.g., using HDAC inhibitors like panobinostat)

  • Combining targeted interventions with chemotherapy

These approaches have revealed that MEF2B regulates key lymphoma drivers including BCL6, MYC, TGFB1, CARD11, NDRG1, RHOB, BCL2, and JUN , and that mutations in MEF2B reduce its capacity to promote expression of target genes that would otherwise regulate cell migration and survival .

How can researchers characterize MEF2B protein complexes using co-immunoprecipitation?

Effective characterization of MEF2B-containing protein complexes requires optimized co-immunoprecipitation (co-IP) protocols:

• Expression system setup:

  • Generate cell lines stably expressing tagged versions (e.g., FLAG-HA-MEF2B)

  • Include both wild-type and mutant variants (e.g., D83V)

  • Use appropriate control cells (empty vector)

• Sequential immunoprecipitation protocol:

  • Prepare whole-cell protein lysates in IP buffer

  • First immunoprecipitation: anti-FLAG antibody-conjugated M2 agarose beads

  • Elution with FLAG peptide (0.25 mg/ml)

  • Second immunoprecipitation: anti-HA antibody-conjugated agarose beads

  • Final elution with HA peptide (0.25 mg/ml)

• Mass spectrometry analysis:

  • Digest eluates with trypsin

  • Load on nano-scale reverse-phase HPLC capillary column

  • Subject to electrospray ionization and LTQ Orbitrap Velos Pro ion-trap mass spectrometry

  • Use Sequest software to match peptide sequences to protein databases

• Data analysis:

  • Prune non-specific interactors detected in negative controls

  • Rank candidate interactors based on peptide counts

  • Compare interactors between wild-type and mutant MEF2B

  • Validate key interactions by reciprocal co-IP or functional assays

This approach has identified important MEF2B interaction partners and revealed how mutations can affect the composition of MEF2B-containing complexes, contributing to understanding of lymphomagenesis mechanisms .

What immunohistochemistry protocols are optimal for MEF2B detection in tissue samples?

For reliable MEF2B detection in tissue samples, researchers should follow these optimized immunohistochemistry protocols:

• Tissue preparation:

  • Use 3-μm-thick formalin-fixed, paraffin-embedded (FFPE) sections

  • Heat-induced epitope retrieval is critical:

    • For MEF2B: citrate buffer (pH 6.0)

    • For co-staining with PNA: EDTA buffer (pH 8.0)

• Antibody incubation:

  • Primary anti-MEF2B antibody: overnight incubation at 4°C

  • Washing: 1X PBS+0.1% Tween20

  • For fluorescent detection:

    • EnVision system with polymer-enhanced HRP-conjugated secondary antibody

    • Tyramide-fluorochrome amplification

• Co-staining options:

  • With BCL6: anti-BCL6 antibody followed by Cy3-conjugated secondary antibody

  • With PNA: biotin-conjugated anti-PNA antibody followed by Cy3-conjugated streptavidin or AEC substrate

• Validation strategies:

  • Include positive controls (germinal center B cells)

  • Include negative controls (tissues known to lack MEF2B expression)

  • Compare staining patterns with validated antibodies targeting different epitopes