At3g23060 Antibody

What is At3g23060/AtBMI1C and what is its functional significance?

At3g23060 (AtBMI1C) is one of three BMI1 homologs in Arabidopsis thaliana, alongside AtBMI1A (At2G30580) and AtBMI1B (AT1G06770). AtBMI1C functions as a component of the Polycomb Repressive Complex 1 (PRC1) and is evolutionarily conserved with Psc and BMI1 proteins found in other organisms . The protein is localized in the nucleus and possesses H2A monoubiquitination activity, which plays a key role in epigenetic gene silencing mechanisms . AtBMI1C specifically participates in flowering time control by regulating the expression of FLOWER LOCUS C (FLC), with overexpression studies demonstrating both FLC suppression and FLOWER LOCUS T (FT) activation, resulting in early flowering phenotypes .

What are the specifications of the At3g23060 Antibody for research applications?

The At3g23060 Antibody (e.g., CSB-PA881793XA01DOA) is a rabbit-raised polyclonal antibody that targets recombinant Arabidopsis thaliana At3g23060 protein . It is supplied in liquid form, non-conjugated, and has been antigen affinity purified to ensure specificity . The antibody is specifically tested for ELISA and Western Blot applications for identifying At3g23060 protein . It is provided in a storage buffer containing 0.03% Proclin 300 as a preservative, 50% glycerol, and 0.01M PBS at pH 7.4 . This formulation helps maintain antibody stability during storage and use in laboratory settings.

How should researchers interpret Western Blot results when using At3g23060 Antibody?

When interpreting Western Blot results with At3g23060 Antibody, researchers should first validate the antibody's specificity by comparing signal patterns between wild-type and AtBMI1C-overexpressing lines. The expected molecular weight of AtBMI1C should be confirmed based on its amino acid sequence (UniProt: Q9LS86) . For H2A monoubiquitination studies, researchers should look for two distinct bands in nuclear histone extracts when using anti-ubiquitin antibodies, with the lower band representing monoubiquitinated H2A (uH2A) . An increase in H2A monoubiquitination should be observed in AtBMI1C-overexpressing lines compared to wild-type plants, while H2B monoubiquitination typically remains unchanged . When analyzing protein-protein interactions, co-immunoprecipitation experiments can validate interactions between AtBMI1C and other PRC1 components such as AtRING1A and AtRING1B.

What are the optimal storage and handling conditions for At3g23060 Antibody?

The At3g23060 Antibody requires careful storage to maintain its activity and specificity. Upon receipt, the antibody should be stored at either -20°C or -80°C, with -80°C being preferred for long-term storage . Repeated freeze-thaw cycles should be strictly avoided as they can degrade the antibody and reduce its effectiveness . For working solutions, small aliquots should be prepared and stored separately to minimize freeze-thaw cycles. When handling the antibody, researchers should use sterile techniques and maintain the cold chain during all procedures. The antibody is provided in a buffer containing 50% glycerol, which helps prevent complete freezing and reduces damage during freeze-thaw transitions . For routine laboratory use, antibody aliquots can be kept at 4°C for up to one week, but longer periods require freezing storage.

How can researchers optimize immunoprecipitation protocols using At3g23060 Antibody?

For successful immunoprecipitation with At3g23060 Antibody, researchers should:

• Start with fresh plant tissue (preferably young tissues where AtBMI1C is actively expressed)

• Use an optimized nuclear protein extraction buffer (containing protease inhibitors and phosphatase inhibitors)

• Pre-clear lysates with protein A/G beads to reduce non-specific binding

• Incubate lysates with At3g23060 Antibody at 4°C overnight using a 1:50 to 1:100 antibody dilution

• Capture antibody-protein complexes with protein A beads (since the antibody is raised in rabbit)

• Include stringent washing steps (at least 4-5 washes) with buffers of increasing stringency

• Validate results with appropriate controls, including:

  • IgG control to assess non-specific binding

  • Input control to confirm target protein presence

  • Reciprocal co-IP with antibodies against known interactors (AtRING1A, AtRING1B)
    This approach has been successfully used to demonstrate the physical interaction between AtBMI1C and other PRC1 components like AtRING1A and AtRING1B .

What methods can researchers use to analyze H2A monoubiquitination mediated by AtBMI1C?

To analyze H2A monoubiquitination mediated by AtBMI1C, researchers should implement the following methodology:

• Extract nuclear histones using acid extraction protocols optimized for plant tissues

• Separate histones using SDS-PAGE with 15-18% polyacrylamide gels

• Transfer proteins to PVDF membranes (preferred over nitrocellulose for histone studies)

• Block membranes with 5% non-fat milk in TBST

• Probe with anti-ubiquitin antibodies capable of detecting monoubiquitinated histones

• Distinguish between uH2A (lower band) and uH2B (upper band) based on molecular weight

• Quantify band intensities using densitometry software

• Normalize against total H2A or H3 levels as loading controls
  Comparative analysis between wild-type plants and AtBMI1C-overexpressing lines should show increased levels of H2A monoubiquitination in the overexpression lines, confirming AtBMI1C's enzymatic activity . This approach can be supplemented with chromatin immunoprecipitation (ChIP) assays to identify specific genomic regions targeted by AtBMI1C.

How does AtBMI1C function as part of the Polycomb Repressive Complex 1?

AtBMI1C functions as an integral component of the Arabidopsis PRC1 complex through several key mechanisms:

• Protein-Protein Interactions: AtBMI1C physically interacts with AtRING1A and AtRING1B, which are established components of PRC1 in Arabidopsis . These interactions have been confirmed through both yeast two-hybrid assays and pull-down experiments .

• Domain Requirements: The N-terminal domain of AtBMI1C, including the conserved RING domain, is required for these protein interactions, suggesting evolutionary conservation of functional domains .

• Enzymatic Activity: As part of PRC1, AtBMI1C exhibits H2A monoubiquitination activity, which contributes to chromatin remodeling and gene silencing . Overexpression of AtBMI1C leads to increased H2A monoubiquitination levels without affecting H2B monoubiquitination .

• Target Specificity: The AtBMI1C-containing PRC1 complex specifically targets and represses FLC chromatin to regulate flowering time, demonstrating functional specificity despite structural similarity to other BMI1 homologs .
  The incorporation of AtBMI1C into PRC1 appears to direct the complex toward specific genomic targets distinct from those regulated by AtBMI1A/B-containing complexes, suggesting functional specialization among PRC1 variants in Arabidopsis .

What is the relationship between AtBMI1C-mediated gene silencing and the canonical PRC2 pathway?

The relationship between AtBMI1C-mediated gene silencing and the canonical PRC2 pathway represents an intriguing deviation from the classical hierarchical model of Polycomb-mediated repression:

• Independent Mechanisms: In AtBMI1C-overexpressing lines, no change in H3K27me3 levels at FLC chromatin was detected, suggesting that AtBMI1C represses FLC independently of PRC2 activity .

• Hierarchical Reversal: This finding contradicts the classical model where PRC2 acts upstream of PRC1. In the case of AtBMI1C-mediated FLC repression, PRC1 activity appears to operate independently of PRC2-mediated H3K27 trimethylation .

• Functional Consequences: The suppression of FLC and activation of FT observed in AtBMI1C-overexpressing lines result in early flowering phenotypes, demonstrating the biological significance of this PRC2-independent pathway .

• Evolutionary Implications: This mechanism suggests that plants may have evolved alternative epigenetic regulatory pathways that differ from the canonical PRC1/PRC2 hierarchical model established in animals .
  This data indicates that in Arabidopsis, AtBMI1C-containing PRC1 complexes can function as primary repressors that do not require prior PRC2 activity, representing an important variation in epigenetic regulatory mechanisms between plants and animals.

How can researchers distinguish the functions of AtBMI1C from other BMI1 homologs in Arabidopsis?

Distinguishing the functions of AtBMI1C from its homologs AtBMI1A and AtBMI1B requires a multifaceted experimental approach:

• Use specific antibodies that can distinguish between the three BMI1 homologs

• Create transgenic lines expressing tagged versions of each protein for comparative localization and ChIP studies

• Perform genome-wide binding studies (ChIP-seq) to identify unique and shared targets

• Conduct comparative transcriptome analyses in single, double, and triple mutant/RNAi backgrounds

• Analyze protein complexes using mass spectrometry to identify homolog-specific interacting partners
  These approaches will help delineate the specific roles of AtBMI1C in contrast to its homologs, providing insights into the functional diversification of BMI1 proteins in plants .

What experimental designs are most effective for studying AtBMI1C function given the challenges with loss-of-function mutants?

Given the challenges in obtaining viable loss-of-function mutants for AtBMI1C , researchers should consider the following alternative experimental strategies:

• Inducible RNAi/CRISPR Systems: Develop temporal and tissue-specific knockdown/knockout systems to circumvent potential lethality or redundancy issues.

• Dominant Negative Approaches: Express truncated or mutated versions of AtBMI1C that retain protein interaction domains but lack catalytic activity, disrupting endogenous complex formation.

• CRISPR Base Editing: Introduce specific amino acid changes in functional domains rather than complete gene disruption.

• Higher-Order Mutants: Generate double or triple mutants with related genes (AtBMI1A, AtBMI1B) to overcome functional redundancy .

• Comparative Overexpression Studies: Systematically overexpress all three BMI1 homologs to identify both shared and unique functions through differential phenotypic analysis .

• Chimeric Protein Approach: Create chimeric proteins exchanging domains between AtBMI1C and its homologs to identify which regions confer specific functions.

• Synthetic Genetic Arrays: Combine AtBMI1C manipulation with mutations in genes from parallel pathways to uncover genetic interactions and redundancies.
  Each of these approaches can help circumvent the limitations encountered with conventional knockout strategies and provide complementary insights into AtBMI1C function.

How should researchers design ChIP experiments to study AtBMI1C binding to chromatin?

Designing effective ChIP experiments to study AtBMI1C binding to chromatin requires careful planning and multiple controls:

• Antibody Validation:

  • Validate At3g23060 Antibody specificity using AtBMI1C-overexpressing lines and wild-type comparisons

  • Use antibody in immunoblotting to confirm it recognizes native AtBMI1C protein

  • Perform peptide competition assays to verify epitope specificity

• Experimental Design:

  • Use young, actively growing tissue where AtBMI1C is expressed

  • Fix tissue with 1% formaldehyde for 10-15 minutes at room temperature

  • Optimize sonication conditions to achieve 200-500bp chromatin fragments

  • Include input, IgG, and positive control (known PRC1 target) samples

• Target Selection:

  • Focus on FLC locus given its established regulation by AtBMI1C

  • Design primers covering different regions of FLC (promoter, first intron, gene body)

  • Include primers for known PRC1 targets as positive controls

  • Include primers for housekeeping genes as negative controls

• Validation and Analysis:

  • Confirm enrichment using qPCR before proceeding to genome-wide methods

  • For ChIP-seq, use paired-end sequencing for better mapping at repetitive regions

  • Apply bioinformatic analyses to identify binding motifs and co-occurring factors

  • Correlate binding with gene expression data and H2A monoubiquitination marks
    This methodical approach allows researchers to identify genuine AtBMI1C binding sites while distinguishing them from technical artifacts or non-specific binding.

How can researchers reconcile the apparent lack of phenotype in AtBMI1C RNAi lines with its role in flowering regulation?

The absence of obvious phenotypes in AtBMI1C RNAi lines despite evidence for its role in flowering regulation presents an interesting scientific puzzle. Researchers can approach this apparent contradiction through several analytical frameworks:

• Functional Redundancy Analysis: Quantify expression levels of AtBMI1A and AtBMI1B in AtBMI1C knockdown lines to determine if compensatory upregulation occurs. Create double or triple knockdown lines to overcome potential redundancy .

• Sensitivity Enhancement: Examine phenotypes under varying environmental conditions (different photoperiods, temperatures, or stress conditions) that might reveal conditional requirements for AtBMI1C function.

• Quantitative Phenotyping: Implement high-precision phenotyping approaches to detect subtle phenotypes that might be missed by visual inspection, such as:

  • Detailed flowering time measurements under various day lengths

  • Quantitative RT-PCR analysis of FLC and FT expression levels

  • ChIP analysis of H2A monoubiquitination at target genes

• Threshold Effects: Consider that RNAi might not reduce AtBMI1C levels below a functional threshold. Analyze the correlation between knockdown efficiency and molecular phenotypes (changes in H2A monoubiquitination levels or target gene expression).

• Developmental Timing: Examine phenotypes throughout development and in multiple generations, as some epigenetic effects may manifest progressively or transgenerationally.
  This multi-faceted approach can help resolve the apparent contradiction and provide insights into the complex regulatory networks involving AtBMI1C.

What are the implications of AtBMI1C's evolutionary divergence from AtBMI1A/B for experimental design?

The evolutionary divergence of AtBMI1C from AtBMI1A/B has significant implications for experimental design when studying these proteins:

• Phylogenetic Context: Conduct comprehensive phylogenetic analysis to position AtBMI1C within the evolutionary context of plant BMI1 proteins to inform functional hypotheses.

• Domain-Specific Studies: Focus on the unique features of AtBMI1C's protein sequence to identify potentially novel functions:

  • Analyze conserved versus divergent domains

  • Identify unique post-translational modification sites

  • Examine species-specific variations in AtBMI1C structure

• Interactome Mapping: Perform comparative interactome analyses to identify:

  • Shared interacting partners among all BMI1 proteins

  • Unique protein interactions specific to AtBMI1C

  • Context-dependent interaction networks in different tissues or conditions

• Target Specificity Analysis: Design experiments to identify the molecular basis of target specificity:

  • Compare genome-wide binding profiles of all three BMI1 proteins

  • Analyze DNA sequence motifs at binding sites

  • Examine chromatin features at AtBMI1C-specific targets

• Structure-Function Analysis: Use structure prediction tools and validation experiments to:

  • Model the three-dimensional structure of AtBMI1C

  • Identify critical residues for function through site-directed mutagenesis

  • Design experiments to test if divergent domains confer novel functions
    These approaches acknowledge the evolutionary distinctiveness of AtBMI1C and can help researchers uncover its unique biological roles beyond what might be inferred from studies of its homologs.

What emerging technologies could enhance the study of AtBMI1C function in chromatin regulation?

Several cutting-edge technologies offer promising avenues for deeper insights into AtBMI1C function:

• Proximity Labeling Techniques: BioID or TurboID fused to AtBMI1C can identify transient or weak interactors in living plant cells, providing more comprehensive interactome data than traditional co-immunoprecipitation.

• Live-Cell Chromatin Imaging: CRISPR-based imaging systems (e.g., dCas9-GFP) combined with fluorescently tagged AtBMI1C can visualize dynamic interactions with specific genomic loci in real-time.

• Single-Cell Epigenomics: Single-cell ChIP-seq or CUT&Tag approaches can reveal cell-type-specific functions of AtBMI1C in complex tissues, potentially uncovering roles masked in whole-tissue analyses.

• Cryo-EM Structure Determination: Structural analysis of AtBMI1C-containing complexes can provide mechanistic insights into how these complexes recognize and modify their chromatin targets.

• Optogenetic Control Systems: Light-inducible AtBMI1C variants allow precise temporal control of protein activity, enabling the study of immediate effects on chromatin and transcription.

• Long-read Sequencing: Technologies like PacBio or Nanopore sequencing combined with chromatin capture methods can provide insights into how AtBMI1C affects higher-order chromatin structure and long-range interactions.

• Integrative Multi-omics: Combining ChIP-seq, RNA-seq, ATAC-seq, and protein-DNA interaction data in systems biology frameworks can reveal how AtBMI1C functions within broader regulatory networks.
  Implementation of these advanced technologies could help resolve current contradictions in AtBMI1C research and uncover novel aspects of its function in plant epigenetic regulation.

How might researchers leverage computational approaches to predict novel functions of AtBMI1C?

Computational approaches offer powerful tools to predict novel functions of AtBMI1C beyond current experimental evidence:

• Co-expression Network Analysis: Identify genes with expression patterns correlated with AtBMI1C across diverse conditions to predict functional associations and regulatory networks.

• Protein Structure Prediction: Use AlphaFold2 or similar tools to generate high-confidence structural models of AtBMI1C, enabling:

  • Identification of potential ligand-binding pockets

  • Prediction of protein-protein interaction interfaces

  • Virtual screening for small molecule modulators

• Machine Learning on Epigenomic Data: Train algorithms on existing ChIP-seq and expression data to:

  • Predict additional genomic targets of AtBMI1C

  • Identify DNA sequence features or motifs associated with binding

  • Forecast effects of AtBMI1C binding on gene expression

• Evolutionary Rate Analysis: Study the evolutionary rate of AtBMI1C across plant species to identify:

  • Rapidly evolving domains that may confer species-specific functions

  • Conserved regions that likely maintain essential functions

  • Coevolution patterns with interacting partners

• Genomic Context Mining: Analyze the genomic neighborhood of AtBMI1C across plant species to identify:

  • Syntenic relationships that suggest functional connections

  • Shared regulatory elements with functionally related genes

  • Evolutionary events like duplications or rearrangements These computational approaches can generate testable hypotheses about AtBMI1C functions beyond its established role in flowering time regulation, guiding future experimental work in unexplored research directions.