RDM4 Antibody
Description
RDM4: Functional Overview
RDM4 (RNA-DIRECTED DNA METHYLATION 4) is a nuclear protein in Arabidopsis that interacts with RNA polymerases Pol II and Pol V. While it is implicated in RNA-directed DNA methylation (RdDM), its role in cold stress resistance operates independently of this pathway .
Key Functions:
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Cold Stress Regulation: RDM4 enhances survival under chilling/freezing conditions by promoting the expression of C-REPEAT-BINDING FACTOR (CBF) genes and their downstream regulon .
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Transcriptional Modulation: Facilitates RNA Pol II occupancy at CBF2 and CBF3 promoters, enabling transcriptional activation during cold exposure .
Experimental Evidence for RDM4’s Role in Cold Stress
Mutational and overexpression studies reveal RDM4’s critical role in cold resilience:
| Parameter | Wild Type (WT) | rdm4 Mutant | RDM4 Overexpression (OE) |
|---|---|---|---|
| Survival Rate (Cold Stress) | ~100% | Reduced significantly | Increased constitutively |
| Electrolyte Leakage | Low | Elevated (membrane injury) | Reduced |
| CBF Gene Expression | Induced by cold | Downregulated (basal and cold) | Constitutively upregulated |
| H₂O₂ Accumulation | Low | Increased (pre/post stress) | Not reported |
Mechanistic Insights:
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RDM4 Mutants: Hypersensitive to cold due to reduced CBF expression and impaired antioxidant defenses (e.g., lower catalase activity) .
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RDM4 OE: Constitutively activates CBF regulon genes, mimicking cold acclimation and enhancing membrane stability .
Independent Function from RdDM Pathway
While RDM4 interacts with Pol V (a key RdDM component), its cold stress role diverges from RdDM-mediated gene silencing:
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nrpe1 Mutants (Pol V subunit defect): Show impaired RdDM but no effect on CBF expression or cold tolerance .
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ROS1-NRPA Mutants: RdDM defects lead to ectopic gene activation but no overlap with CBF downregulation observed in rdm4 .
Transcriptomic and Proteomic Evidence
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Microarray Data: rdm4 mutants exhibit ~1,500 downregulated genes, including CBF, COR15A (cold-responsive), and KIN2 (ABA signaling) .
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ChIP-Seq Results: RDM4 is required for Pol II recruitment to CBF2 and CBF3 promoters, even under non-stress conditions .
No Evidence of RDM4-Specific Antibodies
The provided sources reference antibodies only for unrelated proteins (e.g., rhodopsin ) or general antibody applications in plant biology . No studies describe an antibody targeting RDM4, likely due to its intracellular localization and functional focus in plant research.
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- RDM4 plays a crucial role in Pol II occupancy at the promoters of CBF2 and CBF3. PMID: 26522658
- Research indicates that SHH1(AT1G15215), RDR2, CLASSY1, and RDM4 were co-purified with NRPD1(At1g63020), the largest subunit of Pol-IV. PMID: 21811420
- Evidence suggests that DMS4 might function as a regulatory factor for various RNA polymerases, explaining its diverse roles in the plant. [DMS4] PMID: 20010803
Q&A
FAQs for RDM4 Antibody in Academic Research
What is the functional role of RDM4 in plant stress responses, and how can its antibody validate experimental outcomes?
RDM4 (RNA-DIRECTED DNA METHYLATION 4) is a transcriptional regulator critical for RNA-directed DNA methylation (RdDM) and stress adaptation in Arabidopsis. Its antibody is essential for:
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Validating protein-protein interactions: Confirming associations with RNA Polymerase II (Pol II) and Pol V via co-immunoprecipitation (Co-IP) .
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Tracking cellular localization: Immunofluorescence assays show RDM4 localizes to nuclei, consistent with its role in chromatin remodeling .
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Assessing mutant phenotypes: Western blotting using RDM4 antibodies confirms knockout (rdm4) lines lack the protein, correlating with reduced cold tolerance and altered gene expression .
Key methodological steps:
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Use anti-RDM4 antibodies in Western blotting to compare protein levels in wild-type vs. rdm4 mutants.
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Combine ChIP-PCR with RDM4 antibodies to study Pol II occupancy at CBF2/3 promoters under cold stress .
How does RDM4 contribute to the RdDM pathway, and what experimental approaches resolve its mechanistic ambiguity?
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RdDM-independent functions: In rdm4 mutants, CBF regulon genes are downregulated, but nrpe1 (Pol V subunit) mutants show no such effect, suggesting alternative pathways .
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Transcriptomic profiling: RNA-seq of rdm4 mutants reveals differential expression of 1,200+ genes, including stress-responsive loci .
Experimental design considerations:
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Compare rdm4 and nrpe1 mutants to isolate RdDM-dependent vs. independent roles.
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Use RDM4 antibodies in chromatin fractionation assays to study its interaction with Pol II/Pol V .
How to reconcile contradictions in RDM4’s role in cold stress vs. RdDM pathways?
Conflict: rdm4 mutants impair cold tolerance and CBF expression but do not fully disrupt RdDM .
Resolution strategies:
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Dual-pathway analysis: Perform parallel experiments measuring DNA methylation (bisulfite sequencing) and stress-responsive gene expression (qRT-PCR) in rdm4.
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Time-course assays: Monitor RDM4 protein dynamics during cold acclimation using antibodies to detect temporal expression shifts .
Data example:
| Condition | Pol II occupancy at CBF2 | CBF3 expression | RdDM activity |
|---|---|---|---|
| Wild-type | High | High | Normal |
| rdm4 mutant | Low | Low | Partial loss |
| nrpe1 mutant | No change | No change | Lost |
What methodologies optimize RDM4 antibody specificity in complex plant tissues?
Challenges: Cross-reactivity with homologs (e.g., FRG1/2 in Arabidopsis) or epitope masking in chromatin-bound RDM4 .
Solutions:
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Antigen design: Use full-length RDM4 recombinant protein for antibody production (as in ).
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Pre-clearing steps: Pre-incubate plant lysates with frg1/frg2 mutant extracts to absorb non-specific antibodies.
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Validation: Perform knockout-validated Western blotting (e.g., confirm absence of signal in rdm4 null mutants) .
How to investigate RDM4’s structural interactions using its antibody?
Approaches:
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Crosslinking-MS: Use anti-RDM4 antibodies for immunoprecipitation followed by mass spectrometry to identify interactomes under stress vs. control conditions.
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Cryo-EM integration: Combine antibody-based purification with structural studies to resolve RDM4-Pol II/Pol V complexes .
Critical finding: RDM4’s association with Pol II is essential for recruiting transcriptional machinery to CBF promoters during cold stress, independent of RdDM .
