rnf169 Antibody

What is RNF169 and why is it important in DNA damage research?

RNF169 is a paralog of RNF168 that contains an N-terminal RING finger motif and a C-terminal motif interacting with ubiquitin (MIU) domain . It functions as a negative regulator of the ubiquitin-dependent DNA damage response pathway by competing with repair factors for binding to ubiquitylated chromatin at DNA double-strand breaks (DSBs) . The importance of RNF169 stems from its role in:

• Regulating DNA damage signal transduction

• Influencing repair pathway choice between homologous recombination (HR) and non-homologous end joining (NHEJ)

• Antagonizing the recruitment of 53BP1 and RAP80 to damaged chromatin

• Promoting homology-mediated DSB repair

These functions make RNF169 a critical protein to study in the context of genomic integrity maintenance and DNA repair mechanisms.

What methods can be used to detect RNF169 in experimental settings?

Based on available research tools, RNF169 can be detected through several methodological approaches:

When performing these experiments, it's important to note that while some commercial RNF169 antibodies are suitable for detecting the protein by immunoblotting, they may not be effective for immunofluorescence applications .

How does RNF169 localize within cells and how can this be visualized?

RNF169 shows specific localization patterns that can be visualized experimentally:

• In unperturbed cells: RNF169 is diffusely localized to the nucleoplasm throughout interphase

• After DNA damage: RNF169 becomes concentrated in foci that colocalize with DSB markers, including γ-H2AX and MDC1

• Recruitment to damage sites: RNF169 can be efficiently recruited to sites of microlaser-induced DSBs

To visualize RNF169, researchers typically use GFP-tagged RNF169 constructs rather than antibody-based detection of endogenous RNF169 for immunofluorescence studies. This is because available antibodies, while effective for immunoblotting, have shown limitations in immunofluorescence applications . For optimal visualization:

• Express GFP-tagged RNF169 in cells of interest

• Induce DNA damage using ionizing radiation or laser microirradiation

• Fix cells and counterstain with antibodies against DSB markers (e.g., γ-H2AX)

• Image using confocal microscopy to observe colocalization

How can researchers distinguish between RNF168 and RNF169 functions experimentally?

Despite structural similarities, RNF168 and RNF169 have distinct functions in the DNA damage response pathway. To experimentally distinguish between them:

• Functional Assessment:

  • RNF168 depletion impairs ubiquitin conjugate formation and 53BP1 recruitment to DSB sites

  • RNF169 depletion does not affect initial ubiquitin conjugate formation but enhances 53BP1 recruitment

• E3 Ligase Activity:

  • RNF168 catalyzes robust H2A polyubiquitylation

  • RNF169 displays much weaker E3 ligase activity and does not appreciably ubiquitylate H2A-type histones

• Impact on Repair Pathway:

  • RNF168 knockdown increases HR efficiency

  • RNF169 knockdown decreases HR efficiency

• Experimental Approach:

  • Use siRNA-mediated knockdown of either protein

  • Rescue experiments with siRNA-insensitive constructs

  • Compare phenotypes using readouts such as 53BP1 foci intensity, HR reporter assays, and chromatin immunoprecipitation

These distinctive properties allow researchers to experimentally separate the functions of these two related proteins in the DNA damage response pathway.

What are the key considerations when using RNF169 antibodies for chromatin immunoprecipitation (ChIP) experiments?

When conducting ChIP experiments with RNF169 antibodies, several methodological considerations are critical:

• Antibody Selection:

  • For tagged RNF169, validated ChIP-grade anti-tag antibodies (e.g., anti-Flag M2) show higher specificity

  • For endogenous RNF169, custom antibodies may be required as commercial antibodies have variable ChIP performance

• Experimental Design:

  • Include controls for antibody specificity (e.g., RNF169-depleted cells)

  • Use site-specific DSB systems (e.g., AsiSI-ER system) to map RNF169 distribution along the damaged chromatin

• Distribution Profiling:

  • RNF169 predominantly accumulates at DNA end-proximal regions (0.1-5 kb from break sites)

  • Compare with γH2AX (spreads up to 1 Mb) and 53BP1 profiles to understand relative positioning

• Data Analysis:

  • Normalize to input DNA

  • Compare profiles with other DDR factors (e.g., 53BP1, RAP80) to identify competitive binding relationships

  • Use qPCR primers at various distances from DSB sites to map distribution patterns

These considerations will help ensure reliable and interpretable ChIP results when studying RNF169 chromatin interactions.

How does the MIU domain of RNF169 contribute to its function and how can this be experimentally validated?

The MIU (motif interacting with ubiquitin) domain is critical for RNF169 function, particularly in its recruitment to DNA damage sites. Experimental approaches to validate the MIU domain's contribution include:

• Structural Analysis:

  • The MIU domain of RNF169 recognizes ubiquitin via a hydrophobic surface centered around A673 of RNF169

  • It interacts with the canonical binding interface on ubiquitin (I44, L8, and V70)

• Mutation Studies:

  • Generate RNF169 constructs with point mutations in the MIU domain

  • A *MIU mutant fails to accumulate at DSB sites and does not affect 53BP1 recruitment

  • The MIU domain binds efficiently to both K48- and K63-linked ubiquitin chains in vitro

• Experimental Validation:

  • Ubiquitin binding assays with purified proteins

  • Recruitment dynamics to laser-induced damage tracks

  • Rescue experiments in RNF169-depleted cells

  • Structural studies using methyl-TROSY NMR and cryo-EM

• Functional Outcomes:

  • MIU-mutant RNF169 fails to affect HR efficiency

  • Unlike wild-type RNF169, MIU mutants do not sensitize cells to ionizing radiation

These approaches collectively demonstrate the essential role of the MIU domain in RNF169 function at DNA damage sites.

Why might RNF169 antibodies show different apparent molecular weights in Western blots?

Researchers often observe RNF169 at different molecular weights than predicted. The canonical human RNF169 protein has a reported length of 708 amino acid residues and a theoretical mass of 77.2 kDa , yet it may appear at ~90 kDa in Western blots . Several factors may explain this discrepancy:

• Post-translational modifications:

  • RNF169 undergoes phosphorylation

  • The protein contains a substantial chromatin-bound pool even in the absence of DNA damage

  • These modifications can significantly alter migration patterns

• Technical considerations:

  • SDS-PAGE gel percentage affects migration (5-20% gradient gels are recommended)

  • Running conditions (70V stacking/90V resolving for 2-3 hours shows optimal separation)

  • Sample preparation (reducing conditions are essential)

• Validation approaches:

  • Use positive controls (e.g., cell lines with confirmed RNF169 expression like U20S or K562)

  • Include RNF169-depleted samples as negative controls

  • Consider using tagged RNF169 as a reference point

When troubleshooting Western blot results, these factors should be systematically addressed to ensure accurate detection and characterization of RNF169.

How can researchers quantitatively assess the impact of RNF169 on 53BP1 recruitment to DNA damage sites?

To quantitatively evaluate RNF169's effect on 53BP1 recruitment, researchers can employ several complementary approaches:

• Immunofluorescence-based quantification:

  • RNF169 knockdown increases 53BP1 foci intensity by approximately 1.5-fold

  • Use automated high-content microscopy for unbiased analysis of foci intensity

  • Analyze large cohorts of cells (>100 cells per condition) for statistical robustness

• ChIP-qPCR at defined DSB sites:

  • Utilize site-specific DSB induction systems (e.g., AsiSI-ER)

  • Measure 53BP1 occupancy at various distances from the break site

  • Compare with and without RNF169 manipulation

• Live-cell imaging:

  • Track GFP-53BP1 recruitment kinetics in real-time

  • Compare recruitment rates and plateau levels between control and RNF169-manipulated cells

  • Quantify fluorescence intensity at damage sites over time

• Biochemical fractionation:

  • Isolate chromatin fractions after DNA damage

  • Quantify 53BP1 levels by Western blotting

  • Compare between RNF169-proficient and RNF169-deficient conditions

These approaches provide complementary data on how RNF169 impacts 53BP1 recruitment both spatially and temporally at DNA damage sites.

What controls are essential when using RNF169 antibodies in different experimental applications?

Proper controls are crucial for ensuring the reliability and interpretability of experiments using RNF169 antibodies:

Incorporating these controls systematically ensures that observed results are specifically attributable to RNF169 and not to technical artifacts or off-target effects.

How can researchers investigate the relationship between RNF169 and homology-directed repair in BRCA-deficient contexts?

Investigating RNF169's role in homology-directed repair in BRCA-deficient contexts requires specialized approaches:

• Reporter Assays:

  • Use established cell reporters measuring DSB repair events mediated by HR, SSA, aNHEJ, and NHEJ

  • RNF169 silencing compromises high-fidelity HR repair, single-strand annealing (SSA), and alternative NHEJ (aNHEJ), but not classical NHEJ

  • In BRCA2-deficient or PALB2-deficient cells, RNF169 overexpression substantially elevates SSA repair efficiency

• Experimental Design:

  • Generate BRCA1, BRCA2, or PALB2 knockdown cells

  • Modulate RNF169 levels (overexpression or knockdown)

  • Measure repair pathway utilization using fluorescent reporter systems

  • Quantify repair efficiency by flow cytometry

• Key Findings to Consider:

  • BRCA1 silencing impairs SSA repair

  • BRCA2 and PALB2 inactivation results in hyperactive SSA

  • RNF169 overexpression further elevates SSA in BRCA1/2/PALB2-silenced cells

  • RNF169 inactivation reduces the hyperactivated SSA in BRCA2/PALB2-deficient cells

• Mechanistic Investigation:

  • Examine DSB end resection using BrdU-based assays

  • Analyze RPA loading at DSB sites

  • Measure RAD51 filament formation in different genetic backgrounds

These approaches provide insights into how RNF169 may specifically fine-tune SSA repair in BRCA-deficient cells, with important implications for cancer research.

What methods can be used to study the structural interaction between RNF169 and ubiquitylated nucleosomes?

Studying the structural interaction between RNF169 and ubiquitylated nucleosomes requires advanced biophysical and biochemical approaches:

• Integrative Structural Biology Approach:

  • Methyl-TROSY NMR spectroscopy for detecting specific interactions

  • Mutagenesis studies to identify critical interaction residues

  • Replica-averaged molecular dynamics simulations incorporating experimental restraints

  • Cryo-electron microscopy for validation

• Key Components to Study:

  • The MIU2-ubiquitin interaction (centered on A673 of RNF169)

  • Electrostatic contacts between the LRM2 module of RNF169 and the acidic patch of the nucleosome

  • Critical arginine residues (R689 and R700) that contact the nucleosome surface

• Experimental Design:

  • Generate nucleosomes monoubiquitylated at H2AK13/K15

  • Express and purify RNF169(UDM2) constructs (wild-type and mutants)

  • Measure binding affinities using biophysical techniques

  • Determine structural details through a combination of methods

• Technical Considerations:

  • Optimal protein labeling methods enhance spectral resolution and sensitivity

  • TROSY-based experimental approaches are critical for large complexes

  • Distance restraints from methyl groups serve as key structural information

This integrative approach has revealed that RNF169 specifically recognizes ubiquitylated nucleosomes through a three-pronged interaction involving the MIU2 module, ubiquitin, and the acidic patch of the nucleosome .

What emerging research questions remain to be addressed regarding RNF169 function and regulation?

Despite significant advances in understanding RNF169, several important research questions remain:

• Regulation of RNF169 Expression and Activity:

  • How is RNF169 expression regulated in different cell types and pathological contexts?

  • What post-translational modifications control RNF169 function?

  • Are there additional protein partners that modulate RNF169 activity?

• Therapeutic Implications:

  • Could modulation of RNF169 levels sensitize cancer cells to DNA-damaging therapies?

  • Might RNF169 be a viable target to enhance synthetic lethality approaches in BRCA-deficient cancers?

  • How does RNF169 function relate to resistance mechanisms in PARP inhibitor therapy?

• Mechanistic Details:

  • What is the exact nature of the ubiquitylated species recognized by the MIU domain of RNF169 at DSB sites?

  • How does RNF169 specifically compete with both 53BP1 and RAP80 despite their different binding requirements?

  • What determines the balance between RNF168 and RNF169 at DNA damage sites?

• Physiological Significance:

  • What is the role of RNF169 in development and tissue homeostasis?

  • How does RNF169 function in non-dividing cells with limited HR capacity?

  • What is the evolutionary significance of the RNF168/RNF169 relationship?

Addressing these questions will provide deeper insights into the fundamental biology of DNA damage responses and may reveal new therapeutic opportunities in cancer and other diseases associated with genomic instability.