At2g31400 Antibody

What is AT2G31400 and why is it important in plant research?

AT2G31400 encodes GUN1 (Genomes Uncoupled 1), a chloroplast-localized pentatricopeptide-repeat protein that plays a crucial role in retrograde signaling pathways in plants. GUN1 is fundamentally involved in the regulation of nuclear gene expression in response to chloroplast signals, making it a key component in understanding chloroplast-to-nucleus communication mechanisms. The protein is located on chromosome 2 of Arabidopsis thaliana and contains several functional domains, including multiple pentatricopeptide repeat (PPR) motifs and an Smr domain . GUN1's significance stems from its central role in integrating various retrograde signaling pathways, particularly during chloroplast biogenesis and stress responses. Research on GUN1 has provided valuable insights into how plants coordinate gene expression between organelles and the nucleus, which is essential for understanding plant development and stress adaptation mechanisms .

What are the key protein characteristics of AT2G31400/GUN1?

GUN1 is characterized by several distinct protein features that are important to consider when developing and using antibodies:

The protein contains multiple pentatricopeptide repeat (PPR) domains (InterPro:IPR002885) and an Smr protein/MutS2 C-terminal domain (InterPro:IPR002625) . These structural features are important considerations when selecting epitopes for antibody generation. The protein's plastid localization is highly confident (SUBAcon score of 1.000), which has implications for experimental design when using GUN1 antibodies for localization studies .

How can I validate the specificity of an AT2G31400/GUN1 antibody?

Validating the specificity of a GUN1 antibody requires a multi-faceted approach:

First, perform western blot analysis using wild-type Arabidopsis samples alongside a gun1 mutant line (such as SAIL_290_D09 available from ABRC) . A specific antibody will show a band at approximately 101 kDa in wild-type samples that is absent in the mutant. For example, in studies investigating RH50 protein, researchers validated antibody specificity through western-blot analysis, confirming the antibody's ability to precipitate the target protein .

Second, conduct immunoprecipitation followed by mass spectrometry to confirm that the antibody pulls down GUN1. This technique allows for objective verification of antibody specificity beyond the conventional western blot approach.

Third, perform immunolocalization studies to verify that the antibody-detected signal colocalizes with chloroplasts, which is consistent with GUN1's known subcellular localization. This can be done using confocal microscopy with appropriate chloroplast markers .

Finally, use recombinant GUN1 protein as a positive control and for competition assays. Pre-incubation of the antibody with the recombinant protein should eliminate the signal in western blots if the antibody is specific.

What are the optimal protocols for using AT2G31400/GUN1 antibodies in co-immunoprecipitation experiments?

For successful co-immunoprecipitation (co-IP) experiments with GUN1 antibodies, the following optimized protocol is recommended:

• Sample preparation: Extract total chloroplast proteins from Arabidopsis seedlings (3-4 weeks old) using a chloroplast isolation buffer (0.33 M sorbitol, 50 mM HEPES-KOH pH 7.5, 5 mM MgCl₂, 5 mM EGTA, 1 mM DTT, and protease inhibitor cocktail).

• Solubilization: Solubilize chloroplast membranes with a mild detergent such as 1% n-dodecyl β-D-maltoside (β-DM) or 1% digitonin in IP buffer (50 mM HEPES-KOH pH 7.5, 150 mM NaCl, 10% glycerol, 2 mM EDTA) for 30 minutes on ice.

• Pre-clearing: Incubate the lysate with Protein A/G agarose beads for 1 hour at 4°C to reduce non-specific binding.

• Immunoprecipitation: Incubate the pre-cleared lysate with GUN1 antibody (typically 2-5 μg per 500 μg of protein) overnight at 4°C with gentle rotation, followed by addition of Protein A/G beads for 2-4 hours.

• Washing: Wash the beads 4-5 times with wash buffer (IP buffer with reduced detergent concentration of 0.1%) to remove non-specifically bound proteins.

• Elution: Elute bound proteins using either low pH (0.1 M glycine, pH 2.5) followed by immediate neutralization, or by boiling in SDS-sample buffer.

• Analysis: Analyze the immunoprecipitated proteins by western blotting or mass spectrometry.

When investigating protein interactions, research has shown that careful validation is essential. For example, when RH50 was tested for binding to GUN1 using yeast two-hybrid (Y2H) assays, proper controls were crucial for identifying true interactions versus false positives .

How can AT2G31400/GUN1 antibodies be used to study retrograde signaling pathways?

GUN1 antibodies are valuable tools for investigating retrograde signaling pathways through several experimental approaches:

Retrograde signaling induction experiments:
Treatment with lincomycin (0.5 mM) or norflurazon (5 μM) is commonly used to induce retrograde signaling by inhibiting chloroplast biogenesis or carotenoid synthesis, respectively . Following treatment, GUN1 antibodies can be used to:

• Track GUN1 protein abundance changes: Monitor GUN1 protein levels in response to retrograde signaling activation by quantitative western blot analysis.

• Identify protein interaction dynamics: Use co-immunoprecipitation with GUN1 antibodies before and after treatment to identify proteins that differentially associate with GUN1 during retrograde signaling.

• Chromatin immunoprecipitation (ChIP) studies: If GUN1 associates with DNA, ChIP experiments using GUN1 antibodies can reveal its genomic associations during retrograde signaling.

• Subcellular localization changes: Immunofluorescence microscopy with GUN1 antibodies can track potential relocalization of GUN1 during retrograde signaling activation.

For example, researchers have used a combination of red and blue light at different intensities (130 μmol m⁻² s⁻¹ for standard light and 310 μmol m⁻² s⁻¹ for high light) to study GUN1's involvement in light-responsive retrograde signaling . The protein's interaction with photoreceptors and downstream signaling components can be investigated through co-IP experiments using GUN1 antibodies under these different light conditions.

What considerations are important when using AT2G31400/GUN1 antibodies for immunolocalization studies?

When conducting immunolocalization studies with GUN1 antibodies, several critical factors should be considered for reliable results:

Fixation protocols: Due to GUN1's chloroplast localization, use fixation methods that preserve chloroplast structure. A recommended approach is 4% paraformaldehyde fixation with 0.1% glutaraldehyde in PBS for 30 minutes, followed by permeabilization with 0.1% Triton X-100.

Antibody specificity validation: Always include appropriate controls, including gun1 mutant tissues and pre-immune serum or IgG controls, to confirm signal specificity. The subcellular localization of GUN1 is confirmed to be in the plastid with a SUBAcon score of 1.000, making this validation particularly important .

Antigen retrieval considerations: GUN1's complex PPR domain structure may require antigen retrieval steps (such as heat-induced epitope retrieval using citrate buffer, pH 6.0) to expose epitopes that might be masked during fixation.

Co-localization markers: Use established chloroplast markers (such as antibodies against RbcL or PRPS1) for co-localization studies. Previous studies have used these markers alongside proteins of interest to confirm organellar localization .

Signal amplification techniques: Due to potentially low GUN1
abundance, consider using signal amplification methods such as tyramide signal amplification (TSA) to enhance detection sensitivity while maintaining specificity.

Confocal microscopy settings: Use appropriate excitation and emission settings to minimize chlorophyll autofluorescence interference, which can be particularly challenging when working with chloroplast-localized proteins like GUN1.

How can AT2G31400/GUN1 antibodies be used in RNA immunoprecipitation (RIP) experiments to study RNA-protein interactions?

GUN1 contains multiple pentatricopeptide repeat (PPR) domains, which are known to be involved in RNA binding. To study GUN1-RNA interactions using RNA immunoprecipitation (RIP), the following protocol is recommended:

• Crosslinking: Perform in vivo crosslinking of Arabidopsis seedlings using 1% formaldehyde for 10 minutes to stabilize RNA-protein interactions.

• Tissue homogenization: Grind tissue in liquid nitrogen and extract in RIP buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 4 mM MgCl₂, 1% NP-40, 1 mM DTT, 1× protease inhibitor cocktail, 100 U/ml RNase inhibitor).

• Immunoprecipitation: Incubate the cleared lysate with GUN1 antibody coupled to magnetic beads overnight at 4°C.

• Stringent washing: Perform at least 5 washes with high-stringency buffers to remove non-specifically bound RNAs.

• RNA isolation: Reverse crosslinks and isolate bound RNAs using TRIzol or commercial RNA isolation kits.

• RNA analysis: Identify bound RNAs using RT-PCR for candidate approaches or RNA-seq for unbiased discovery.

When analyzing RNA gel-blot data, follow established protocols for stringent conditions as described in previous studies . For quantitative analysis, signals can be measured using ImageJ software (http://imagej.nih.gov/ij/index.html), similar to how researchers have quantified immunoblot signals in related studies .

This approach has proven valuable for studying RNA-binding proteins in chloroplasts. For instance, researchers investigating RH50 (a DEAD-box RNA helicase) used similar techniques to demonstrate its association with RNA-containing particles, which were sensitive to RNase treatment .

What are the key challenges in generating phospho-specific antibodies against AT2G31400/GUN1?

Generating phospho-specific antibodies against GUN1 presents several unique challenges that researchers should address:

Prediction of phosphorylation sites: Use bioinformatic tools to predict potential phosphorylation sites on GUN1. Consider evolutionary conservation of these sites across different plant species to identify functionally important phosphorylation sites.

Peptide design considerations:

• Select peptides containing 10-15 amino acids surrounding the phosphorylation site

• Ensure peptide solubility by including charged residues

• Add a terminal cysteine for conjugation if not naturally present

• Purify phosphopeptides to >90% purity by HPLC

• Confirm phosphopeptide identity by mass spectrometry

Antibody validation requirements:

• Test antibody recognition using both phosphorylated and non-phosphorylated peptides in ELISA

• Validate with western blots using phosphatase-treated samples as controls

• Verify specificity using gun1 mutant extracts

• Confirm signal changes in response to treatments known to alter chloroplast signaling (e.g., high light, lincomycin treatment)

Potential cross-reactivity issues: The antibody might cross-react with other PPR proteins that share sequence similarity with GUN1. Therefore, comprehensive specificity testing against recombinant proteins of related PPR family members is necessary.

Phosphorylation dynamics: GUN1 phosphorylation status may change rapidly in response to environmental conditions. Consider using phosphatase inhibitors (e.g., PhosSTOP) during all extraction procedures and rapid tissue harvesting to preserve phosphorylation status.

How can mass spectrometry be used alongside AT2G31400/GUN1 antibodies to study the GUN1 interactome?

Combining immunoprecipitation using GUN1 antibodies with mass spectrometry provides a powerful approach to characterize the GUN1 interactome:

Sample preparation protocol:

• Isolate intact chloroplasts from Arabidopsis seedlings

• Fractionate into stromal, thylakoid, and envelope fractions

• Solubilize proteins using a non-denaturing detergent (1% digitonin)

• Immunoprecipitate using GUN1 antibodies

• Perform on-bead tryptic digestion

• Analyze peptides by LC-MS/MS

Mass spectrometry approach:
For comprehensive interactome analysis, use a label-free quantitative (LFQ) proteomics approach comparing GUN1 immunoprecipitates to IgG controls. Alternatively, more quantitative approaches using SILAC or TMT labeling can be employed for comparative studies under different conditions.

Validation of protein interactions:
Confirm key interactions using alternative methods such as yeast two-hybrid assays, bimolecular fluorescence complementation, or reverse co-IP. Previous research has used Y2H assays to test GUN1 interactions with proteins like RH50, where GUN1 was employed as prey protein in Ad vectors .

Functional characterization of interactors:
Group identified proteins by functional categories and identify enriched biological processes using GO term analysis. Previous studies have identified GUN1's role in megadalton complexes, comigrating with ribosomal particles, as demonstrated by immunodetection using specific antibodies like RH50-, PRPL11-, and PRPS5-specific antibodies .

Dynamic interactome analysis:
Investigate how the GUN1 interactome changes in response to:

• Light intensity changes (e.g., 130 μmol m⁻² s⁻¹ vs. 310 μmol m⁻² s⁻¹)

• Retrograde signaling activation (e.g., 0.5 mM lincomycin or 5 μM norflurazon treatment)

• Developmental stages

• Abiotic stress conditions

This approach has successfully identified interaction networks of chloroplast proteins. For example, size-exclusion chromatography of chloroplast stroma extracts revealed that certain proteins associate with RNA-containing and RNase-sensitive particles, including ribosomal proteins .

How can non-specific binding issues with AT2G31400/GUN1 antibodies be addressed?

Non-specific binding is a common challenge when working with antibodies against chloroplast proteins like GUN1. These approaches can help minimize such issues:

Optimization of blocking conditions:
Test different blocking agents including 5% non-fat dry milk, 3-5% BSA, or commercial blocking reagents. For GUN1 antibodies, 3% BSA in TBST (TBS with 0.1% Tween-20) for 1 hour at room temperature often provides optimal blocking.

Antibody dilution optimization:
Titrate the GUN1 antibody to determine the optimal concentration that provides specific signal with minimal background. Typically, starting with 1:1000 dilution and testing up to 1:5000 is recommended for western blots.

Pre-adsorption protocol:
If background persists, consider pre-adsorbing the antibody:

• Express and purify a GST-tagged fragment of GUN1 that doesn't contain the epitope

• Incubate diluted antibody with this protein (10-50 μg/ml) for 2 hours at room temperature

• Proceed with the pre-adsorbed antibody for your application

Alternative extraction methods:
Different extraction buffers can affect antibody specificity. Compare TCA-acetone precipitation, phenol extraction, and direct SDS extraction to determine which provides the cleanest results with your GUN1 antibody.

Increased washing stringency:
For persistent background in immunoblots or IPs, increase the stringency of wash buffers by:

• Increasing salt concentration (up to 500 mM NaCl)

• Adding low concentrations of SDS (0.1%)

• Increasing Tween-20 concentration (up to 0.3%)

• Extending wash times and number of washes

Cross-validation with multiple antibodies:
If available, use antibodies against different epitopes of GUN1 to confirm specificity. Previous studies have used specifically generated antibodies against defined peptides, such as the RH50 antibody raised against the peptide CDNERGLRGGSHSKG .

What are the best approaches for quantifying AT2G31400/GUN1 protein levels in different experimental conditions?

Accurate quantification of GUN1 protein levels requires careful experimental design and appropriate controls:

Western blotting optimization for quantification:

• Use gradient gels (4-12% or 4-20%) to ensure optimal resolution of GUN1's 101 kDa band

• Include a dilution series of recombinant GUN1 protein to generate a standard curve

• Ensure linear detection range by using fluorescent secondary antibodies rather than chemiluminescence

• Load equal total protein amounts (15-20 μg) verified by Ponceau S staining or normalization to housekeeping proteins

Recommended normalization controls:
For chloroplast proteins like GUN1, normalize to chloroplast housekeeping proteins rather than cellular housekeeping genes. RbcL (Rubisco large subunit) is often used as a loading control for chloroplast proteins .

Image acquisition and analysis:
Capture images using a digital imaging system with a wide dynamic range. Quantify band intensities using ImageJ software as described in previous studies . Ensure background subtraction is performed consistently.

Sample preparation considerations:
GUN1 protein may be sensitive to degradation. Use fresh tissue whenever possible and include protease inhibitors in all extraction buffers. Process samples quickly and maintain cold temperatures throughout.

Statistical analysis:
When comparing GUN1 protein levels across conditions, perform at least three biological replicates and analyze data using appropriate statistical tests. Previous studies have used one-way analysis of variance with Tukey-b post hoc multiple comparison tests (for P values <0.05) or Student's t-tests depending on the experimental design .

How can epitope masking issues be addressed when using AT2G31400/GUN1 antibodies?

Epitope masking can occur due to protein-protein interactions, post-translational modifications, or conformational changes in GUN1, limiting antibody accessibility. These approaches can help overcome such challenges:

Denaturing conditions:
For applications like western blotting, ensure complete denaturation by:

• Using strong reducing agents (e.g., 100 mM DTT)

• Including 2% SDS in sample buffer

• Heating samples at 95°C for 5-10 minutes

Epitope retrieval techniques for fixed tissues:
For immunohistochemistry or immunofluorescence:

• Heat-induced epitope retrieval using citrate buffer (pH 6.0) at 95°C for 20 minutes

• Enzymatic epitope retrieval using proteinase K (10 μg/ml for 10-15 minutes)

• Combined approaches for difficult samples

Modified extraction conditions:
Test different extraction buffers that may preserve epitope accessibility:

• High pH buffers (pH 8.5-9.0)

• Inclusion of chaotropic agents (low concentrations of urea, 1-2 M)

• Different detergents (CHAPS, deoxycholate, or SDS at low concentrations)

Alternative antibody clones:
If available, test antibodies raised against different epitopes of GUN1, as some may be less susceptible to masking effects.

Pre-treatment of samples:
For proteins involved in large complexes like GUN1, which has been shown to exist in megadalton complexes comigrating with ribosomal particles , consider:

• Sonication to disrupt protein complexes

• Limited proteolysis to expose hidden epitopes

• DNase/RNase treatment if nucleic acid binding is causing masking

Verification with recombinant protein:
Express different domains of GUN1 as recombinant proteins to identify which regions might be subject to masking and optimize detection conditions accordingly.

How can AT2G31400/GUN1 antibodies be used in single-cell proteomics approaches?

While single-cell proteomics is still emerging for plant systems, GUN1 antibodies can be adapted for this cutting-edge approach:

Sample preparation considerations:

• Optimize protoplast isolation protocols to maintain GUN1 protein integrity

• Utilize gentle cell sorting methods (FACS with appropriate parameters)

• Implement nano-scale protein extraction techniques

• Consider proximity labeling approaches using engineered GUN1 fusions

Antibody-based enrichment strategies:
For single-cell applications, standard immunoprecipitation volumes are prohibitive. Instead:

• Develop microfluidic immunocapture devices coated with GUN1 antibodies

• Use paramagnetic nanobeads conjugated to GUN1 antibodies for small-volume IPs

• Consider proximity extension assays (PEA) for sensitive detection

Mass spectrometry adaptations:

• Implement carrier protein strategies to enhance detection of low-abundance proteins

• Utilize nanoPOTS (Nanodroplet Processing in One pot for Trace Samples) technology

• Apply TMT labeling for multiplexed analysis of single cells across conditions

Validation approaches:

• Correlate findings with single-cell RNA-seq data for the same cell types

• Perform pseudo-bulk analysis as a bridge to conventional proteomics

• Use immunofluorescence on fixed single cells to validate key findings

Data analysis considerations:

• Apply specialized computational pipelines designed for sparse data

• Implement imputation strategies appropriate for single-cell proteomics

• Use dimensionality reduction and clustering approaches that account for technical noise

This approach can provide unprecedented insights into cell-type-specific roles of GUN1 in retrograde signaling, particularly in heterogeneous tissues or developmental contexts.

What are the considerations for using AT2G31400/GUN1 antibodies in chromatin immunoprecipitation (ChIP) experiments?

Although GUN1 is primarily a chloroplast protein, recent research suggests nuclear components of retrograde signaling pathways might interact with GUN1 or that GUN1 might have nuclear functions under specific conditions. For researchers exploring this possibility, these considerations for ChIP experiments are important:

Crosslinking optimization:

• Test different formaldehyde concentrations (0.5-3%) and incubation times (5-20 minutes)

• Consider dual crosslinking with DSG (disuccinimidyl glutarate) followed by formaldehyde

• Optimize quenching conditions (125-250 mM glycine)

Chromatin fragmentation:

• Determine optimal sonication conditions (amplitude, pulse duration, cycle number)

• Aim for DNA fragments of 200-500 bp

• Verify fragmentation efficiency by agarose gel electrophoresis

Antibody specificity concerns:

• Perform ChIP in gun1 mutant plants as negative control

• Include IgG control immunoprecipitations

• Validate with antibodies against known DNA-binding proteins as positive controls

ChIP-qPCR design:

• Select candidate target regions based on promoter analysis of genes affected in gun1 mutants

• Design primers for qPCR with amplicon sizes of 80-150 bp

• Include primers for negative control regions (typically housekeeping genes)

Data analysis approach:

• Calculate percent input or fold enrichment relative to IgG control

• Perform statistical analysis using Student's t-tests as described in previous studies

• For genome-wide approaches (ChIP-seq), implement peak calling algorithms suitable for transcription factors

Previous studies have identified DNA-binding motifs in promoters of genes regulated by pathways involving GUN1, which could guide the selection of candidate regions for ChIP-qPCR. For example, researchers have used tools like TAIR's 'Motif Analysis' for statistically overrepresented 6-mer motifs in promoter regions .

What are the most promising future applications of AT2G31400/GUN1 antibodies in plant biology research?

Several emerging technologies hold great promise for expanding the utility of GUN1 antibodies in plant research:

Spatial proteomics approaches:
Advances in spatial proteomics could enable mapping GUN1's distribution within chloroplast subcompartments and tracking its relocalization during retrograde signaling. This would provide unprecedented insights into how GUN1's spatial organization correlates with its function in integrating signals.

Multi-omics integration:
Combining GUN1 antibody-based proteomics with transcriptomics and metabolomics will allow researchers to build comprehensive models of retrograde signaling networks. Previous studies have already begun integrating microarray data to understand GUN1's role in gene expression regulation .

Structural biology applications:
GUN1 antibodies could aid in protein complex purification for structural studies using cryo-EM, potentially revealing the molecular architecture of GUN1-containing complexes and providing mechanistic insights into its function.

In vivo dynamics:
Development of intrabodies (intracellular antibodies) derived from GUN1 antibodies could enable real-time tracking of GUN1 protein dynamics in living plants, providing insights into its behavior during environmental responses.

Synthetic biology applications:
GUN1 antibodies could facilitate the development of synthetic retrograde signaling circuits by allowing precise monitoring of engineered GUN1 variants and their interaction partners.

These applications will be particularly valuable for understanding how GUN1 integrates multiple signaling pathways, as suggested by studies showing its involvement in both light signaling and chloroplast development pathways .

What are the current limitations of AT2G31400/GUN1 antibodies and how might they be addressed in future research?

Current GUN1 antibody applications face several limitations that future research could address:

Specificity limitations:
GUN1 belongs to the PPR protein family, which has numerous members with similar sequence motifs. Future approaches should include:

• Development of monoclonal antibodies targeting unique regions of GUN1

• Creation of synthetic antibodies using phage display technology

• Implementation of CRISPR-based epitope tagging of endogenous GUN1

Detection sensitivity:
GUN1 may be expressed at low levels or only under specific conditions, making detection challenging. Future improvements could include:

• Signal amplification methods such as tyramide signal amplification

• Development of more sensitive detection systems like proximity ligation assays

• Implementation of highly sensitive mass spectrometry approaches such as PRISM (high-pressure, high-resolution separations with intelligent selection and multiplexing)

Post-translational modification detection:
Current antibodies may not discriminate between modified forms of GUN1. Future development of modification-specific antibodies against phosphorylated, acetylated, or ubiquitinated GUN1 would provide valuable insights into its regulation.

Temporal resolution:
Current methods often provide snapshots rather than dynamic information. Development of biosensors based on GUN1 antibody fragments could enable real-time monitoring of GUN1 activity or interactions.

Species limitation:
Most research on GUN1 has been conducted in Arabidopsis thaliana. Development of cross-species reactive antibodies would enable comparative studies across different plant species, including crops, expanding our understanding of retrograde signaling evolution.

By addressing these limitations, researchers will gain more powerful tools to unravel the complex functions of GUN1 in integrating chloroplast signals and regulating nuclear gene expression, ultimately advancing our understanding of chloroplast-to-nucleus communication in plants.

Practical Considerations for AT2G31400/GUN1 Antibody Generation

Researchers planning to generate new antibodies against GUN1 should consider several critical factors for successful development:

What are the optimal epitope selection strategies for generating AT2G31400/GUN1 antibodies?

When selecting epitopes for GUN1 antibody generation, researchers should consider the protein's structural features and accessibility:

• Avoid conserved PPR motifs: GUN1 contains multiple pentatricopeptide repeat (PPR) domains that share sequence similarity with other PPR proteins. Target unique regions outside these conserved domains to minimize cross-reactivity .

• Target protein-specific regions: The C-terminal region containing the Smr domain (InterPro:IPR002625) offers a relatively unique sequence that may provide higher specificity .

• Consider protein topology: Based on GUN1's chloroplast localization and predicted membrane associations, select epitopes that are likely to be surface-exposed and accessible to antibodies .

• Evaluate sequence conservation: If cross-species reactivity is desired, align GUN1 sequences from multiple plant species and select epitopes conserved across target species.

• Peptide properties: For synthetic peptide antigens, select regions with balanced hydrophobicity, avoid extensive stretches of hydrophobic residues, and ensure adequate solubility for conjugation to carrier proteins.

For example, in previous studies, researchers have successfully generated antibodies against specific peptides from target proteins, such as the peptide CDNERGLRGGSHSKG for RH50 antibody production . Similar approaches could be applied to GUN1-specific regions.