ATXR6 Antibody

What is ATXR6 and what is its primary function in plants?

ATXR6 (ARABIDOPSIS TRITHORAX-RELATED PROTEIN 6) is a plant-specific histone methyltransferase that catalyzes monomethylation of histone H3 at lysine 27 (H3K27me1). It functions as part of a critical epigenetic regulatory system in plants, particularly in Arabidopsis thaliana. ATXR6, along with its paralog ATXR5, plays an essential role in heterochromatin formation and maintenance. These proteins specifically catalyze H3K27 monomethylation at constitutive heterochromatin regions, which is crucial for proper chromatin condensation and transcriptional gene silencing of heterochromatic elements such as transposons and DNA repeats . Unlike the well-known E(Z) homologs (MEA, CLF, and SWN) that mediate H3K27 di- and trimethylation, ATXR5 and ATXR6 comprise a distinct class of H3K27 methyltransferases that specifically generate the monomethylated form .

Why are antibodies against ATXR6 important for plant epigenetics research?

ATXR6 antibodies are critical research tools for investigating heterochromatin formation and epigenetic regulation in plants. These antibodies enable researchers to track ATXR6 protein localization, quantify expression levels, and perform chromatin immunoprecipitation (ChIP) experiments to identify genomic regions where ATXR6 binds. Since ATXR6 functions in a pathway distinct from but complementary to DNA methylation and H3K9 methylation pathways for silencing heterochromatic elements, antibodies against this protein help researchers dissect the specific contribution of H3K27me1 to gene silencing and chromatin organization . Additionally, ATXR6 antibodies facilitate studies examining how this protein interacts with cell cycle regulation through its PCNA-interacting protein (PIP) box, providing insights into the mechanisms of epigenetic inheritance during DNA replication .

How do ATXR5 and ATXR6 differ structurally and functionally?

ATXR5 and ATXR6 share significant sequence homology and similar domain structure, each containing a plant homeo-domain (PHD) and a SET domain responsible for catalyzing H3K27 monomethylation. Both proteins also contain a PCNA-interacting protein (PIP) box, suggesting involvement in DNA replication processes . The key structural regions of ATXR6 include amino acids 25-349 encompassing the PHD-SET domains, with the PHD domain specifically located at amino acids 25-103 .

What are the recommended protocols for using ATXR6 antibodies in chromatin immunoprecipitation (ChIP) experiments?

For effective ChIP experiments using ATXR6 antibodies, researchers should follow these methodological guidelines:

• Crosslinking and Chromatin Preparation:

  • Fix plant tissue (typically seedlings or leaves) with 1% formaldehyde for 10-15 minutes at room temperature

  • Quench with 0.125M glycine for 5 minutes

  • Isolate nuclei using extraction buffer (0.25M sucrose, 10mM Tris-HCl pH 8.0, 10mM MgCl₂, 1% Triton X-100, protease inhibitors)

  • Sonicate chromatin to fragments of 200-500bp (conditions must be optimized for specific sonicator)

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Incubate cleared chromatin with 2-5μg ATXR6 antibody overnight at 4°C

  • Add protein A/G beads and incubate for 2-3 hours

  • Perform stringent washes to remove non-specific binding

• Target Validation:

  • Include positive controls by testing known ATXR6 targets like TSI, Ta3, and CACTA transposon sequences

  • Use IgG antibodies as negative control

  • Include input samples (non-immunoprecipitated chromatin) for normalization

When analyzing ChIP data, researchers should normalize to input and calculate enrichment relative to a control region. For investigating ATXR6's relationship with its target H3K27me1 mark, parallel ChIP experiments using H3K27me1-specific antibodies on wild-type and atxr5 atxr6 mutant tissues are recommended to correlate ATXR6 binding with H3K27me1 deposition .

How can I validate the specificity of an ATXR6 antibody for immunostaining experiments?

Validating ATXR6 antibody specificity for immunostaining requires several complementary approaches:

• Genetic Controls:

  • Perform parallel immunostaining in wild-type and atxr6 mutant tissues (ideally in atxr5 atxr6 double mutants to account for redundancy)

  • Expect significantly reduced or absent signal in mutant samples

• Peptide Competition Assay:

  • Pre-incubate the antibody with excess ATXR6 peptide antigen

  • Perform immunostaining with both blocked and unblocked antibody

  • Specific staining should be eliminated or greatly reduced in the blocked sample

• Western Blot Validation:

  • Confirm antibody recognizes a band of the expected molecular weight (approximately 39kDa for ATXR6) in plant extracts

  • Verify band intensity is reduced in mutant samples or RNAi lines

• Co-localization Studies:

  • Perform dual immunostaining with ATXR6 antibody and antibodies against H3K27me1

  • Expect substantial overlap in chromocenter regions, as ATXR6 is responsible for H3K27me1 deposition at these locations

• Recombinant Protein Controls:

  • Test antibody reactivity against purified recombinant ATXR6 protein

  • Confirm specificity by demonstrating lack of cross-reactivity with ATXR5 despite sequence similarity

When conducting immunostaining experiments, researchers should carefully optimize fixation conditions (typically 4% paraformaldehyde), antibody dilution (usually starting with 1:100 to 1:500), and include appropriate blocking steps to minimize background staining.

What are the optimal conditions for detecting ATXR6 in Western blot analyses?

For optimal detection of ATXR6 in Western blot analyses, researchers should follow these methodological considerations:

• Sample Preparation:

  • Extract proteins from plant nuclear fractions using buffer containing 50mM Tris-HCl pH 7.5, 150mM NaCl, 5mM EDTA, 0.1% Triton X-100, 10% glycerol, and protease inhibitors

  • Add phosphatase inhibitors if investigating potential post-translational modifications

  • Use fresh tissue whenever possible or flash-freeze in liquid nitrogen

• Gel Electrophoresis and Transfer:

  • Use 12-15% SDS-PAGE gels for optimal resolution of ATXR6 (approximately 39kDa)

  • Transfer proteins to PVDF membrane at 100V for 60-90 minutes in standard transfer buffer

  • Verify transfer efficiency with reversible protein stain

• Antibody Incubation:

  • Block membrane with 5% non-fat dry milk in TBST for 1 hour at room temperature

  • Incubate with ATXR6 primary antibody (1:1000 to 1:5000 dilution) overnight at 4°C

  • Wash extensively with TBST (at least 3 x 10 minutes)

  • Incubate with HRP-conjugated secondary antibody (1:5000 to 1:10000) for 1 hour at room temperature

• Detection and Controls:

  • Use enhanced chemiluminescence (ECL) detection systems with exposure times of 30 seconds to 5 minutes

  • Include recombinant ATXR6 protein as positive control

  • Include samples from atxr6 mutants as negative controls

  • For loading control, reprobe with antibodies against histone H3 or other nuclear proteins

When interpreting Western blot results, researchers should be aware that ATXR6 signal intensity may vary across different tissues and developmental stages, reflecting its differential expression patterns. Additionally, due to functional redundancy with ATXR5, phenotypic effects may only be apparent in double mutants despite successful detection of ATXR6 protein reduction in single mutants .

How can I determine if ATXR6 directly interacts with specific heterochromatic regions in my plant model?

To determine direct interactions between ATXR6 and specific heterochromatic regions, researchers should implement a multi-faceted experimental approach:

• Chromatin Immunoprecipitation followed by Sequencing (ChIP-seq):

  • Perform ChIP using validated ATXR6 antibodies

  • Construct sequencing libraries from immunoprecipitated DNA

  • Map sequence reads to the reference genome

  • Analyze enrichment patterns, focusing on heterochromatic regions, transposons, and repetitive elements

  • Look for enrichment at known targets such as TSI, Ta3, and CACTA transposon sequences

• Parallel H3K27me1 ChIP-seq:

  • Conduct parallel H3K27me1 ChIP-seq experiments

  • Compare ATXR6 binding sites with H3K27me1 enrichment patterns

  • Quantify correlation between ATXR6 occupancy and H3K27me1 levels

• Genetic Validation:

  • Perform H3K27me1 ChIP-seq in wild-type and atxr5 atxr6 double mutants

  • Identify genomic regions with reduced H3K27me1 in the mutants

  • These regions represent likely direct ATXR6/ATXR5 targets

• In Vitro Binding Assays:

  • Conduct electrophoretic mobility shift assays (EMSAs) with recombinant ATXR6 protein and candidate DNA sequences

  • Test binding to different heterochromatic sequences to determine sequence preferences

• Targeted Chromatin Analysis:

  • Design primers for specific heterochromatic regions of interest

  • Perform targeted ChIP-qPCR to quantify ATXR6 enrichment

  • Compare enrichment levels between constitutive heterochromatin and euchromatic regions

When interpreting results, researchers should consider that ATXR6 may not bind DNA directly but rather interact with chromatin through its PHD domain recognizing specific histone modifications, or through interactions with other chromatin-associated proteins . The PCNA-interacting motif in ATXR6 suggests its binding may also be influenced by DNA replication status, potentially showing cell cycle-dependent association patterns .

What is the relationship between ATXR6 activity and other epigenetic marks in heterochromatin maintenance?

The relationship between ATXR6 activity and other epigenetic marks involves complex interactions but maintains distinct functional pathways:

• Independence from DNA Methylation and H3K9me2:

  • ATXR6 (with ATXR5) functions in a pathway parallel to DNA methylation and H3K9me2

  • In atxr5 atxr6 double mutants, DNA methylation and H3K9me2 levels remain unchanged despite transcriptional activation of silenced elements

  • Conversely, H3K27me1 levels were found to be unchanged in DNA methylation or H3K9 methyltransferase mutants

• Synergistic Effects on Gene Silencing:

  • Both H3K27me1 (via ATXR5/6) and DNA methylation/H3K9me2 pathways are required for complete silencing of heterochromatic elements

  • Loss of either pathway results in partial reactivation of silenced elements

  • The two pathways appear to cooperate in maintaining heterochromatin structure and function

• Chromatin Structural Organization:

  • Both pathways contribute to chromocenter organization

  • atxr5 atxr6 double mutants show partial heterochromatin decondensation

  • This suggests that H3K27me1 plays a structural role in organizing heterochromatin

• Cell Cycle Regulation Interface:

  • ATXR6 contains a PCNA-interacting protein (PIP) box

  • This suggests a potential role in maintaining H3K27me1 through DNA replication

  • May provide a mechanism for epigenetic inheritance distinct from the DNA methylation maintenance pathway

This evidence establishes ATXR6-mediated H3K27me1 as an essential and independent epigenetic pathway that works in parallel with DNA methylation and H3K9me2 to maintain heterochromatin integrity and silence transposable elements. While these pathways operate independently at the biochemical level, they converge functionally to ensure proper heterochromatin structure and transcriptional silencing .

How do mutations in ATXR6 affect genome stability and heterochromatin organization?

Mutations in ATXR6, particularly in combination with ATXR5 mutations, significantly impact genome stability and heterochromatin organization through several mechanisms:

The combined evidence demonstrates that ATXR6, along with ATXR5, establishes an essential epigenetic pathway required for heterochromatin maintenance and genome stability. Their H3K27 monomethyltransferase activity provides a separate layer of epigenetic control that works alongside but independently of DNA methylation and H3K9me2 pathways .

Why might I observe inconsistent immunostaining results with ATXR6 antibodies in different plant tissues?

Inconsistent immunostaining results with ATXR6 antibodies across different plant tissues can stem from several methodological and biological factors:

• Differential Expression Patterns:

  • ATXR5 and ATXR6 show partially overlapping but distinct expression patterns across tissues

  • Tissues with naturally lower ATXR6 expression will show reduced signal intensity

  • Expression levels may vary with developmental stage and environmental conditions

• Fixation and Permeabilization Variability:

  • Different tissue types have varying cell wall and membrane compositions

  • This affects penetration of fixatives and antibodies

  • Optimize fixation time (typically 15-30 minutes) and concentration (3-4% paraformaldehyde) for each tissue type

  • Consider enzymatic cell wall digestion for tissues with thicker cell walls

• Epitope Accessibility Issues:

  • Chromatin compaction state varies between tissues and can mask epitopes

  • ATXR6 association with heterochromatin may make the protein less accessible in certain cell types

  • Try antigen retrieval methods (heat or pH-based) to improve epitope exposure

• Functional Redundancy with ATXR5:

  • ATXR5 and ATXR6 have redundant functions

  • Tissues with higher ATXR5 expression may show reduced phenotypic effects of ATXR6 absence

  • For clearer results, use atxr5 atxr6 double mutants as negative controls

• Cell Cycle Dependence:

  • ATXR6 contains a PCNA-interacting protein (PIP) box suggesting cell cycle regulation

  • Signal intensity may vary with cell cycle stage

  • Actively dividing tissues may show different localization patterns

  • Consider synchronizing cells or co-staining with cell cycle markers

To address these issues, researchers should implement standardized protocols with tissue-specific optimizations, include appropriate controls (both positive and negative), and interpret results in the context of known ATXR6 biology. When possible, complement immunostaining with other detection methods such as Western blotting or RT-qPCR to verify expression patterns across tissues.

How do I interpret ChIP-seq data when analyzing ATXR6 binding in relation to H3K27me1 distribution?

Interpreting ChIP-seq data for ATXR6 binding in relation to H3K27me1 distribution requires careful analysis and consideration of several key factors:

This analytical framework allows researchers to distinguish direct ATXR6 targets from regions where H3K27me1 might be deposited through other mechanisms. When presenting ChIP-seq data, include genome browser tracks showing ATXR6 binding, H3K27me1 distribution, and other relevant chromatin marks to visualize spatial relationships and overlap patterns.

What controls should I include when studying the effects of ATXR6 antibody in histone methyltransferase assays?

For rigorous histone methyltransferase assays with ATXR6 antibodies, researchers should implement comprehensive controls addressing enzymatic activity, antibody specificity, and experimental validity:

• Enzyme Activity Controls:

  • Positive Control: Include GST-tagged ATXR6 recombinant protein with known activity

  • Negative Control: Use catalytically inactive ATXR6 mutant (mutation in SET domain)

  • Substrate Specificity Control: Test activity on different histone proteins (H3 should show activity while other histones should not)

• Substrate Verification Controls:

  • Wild-type H3 Control: Should show methylation by ATXR6

  • H3K27A Mutant Control: Mutation of lysine 27 to alanine should eliminate methylation

  • Include both calf thymus histones and recombinant H3 as substrates to compare native and recombinant contexts

• Methylation State Controls:

  • Test reactivity with antibodies specific for H3K27me1, H3K27me2, and H3K27me3

  • Expect positive signal only with H3K27me1 antibody, confirming ATXR6's monomethyltransferase specificity

  • Extended incubation time control to verify ATXR6 cannot add second or third methyl groups

• Assay Validation Controls:

  • No-enzyme control: Reaction mixture without ATXR6 protein

  • No-SAM control: Reaction mixture without S-adenosyl methionine cofactor

  • Time course: Multiple time points to establish reaction kinetics

  • Temperature sensitivity: Test activity at different temperatures (typically 30°C optimal)

• Detection Controls for Western Blot Analysis:

  • Include methylated H3 peptide standards for antibody validation

  • Test antibody cross-reactivity with differentially methylated H3K27 peptides

  • Include both radioisotope-labeled (14C-SAM) and unlabeled SAM detection methods for confirmation

When designing a histone methyltransferase assay, researchers should use standardized conditions as described in the literature: 50mM Tris-HCl pH 8.5, 20mM KCl, 10mM MgCl2, 10mM β-mercaptoethanol, and 250mM sucrose buffer, with incubation at 30°C for 3 hours . These conditions have been demonstrated to support optimal ATXR6 enzymatic activity.

How do the functions of ATXR6 in plants compare to histone methyltransferases in other organisms?

ATXR6 represents a unique class of histone methyltransferase with distinct evolutionary and functional characteristics compared to methyltransferases in other organisms:

• Evolutionary Distinctiveness:

  • ATXR5 and ATXR6 are plant-specific H3K27 methyltransferases not related to the Drosophila E(Z) protein

  • They represent the first identified eukaryotic H3K27 methyltransferases distinct from the well-characterized Polycomb Repressive Complex 2 (PRC2) enzymes

  • This suggests independent evolution of H3K27 methylation systems in plants

• Methylation State Specificity:

  • ATXR6 exclusively catalyzes monomethylation of H3K27

  • In contrast, PRC2 complexes in animals and the plant E(Z) homologs (MEA, CLF, SWN) primarily catalyze H3K27me2/3

  • This specialized activity distinguishes ATXR6 functionally from other H3K27 methyltransferases

• Genomic Targeting:

  • ATXR6 targets constitutive heterochromatin in plants

  • In contrast, animal PRC2 complexes primarily target facultative heterochromatin in euchromatic regions

  • Plant PRC2 complexes (containing E(Z) homologs) also primarily target euchromatic regions

• Structural Features:

  • ATXR6 contains a PHD domain and PCNA-interacting motif

  • The PCNA interaction suggests a direct connection to DNA replication, similar to mammalian SETDB1 and G9A histone methyltransferases

  • This structural organization differs from PRC2 components, which function in multi-protein complexes

• Functional Impact:

  • In mammals, H3K27me1 is enriched at pericentromeric heterochromatin (similar to plants) but also broadly distributed in euchromatin

  • Gymnosperms show uniform H3K27me1 distribution along chromosomes

  • These differences suggest that H3K27me1 function and distribution have evolved differently across lineages

This comparative analysis highlights ATXR6 as part of a plant-specific epigenetic regulatory system that parallels but differs mechanistically from systems in other organisms. The distinct evolutionary origin and specialized function of ATXR6 make it a particularly interesting subject for studying convergent evolution of epigenetic mechanisms across kingdoms.

What are the emerging applications of ATXR6 antibodies in plant biotechnology and crop improvement?

Emerging applications of ATXR6 antibodies in plant biotechnology and crop improvement span several innovative research directions:

• Epigenome Engineering:

  • ATXR6 antibodies enable precise mapping of heterochromatin dynamics during stress responses

  • This knowledge informs targeted epigenetic modifications to improve stress tolerance

  • Researchers can monitor changes in H3K27me1 distribution when introducing modified ATXR6 variants

  • The independent nature of the H3K27me1 pathway from DNA methylation provides an additional lever for epigenetic manipulation

• Transposon Activity Management:

  • ATXR6 antibodies help track heterochromatin stability in crop breeding programs

  • Since ATXR6 regulates transposon silencing, its antibodies can monitor potential transposon reactivation events

  • This application is particularly valuable for wide-cross hybrids where genome compatibility issues may destabilize heterochromatin

• Cell Cycle-Specific Chromatin Dynamics:

  • The PCNA-interacting motif in ATXR6 connects its function to DNA replication

  • Antibodies against ATXR6 allow researchers to track how heterochromatin marks are maintained through cell division

  • This knowledge informs strategies for maintaining desirable epigenetic states across generations

• Chromatin State Diagnostics:

  • ATXR6 antibodies provide a tool to assess heterochromatin integrity in plant tissues

  • Changes in ATXR6 localization or H3K27me1 patterns may serve as early indicators of epigenomic instability

  • This application could help identify varieties with more stable epigenomes for breeding programs

• Comparative Epigenomics:

  • ATXR6 antibodies facilitate cross-species comparison of heterochromatin organization

  • This information helps translate knowledge from model plants to crops

  • Researchers can establish how heterochromatin mechanisms differ between species with varying genome sizes and repetitive element content

These applications leverage ATXR6 antibodies as powerful tools for understanding and manipulating plant epigenomes. As research progresses, these applications will likely expand to include more sophisticated approaches to crop improvement through targeted epigenetic modifications focusing on heterochromatin stability and transposon control.

What gene expression changes are associated with ATXR6 dysfunction, and how might this impact plant development?

ATXR6 dysfunction, particularly in combination with ATXR5 mutation, leads to specific patterns of gene expression changes with significant developmental implications:

The developmental impact of ATXR6 dysfunction demonstrates how specialized epigenetic pathways contribute to normal plant growth and development. The connection between heterochromatin maintenance and proper growth regulation reveals the essential nature of this epigenetic pathway, despite its independence from the more extensively studied DNA methylation and H3K9me2 pathways .