At4g35335 Antibody

What is the At4g35335 gene and what protein does it encode?

At4g35335 is an Arabidopsis thaliana gene that encodes HDA9 (Histone Deacetylase 9), which belongs to the RPD3-like family of histone deacetylases. HDA9 plays a pivotal role in promoting the onset of leaf senescence and is critical for the deacetylation of histone H3K9 and H3K27 in vivo . Unlike general assumptions about histone deacetylases (HDACs) being exclusively associated with gene silencing, HDA9 shows preferential enrichment in promoters of active genes rather than silent genes, suggesting a more complex regulatory function .

What are the key protein interactions of HDA9 revealed through antibody-based research?

Immunoprecipitation followed by mass spectrometry (IP-MS) research has identified several key HDA9 protein interactions. Most notably, HDA9 strongly interacts with POWERDRESS (PWR), a SANT domain-containing protein, as evidenced by the co-purification of 27 unique peptides of PWR with HDA9 . Additionally, HDA9 interacts with the WRKY53 transcription factor, which is induced during early leaf senescence and promotes senescence onset. This interaction has been confirmed through GST pull-down assays where HDA9-FLAG was successfully pulled down by GST-WRKY53 but not by GST alone . These interactions suggest that HDA9 functions within a complex protein network to regulate gene expression during developmental processes.

What epitope tags are commonly used with At4g35335/HDA9 antibodies?

For detection and purification of HDA9 in research settings, several epitope tags have been successfully employed. The most commonly used tags include FLAG and HA epitopes, which can be detected with horseradish peroxidase (HRP) conjugated anti-FLAG (Sigma, A8592) and anti-HA (Roche, 12013819001) antibodies, respectively . Specifically, transgenic Arabidopsis plants expressing HDA9-3xFLAG driven by the native HDA9 promoter (pHDA9::HDA9-3xFLAG) have been successfully generated and utilized for functional studies and protein interaction analyses .

How should I design experiments to study HDA9 chromatin binding patterns?

To study HDA9 chromatin binding patterns, researchers should implement Chromatin Immunoprecipitation followed by sequencing (ChIP-seq) using epitope-tagged HDA9. First, generate transgenic plants expressing HDA9-FLAG driven by its native promoter in the hda9 mutant background to ensure functionality (confirm this by phenotypic rescue) . For ChIP-seq library preparation, the Ovation Ultralow DR Multiplex System (NuGEN, #0330) has proven effective . Include appropriate controls by performing parallel ChIP-seq in wild-type plants without the tagged protein. For data analysis, align reads to the Arabidopsis reference genome (TAIR10) using Bowtie2 (v2.1) and identify enriched regions . Specifically analyze the distribution of binding sites across genomic features (promoters, gene bodies, etc.) and correlate binding patterns with gene expression data to determine functional relationships between HDA9 binding and transcriptional activity .

What is the optimal protocol for immunoprecipitation of HDA9 and its interacting partners?

For optimal immunoprecipitation of HDA9 and its interacting partners, researchers should:

• Generate transgenic plants expressing epitope-tagged HDA9 (HDA9-FLAG) under its native promoter

• Harvest appropriate tissue (considering developmental stage and tissue specificity)

• Extract nuclear proteins using a buffer system that preserves protein-protein interactions

• Perform immunoprecipitation using anti-FLAG beads

• For reciprocal confirmation of interactions, conduct parallel IP with tagged versions of putative interacting partners (e.g., PWR-FLAG)

• Validate interactions through co-immunoprecipitation in plants expressing both HA-tagged HDA9 and FLAG-tagged interacting partners

• Conduct in vitro validation through methods such as GST pull-down assays using purified proteins

This methodology has successfully identified interactions between HDA9 and PWR, as well as between HDA9 and WRKY53 transcription factor .

How can I assess the functional specificity of HDA9 antibodies in histone modification studies?

To assess the functional specificity of HDA9 antibodies in histone modification studies, implement a comprehensive validation approach:

• Genetic controls: Compare antibody signals between wild-type and hda9 mutant plants

• Histone modification antibody panel: Use a diverse panel of histone antibodies including H3K9ac (Millipore, 07-352), H3K27ac (Active Motif, 39133), H3ac (Active Motif, 39139), H4K8ac (Millipore, 07-328), H4K12ac (Millipore, 07-595), H4K16ac (Millipore, 07-329), and H4ac (Active Motif, 39243)

• Control for total histone levels: Include antibodies against unmodified histones like H3 (Abcam, ab1791) and H4 (Abcam, ab7311) for normalization

• Functional validation: Correlate antibody-detected changes with expected biological outcomes (e.g., increased H3K9ac and H3K27ac levels in hda9 mutants)

• Detection methods: Use ECL Plus Western Blotting Detection System (GE healthcare, RPN2132) for consistent results

How does HDA9 binding correlate with chromatin accessibility and gene expression patterns?

HDA9 exhibits a counterintuitive binding pattern that challenges conventional understanding of histone deacetylases as transcriptional repressors. ChIP-seq analysis of HDA9 binding in Arabidopsis has revealed that HDA9 is highly enriched in gene-rich euchromatic regions but depleted in repeat-rich centromeric heterochromatin . Importantly, approximately 69% of the 9,489 identified HDA9 binding peaks were located in promoter regions .

Contrary to expectations for a histone deacetylase, HDA9 is preferentially enriched in the promoters of actively transcribed genes rather than silent genes. HDA9-bound genes show significantly higher expression than the average expression levels of all genes . This pattern is further supported by the co-localization between HDA9 binding sites and DNase I hypersensitive sites, which are generally associated with accessible chromatin states .

Several hypotheses may explain this pattern:

• HDA9 may be recruited to active gene promoters to prevent promiscuous cryptic transcription

• HDA9 may compete with more active histone deacetylases for binding to similar genomic regions

• Silent genes in Arabidopsis tend to have high promoter DNA methylation and may be repressed by DNA methylation rather than histone deacetylation

This unique binding pattern suggests a more complex role for HDA9 in transcriptional regulation than simply acting as a repressor.

What are the implications of the HDA9-WRKY53 interaction for senescence research?

The physical interaction between HDA9 and WRKY53 transcription factor has significant implications for senescence research. WRKY53 is known to be induced at the early stage of leaf senescence and promotes the onset of senescence . This interaction is particularly important because:

• HDA9 binding peaks are significantly enriched for WRKY binding motifs, suggesting a mechanistic basis for their co-localization on chromatin

• WRKY53 can function as either a transcriptional activator or repressor in leaf senescence processes

• The interaction suggests WRKY53 may recruit HDA9 to active genes to remove acetylation marks added by histone acetyltransferases (HATs) to maintain proper expression levels during senescence

This interaction represents a molecular mechanism by which transcription factor-guided histone deacetylation may fine-tune gene expression during developmental transitions like senescence. Future research using HDA9 antibodies should investigate the temporal dynamics of this interaction and its regulation under different environmental conditions that affect senescence.

How can cell-penetrating antibody technology be adapted for plant research on nuclear proteins like HDA9?

Adapting cell-penetrating antibody technology for plant research on nuclear proteins like HDA9 represents a frontier opportunity, drawing inspiration from recent advancements in mammalian systems. Traditional antibodies face significant barriers in targeting intracellular proteins in living cells, but the emergence of cell-penetrating antibodies like 3E10 in cancer research offers promising directions for plant science adaptation .

For HDA9 research, consider:

• Antibody engineering: Modify plant-specific antibodies with cell-penetrating peptides (CPPs) or leverage naturally cell-penetrating antibody frameworks

• Plant cell wall considerations: Develop methods to overcome the additional barrier of plant cell walls, potentially through:

  • Protoplast-based intermediate systems

  • Nanoparticle conjugation for cell wall penetration

  • Microinjection techniques for direct delivery

• Validation protocols: Establish clear subcellular localization confirmation through:

  • Confocal microscopy with fluorescently-tagged antibodies

  • Biochemical fractionation followed by Western blotting

  • Split-reporter systems activated upon successful nuclear targeting

Such approaches would enable novel experimental paradigms, including real-time tracking of HDA9 dynamics and direct intervention in protein-protein interactions like HDA9-WRKY53 binding during senescence progression.

How should researchers interpret contradictory results between ChIP-seq and RNA-seq data involving HDA9?

When encountering contradictory results between ChIP-seq and RNA-seq data involving HDA9, researchers should apply a systematic analytical framework:

The observation that HDA9 preferentially binds to active genes contradicts the conventional view of HDACs as strict repressors, highlighting the need for nuanced interpretation of epigenetic regulator functions .

What controls should be included when validating HDA9 antibody specificity in plant tissues?

When validating HDA9 antibody specificity in plant tissues, a comprehensive set of controls should be included:

• Genetic Controls:

  • Wild-type plants (positive control)

  • hda9 knockout mutants (negative control)

  • HDA9 complementation lines (rescue control)

  • Transgenic plants expressing epitope-tagged HDA9 (reference control)

• Specificity Controls:

  • Pre-adsorption control (pre-incubating antibody with purified antigen)

  • Cross-reactivity assessment with closely related HDAC family members

  • Secondary antibody-only controls to assess non-specific binding

• Technical Controls:

  • Multiple antibody dilutions to establish optimal signal-to-noise ratios

  • Different tissue types and developmental stages to assess context-dependent specificity

  • Parallel detection with independent antibodies targeting different epitopes of HDA9

• Validation Methods:

  • Western blotting using nuclear extracts

  • Immunoprecipitation followed by mass spectrometry

  • Immunohistochemistry correlated with known expression patterns

  • ChIP-qPCR at known HDA9 target loci prior to genome-wide analyses

These controls ensure that observed signals truly represent HDA9 and not experimental artifacts or cross-reactive proteins.

What are the critical considerations when tracking the dynamics of HDA9-protein interactions during leaf senescence?

When tracking the dynamics of HDA9-protein interactions during leaf senescence, researchers should consider these critical factors:

• Developmental staging: Precisely define leaf senescence stages using established markers (chlorophyll content, senescence-associated gene expression) to ensure reproducible sampling across experiments

• Time-course design: Implement a fine-grained temporal sampling approach capturing pre-senescence, early senescence, mid-senescence, and late senescence phases to track dynamic changes in interaction patterns

• Interactome shifts: Account for the changing cellular environment during senescence, including:

  • The upregulation of WRKY53 expression in early senescence stages

  • Potential competition between different WRKY transcription factors for HDA9 binding

  • Changes in subcellular localization of interaction partners

• Integration with functional data:

  • Correlate interaction dynamics with changes in histone acetylation patterns at target loci

  • Track corresponding gene expression changes at HDA9-bound genes

  • Assess phenotypic progression of senescence in plants with mutations affecting the interaction

• Methodological considerations:

  • Use techniques capable of detecting transient and weak interactions

  • Consider native protein concentration levels when interpreting in vitro interaction studies

  • Validate key interactions using multiple methodologies (co-IP, BiFC, FRET)

This comprehensive approach will provide insights into how HDA9-protein interaction networks reconfigure during the senescence process and how these reconfigurations relate to the epigenetic regulation of senescence-associated genes.

How can tracking technologies used in Google Analytics be adapted to monitor At4g35335/HDA9 antibody binding in live cell imaging?

The principles behind Google Analytics tracking methodologies, particularly those used for tracking "People Also Ask" features, can be creatively adapted for monitoring HDA9 antibody binding in live cell imaging contexts . Just as Google's text fragment identifiers (#:~:text=) allow precise targeting of specific content on webpages, advanced microscopy techniques can be developed to track specific binding events of antibodies to HDA9 protein in living plant cells.

This approach could involve:

• Fragment-based tracking system: Develop a molecular equivalent to URL text fragments by creating a system where specific antibody-binding events generate unique spectral signatures that can be tracked over time

• Event-based detection: Similar to how Google Analytics uses JavaScript variables to detect specific URL fragments (#:~:text=), researchers could implement fluorescent reporter systems that activate only upon specific binding events between antibodies and HDA9 target sites

• Data capture methodology: Create a unified data acquisition pipeline that captures:

  • Spatiotemporal binding patterns

  • Binding duration metrics

  • Co-occurrence with other labeled proteins

  • Correlation with cell physiological states

• Variable binding context: Account for different binding environments within the nucleus, similar to how tracking systems detect different user contexts in analytics

This innovative cross-disciplinary approach would enable researchers to generate dynamic maps of HDA9 interactions in living cells during developmental transitions like senescence.

What potential exists for developing cell-penetrating HDA9 antibodies for in vivo chromatin remodeling studies?

Recent breakthroughs in cell-penetrating monoclonal antibody technologies present exciting opportunities for developing tools to study HDA9 function in living plants . The development of such antibodies could revolutionize chromatin remodeling studies by enabling:

• Direct targeting of nuclear HDA9: By engineering HDA9 antibodies with cell-penetrating capabilities similar to the 3E10 antibody used in cancer research, researchers could directly access intracellular HDA9 in intact plant cells

• Functional interference studies: Cell-penetrating antibodies could be designed to specifically block the interaction between HDA9 and key partners like WRKY53 or PWR, allowing for acute disruption of function without genetic manipulation

• Payload delivery systems: Similar to how 3E10 antibodies can carry therapeutic molecules into cancer cells, cell-penetrating HDA9 antibodies could be used to deliver:

  • Chromatin modifiers to specific genomic loci

  • DNA or RNA molecules for site-specific genome editing

  • Fluorescent probes for real-time monitoring of chromatin dynamics

• Comparative systems: The development of plant-specific cell-penetrating antibody frameworks would enable comparative studies between plant and animal chromatin remodeling mechanisms

This approach would bridge the gap between in vitro biochemical studies and in vivo genetic approaches by providing a direct intervention tool for studying HDA9 function in intact cells.

How might the integration of HDA9 ChIP-seq data with DNase hypersensitivity mapping advance our understanding of plant epigenetic regulation?

The integration of HDA9 ChIP-seq data with DNase hypersensitivity mapping represents a powerful approach to uncover novel insights into plant epigenetic regulation. Current research has already revealed an unexpected correlation between HDA9 binding and DNase I hypersensitive sites in gene promoters, suggesting HDA9 preferentially associates with accessible chromatin regions .

Advanced integration strategies could include:

• Temporal correlation analysis: Track changes in both HDA9 binding and chromatin accessibility during developmental transitions like leaf senescence to identify:

  • Regions where HDA9 binding precedes changes in accessibility

  • Regions where accessibility changes precede HDA9 recruitment

  • Stable HDA9-bound accessible regions that serve as regulatory anchors

• Multi-omics integration: Combine HDA9 ChIP-seq, DNase-seq, RNA-seq, and histone modification ChIP-seq data sets to create comprehensive regulatory maps that define:

  • Classes of HDA9-regulated genes based on their chromatin signatures

  • Feedback loops between transcription and chromatin structure

  • Pioneer factor activities that may precede HDA9 binding

• Comparative genomics approach: Analyze the conservation of the HDA9-accessibility relationship across:

  • Different plant species with varying genome complexity

  • Various developmental contexts and stress responses

  • Evolutionary lineages to identify core regulatory principles

This integrated approach would move beyond correlative observations to establish mechanistic understanding of how HDA9 functions within the dynamic chromatin environment to regulate gene expression during plant development.