GATAD2B Antibody

What is GATAD2B and why is it significant in neurodevelopmental research?

GATAD2B (GATA zinc finger domain containing 2B) is a critical component of the Nucleosome Remodeling and Histone Deacetylase (NuRD) complex that regulates transcriptional programs essential for proper neurodevelopment. Variants in the GATAD2B gene are associated with GATAD2B-associated neurodevelopmental disorder (GAND), characterized by intellectual disability, infantile hypotonia, apraxia of speech, epilepsy, macrocephaly, and distinctive facial features . Mouse models with Gatad2b mutations display behavioral abnormalities resembling GAND clinical features, along with abnormal cortical patterning and cell-specific alterations in the developmental transcriptome. Single-cell RNA sequencing of embryonic cortex has identified misexpression of genes crucial for corticogenesis such as Bcl11b, Nfia, H3f3b, and Sox5, highlighting the fundamental role of GATAD2B in brain development .

What are the primary cellular functions of the GATAD2B-NuRD complex?

The GATAD2B-NuRD complex serves as a key regulator in the interplay between transcription and chromatin dynamics, particularly during DNA damage response. Recent research demonstrates that GATAD2B-NuRD forms a critical boundary between open and closed chromatin at sites of DNA double-strand breaks (DSBs) . This complex associates with DSBs in a manner dependent on transcription and DNA:RNA hybrids (R-loops), facilitating histone deacetylation and chromatin condensation . The absence of GATAD2B-NuRD results in chromatin hyper-relaxation and excessive DNA end resection, which leads to failure in homologous recombination (HR) repair . This mechanism is essential for genomic stability and proper cellular function.

How does GATAD2B function in early embryonic development?

GATAD2B plays a crucial role in pre-implantation embryonic development. Research using knockdown experiments targeting GATAD2B mRNA at the one-cell stage has demonstrated significant developmental implications . The proportion of four-cell embryos in GATAD2B-knockdown groups shows notable decrease (70% compared to 98% in controls). More dramatically, blastocyst formation is significantly impaired, with only 50% of GATAD2B-knockdown embryos reaching the blastocyst stage compared to over 90% in control groups . This indicates GATAD2B's essential function in early developmental processes and cellular differentiation.

What criteria should be considered when selecting a GATAD2B antibody for research applications?

When selecting a GATAD2B antibody for research, consider these methodological criteria:

• Antibody specificity: Validate using positive controls (tissues/cells known to express GATAD2B) and negative controls (GATAD2B-knockout samples or siRNA-treated cells)

• Host species and clonality: Choose based on experimental design (rabbit monoclonal antibodies often provide higher specificity; polyclonal antibodies may offer greater epitope recognition)

• Validated applications: Ensure the antibody has been validated for your specific application (Western blot, immunoprecipitation, ChIP, immunofluorescence)

• Epitope location: Select antibodies targeting conserved regions if studying homologs across species

• Publications: Prioritize antibodies with demonstrated performance in peer-reviewed literature

The search results mention specific GATAD2B antibodies used successfully in research applications, including rabbit anti-Gatad2b (A301-2882A, Bethyl) and GATAD2B antibody (Cat no: 25679-1-AP; Proteintech) .

How can GATAD2B antibodies be validated for specificity in immunoprecipitation experiments?

Validation of GATAD2B antibodies for immunoprecipitation requires a systematic approach:

• Western blot confirmation: First validate that the antibody detects a band of the expected molecular weight (~65 kDa for human GATAD2B)

• Knockdown controls: Compare immunoprecipitation results between normal samples and those with GATAD2B knocked down via siRNA or CRISPR/Cas9

• Mass spectrometry validation: Perform mass spectrometry analysis of immunoprecipitated proteins to confirm GATAD2B presence and identify interacting partners

• Co-immunoprecipitation of known interactors: Verify pull-down of established GATAD2B-interacting proteins (e.g., MBD3 and HDAC1 from the NuRD complex)

• Reciprocal immunoprecipitation: Confirm results using antibodies against known interacting partners to pull down GATAD2B

Research has successfully used immunoprecipitation with GATAD2B antibodies followed by mass spectrometry to identify interacting proteins in mouse ovarian tissue, demonstrating a methodological approach to validating antibody performance in complex biological samples .

What are the optimal protocols for using GATAD2B antibodies in proximity ligation assays (PLA)?

For optimal proximity ligation assays using GATAD2B antibodies:

• Primary antibody selection: Use validated GATAD2B antibodies from different host species than the second target protein antibody (e.g., rabbit anti-GATAD2B with mouse anti-γH2AX)

• Controls: Include these critical controls:

  • Single antibody controls to establish background signal levels

  • Positive controls with known interacting partners (e.g., MBD3)

  • Negative controls with non-interacting proteins (e.g., HOXD11)

• Signal validation: Confirm specificity through:

  • RNaseH1 overexpression (when studying R-loop-dependent interactions)

  • Transcription inhibitors (triptolide or DRB) when examining transcription-dependent interactions

  • Depletion controls (siRNA knockdown)

• Signal quantification: Count PLA foci per cell and perform statistical analysis across multiple biological replicates

This methodology has successfully demonstrated GATAD2B interactions with DNA:RNA hybrids and other proteins at sites of DNA damage, revealing its functional associations .

How can GATAD2B antibodies be effectively utilized in chromatin immunoprecipitation (ChIP) experiments?

For effective GATAD2B ChIP experiments:

• Crosslinking optimization: Titrate formaldehyde concentration (typically 1-1.5%) and crosslinking time (8-15 minutes) to preserve GATAD2B-DNA interactions

• Sonication parameters: Optimize to achieve chromatin fragments of 200-500 bp

• Antibody amounts: Titrate antibody concentration (typically 2-5 μg per ChIP reaction)

• Controls:

  • Input control (non-immunoprecipitated chromatin)

  • IgG control (non-specific antibody of same isotype)

  • GATAD2B-depleted cells as negative control

• Validation of enrichment:

  • qPCR of known target loci

  • Sequencing (ChIP-seq) for genome-wide binding profile

For DNA damage studies, the DIvA cell system (with AsiSI restriction enzyme) provides a powerful model to study GATAD2B recruitment to specific DNA break sites in a controlled manner .

What is the methodology for studying GATAD2B recruitment to DNA damage sites using immunofluorescence?

To study GATAD2B recruitment to DNA damage sites:

• Cell preparation: Transfect cells with GFP-tagged GATAD2B constructs or prepare for immunofluorescence of endogenous GATAD2B

• DNA damage induction:

  • Laser micro-irradiation (405 nm laser) for localized damage

  • Ionizing radiation (IR) for widespread damage

  • DIvA cell system with 4-hydroxytamoxifen treatment for sequence-specific breaks

• Time-course analysis: Perform live-cell imaging or fixed-cell immunofluorescence at multiple time points (30 seconds to several hours post-damage)

• Co-localization studies: Use γH2AX as a marker of DNA damage sites

• Mechanism dissection:

  • Transcription inhibition (triptolide, DRB)

  • R-loop resolution (RNaseH1 overexpression)

  • DDR pathway inhibition (ATM or PARP1 inhibitors)

This approach has revealed that GATAD2B is rapidly recruited to DNA damage sites (<30 seconds) in a transcription and R-loop dependent manner .

How can GATAD2B antibodies be used to investigate chromatin remodeling dynamics at DNA damage sites?

For investigating GATAD2B-mediated chromatin remodeling:

• Histone modification analysis: Perform sequential ChIP (Re-ChIP) with GATAD2B antibodies followed by antibodies against:

  • Histone acetylation marks (e.g., H3K9ac, H4ac)

  • Histone methylation marks (e.g., H3K4me3, H3K9me3)

• Chromatin accessibility assessment:

  • ATAC-seq at GATAD2B-bound regions

  • Micrococcal nuclease sensitivity assays

• Protein complex analysis:

  • Proximity ligation assays (PLA) to detect GATAD2B interactions with histone deacetylases (HDAC1)

  • Co-immunoprecipitation followed by western blotting for NuRD complex components

• Functional assays:

  • DNA end resection measurements using RPA or BrdU incorporation

  • Homologous recombination repair efficiency assays

Research demonstrates that GATAD2B-NuRD promotes histone deacetylation and chromatin condensation at DNA damage sites, creating a temporal boundary between open and closed chromatin that is necessary for proper DNA repair .

What methods can be used to study GATAD2B interactions with R-loops and DNA:RNA hybrids?

To study GATAD2B interactions with R-loops:

• Proximity ligation assay (PLA):

  • Use S9.6 antibody (specific for DNA:RNA hybrids) and GATAD2B antibody

  • Include RNaseH1 overexpression controls to confirm R-loop specificity

• DNA:RNA immunoprecipitation (DRIP):

  • Immunoprecipitate DNA:RNA hybrids with S9.6 antibody

  • Perform western blot for GATAD2B

• R-loop mapping:

  • DRIP-seq to identify genome-wide R-loop distribution

  • Cross-reference with GATAD2B ChIP-seq data

• Functional analysis:

  • Manipulate R-loops with RNaseH1 overexpression

  • Assess GATAD2B recruitment to DNA damage sites

Research has shown that GATAD2B binds to DNA:RNA hybrids at DNA damage sites, and this interaction increases following DNA damage induction. The interaction is sensitive to transcription inhibition and RNaseH1 overexpression, confirming the R-loop dependency .

How should researchers interpret conflicting GATAD2B antibody results across different experimental conditions?

When encountering conflicting GATAD2B antibody results:

• Antibody epitope considerations:

  • Different antibodies may recognize distinct epitopes that could be masked in certain contexts

  • Some epitopes may be inaccessible in protein complexes or following post-translational modifications

• Experimental condition variables:

  • Cell/tissue type differences (expression levels vary across tissues)

  • Fixation methods (formaldehyde vs. methanol) affect epitope accessibility

  • Buffer compositions (detergent types and concentrations)

• Validation approaches:

  • Use multiple antibodies targeting different GATAD2B epitopes

  • Implement genetic controls (siRNA, CRISPR knockout)

  • Compare results with tagged GATAD2B constructs

• Context-dependent interactions:

  • GATAD2B function changes based on cellular context (DNA damage, developmental stage)

  • Interactions may be regulated by DDR signaling pathways (e.g., PARP1-dependent, ATM-independent)

For example, research shows that GATAD2B binding to R-loops is significantly decreased by PARP1 inhibition but not affected by ATM inhibition, revealing pathway-specific regulation of GATAD2B functions .

What controls are essential when analyzing GATAD2B localization patterns in response to DNA damage?

Essential controls for GATAD2B localization studies include:

• Damage verification controls:

  • γH2AX immunofluorescence to confirm DNA damage induction

  • 53BP1 or other DSB markers as independent verification

• Antibody specificity controls:

  • GATAD2B-depleted cells as negative control

  • Single antibody controls in co-localization or PLA experiments

• Mechanism dissection controls:

  • Transcription inhibitors (triptolide, DRB) to test transcription dependency

  • RNaseH1 overexpression to assess R-loop dependency

  • DNA damage response pathway inhibitors (ATM, PARP1)

• Non-relevant protein controls:

  • Proteins not involved in DNA damage response (e.g., HOXD11)

These controls are crucial for establishing the specificity and mechanism of GATAD2B recruitment to DNA damage sites, as demonstrated in studies showing transcription and R-loop dependent localization of GATAD2B to double-strand breaks .

How can researchers differentiate between direct and indirect interactions of GATAD2B with chromatin and other proteins?

To differentiate between direct and indirect GATAD2B interactions:

• Biochemical approaches:

  • In vitro binding assays with purified recombinant proteins

  • Domain mapping using truncated protein constructs

  • Cross-linking mass spectrometry (XL-MS) to identify direct interaction interfaces

• Proximity-based methods:

  • BioID or APEX proximity labeling to identify proteins in close proximity to GATAD2B

  • Compare standard co-IP with stringent co-IP conditions (high salt, detergents)

• Sequential ChIP (Re-ChIP):

  • First IP with GATAD2B antibody followed by IP with antibody against potential interactor

  • Confirms co-occupancy at the same genomic loci

• Functional validation:

  • Mutational analysis of interaction domains

  • Competition assays with peptides or small molecules

Research has employed multiple approaches to characterize GATAD2B interactions, including PLA with specific controls to distinguish direct interactions from coincidental co-localization, and ChIP in the DIvA cell system to analyze recruitment specificity .

What methodologies are most effective for studying GATAD2B function in neurodevelopmental processes?

For studying GATAD2B in neurodevelopment:

• Animal models:

  • Characterize mouse models with inactivating mutations in Gatad2b

  • Analyze behavioral phenotypes resembling GAND patient features

  • Examine cortical patterning and cellular proportions

• Transcriptomic analyses:

  • Single-cell RNA sequencing (scRNAseq) of embryonic cortex

  • Identify misexpression of genes critical for corticogenesis

  • Compare with gene expression patterns in GAND patients

• Immunohistochemistry panel:

  • Use specific markers: SATB2, CTIP2, TBR1 (cortical layer markers)

  • Combine with GATAD2B antibodies to analyze expression patterns

  • Quantify neuronal and glial cell proportions

• Functional rescue experiments:

  • Reintroduce wild-type GATAD2B in knockout models

  • Test specific domains for functional complementation

Research using these approaches has revealed that haploinsufficient Gatad2b mice exhibit behavioral abnormalities resembling GAND clinical features, along with altered expression of key neurodevelopmental genes including Bcl11b, Nfia, and Sox5 .

How can GATAD2B antibodies be used to investigate its role in embryonic development?

To investigate GATAD2B in embryonic development:

• Expression analysis:

  • Immunofluorescence to track GATAD2B protein levels across developmental stages

  • Western blot quantification during one-cell to blastocyst transition

• Knockdown studies:

  • siRNA injection at zygote stage

  • Monitor developmental progression through cleavage stages

  • Quantify blastocyst formation rates

• Protein interaction studies:

  • Immunoprecipitation with GATAD2B antibodies from embryonic tissues

  • Mass spectrometry to identify stage-specific interaction partners

• Chromatin association analysis:

  • ChIP-seq to map GATAD2B binding sites during developmental transitions

  • Correlation with gene expression changes

Research has demonstrated that GATAD2B knockdown at the zygote stage results in developmental arrest, with only 50% of embryos reaching the blastocyst stage compared to over 90% in control groups, highlighting its critical role in early embryonic development .

What bioinformatic approaches are recommended for analyzing GATAD2B ChIP-seq data in the context of DNA damage response?

For analyzing GATAD2B ChIP-seq in DNA damage contexts:

• Experimental design considerations:

  • Compare damaged (IR, AsiSI-induced breaks) vs. undamaged conditions

  • Include transcription inhibition and R-loop resolution controls

  • Analyze GATAD2B binding in relation to DNA repair pathway choice

• Peak calling and annotation:

  • Use specialized peak callers optimized for transcription factors

  • Annotate peaks relative to genomic features (promoters, enhancers, gene bodies)

  • Compare with known DSB sites (e.g., AsiSI cut sites in DIvA cells)

• Integration with other datasets:

  • Histone modification ChIP-seq (H3K9ac, H3K4me3)

  • Chromatin accessibility data (ATAC-seq)

  • R-loop mapping data (DRIP-seq)

  • Transcription data (RNA-seq, GRO-seq)

• Motif analysis:

  • Identify DNA sequence motifs enriched at GATAD2B binding sites

  • Analyze relationship to R-loop forming sequences

• Visualization and statistical analysis:

  • Generate heat maps and average profile plots around DSB sites

  • Perform differential binding analysis between conditions

This approach has revealed that GATAD2B associates with transcriptionally active and homologous recombination-prone DSBs in a manner dependent on transcription and R-loops .

How should researchers integrate GATAD2B functional data across different experimental systems?

For integrating GATAD2B data across experimental systems:

• Cross-platform normalization:

  • Develop standardized controls applicable across different techniques

  • Use relative quantification approaches rather than absolute values

  • Apply batch correction methods when combining datasets

• Multi-omics data integration:

  • Correlate ChIP-seq, RNA-seq, and proteomics data

  • Use network analysis to identify functional modules

  • Apply machine learning approaches to identify patterns across datasets

• Model systems comparison:

  • Map orthologous genes/proteins across species

  • Compare phenotypic outcomes of GATAD2B perturbation

  • Identify conserved vs. divergent mechanisms

• Pathway enrichment analysis:

  • Apply gene set enrichment analysis to identify biological processes

  • Compare enriched pathways across experimental systems

  • Contextualize within known NuRD complex functions

• Visualization techniques:

  • Use dimensional reduction methods (PCA, t-SNE) to visualize similarities

  • Create integrated network visualizations

  • Develop interactive dashboards for data exploration

This integrated approach helps reconcile findings from diverse experimental systems, such as the roles of GATAD2B in neurodevelopment , DNA damage response , and embryonic development .