GTF2IRD1 Antibody

What is GTF2IRD1 and what biological functions does it serve?

GTF2IRD1 is a transcription factor involved in chromatin remodeling and gene expression regulation. It plays critical roles in cell-cycle progression and skeletal muscle differentiation, and may repress GTF2I transcriptional functions by preventing its nuclear residency or inhibiting its transcriptional activation .

GTF2IRD1 is among 28 genes deleted in Williams-Beuren Syndrome (WBS) and has been implicated in the syndrome's neurocognitive profile and craniofacial abnormalities . In the developing brain, GTF2IRD1 binds to enhancer and promoter regions in rhodopsin, M- and S-opsin genes, regulating them differentially to maintain photoreceptor cell identity and function .

What is the molecular structure and characteristics of GTF2IRD1?

GTF2IRD1 is characterized by the following molecular properties:

The protein often shows multiple bands in Western blot analyses, commonly at 120-145 kDa, which may reflect post-translational modifications or alternative splicing variants .

What antibody types are available for GTF2IRD1 research?

Several types of GTF2IRD1 antibodies are available for research applications:

These antibodies target different epitopes of GTF2IRD1 and have been validated for various applications, allowing researchers to select the most appropriate reagent based on their experimental requirements.

What are the optimal protocols for Western blot detection of GTF2IRD1?

For optimal Western blot detection of GTF2IRD1:

• Sample preparation: Prepare nuclear and cytoplasmic extracts using reagents like NE-PER Nuclear and Cytoplasmic Reagents as GTF2IRD1 is predominantly nuclear-localized .

• Protein loading: Use 10 μg of protein for nuclear fraction analysis .

• Antibody selection and dilution:

  • 17052-1-AP: 1:500-1:3000

  • 68899-1-Ig: 1:2000-1:10000

  • ab64805: 1:500

  • CAB6613: 1:500-1:2000

• Detection system: Use HRP-conjugated secondary antibodies (typically 1:2000 dilution) and chemiluminescent substrates like SuperSignal West Pico .

• Expected band size: Look for bands at 106-145 kDa, with common observations at 120-145 kDa .

Always include appropriate positive controls (e.g., HeLa cells, mouse brain tissue) and negative controls to validate specificity .

How should GTF2IRD1 antibodies be used for immunohistochemistry and immunofluorescence?

For immunohistochemistry (IHC) and immunofluorescence (IF) detection of GTF2IRD1:

• Tissue preparation: Fix tissues with 4% paraformaldehyde in 0.1M phosphate buffer for 2 hours at 4°C .

• Antigen retrieval: Use TE buffer pH 9.0 as the preferred method, or alternatively, citrate buffer pH 6.0 .

• Antibody dilution: For 17052-1-AP antibody, use a dilution of 1:50-1:500 for both IHC and IF/ICC applications .

• Validation in specific tissues: GTF2IRD1 has been successfully detected in various human tissues including testis, spleen, kidney, heart, ovary, skin, brain, and liver tissues , as well as in retinal tissues .

• Cellular localization: GTF2IRD1 is predominantly localized in the nucleus, which should be considered when evaluating staining patterns .

For IF applications, HeLa cells serve as a reliable positive control .

How can I validate GTF2IRD1 antibody specificity for my experiments?

To validate GTF2IRD1 antibody specificity:

• Western blot analysis: Compare observed band size (106-145 kDa) with the expected molecular weight. Multiple bands may represent different isoforms or post-translational modifications .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to samples. Signal elimination confirms specificity, as demonstrated with ab64805 antibody .

• Knockout/knockdown controls: Use GTF2IRD1-knockout or knockdown samples as negative controls. For example, Gtf2ird1 knockout mice samples can serve as specificity controls .

• Cross-reactivity assessment: Test the antibody across multiple species if working with non-human models. Several antibodies show reactivity with human, mouse, and rat samples .

• Immunoprecipitation validation: For antibodies used in ChIP or IP experiments, verify target pulldown through Western blot analysis with a separate antibody targeting a different epitope of GTF2IRD1 .

How is GTF2IRD1 involved in transcriptional regulation and what techniques can identify its binding targets?

GTF2IRD1 functions as a transcription factor with roles in:

• Transcriptional modulation: GTF2IRD1 can both enhance and repress gene expression depending on context. It enhances M-opsin and rhodopsin expression while suppressing S-opsin expression in photoreceptor cells .

• Binding preferences: GTF2IRD1 predominantly binds at promoters and gene bodies, with significant enrichment at promoters (94% in H3K4me3 regions versus only 11% in H3K27me3 regions), suggesting it functions more in activation than repression .

• Binding target functions: GTF2IRD1-bound regions are enriched for genes involved in transcription regulation, chromatin organization, and protein ubiquitination .

To identify GTF2IRD1 binding targets:

• ChIP-seq methodology: This approach has successfully identified 1410 peaks enriched in GTF2IRD1 IP samples compared to input controls .

• Conservation analysis: GTF2IRD1-bound peaks demonstrate higher conservation than random genomic targets and random promoter regions, suggesting functional importance .

• Interaction studies: Co-immunoprecipitation (Co-IP) with transcription factors like CRX can reveal functional complexes. In vitro co-immunoprecipitation has shown GTF2IRD1 interactions with CRX and other transcription factors .

What is known about GTF2IRD1's role in neurodevelopmental disorders, particularly Williams-Beuren Syndrome?

GTF2IRD1 has been implicated in the pathophysiology of Williams-Beuren Syndrome (WBS):

• Genetic basis: GTF2IRD1 is one of approximately 28 genes deleted in the 1.5-1.8 Mb hemizygous deletion in chromosome 7q11.23 that causes WBS .

• Neurological contributions: GTF2IRD1 has been implicated as a contributor to the unique neurocognitive profile seen in WBS patients .

• Auditory pathology: Studies using Gtf2ird1 knockout mice demonstrate its expression in multiple cell types within the cochlea. These mice exhibit higher auditory thresholds (hypoacusis) in both ABR and DPOAE hearing assessments, suggesting GTF2IRD1 deficiency contributes to the sensorineural hearing loss (SNHL) observed in WBS patients .

• Behavioral phenotypes: Mouse models with mutations in Gtf2ird1 show balance deficits, altered marble burying behavior, and differences in conditioned fear responses, potentially correlating with behavioral aspects of WBS .

• Research methodology: Analysis of GTF2IRD1 function in WBS has utilized:

  • In situ hybridization to determine expression patterns

  • Auditory brainstem response (ABR) testing

  • Distortion product of otoacoustic emissions (DPOAE) assessments

  • Behavioral testing paradigms

How is GTF2IRD1 involved in retinal development and visual function?

GTF2IRD1 plays critical roles in retinal development and photoreceptor function:

• Cell-fate determination: GTF2IRD1 is involved in maintaining medium-wave-sensitive (M) cone cell identity and rod photoreceptor function .

• Transcriptional regulation: GTF2IRD1 binds to enhancer and promoter regions in rhodopsin, M- and S-opsin genes, regulating their expression differentially :

  • Enhances M-opsin expression through interaction with CRX and thyroid hormone receptor β2

  • Suppresses S-opsin expression

  • Enhances rhodopsin expression through interaction with CRX and NRL

• Expression pattern: GTF2IRD1 is localized in the nucleus of photoreceptors in the mouse retina, with expression detected in cell-fate determined photoreceptors at postnatal day 10 .

• Research approaches:

  • Western blotting of nuclear and cytoplasmic fractions to confirm nuclear localization

  • Immunohistochemistry on retinal sections

  • In situ hybridization to visualize mRNA expression patterns

  • Laser capture microdissection (LCM) to isolate specific retinal layers including the outer nuclear layer (ONL) where photoreceptors reside

How can I resolve multiple band patterns in Western blot detection of GTF2IRD1?

Multiple bands when detecting GTF2IRD1 by Western blot are common and can be addressed as follows:

• Understand expected patterns: GTF2IRD1 typically shows bands at calculated MW of 106 kDa but observed MW varies from 106-145 kDa across different antibodies and samples . Common observations include bands at 120-145 kDa .

• Verify specificity:

  • Conduct peptide competition assays to confirm that bands are specific to GTF2IRD1

  • Test in knockout/knockdown samples to identify non-specific bands

  • Compare patterns across different GTF2IRD1 antibodies targeting distinct epitopes

• Optimize sample preparation:

  • Use fresh protease inhibitors to prevent degradation

  • Consider nuclear extraction since GTF2IRD1 is predominantly nuclear-localized

  • Test different lysis buffers if protein solubilization is an issue

• Adjust gel conditions:

  • Use lower percentage gels (6-8%) for better resolution of high molecular weight proteins

  • Consider gradient gels for simultaneous resolution of multiple isoforms

• Optimize antibody concentration: Test a range of dilutions (e.g., 1:500-1:3000 for 17052-1-AP or 1:2000-1:10000 for 68899-1-Ig) to improve signal-to-noise ratio

What are the critical considerations for successful immunoprecipitation of GTF2IRD1?

For successful immunoprecipitation (IP) of GTF2IRD1:

• Antibody selection: Not all GTF2IRD1 antibodies work equally well for IP. 17052-1-AP has been validated for IP in mouse liver tissue .

• Antibody amount: Use 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate .

• Sample preparation:

  • For tissue samples, ensure proper homogenization and protein extraction

  • Consider nuclear extraction protocols since GTF2IRD1 is predominantly nuclear

  • Use fresh protease inhibitors to prevent degradation

• Controls:

  • Include IgG control to identify non-specific binding

  • Include input sample to verify target protein presence

  • If available, use GTF2IRD1-deficient samples as negative controls

• Validation:

  • Confirm successful IP by Western blot using a different GTF2IRD1 antibody

  • For co-IP experiments, analyze both GTF2IRD1 and potential interacting partners (e.g., CRX, thyroid hormone receptor β2, NRL)

• Cross-linking consideration: For chromatin IP (ChIP) applications, optimize formaldehyde cross-linking conditions to preserve GTF2IRD1-DNA interactions while allowing efficient immunoprecipitation

How do GTF2IRD1 and GTF2I cooperate or compete in transcriptional regulation networks?

The relationship between GTF2IRD1 and GTF2I in transcriptional networks reveals complex interactions:

• Binding site overlap: GTF2IRD1 and GTF2I binding sites show moderate overlap, suggesting potential epistatic effects. They share 148 common gene targets at promoters (OR = 1.4, P = 0.00015 FET), indicating functional interaction .

• Distinct binding preferences:

  • Both TFs are highly enriched at promoters

  • Both strongly overlap with CTCF binding and topological associating domain boundaries

  • GTF2IRD1 has a higher proportion of peaks at promoters compared to GTF2I

  • GTF2I has more peaks at intergenic regions

  • GTF2IRD1-bound peaks are significantly more conserved than GTF2I-bound peaks (t = 7.81, df = 2736.5, P = 8.2 × 10^-15)

• Functional distinctions:

  • GTF2IRD1 binding targets are enriched for transcriptional and chromatin regulators and mediators of ubiquitination

  • GTF2I targets are enriched for signal transduction proteins, including regulators of phosphorylation and WNT pathway components

• Combinatorial effects: Shared targets between GTF2IRD1 and GTF2I are enriched for reactive oxygen species-responsive genes, synaptic proteins, and transcription regulators including chromatin modifiers. These targets include a significant number of highly constrained genes and known autism spectrum disorder (ASD) genes .

• Mutation studies: Research comparing single-gene Gtf2ird1 mutants to double mutants (Gtf2i and Gtf2ird1) did not reveal more severe transcriptional changes or behavioral phenotypes, suggesting complex rather than simply additive interactions .

What are the technical considerations for performing ChIP-seq with GTF2IRD1 antibodies?

For successful ChIP-seq experiments with GTF2IRD1 antibodies:

• Antibody validation: Verify antibody specificity through Western blot and immunoprecipitation before ChIP-seq. Confirm the antibody pulls down GTF2IRD1 specifically and doesn't cross-react with related proteins like GTF2I .

• Chromatin preparation:

  • Optimize cross-linking conditions (typically 1% formaldehyde for 10 minutes)

  • Test different sonication parameters to achieve chromatin fragments of 200-500 bp

  • Verify fragmentation efficiency by agarose gel electrophoresis

• Experimental controls:

  • Include input chromatin as control for normalization

  • Use IgG control for assessing non-specific binding

  • Consider including GTF2IRD1-deficient samples as negative controls

• Sequencing depth: Aim for at least 20 million uniquely mapped reads for sufficient coverage of binding sites

• Data analysis considerations:

  • GTF2IRD1 predominantly binds at promoters and gene bodies

  • Examine overlap with histone modifications (94% of GTF2IRD1 peaks found in H3K4me3 regions versus only 11% in H3K27me3 regions)

  • Perform motif analysis to identify binding sequences

  • Consider integrating with gene expression data to correlate binding with transcriptional effects

  • Analyze conservation of binding sites as GTF2IRD1-bound peaks show higher conservation than random genomic regions

• Validation of binding sites: Confirm key binding targets through ChIP-qPCR using different GTF2IRD1 antibodies or testing in different cell types/tissues

How does GTF2IRD1 function differ across developmental stages and tissue contexts?

GTF2IRD1 exhibits context-dependent functions across development and tissues:

• Developmental expression patterns:

  • In photoreceptors, GTF2IRD1 is expressed in cell-fate determined cells at postnatal day 10

  • Expression patterns may differ between embryonic and adult tissues as evidenced by limited overlap between E10.5 craniofacial and E13.5 brain ChIP datasets

• Tissue-specific functions:

  • Neural tissues: GTF2IRD1 regulates genes involved in transcription and chromatin organization in the developing brain

  • Retina: Maintains M cone photoreceptor identity and rod function through differential regulation of opsin genes

  • Cochlea: Expressed in multiple cell types within the cochlea, with mutation leading to auditory deficits

  • Craniofacial development: Implicated in craniofacial abnormalities in Williams-Beuren Syndrome

• Cell-type specificity:

  • Successfully detected in multiple cell lines including HeLa, SH-SY5Y, hTERT-RPE1, TT, and MKN-45 cells

  • Detected in various human tissues including testis, spleen, kidney, heart, ovary, skin, brain, and liver

• Temporal dynamics:

  • Binding patterns may vary across developmental stages as evidenced by limited overlap between GTF2I ChIP peaks identified in E13.5 brain versus E10.5 craniofacial tissue (only 12 genes in common)

  • Conversely, GTF2IRD1 peaks at gene promoters in E13.5 brain showed significant overlap with E10.5 craniofacial peak dataset (128 genes in common, P < significant)

• Methodological approaches for studying context-dependent functions:

  • Tissue-specific knockout models

  • Temporal gene expression analysis

  • ChIP-seq across different developmental stages

  • Single-cell approaches to resolve cell-type specific functions

  • Comparative genomics to identify conserved versus context-dependent binding sites