GTF2H5 Antibody

What is GTF2H5 and what are its primary cellular functions?

GTF2H5 (General Transcription Factor IIH Subunit 5) functions as a critical component of the transcription/repair factor TFIIH complex, which serves dual roles in gene transcription and DNA repair. Specifically, GTF2H5:

• Participates in general and transcription-coupled nucleotide excision repair (NER) of damaged DNA

• Contributes to RNA transcription by RNA polymerase II when complexed with CAK

• Facilitates DNA opening around lesions during NER, allowing excision of damaged oligonucleotides and replacement with new DNA fragments

• Plays an essential role in transcription initiation, promoter opening, and promoter escape

• Maintains stability of the TFIIH complex and ensures normal cellular levels of TFIIH

GTF2H5 mutations are associated with trichothiodystrophy (TTD), an autosomal recessive disease characterized by sulfur-deficient brittle hair and variable multisystem abnormalities ranging from mild to severe forms with developmental defects .

What experimental applications are GTF2H5 antibodies validated for?

Current commercially available GTF2H5 antibodies have been validated for multiple applications:

Most GTF2H5 antibodies detect endogenous levels of total GTF2H5 protein. When selecting an antibody for specific applications, researchers should consider the host species (typically rabbit), clonality (monoclonal vs. polyclonal), and validated reactivity with the experimental model organism .

What is the molecular structure and characteristics of the GTF2H5 protein?

GTF2H5 is a relatively small protein with the following characteristics:

• Calculated molecular weight: 8 kDa

• Human GTF2H5 UniProt ID: Q6ZYL4

• Mouse GTF2H5 UniProt ID: Q8K2X8

• Belongs to the TFB5 protein family

• Contains a conserved region from amino acids 16-55 that is retained in all human truncated, partially functional mutant TTDA proteins

The protein's small size makes it challenging to detect in some experimental contexts, requiring optimized protocols for reliable visualization in techniques like Western blotting.

How does GTF2H5 contribute to TFIIH complex stability across different tissues?

Research has demonstrated that GTF2H5 plays a crucial role in maintaining TFIIH stability across various tissues:

These findings support an evolutionarily conserved function of GTF-2H5 in maintaining steady-state levels of TFIIH across various tissues in vivo.

What role does GTF2H5 play in cancer chemoresistance mechanisms?

Recent research has identified GTF2H5 as a potential therapeutic target in chemoresistant colorectal cancer (CRC):

• Multi-omic analysis uncovered relationships between synthetic lethal (SL) genes, including GTF2H5, mutations (particularly TP53 and KRAS), and poor patient prognosis in CRC .

• Time series analysis revealed that GTF2H5 expression gradually increases during the transition from sensitivity to resistance in CRC cells .

• Experimental validation showed:

  • GTF2H5 expression was significantly elevated after FOLFOX treatment in both sensitive and resistant CRC cells at both mRNA and protein levels

  • Knockdown of GTF2H5 significantly inhibited cell proliferation in CRC cells (as determined by CCK8 assays)

  • In a 3D gelatin-based co-culture system with fibroblasts, downregulation of GTF2H5 notably increased cell death under FOLFOX treatment

• Mechanistically, researchers propose that GTF2H5 activation protects tumor cells by facilitating DNA repair, allowing survival despite chemotherapy-induced DNA damage .

• In contrast to CRC, low levels of GTF2H5 were associated with favorable prognosis in high-grade serous ovarian cancer, and GTF2H5 silencing contributed to cisplatin sensitization in ovarian cancer patients .

These findings suggest GTF2H5 as a promising synthetic lethal target to counteract chemoresistance in CRC, particularly when targeted in combination with conventional chemotherapeutic agents.

What are the differential functions of GTF2H5 in transcription versus DNA repair?

GTF2H5 serves specialized functions in both transcription and DNA repair:

In Transcription:

• Functions as part of the TFIIH complex in RNA polymerase II-mediated transcription

• Essential for transcription initiation, promoter opening, and promoter escape

• Works with the CAK complex to phosphorylate the C-terminal domain (CTD) of RNA polymerase II's largest subunit, controlling transcription initiation

In DNA Repair:

• Critical component of nucleotide excision repair (NER) pathways

• Facilitates opening of DNA around lesions to allow excision of damaged oligonucleotides

• Promotes recruitment of TFIIH to DNA damage sites

• C. elegans studies demonstrate GTF-2H5 is indispensable for NER function

Interestingly, studies in C. elegans revealed that while GTF-2H5 deficiency is compatible with life (in contrast to depletion of other TFIIH subunits), gtf-2H5 embryos die when transcription is challenged, suggesting a context-dependent requirement for GTF2H5 in transcription .

This dual functionality explains why GTF2H5 mutations in humans can lead to trichothiodystrophy, with features thought to be caused by subtle transcription defects .

What are the optimal protocols for immunohistochemistry using GTF2H5 antibodies?

For optimal immunohistochemistry (IHC) results with GTF2H5 antibodies, researchers should consider:

Recommended Dilutions:

• General range: 1/20 - 1/200 (optimal dilutions should be determined by the end user)

• For specific antibodies like 14539-1-AP, refer to manufacturer protocols

Protocol Considerations:

• Fixation: Standard formalin fixation and paraffin embedding techniques are compatible with GTF2H5 antibodies

• Antigen Retrieval: Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) is typically effective

• Blocking: Use appropriate blocking sera (e.g., 10% normal goat serum) to minimize background staining

• Controls: Include positive control tissues with known GTF2H5 expression and negative controls (omitting primary antibody)

• Detection System: Both chromogenic (DAB) and fluorescent secondary detection systems are compatible

Validation of Results:

• Successful staining typically shows nuclear localization of GTF2H5

• Expression is observed in various tissues but may show variable levels between tissues

• Nuclear staining intensity can be used to assess expression levels

GTF2H5 antibodies have been successfully used in IHC applications for human, mouse, and rat tissues . Published applications demonstrate utility in cancer research, particularly in studies of high-grade serous ovarian cancer and colorectal cancer .

What experimental approaches can quantify TFIIH complex stability and GTF2H5 concentration in cells?

Researchers have employed various techniques to quantify TFIIH complex stability and GTF2H5 concentration:

1. Quantification of Nuclear Concentrations:
In C. elegans studies, researchers determined GTF-2H5 and GTF-2H1 concentrations by comparing nuclear fluorescence signals to known GFP concentrations, revealing:

• Comparable concentrations of approximately 0.2 μM for GTF-2H1 across multiple tissue types

• Similar but more variable concentrations for GTF-2H5

• Lower GTF-2H5 concentration specifically in muscle cells

2. Immunoprecipitation-Based Complex Analysis:

• Pull-down of TFIIH components using tagged subunits (e.g., GFP-tagged GTF-2H1)

• Mass spectrometry analysis of co-immunoprecipitated proteins

• Comparison of TFIIH subunit abundance between wild-type and mutant/knockdown cells

3. Western Blot Quantification:

• Measurement of protein levels after treatment with various concentrations of factors affecting GTF2H5 expression

• Comparison between sensitive and resistant cancer cell lines

4. Real-Time qPCR:

• Quantification of GTF2H5 mRNA levels in response to treatments

• Comparative analysis between different cell types or conditions

5. Fluorescence-Based Approaches:

• Tagging GTF2H5 with GFP or other fluorescent markers

• Using auxin-inducible degradation (AID) systems for controlled depletion and quantification

The choice of method depends on research questions, with fluorescence-based approaches providing spatial information, while biochemical methods like Western blotting and mass spectrometry provide more precise quantitative measurements of complex composition.

What 3D cell culture systems are most effective for studying GTF2H5 function in cancer models?

Recent research has demonstrated the utility of specialized 3D culture systems for studying GTF2H5 function in cancer:

Gelatin-Based 3D Co-Culture System:

• Successfully employed in colorectal cancer research to simulate in vivo cellular growth and extracellular matrix environment

• Incorporates co-culture with fibroblasts to better recapitulate tumor microenvironment

• Effectively demonstrates the synergistic effect of GTF2H5 inhibition with chemotherapeutic agents like FOLFOX

Key Components and Methodology:

• Scaffold Material: Gelatin-based matrix providing structural support

• Cell Types: Cancer cells (e.g., HCT8, HCT15) co-cultured with fibroblasts

• Drug Treatment: Enables assessment of chemotherapeutic effects (e.g., FOLFOX)

• Genetic Manipulation: Compatible with knockdown approaches (e.g., siRNA targeting GTF2H5)

• Analysis Methods: Supports cell viability assessment and death quantification

Advantages Over 2D Culture:

• Better simulates the cellular growth patterns observed in vivo

• Incorporates extracellular matrix components affecting drug penetration

• Allows cell-cell interactions between tumor cells and stromal components

• More accurately predicts therapeutic responses

This 3D co-culture approach has proven valuable for demonstrating that GTF2H5 inhibition combined with FOLFOX treatment produces a synergistic effect increasing cancer cell death, highlighting its potential as a therapeutic target in chemoresistant colorectal cancer .

How can researchers optimize experimental design for GTF2H5 knockdown studies?

For effective GTF2H5 knockdown studies, researchers should consider the following experimental design considerations:

Knockdown Approaches:

• siRNA Transfection: Effective for transient knockdown in various cell types

• shRNA Expression: For stable, long-term knockdown experiments

• CRISPR/Cas9: For complete knockout studies where applicable

Essential Controls:

• Negative Control: Non-targeting siRNA/shRNA with similar chemical modifications

• Positive Control: siRNA targeting a housekeeping gene with known knockdown phenotype

• Phenotype Rescue: Expression of siRNA-resistant GTF2H5 to confirm specificity

Validation of Knockdown:

• mRNA Level: qRT-PCR to confirm reduction in GTF2H5 transcripts

• Protein Level: Western blotting to confirm reduction in GTF2H5 protein

  • Example: Knockdown verification as performed in colorectal cancer cell studies

Functional Assays:

• Cell Proliferation: CCK8 assays revealed significant inhibition of proliferation in CRC cells following GTF2H5 knockdown

• Apoptosis Assessment: Flow cytometry analysis to determine whether GTF2H5 knockdown affects apoptosis rates

  • Note: GTF2H5 knockdown did not markedly elevate apoptosis levels in CRC cells but did in glioma cells, suggesting tissue-specific effects

• Drug Sensitivity: 3D culture experiments showed increased sensitivity to FOLFOX treatment following GTF2H5 knockdown

Key Considerations:

• Cell type-specific responses may occur (effects differ between cancer types)

• Timing of analysis is critical (immediate vs. long-term effects)

• Combined treatment approaches may reveal synthetic lethal interactions not visible with knockdown alone

How does GTF2H5 function differ between species models, and what are the implications for translational research?

Comparative analysis of GTF2H5 function across species reveals important evolutionary conservation and divergence:

C. elegans vs. Mammalian Models:

• Viability: GTF-2H5 deficiency is compatible with life in C. elegans, unlike in mammalian models where TTDA/GTF2H5 knockout mice are not viable

• TFIIH Stability: In both C. elegans and mammalian cells, GTF2H5 promotes TFIIH stability, with loss leading to reduced levels of other TFIIH subunits

• DNA Repair Function: GTF-2H5 is indispensable for nucleotide excision repair in C. elegans, facilitating recruitment of TFIIH to DNA damage, consistent with mammalian studies

• Transcription Role: When transcription is challenged, gtf-2H5 embryos die due to intrinsic TFIIH fragility, supporting the idea that TTDA/GTF2H5 mutations cause transcription impairment underlying trichothiodystrophy

Concentration Comparisons:

• GTF-2H1 concentration in C. elegans tissues (approximately 0.2 μM) is strikingly similar to concentration estimations of Pol II in human fibroblasts (0.18 μM)

• This conservation suggests fundamental constraints on the concentration of these transcriptional machinery components across species

Research Implications:

• C. elegans provides a valuable model for studying TTD pathogenesis due to the viability of GTF-2H5 mutants

• The differences in viability between species highlight potential compensatory mechanisms in C. elegans that could inform therapeutic approaches

• Functional conservation supports the translational relevance of findings from invertebrate models to human disease

These comparative insights help researchers select appropriate model systems and understand the limitations when extrapolating findings from one species to another.

What is the significance of GTF2H5 in trichothiodystrophy and other human diseases?

GTF2H5 mutations are causally linked to trichothiodystrophy and may play roles in other diseases:

Trichothiodystrophy 3, Photosensitive (TTD3):

• Autosomal recessive disease characterized by sulfur-deficient brittle hair and variable multisystem abnormalities

• Clinical spectrum ranges from mild disease with only hair involvement to severe forms with cutaneous, neurologic, and developmental defects

• Common manifestations include ichthyosis, intellectual disabilities, decreased fertility, abnormal birth characteristics, ocular abnormalities, short stature, and increased infections

• Both photosensitive and non-photosensitive forms exist

Cancer Relevance:

• Colorectal Cancer (CRC):

  • Identified as a synthetic lethal target in chemoresistant CRC

  • Expression increases during transition from chemosensitivity to resistance

  • Targeting GTF2H5 enhances efficacy of FOLFOX treatment

• Ovarian Cancer:

  • Low levels of GTF2H5 associated with favorable prognosis in high-grade serous ovarian cancer

  • Silencing GTF2H5 expression contributes to cisplatin sensitization

• Glioma:

  • Highly expressed in glioma tissues

  • Promotes cell proliferation

  • Mechanistically weakens pro-apoptotic signaling through the p53-Bax/Bcl2 mitochondrial pathway

Mechanistic Understanding:

• Transcription defects: Some TTD features thought to be caused by subtle transcription defects

• DNA repair impairment: Compromised NER function increases UV sensitivity in photosensitive forms

• TFIIH stability: GTF2H5 maintains stability of the TFIIH complex, with mutations leading to reduced TFIIH levels

These disease associations highlight GTF2H5 as both a diagnostic marker and potential therapeutic target, with implications extending beyond rare genetic disorders to common cancers.

What are the emerging therapeutic applications targeting GTF2H5 in cancer treatment?

Research has revealed promising therapeutic applications targeting GTF2H5, particularly in cancer treatment:

1. Synthetic Lethality in Colorectal Cancer:

• GTF2H5 has been identified as a crucial synthetic lethal target to counteract chemoresistance in colorectal cancer

• Multi-omic analysis revealed GTF2H5 as one of five key synthetic lethal genes associated with chemoresistance

• Expression of GTF2H5 was highly elevated in the presence of TP53 or KRAS mutations, correlating with poor prognosis

2. Combination Therapy Approaches:

• Experimental evidence shows that inhibition of GTF2H5 combined with FOLFOX chemotherapy results in synergistic killing of resistant CRC cells

• In 3D co-culture systems mimicking in vivo conditions, GTF2H5 knockdown significantly increased tumor cell mortality when treated with chemotherapeutic agents

3. Potential Mechanisms for Therapeutic Exploitation:

• GTF2H5 activation protects tumor cells from chemotherapy-induced DNA damage

• Inhibition of GTF2H5 may prevent effective DNA repair in cancer cells, enhancing chemotherapy efficacy

• Similar to other DNA damage repair-related targets being developed (ATR, DDR, USP1 inhibitors)

4. Tissue-Specific Considerations:

• GTF2H5 appears to play opposite roles in different cancer types:

  • In glioma: Promotes proliferation via p53-Bax/Bcl2 pathway

  • In ovarian cancer: Low levels associated with better prognosis

  • In colorectal cancer: Associated with chemoresistance

These findings suggest GTF2H5 as a promising therapeutic target, particularly for patients with chemoresistant colorectal cancer harboring specific mutations. Future research directions include developing specific inhibitors of GTF2H5 and exploring combination therapies for multiple cancer types.

What are the critical factors for proper handling and storage of GTF2H5 antibodies?

Proper handling and storage are crucial for maintaining GTF2H5 antibody activity and specificity:

Storage Conditions:

• Temperature: Store at -20°C

• Stability: Typically stable for one year after shipment if properly stored

• Aliquoting: While some manufacturers indicate aliquoting is unnecessary for -20°C storage, dividing into small aliquots is generally recommended to avoid freeze-thaw cycles

• Buffer: Typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• BSA content: Some preparations contain 0.1% BSA (e.g., 20µl sizes)

Handling Precautions:

• Freeze-Thaw Cycles: Avoid repeated freeze/thaw cycles that can degrade antibody performance

• Working Dilution: Prepare fresh working dilutions on the day of use

• Temperature Transitions: Allow antibodies to equilibrate to room temperature before opening vials

• Centrifugation: Briefly centrifuge vials before opening to collect all material at the bottom

Shipping Considerations:

• Typically shipped on ice packs or dry ice

• Allow adequate time for temperature equilibration before opening upon receipt

• Verify antibody activity after shipping with appropriate controls

Long-term Stability:

• Shelf life: Typically 12 months from date of receipt when stored properly

• Activity testing: Consider validation experiments if using antibodies approaching expiration dates

• Physical appearance: Clear solution without visible precipitates indicates proper storage

Adherence to these storage and handling guidelines ensures optimal antibody performance and experimental reproducibility.

How can researchers validate GTF2H5 antibody specificity for their particular experimental system?

Thorough validation of GTF2H5 antibody specificity is essential for reliable experimental results:

Recommended Validation Approaches:

1. Genetic Controls:

• Compare staining between wild-type samples and GTF2H5 knockout/knockdown samples

• C. elegans studies demonstrated absence of GTF-2H5 signal in gtf-2H5 mutants

• siRNA knockdown verification as shown in colorectal cancer studies

2. Peptide Competition Assay:

• Pre-incubate antibody with excess immunizing peptide

• Compare staining with and without peptide competition

• Specific signal should be eliminated or significantly reduced

3. Molecular Weight Verification:

• Western blot should show a band at the expected molecular weight (8 kDa for GTF2H5)

• Note that the small size of GTF2H5 may require optimized gel conditions for proper resolution

4. Multiple Antibody Concordance:

• Use multiple antibodies targeting different epitopes of GTF2H5

• Compare staining patterns to ensure consistency

• Commercial offerings include antibodies raised against different immunogens

5. Subcellular Localization:

• Verify nuclear localization consistent with GTF2H5's known function

• Both GTF-2H5 and GTF-2H1 show exclusive nuclear expression in various tissues

6. Positive Control Tissues/Cells:

• Include samples known to express GTF2H5

• GTF2H5 concentrations vary across tissues, with some showing stronger expression than others

7. Recombinant Protein Controls:

• Commercial recombinant GTF2H5 proteins are available from multiple expression systems

• These can serve as positive controls for antibody binding specificity

Comprehensive validation using multiple approaches provides the strongest evidence for antibody specificity, ensuring confidence in experimental results.

What troubleshooting strategies should be employed when GTF2H5 antibodies yield unexpected results?

When GTF2H5 antibody experiments produce unexpected results, researchers should consider several troubleshooting approaches:

1. Inconsistent or Absent Signal in Western Blots:

• Challenge: GTF2H5's small size (8 kDa) makes detection difficult

• Solutions:

  • Use higher percentage gels (15-20%) for better resolution of small proteins

  • Optimize transfer conditions (consider semi-dry transfer for small proteins)

  • Verify protein extraction method preserves nuclear proteins

  • Use positive controls (tissues known to have high GTF2H5 expression)

  • Consider detection method sensitivity (chemiluminescence vs. fluorescence)

2. Unexpected Tissue Expression Patterns:

• Challenge: GTF2H5 concentrations vary across tissues

• Solutions:

  • Compare to literature-reported expression patterns

  • Note that neuronal nuclei often show lower expression levels

  • Verify antibody specificity using knockout/knockdown controls

  • Consider tissue-specific post-translational modifications affecting epitope recognition

3. Discrepancies Between Protein and mRNA Levels:

• Challenge: GTF2H5 protein levels can be affected by TFIIH complex stability

• Solutions:

  • Compare protein detection with mRNA quantification (qPCR)

  • Note that in GTF2H5-deficient conditions, TFIIH subunit mRNA levels are unaffected despite lowered protein levels

  • Assess other TFIIH complex members as additional readouts

4. Unexpected Subcellular Localization:

• Challenge: GTF2H5 should show exclusive nuclear localization

• Solutions:

  • Check fixation protocols (improper fixation can affect nuclear retention)

  • Verify antibody specificity using peptide competition or knockdown controls

  • Compare with other nuclear markers to confirm proper nuclear visualization

5. Species Cross-Reactivity Issues:

• Challenge: Antibody may not perform equally across all claimed reactive species

• Solutions:

  • Verify species reactivity claims with positive controls

  • Consider using species-specific antibodies for critical experiments

  • Check sequence homology of the immunizing peptide across species

6. Assay-Specific Optimization:

• For IHC: Test different antigen retrieval methods and dilutions (1/20 - 1/200 range)

• For IF: Optimize fixation methods and permeabilization conditions

• For ELISA: Test different coating buffers and blocking reagents