GTF2H5 Human

What is GTF2H5 and what is its role in human cells?

GTF2H5, also known as TTDA, TFB5, TTD-A, and several other aliases, is the smallest subunit (8kD protein) of the 10-subunit TFIIH complex. This evolutionary conserved complex plays essential roles in both transcription initiation and nucleotide excision repair (NER) . In transcription, GTF2H5 is part of the TFIIH complex that enables RNA polymerase II-driven transcription initiation by facilitating promoter escape and RNA synthesis . In DNA repair, GTF2H5 stimulates ERCC3/XPB ATPase activity to trigger DNA opening during the repair process and is particularly important for the removal of interstrand adducts .

Where is the GTF2H5 gene located in the human genome?

The GTF2H5 gene is located on chromosome 6 at position q25.3 (6q25.3). According to the genomic context information, it spans the region 158168350 to 158199344 on chromosome 6 (NC_000006.12) and contains 3 exons in total . This precise genomic localization is important for researchers conducting genetic studies or designing targeted gene manipulation experiments.

What disorders are associated with mutations in GTF2H5?

Mutations in the GTF2H5 gene result in trichothiodystrophy 3, photosensitive (TTD3), a rare autosomal recessive developmental disorder . This condition is characterized by brittle hair, developmental abnormalities, and photosensitivity . The disease phenotype is thought to be caused by subtle transcription or gene expression defects resulting from impaired TFIIH function when GTF2H5 is mutated .

How does GTF2H5 contribute to TFIIH stability in vivo?

GTF2H5 plays a crucial role in maintaining the stability of the TFIIH complex in vivo. Research using C. elegans has shown that loss of GTF-2H5 (the C. elegans ortholog) leads to reduced levels of other TFIIH components by more than 50% across multiple tissues . This is consistent with observations in patient and mouse fibroblasts where TFIIH protein levels are lowered while mRNA levels of TFIIH subunits remain unaffected . These findings indicate that GTF2H5 has an evolutionary conserved function in maintaining steady-state levels of TFIIH in various tissues in vivo, likely by stabilizing the complex rather than affecting transcription of its components .

Why is C. elegans a valuable model for studying GTF2H5 function compared to mouse models?

C. elegans presents a unique advantage for studying GTF2H5 function because, unlike in mice where TTDA/GTF2H5 knockout is lethal, GTF-2H5 deficiency in C. elegans is compatible with life and growth . This difference allows researchers to investigate how GTF2H5 promotes transcription in vivo in ways that would be impossible in mouse models. Additionally, C. elegans enables visualization and quantification of TFIIH components in different tissues through fluorescent tagging, permitting direct observation of GTF2H5's impact on TFIIH stability across different cell types . The C. elegans model thus establishes a valuable system for studying the pathogenesis of trichothiodystrophy and the fundamental roles of GTF2H5 in transcription and DNA repair .

What are the methodological approaches to measure TFIIH concentration in different tissues?

Researchers have developed sophisticated approaches to measure TFIIH concentration in different tissues. In C. elegans studies, scientists determined GTF-2H5 and GTF-2H1 concentrations by comparing average nuclear fluorescence signals of fluorescently tagged proteins to known GFP concentrations . This methodology revealed comparable concentrations of around 0.2 μM for GTF-2H1 in oocyte, hypodermal, intestinal, and muscle nuclei, which is strikingly similar to concentration estimations of Pol II in human fibroblasts (0.18 μM) .

GTF-2H5 concentrations showed greater variation between individual nuclei and lower concentration in muscle cells, reflecting its dynamic association with the TFIIH complex. Similar analyses in mouse models using XPB-YFP fluorescence levels showed that TFIIH levels vary depending on the cell type, which correlates with transcriptional activity . These methods provide quantitative biomarkers of transcriptional activity in different tissues and cell types.

How does GTF2H5 expression impact cancer prognosis, particularly in ovarian cancer?

Research has shown that GTF2H5 expression levels have significant prognostic implications in high-grade serous ovarian cancer (HGSOC). Low expression of GTF2H5 is associated with longer 5-year survival of patients at both the protein level (hazard ratio [HR], 0.52; 95% CI, 0.29 to 0.93; p=0.024) and transcriptional level (HR, 0.80; 95% CI, 0.65 to 0.97; p=0.023) .

What methodologies are used to evaluate the relationship between GTF2H5 expression and patient outcomes?

Researchers employ multiple methodological approaches to evaluate the relationship between GTF2H5 expression and patient outcomes:

• Immunohistochemical (IHC) staining: Used to assess protein levels of GTF2H5 in patient tissue samples. In ovarian cancer studies, IHC staining was performed on 139 high-grade serous ovarian carcinomas included in tissue microarrays. Cases were stratified into high- and low-GTF2H5 staining categories based on median staining intensity .

• Transcriptional analysis using public databases: The Kaplan-Meier plotter tool has been used to validate findings at the gene expression level. This online tool incorporates gene expression data and survival information from multiple datasets (13 different datasets comprising 855 HGSOC cases) . The data is typically dichotomized at automatically selected best-fitted cut-offs into high and low expressing groups.

• Statistical analysis: Kaplan-Meier analysis and log-rank tests are employed to estimate patients' survival and assess statistical differences between expression groups. Cox proportional hazard models are used to estimate hazard ratios and 95% confidence intervals .

• Functional validation: Experimental approaches such as stable short hairpin RNA (shRNA)-mediated GTF2H5 downregulation in cancer cell lines are used to evaluate whether GTF2H5 modulates response to treatment (e.g., cisplatin sensitivity) .

How does GTF2H5 affect sensitivity to platinum-based chemotherapy in cancer cells?

GTF2H5 appears to influence sensitivity to platinum-based chemotherapy, particularly cisplatin, in cancer cells. Functional experiments involving stable shRNA-mediated GTF2H5 downregulation in ovarian cancer cell lines (SKOV3 and COV504) have shown that GTF2H5-silencing induces a decrease in the half maximal inhibitory concentration (IC50) upon cisplatin treatment .

This increased sensitivity to cisplatin in GTF2H5-silenced cells is likely related to GTF2H5's role in nucleotide excision repair, particularly in the removal of interstrand adducts—the type of DNA damage caused by platinum compounds . Since GTF2H5 participates in DNA repair pathways that remove cisplatin-induced DNA damage, its downregulation compromises the cell's ability to repair this damage, thereby enhancing cisplatin's cytotoxic effects.

What are the optimal methods for studying GTF2H5's role in nucleotide excision repair?

To effectively study GTF2H5's role in nucleotide excision repair (NER), researchers should consider multiple complementary approaches:

• Model organism studies: C. elegans provides a valuable model system since GTF-2H5 deficiency is compatible with life, unlike in mice. This allows for in vivo assessment of NER function in the absence of GTF2H5 .

• Recruitment assays: Techniques to visualize and quantify the recruitment of TFIIH components to sites of DNA damage can reveal how GTF2H5 facilitates the assembly of the NER machinery. This can be achieved through fluorescently tagged TFIIH subunits and localized UV damage induction .

• DNA damage sensitivity assays: Comparing survival and growth of wild-type and GTF2H5-deficient cells or organisms after exposure to DNA damaging agents (e.g., UV radiation) provides functional evidence of NER deficiency .

• Repair kinetics measurements: Quantitative assessment of repair rates for specific DNA lesions can be performed using techniques such as immunoassays that detect specific DNA adducts or unscheduled DNA synthesis assays .

• Protein-protein interaction studies: Techniques such as co-immunoprecipitation, yeast two-hybrid, or proximity ligation assays can help characterize how GTF2H5 interacts with other TFIIH components and facilitates the recruitment and stability of the complex .

How can researchers quantitatively assess TFIIH stability in the absence of GTF2H5?

Researchers can quantitatively assess TFIIH stability in the absence of GTF2H5 through several methodological approaches:

• Fluorescent tagging and live imaging: By tagging other TFIIH subunits with fluorescent proteins (such as AG::GTF-2H1 in C. elegans studies), researchers can directly visualize and quantify the levels of these subunits in the presence or absence of GTF2H5 . This approach revealed that loss of GTF-2H5 led to reduced levels of AG::GTF2H1 in all tested tissues by more than 50% .

• Western blot analysis: Quantification of TFIIH subunit levels by western blotting can provide biochemical evidence of reduced TFIIH stability in GTF2H5-deficient cells or tissues.

• Immunofluorescence: Antibody staining against TFIIH components can be used to assess their levels and nuclear localization in fixed cells or tissues.

• Protein half-life studies: Pulse-chase experiments with protein synthesis inhibitors can determine whether the reduced levels of TFIIH components are due to increased degradation rather than decreased synthesis.

• mRNA expression analysis: RNA-seq or qPCR can confirm that reduced TFIIH protein levels are not due to transcriptional downregulation of TFIIH subunit genes, supporting a post-transcriptional stabilizing role for GTF2H5 .

What experimental designs are most effective for investigating GTF2H5's role in transcription under challenged conditions?

To investigate GTF2H5's role in transcription under challenged conditions, several experimental designs have proven effective:

• Reporter gene assays under transcriptional stress: By crossing GTF2H5-deficient animals with those expressing fluorescent reporters (e.g., GFP under control of specific promoters), researchers can quantify transcriptional output as a proxy for transcriptional competence under normal and stressed conditions .

• Limiting availability of other transcription factors: Compromising transcription by limiting the availability of other transcription initiation factors (without compromising NER) can reveal synthetic interactions with GTF2H5 deficiency. This approach has shown that GTF2H5-deficient C. elegans embryos die when transcription is challenged due to the intrinsic TFIIH fragility in the absence of GTF2H5 .

• Temperature sensitivity assays: Using temperature-sensitive alleles of transcription factors combined with GTF2H5 deficiency can reveal conditional requirements for GTF2H5 in transcription.

• Nascent RNA labeling: Although challenging in some systems, techniques like 5-ethynyl uridine labeling can potentially measure active transcription directly in different tissues and under different conditions .

• Transcriptome analysis under stress conditions: RNA-seq of GTF2H5-deficient versus wild-type cells or tissues under various stressors (heat shock, DNA damage, etc.) can reveal global transcriptional defects that may only manifest under challenging conditions.

How do recent findings about GTF2H5 in model organisms inform our understanding of human diseases?

Recent findings about GTF2H5 in model organisms, particularly C. elegans, have significantly advanced our understanding of human diseases associated with GTF2H5 mutations. The discovery that GTF-2H5 deficiency in C. elegans is compatible with life, unlike in mice, provides a unique opportunity to study the molecular mechanisms underlying trichothiodystrophy .

Studies in C. elegans have shown that GTF-2H5 promotes TFIIH stability in multiple tissues and is indispensable for nucleotide excision repair, in which it facilitates recruitment of TFIIH to DNA damage . Importantly, these studies revealed that when transcription is challenged, GTF-2H5-deficient embryos die due to the intrinsic TFIIH fragility in the absence of GTF-2H5 .

These findings support the idea that TTDA/GTF2H5 mutations cause trichothiodystrophy through transcription impairment, particularly under conditions of transcriptional stress . This helps explain why patients with GTF2H5 mutations can survive despite the gene's essential functions—normal conditions may allow sufficient transcription, but any additional stress may exceed the capacity of the compromised system.

What are the emerging techniques for studying GTF2H5 function in different cellular contexts?

Several emerging techniques are advancing our ability to study GTF2H5 function in different cellular contexts:

• CRISPR-Cas9 gene editing: Precise manipulation of GTF2H5 in human cells and model organisms allows for detailed functional studies and disease modeling.

• Single-cell transcriptomics: This technology enables researchers to assess how GTF2H5 deficiency affects transcription at the single-cell level, potentially revealing cell type-specific effects that might be masked in bulk analysis.

• Quantitative live-cell imaging: Advanced microscopy techniques combined with fluorescent tagging allow real-time visualization of TFIIH dynamics and GTF2H5's role in complex stability and function.

• Cryo-electron microscopy: Structural studies of TFIIH with and without GTF2H5 are providing insights into how this small subunit stabilizes the complex and influences its conformational changes during transcription and repair.

• Patient-derived iPSCs and organoids: These models allow the study of GTF2H5 mutations in relevant human cellular contexts and tissue-specific effects that might contribute to the complex phenotypes observed in trichothiodystrophy.

What contradictions exist in the current literature regarding GTF2H5's role in transcription?

The literature presents some notable contradictions regarding GTF2H5's role in transcription:

These contradictions highlight the complexity of GTF2H5 function and emphasize the need for continued research to fully understand its role in different contexts and organisms.