SETX Antibody

What is senataxin (SETX) and what are its primary cellular functions?

Senataxin is a large protein (calculated molecular weight of 303 kDa) involved in several critical cellular processes. SETX plays a significant role in DNA double-strand breaks damage response generated by oxidative stress . The protein possesses both RNA binding capability and ATPase/helicase activity, which are essential for its biological functions . Additionally, SETX appears to participate in the development and maturation of germ cells . Research has revealed its involvement in controlling the antiviral response by regulating RNA polymerase II (RNAPII) activity at infection-responsive genes . The gene encoding SETX is identified by NCBI Gene ID 23064, with the UniProt ID Q7Z333 .

What is the mechanism of action for SETX in transcriptional regulation?

SETX functions primarily by promoting premature termination of transcription, particularly at antiviral genes. Mechanistically, SETX binds to the 5' end of nascent RNA through its RNA binding domain and uses its ATPase/helicase activity to promote RNAPII early termination . This process leads to increased production of transcription start site-associated short RNAs (tssRNAs) at IRF3-dependent genes . The mechanism is similar to how Sen1, the yeast ortholog of human SETX, controls termination at small nucleolar RNAs (sno-RNAs) and is reminiscent of RhoA helicase-mediated termination in bacteria .

What are the technical specifications of commercially available SETX antibodies?

SETX antibodies, such as the 13837-1-AP, are typically available as rabbit polyclonal antibodies that target specific epitopes of the human SETX protein . The antibody product information reveals:

Researchers should note that these antibodies are typically unconjugated and validated for Western Blot (WB), Immunohistochemistry (IHC), and ELISA applications .

What are the optimal dilutions and conditions for using SETX antibody in various applications?

Based on validated protocols, SETX antibody should be used at specific dilutions depending on the application:

For IHC applications, antigen retrieval with TE buffer pH 9.0 is suggested, although citrate buffer pH 6.0 may alternatively be used . The antibody has been positively tested in Western blot detection of SETX in HeLa cells and in IHC applications with human ovary cancer tissue . It's important to note that optimal dilutions may be sample-dependent, and researchers should titrate the antibody in each testing system to obtain optimal results .

How should SETX antibody be stored and handled for maximum stability and performance?

SETX antibody should be stored at -20°C, where it remains stable for one year after shipment . The antibody is supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which helps maintain stability during storage . Smaller aliquots (20μl) may contain 0.1% BSA for additional stabilization . According to manufacturer guidelines, aliquoting is unnecessary for -20°C storage, which simplifies handling procedures in laboratory settings .

What experimental approaches can be used to study SETX's role in transcriptional regulation?

To investigate SETX's function in transcriptional regulation, researchers can employ several experimental approaches that have been validated in published studies:

• Chromatin Immunoprecipitation (ChIP): This technique is effective for assessing SETX and RNA polymerase II (RNAPII) loading at promoters of viral-induced genes. ChIP experiments have shown that wild-type SETX causes a reduction of RNAPII at promoters, dependent on its ATPase and RNA binding activity .

• RNA Immunoprecipitation: This approach can determine SETX binding to nascent RNAs, particularly at the 5' end of antiviral genes. Studies have shown that wild-type SETX, but not RNA binding or ATPase mutants, can efficiently bind these RNAs .

• Transcription Start Site associated RNA (tssRNA) assay: This method can quantify the levels of prematurely terminated transcripts generated by SETX activity. The protocol involves separating large (>200 nt) and short (<200 nt) RNAs, followed by oligo-adenylation and cDNA synthesis of short RNA using Superscript III and an oligo-dT primer with a 5' adaptor sequence . qPCR analysis can then be performed with forward primers specific to the 5'UTR of the gene of interest and a universal reverse primer matching the 5' adaptor sequence, with 5s rRNA as a normalization standard .

What is the specific role of SETX in controlling antiviral gene expression?

SETX functions as a negative regulator of the antiviral response by controlling the expression levels of IFN-β and other key antiviral genes . Multiple experimental approaches have confirmed this regulatory role:

• Depletion of SETX results in increased expression of early infection-induced genes, including key cytokines (e.g., IFN-β) and antiviral mediators (e.g., IFI6, IFIT1) .

• Overexpression of SETX counteracts IRF3-dependent activation of antiviral genes .

• Human cells derived from patients with loss-of-function mutations in the SETX gene display upregulation of virally induced genes upon infection compared to wild-type cells .

• SETX-deficient cells inhibit viral biogenesis, similar to SETX-depleted cells .

• Setx−/− mice express higher amounts of inflammatory mediators upon immune stimulation compared to wild-type mice .

How do SETX mutations affect antiviral response and what are the implications for disease?

SETX mutations associated with neurodegenerative diseases like ALS4 and AOA2 alter normal SETX function, which can dysregulate the antiviral response . Cells derived from patients with SETX mutations exhibit enhanced expression of antiviral genes upon infection, suggesting a loss of the normal regulatory function of SETX .

This dysregulation may have significant implications for disease pathogenesis. Unlike congenital diseases that display constitutive expression of inflammatory mediators, SETX-driven changes to the innate immune response manifest experimentally only upon infection . Researchers have postulated that the inducible phenotypes observed in SETX deficiency may be environmentally triggered by exposure to viral agents . Transient states of excessive inflammation resulting from such exposures may, over time, lead to local dysregulation of immune homeostasis and progressive deterioration of affected tissues .

This connection suggests that infection may play an important role in the initiation or progression of AOA2 and ALS4, providing a potential mechanism linking SETX mutations to neurodegeneration through dysregulated inflammatory responses .

What experimental models are available for studying SETX function in antiviral immunity?

Several experimental models have been validated for investigating SETX function in antiviral immunity:

• Cell Culture Systems: Human cell lines with SETX depletion (via siRNA or CRISPR) provide a straightforward model for studying SETX function . Additionally, lymphoblastoid and fibroblast cells derived from patients with SETX mutations (particularly those associated with AOA2) offer valuable disease-relevant models .

• Expression Systems: Transient expression of constitutively active forms of transcription factor IRF3 (dominant active IRF3; daIRF3) can be used to induce IFN-β and ISG expression in non-infected cells, allowing researchers to study SETX's effects on IRF3-dependent transcription .

• SETX Mutant Constructs: SETX mutants lacking enzymatic (helicase and translocase) activity (K1969Q; MUT1) or RNA-binding capability (G2343D; MUT2) can be generated to dissect the molecular requirements for SETX function .

• Mouse Models: Setx−/− mice provide an in vivo system for studying how SETX deficiency affects the organismal response to immune stimulation . These mice can be challenged with viral components, such as in vitro transcribed SeV DI RNA, to assess antiviral responses .

How does SETX contribute to DNA repair mechanisms, and how can this be experimentally assessed?

SETX is involved in DNA double-strand breaks damage response, particularly in response to oxidative stress . While the search results don't provide detailed experimental protocols for assessing this function, researchers typically use techniques such as comet assays, γH2AX foci formation, and double-strand break repair assays to evaluate SETX's role in DNA repair.

SETX's helicase activity is crucial for resolving RNA/DNA hybrids (R-loops) that can form at sites of transcription-associated DNA damage . R-loops can be detected and quantified using techniques such as DRIP (DNA-RNA Immunoprecipitation) assays with antibodies specific for RNA/DNA hybrids. Additionally, sensitivity to DNA-damaging agents can be compared between wild-type and SETX-deficient cells to assess SETX's contribution to DNA repair pathways.

What is the relationship between SETX mutations and neurodegenerative diseases?

SETX mutations are directly implicated in two neurodegenerative disorders:

• Amyotrophic Lateral Sclerosis Type 4 (ALS4): A rare autosomal dominant form of ALS characterized by juvenile onset, slow progression, and distal muscle weakness .

• Ataxia with Oculomotor Apraxia Type 2 (AOA2): An autosomal recessive cerebellar ataxia characterized by progressive cerebellar atrophy, peripheral neuropathy, oculomotor apraxia, and elevated alpha-fetoprotein levels .

The research presents a novel hypothesis connecting SETX mutations, antiviral response dysregulation, and neurodegeneration. According to this model, SETX mutations lead to excessive expression of inflammatory mediators during viral infections . These transient states of excessive inflammation may, over time, result in local dysregulation of immune homeostasis and progressive deterioration of affected neural tissues . This suggests that environmental factors, particularly viral infections, may trigger or exacerbate neurodegeneration in individuals with SETX mutations .

How can researchers experimentally differentiate between SETX's roles in transcription termination and DNA damage response?

To differentiate between SETX's roles in transcription termination and DNA damage response, researchers should design experiments that specifically isolate each function:

• For transcription termination: Techniques like RNA-seq, nascent RNA sequencing (e.g., GRO-seq), and tssRNA assays can be used to measure transcription termination events without inducing DNA damage . Chromatin immunoprecipitation (ChIP) of RNA polymerase II at different genes can also reveal termination defects .

• For DNA damage response: Inducing DNA damage with agents like hydrogen peroxide (for oxidative stress) while measuring SETX recruitment to damage sites can isolate this function. Comet assays and immunofluorescence for DNA repair factors can assess repair efficiency without directly measuring transcription.

• Using specific mutants: SETX mutants that selectively affect one function but not the other can help distinguish between these roles. For instance, the search results indicate that mutations affecting ATPase activity (K1969Q) or RNA binding (G2343D) impair transcription termination . Testing whether these same mutations affect DNA damage response could help separate these functions.

• Cell cycle synchronization: DNA repair predominantly occurs in specific cell cycle phases, while transcription occurs throughout the cell cycle. Synchronizing cells and assessing SETX activity in different phases could help distinguish between these functions.

What are the most common technical challenges when working with SETX antibodies, and how can they be addressed?

Working with SETX antibodies presents several technical challenges due to the large size of the protein (303 kDa) and potential cross-reactivity issues. Though not explicitly stated in the search results, based on general antibody usage principles and the specific information provided about SETX antibody, researchers should consider:

• Protein extraction efficiency: The large size of SETX (303 kDa) can make complete extraction challenging. Using stronger lysis buffers (containing SDS or urea) and mechanical disruption methods can improve extraction efficiency.

• Transfer efficiency in Western blotting: Large proteins transfer poorly to membranes. Researchers should use low percentage gels (6-7%), extend transfer times, add SDS to transfer buffer, and consider wet transfer systems rather than semi-dry methods.

• Antibody specificity verification: Use appropriate positive controls (such as HeLa cells, which have been confirmed to express SETX) and negative controls (SETX-depleted or knockout cells) . When possible, validate results with multiple antibodies targeting different epitopes.

• Sample preparation for IHC: The search results indicate specific antigen retrieval conditions (TE buffer pH 9.0 or citrate buffer pH 6.0) for optimal IHC results with SETX antibodies . Proper tissue fixation and processing are crucial for preserving SETX antigenicity.

How can researchers effectively study the interaction between SETX's enzymatic activities and its cellular functions?

To investigate the relationship between SETX's enzymatic activities and its cellular functions, researchers can employ several strategic approaches:

• Structure-function analysis using mutants: Generate SETX mutants with specifically altered functional domains to dissect which activities are essential for which cellular functions. For example, the K1969Q mutation in the Walker A motif disrupts ATPase/helicase activity, while the G2343D mutation in motif IV impairs RNA binding . These mutants can be used in rescue experiments in SETX-depleted or knockout systems.

• In vitro biochemical assays: Purify recombinant SETX or specific domains to directly test helicase, ATPase, and RNA binding activities in controlled conditions. The search results mention confirmation that purified human SETX possesses helicase activity .

• Domain-specific interaction studies: Identify proteins that interact specifically with SETX's functional domains using techniques like co-immunoprecipitation followed by mass spectrometry. This can reveal how different domains contribute to various cellular functions.

• Inducible expression systems: Use doxycycline-inducible or similar systems to express wild-type or mutant SETX in a controlled manner, allowing for temporal analysis of how SETX enzymatic activities affect cellular processes over time.

• Structural biology approaches: Although challenging due to SETX's large size, structural studies of individual domains can provide insights into how enzymatic activities are regulated and how mutations affect these activities.

What are the current technical limitations in studying SETX in neurodegeneration models?

Studying SETX in neurodegeneration models presents several technical challenges:

• Complexity of neurodegeneration pathways: Neurodegenerative diseases involve multiple complex pathways, making it difficult to isolate SETX's specific contributions. Researchers must design experiments that can distinguish between primary effects of SETX dysfunction and secondary consequences.

• Temporal aspects of disease progression: Neurodegenerative diseases develop over long periods, whereas laboratory models often focus on acute effects. Developing age-appropriate models that capture the progressive nature of SETX-related neurodegeneration is technically challenging.

• Cell-type specificity: Different neural cell types may be differentially affected by SETX dysfunction. Techniques that allow cell-type-specific analysis, such as single-cell RNA-seq or conditional knockout models, are necessary but technically demanding.

• Combined environmental and genetic factors: The search results suggest that environmental triggers (like viral infections) may interact with SETX mutations to promote disease . Modeling this interaction requires sophisticated experimental designs that can incorporate both genetic and environmental variables.

• Translation between model systems and human disease: Findings in cellular or animal models may not directly translate to human disease. Validation in patient-derived cells or tissues is important but limited by sample availability and heterogeneity.