ATRX Antibody

What is ATRX and why is it significant in research?

ATRX (Alpha Thalassemia/Mental Retardation Syndrome X-linked) is a protein encoded by the ATRX gene in humans, also known as ATP-dependent helicase ATRX, X-linked helicase II, or X-linked nuclear protein (XNP) . It belongs to the SNF2 family of helicase/ATPases that regulate gene expression through effects on chromatin structure and function . ATRX is significant in research because mutations in the ATRX gene are associated with X-linked mental retardation syndrome often accompanied by alpha-thalassemia (ATR-X syndrome) . Additionally, loss of ATRX expression has been documented in various cancers, particularly in gliomas, making it an important diagnostic and prognostic marker in neuro-oncology . Research has also revealed ATRX's role in immune response pathways, as it interacts with interferon regulatory factor 3 (IRF3) and facilitates type I interferon transcription .

What cellular localization pattern does ATRX exhibit and how is this relevant to experimental design?

ATRX demonstrates a distinct nuclear localization pattern that varies during the cell cycle, which is crucial to consider when designing immunocytochemistry experiments. During interphase, ATRX associates with the nuclear matrix and exhibits a nonuniform pattern of staining in the nucleus with discrete areas of dense staining against a more diffuse distribution . In mouse cells, ATRX protein is concentrated at or close to the centromeres, where it associates with signals detected by CREST serum, which recognizes the centromeric protein CENP-C . In human cells, the relationship to pericentromeric heterochromatin is less clear, but quantitation by confocal microscopy showed that in unsynchronized HeLa cells, between 39-95% of centromeric signals were closely associated with ATRX .

During mitosis, particularly at metaphase and telophase, ATRX localizes at centromeres, though this localization can be more sensitive to preparation methods . A striking observation in metaphase preparations is that ATRX antibodies consistently localize to the short arms of acrocentric chromosomes, appearing adjacent to the centromere on the stalks of the short arms where rDNA arrays are found . This dynamic localization pattern means researchers should carefully consider fixation methods and cell cycle stage when interpreting ATRX staining patterns.

How does ATRX loss correlate with different types of gliomas, and what are the implications for using ATRX antibody as a diagnostic marker?

Studies have demonstrated significant variation in ATRX loss across different glioma subtypes, making ATRX immunohistochemistry a valuable diagnostic tool. Loss of ATRX expression has been detected in 45% of anaplastic astrocytomas (AA), 27% of anaplastic oligoastrocytomas (AOA), and 10% of anaplastic oligodendrogliomas (AO) . In grade II/III gliomas, ATRX loss was observed in 54.5% of astrocytomas, 30.8% of oligoastrocytomas, and 0.0% of oligodendrogliomas, while only 12.7% of glioblastomas showed ATRX loss .

The diagnostic significance lies in the relationship between ATRX status and other molecular markers. Survival analysis has shown that IDH mutant astrocytic tumors can be separated into two prognostic groups based on ATRX status: tumors with ATRX loss demonstrated a significantly better prognosis . This finding has led to the development of diagnostic algorithms using stepwise analysis starting with immunohistochemistry for ATRX and IDH1-R132H, followed by 1p/19q analysis and IDH sequencing . This approach reduces the number of molecular analyses required while providing better association with patient outcomes. When designing studies using ATRX antibody as a diagnostic marker, researchers should consider that ATRX immunoreactivity is typically either almost totally absent or completely retained in tumor cells, with perfect concordance between immunohistochemistry results and ATRX mutation status .

How does ATRX function in the regulation of innate immunity, and what are the experimental considerations when studying this relationship?

Recent research has uncovered ATRX's significant role in innate immunity, particularly in the regulation of interferon pathways. ATRX depletion results in suppressed activation of DNA and RNA sensing pathways, leading to decreased transcription and secretion of type I interferons, followed by reduced expression of interferon-stimulated genes (ISGs) . Mechanistically, ATRX interacts with the transcription factor interferon regulatory factor 3 (IRF3) and associates with the IFN-β promoter to facilitate transcription .

When designing experiments to study ATRX's role in immunity, researchers should consider that ATRX positively modulates chromatin accessibility specifically upon interferon signaling, affecting promoter regions with recognition motifs for AP-1 family transcription factors . This suggests a co-activating function in innate immunity through regulation of chromatin accessibility. Experiments should therefore include chromatin accessibility assays (such as ATAC-seq) before and after interferon stimulation in both ATRX-expressing and ATRX-depleted conditions.

Furthermore, researchers should be aware that ATRX antagonization by viral proteins and ATRX mutations in tumors may represent strategies to compromise both intrinsic and innate immune responses . This has important implications for studies of viral infections or cancer immunology. When designing such studies, consider including conditions that compare wild-type ATRX with mutant variants to assess differential effects on immune pathway activation.

What is the relationship between ATRX and alternative lengthening of telomeres (ALT) in cancer research, and how can ATRX antibodies be utilized to study this phenomenon?

Although not directly mentioned in the search results, the relationship between ATRX and ALT is an important research area. ATRX loss is strongly associated with the alternative lengthening of telomeres (ALT) phenotype in various cancers, particularly in astrocytomas. When designing experiments to investigate this relationship, researchers should consider combining ATRX immunohistochemistry with telomere FISH or C-circle assays to correlate ATRX loss with ALT activation.

The experimental approach should include careful selection of cell lines or patient samples with documented ATRX status. For immunofluorescence experiments, co-staining of ATRX with ALT-associated PML nuclear bodies (APBs) can provide insight into the spatial relationship between ATRX and telomere maintenance mechanisms. When analyzing patient samples, it's crucial to correlate ATRX immunohistochemistry results with other molecular features such as IDH mutation status, as the prognostic significance of ATRX loss may depend on these interactions.

Researchers should also consider chromatin immunoprecipitation experiments to investigate ATRX binding at telomeric regions. When interpreting results, remember that ATRX loss may not be the sole determinant of ALT activation, as additional factors like DAXX mutations may contribute to the phenotype.

How should researchers interpret discrepancies between ATRX immunohistochemistry results and genetic analyses of ATRX mutations?

When faced with discrepancies between ATRX protein expression (detected by immunohistochemistry) and genetic analyses, researchers should consider several factors that might explain these differences. One study found "perfect concordance between the IHC results and ATRX mutation status" , but this may not always be the case in all research settings.

First, consider antibody specificity and the epitope being recognized. Different antibodies target different regions of the ATRX protein, so mutations affecting one region may not impact antibody binding to another region. For instance, if using an N-terminal antibody like D-5 or BSB-108 , C-terminal mutations might not affect immunoreactivity.

Second, evaluate post-transcriptional and post-translational mechanisms. Some mutations may affect protein stability rather than expression, leading to decreased protein levels despite normal mRNA expression. Conversely, epigenetic silencing could reduce ATRX expression without detectable mutations in the coding sequence.

Third, consider technical factors such as fixation methods, antigen retrieval protocols, and antibody concentration, which can significantly impact immunohistochemistry results. When encountering discrepancies, researchers should validate findings using multiple techniques, such as combining immunohistochemistry with Western blotting or using different antibodies targeting distinct epitopes of ATRX. Additionally, comprehensive genetic analysis including evaluation of large deletions or rearrangements may reveal genetic alterations missed by standard sequencing approaches.

What are the optimal fixation and antigen retrieval methods for ATRX immunohistochemistry in different tissue types?

The fixation and antigen retrieval methods for ATRX immunohistochemistry should be carefully optimized based on tissue type to ensure reliable and reproducible results. For cultured cells, a standard approach involves fixation in 3% paraformaldehyde/PBS for 15 minutes followed by permeabilization in 0.1% Triton X-100/PBS for 10 minutes . For adherent cells, culturing directly on glass coverslips is recommended, while suspension cells should be incubated on coverslips treated with poly-l-lysine .

For formalin-fixed, paraffin-embedded (FFPE) tissues, which are commonly used in diagnostic pathology, heat-induced epitope retrieval methods are typically required. While the search results don't specify exact conditions, standard protocols for nuclear antigens often use citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) for heat-induced antigen retrieval. When optimizing the protocol, researchers should compare both buffers to determine which provides optimal staining with minimal background.

The antibody concentration and incubation conditions should also be optimized for each tissue type. For immunofluorescence on cultured cells, primary antibodies (such as anti-ATRX) can be diluted in block buffer (10% FCS/PBS-T) and incubated on cells at room temperature for 45 minutes . Multiple washing steps in PBS-T are crucial to reduce background staining. For challenging tissues or when working with new antibody lots, a titration experiment comparing different antibody dilutions is recommended to determine optimal signal-to-noise ratio.

What controls should be included when validating a new ATRX antibody for research applications?

Proper validation of ATRX antibodies requires a comprehensive set of controls to ensure specificity, sensitivity, and reproducibility. Positive controls should include tissues or cell lines known to express ATRX, while negative controls should include samples with confirmed ATRX mutations or deletions that eliminate the epitope recognized by the antibody.

For Western blotting validation, compare cellular extracts from normal individuals and patients with previously defined mutations in the ATRX gene . A properly validated antibody should detect a band of approximately 280 kDa in normal samples, consistent with the predicted size of the ATRX protein . In samples from patients with ATRX mutations, the antibody may detect a smaller band (if truncation occurs) or no band (if the epitope is lost) .

For immunohistochemistry validation, include known positive tissues alongside samples from ATR-X patients expressing ATRX protein lacking the epitopes detected by these antibodies . Additionally, include technical negative controls by omitting the primary antibody to assess non-specific binding of the secondary antibody.

When validating a new antibody, compare its performance against established antibodies targeting the same protein. For example, if validating a new antibody, compare its staining pattern with well-characterized antibodies like D-5 (sc-55584) or BSB-108 . The staining pattern should show nuclear localization with concentration at pericentromeric heterochromatin in mouse cells and a similar but less distinct pattern in human cells .

What are the key considerations when using ATRX antibodies for different experimental techniques (Western blot, IHC, IF, IP)?

Different experimental techniques require specific considerations when using ATRX antibodies to ensure optimal results. Here's a breakdown of key factors for each technique:

Western Blotting (WB):

• Sample preparation is crucial due to ATRX's high molecular weight (~280 kDa). Use optimized extraction buffers to ensure complete protein denaturation.

• Employ low percentage (6-8%) SDS-PAGE gels to properly resolve high molecular weight proteins.

• Extended transfer times may be necessary due to ATRX's size.

• When analyzing Western blot results, be aware that mutations can result in truncated proteins of different sizes .

Immunohistochemistry (IHC):

• ATRX staining should be evaluated in cell nuclei, with normal endothelial cells, inflammatory cells, and non-neoplastic glial cells serving as internal positive controls.

• Loss of ATRX expression is typically considered when tumor cell nuclei show complete absence of staining while internal non-neoplastic cells remain positive .

• ATRX immunoreactivity in tumor cells is generally either almost totally absent or completely retained, with rare cases showing mosaic patterns .

Immunofluorescence (IF):

• For examining ATRX localization, dual-staining experiments with markers of nuclear structures can provide valuable insights. For example, dual-staining with CREST serum can demonstrate ATRX association with centromeres .

• The pattern of ATRX staining varies between mouse and human cells and during different cell cycle phases, which should be considered when interpreting results .

• The localization of ATRX to the short arms of acrocentric chromosomes in metaphase preparations is a distinctive feature that can be used to confirm antibody specificity .

Immunoprecipitation (IP):

• Given ATRX's role in protein complexes, mild lysis conditions may be preferable to preserve protein-protein interactions.

• For studying ATRX interactions with transcription factors like IRF3, consider cross-linking before immunoprecipitation to capture transient interactions .

• Validation of immunoprecipitation results may require reciprocal IP experiments and controls using samples lacking ATRX expression.

How can ATRX immunohistochemistry be integrated into the molecular classification of gliomas alongside other markers?

ATRX immunohistochemistry has become an integral component of the molecular classification of diffuse gliomas, particularly when used in conjunction with other molecular markers. Recent studies have proposed diagnostic algorithms that employ a stepwise analysis beginning with immunohistochemistry for ATRX and IDH1-R132H, followed by 1p/19q analysis and IDH sequencing . This approach has been shown to reduce the number of required molecular analyses while providing stronger associations with patient outcomes.

The implementation should follow specific testing sequences. First, ATRX and IDH1-R132H immunohistochemistry should be performed simultaneously. In cases with ATRX loss and IDH1-R132H positivity, the diagnosis leans toward astrocytoma, and 1p/19q testing may be unnecessary. Conversely, in cases with retained ATRX expression and IDH1-R132H positivity, 1p/19q codeletion testing becomes essential to differentiate between oligodendroglioma and astrocytoma. For cases negative for IDH1-R132H by immunohistochemistry, IDH sequencing is recommended to detect other less common IDH mutations.

ATRX loss has been observed in varying frequencies across different glioma subtypes: 54.5% of grades II/III astrocytomas, 30.8% of oligoastrocytomas, and 0.0% of oligodendrogliomas, with 12.7% of glioblastomas also showing ATRX loss . These differential frequencies help refine molecular classification, particularly for histologically ambiguous cases. ATRX status also has prognostic significance, as tumors with ATRX loss in the context of IDH mutation have been shown to have a significantly better prognosis compared to those with retained ATRX expression .

What are the most common technical challenges in ATRX immunohistochemistry interpretation, and how can they be addressed in research settings?

Interpretation of ATRX immunohistochemistry presents several technical challenges that researchers should be aware of to avoid misclassification of samples. One common challenge is distinguishing true ATRX loss in tumor cells from technical artifacts. ATRX immunoreactivity should be evaluated specifically in cell nuclei, and normal endothelial cells, inflammatory cells, and non-neoplastic glial cells within the sample serve as essential internal positive controls .

Fixation and processing variables can significantly impact staining quality. Overfixation or inadequate antigen retrieval may result in false negative staining. To address this, researchers should optimize antigen retrieval protocols for each antibody and tissue type. Testing multiple antigen retrieval methods (heat-induced epitope retrieval with different pH buffers) can identify optimal conditions for specific antibodies.

Interpretation of heterogeneous staining patterns presents another challenge. While ATRX immunoreactivity in tumor cells is typically either almost totally absent or completely retained , mosaic patterns may occasionally occur. These patterns must be carefully evaluated to determine whether they represent true biological heterogeneity or technical artifacts. In research settings, validating equivocal cases with additional molecular methods, such as ATRX sequencing or alternative antibodies targeting different epitopes, can clarify ambiguous results.

Antibody selection is crucial, as different antibodies may have varying sensitivities and specificities. Comparing results from multiple antibodies (such as BSB-108 and D-5) targeting different epitopes can provide more reliable assessments of ATRX status . Additionally, implementing digital pathology approaches with standardized quantification algorithms could help overcome subjectivity in interpretation and improve reproducibility across different laboratories.

How does ATRX function in the regulation of immune responses, and what experimental approaches can be used to study this role?

ATRX has recently been identified as a significant regulator of immune responses, particularly in the context of type I interferon signaling. Depletion of ATRX results in suppressed activation of DNA and RNA sensing pathways, leading to decreased transcription and secretion of type I interferons, followed by reduced expression of interferon-stimulated genes (ISGs) . This finding reveals a novel co-activating function of ATRX in innate immunity.

At the molecular level, ATRX interacts with the transcription factor interferon regulatory factor 3 (IRF3) and associates with the IFN-β promoter to facilitate transcription . Furthermore, whole transcriptome sequencing has revealed that ATRX is required for efficient interferon-induced expression of a distinct set of ISGs . Mechanistically, ATRX positively modulates chromatin accessibility specifically upon interferon signaling, affecting promoter regions with recognition motifs for AP-1 family transcription factors .

To study ATRX's role in immune regulation, researchers can employ several experimental approaches:

• ATRX knockdown/knockout experiments followed by stimulation of DNA or RNA sensing pathways to assess the impact on interferon production and signaling.

• Chromatin immunoprecipitation (ChIP) assays to investigate ATRX association with the IFN-β promoter and other ISG promoter regions.

• Co-immunoprecipitation studies to confirm and characterize the interaction between ATRX and IRF3.

• ATAC-seq analysis in ATRX-proficient versus ATRX-deficient cells before and after interferon stimulation to map changes in chromatin accessibility.

• Reporter assays using the IFN-β promoter to quantify the impact of ATRX manipulation on transcriptional activity.

These approaches would provide comprehensive insights into ATRX's role in immune regulation, with implications for both viral infections and cancer immunology, as ATRX antagonization by viral proteins and ATRX mutations in tumors may represent important strategies to compromise both intrinsic and innate immune responses .

What is the significance of ATRX in viral infections, and how can researchers design experiments to study ATRX-virus interactions?

ATRX plays a crucial role in intrinsic immunity against several viruses, including human cytomegalovirus (HCMV), as a component of promyelocytic leukemia nuclear bodies (PML-NBs) . These nuclear structures are known to mediate intrinsic antiviral defenses. The significance of ATRX in viral infections is underscored by the fact that viruses have evolved different mechanisms to antagonize ATRX, such as displacement from PML-NBs or degradation .

When designing experiments to study ATRX-virus interactions, researchers should consider several approaches:

These experimental approaches would provide valuable insights into the mechanisms by which ATRX contributes to intrinsic immunity and how viruses have evolved to counteract this defense system, potentially leading to new antiviral strategies targeting this interaction.

How might single-cell analysis techniques enhance our understanding of ATRX function in heterogeneous tumor populations?

Single-cell analysis techniques offer unprecedented opportunities to investigate ATRX function in heterogeneous tumor populations, potentially revealing insights not accessible through bulk tissue analysis. Although not directly addressed in the search results, these emerging approaches can resolve several key questions in ATRX research.

Single-cell RNA sequencing (scRNA-seq) could identify cell-specific transcriptional programs regulated by ATRX in tumors with mosaic ATRX expression patterns. This approach might reveal whether ATRX-positive and ATRX-negative cells within the same tumor have distinct gene expression profiles and cellular states. Furthermore, trajectory analysis of scRNA-seq data could elucidate how ATRX loss influences cellular differentiation and tumor evolution.

Single-cell ATAC-seq (scATAC-seq) would be particularly valuable given ATRX's role in chromatin remodeling and its effect on chromatin accessibility during interferon signaling . This technique could map cell-specific changes in chromatin accessibility associated with ATRX status and identify regulatory elements that depend on ATRX for proper function in specific cell populations.

Combined single-cell multi-omics approaches, integrating RNA, ATAC, and protein data from the same cells, could provide comprehensive insights into how ATRX coordinates chromatin structure, transcription, and cellular phenotypes at the single-cell level. This might reveal how ATRX loss contributes to tumor heterogeneity and adaptation.

When designing single-cell experiments to study ATRX, researchers should consider several methodological aspects. Fresh tissue preparation is crucial to maintain cellular integrity, and enzymatic dissociation protocols should be optimized to minimize stress responses. Antibody-based enrichment strategies might be necessary to capture rare cell populations. Data analysis should incorporate strategies to distinguish technical artifacts from true biological heterogeneity, particularly when analyzing chromatin features that might be sensitive to cell cycle effects.

What are the emerging therapeutic implications of ATRX status in cancers, and how can researchers design experiments to explore these potential applications?

The unique molecular and clinical characteristics associated with ATRX alterations in cancers suggest several potential therapeutic implications that warrant exploration. Although not explicitly covered in the search results, understanding ATRX's role in chromatin remodeling, telomere maintenance, and immune regulation opens avenues for targeted therapeutic approaches.

One promising direction involves exploiting synthetic lethality interactions in ATRX-deficient cancers. Researchers could design CRISPR-based synthetic lethality screens in isogenic cell lines with and without ATRX to identify genes or pathways that, when inhibited, selectively kill ATRX-deficient cells. High-throughput drug screens comparing sensitivity profiles of ATRX-proficient versus ATRX-deficient cells could identify existing compounds with selective activity against ATRX-deficient tumors.

Given ATRX's newly discovered role in interferon signaling and innate immunity , immunotherapeutic approaches might be differentially effective based on ATRX status. Researchers could design preclinical studies evaluating immune checkpoint inhibitors, interferon therapy, or other immunomodulatory approaches in ATRX-stratified cancer models. Patient-derived xenograft (PDX) models or genetically engineered mouse models (GEMMs) with controlled ATRX alterations would be valuable for these investigations.

The association between ATRX loss and alternative lengthening of telomeres (ALT) suggests potential vulnerabilities in telomere maintenance. Experimental approaches could include testing ALT-targeting compounds in ATRX-deficient models or exploring combinations of telomerase inhibitors with drugs targeting DNA damage responses.