Recombinant Mouse BRCA1-associated ATM activator 1 (Brat1), partial

What is BRAT1 and what are its primary functions in cellular processes?

BRAT1 (BRCA1-Associated ATM Activator-1) was initially identified as a DNA damage response (DDR) protein that interacts with BRCA1. Subsequent research has revealed its broader functions in cellular processes. BRAT1 binds to multiple DDR proteins including ATM, DNA-PK, and SMC1, playing essential roles in DNA repair and cell cycle regulation . Beyond DDR activation, BRAT1 is critical for cell growth regulation and prevention of constitutive apoptosis, as demonstrated by knockdown studies in mouse embryonic fibroblasts and human osteosarcoma cell lines .

BRAT1 also functions as a key regulator of neuronal differentiation by controlling the occupancy of REST (Repressor element-1 silencing transcription factor) on critical neuronal genes . Through formation of a distinct trimeric complex with INTS11 and INTS9 subunits of the Integrator complex, BRAT1 activates neuronal genes required for proper differentiation .

How does BRAT1 interact with the DNA damage response pathway?

BRAT1 serves as a critical scaffolding protein within the DNA damage response pathway through multiple protein interactions. It functions by:

• ATM Activation: BRAT1 is essential for the phosphorylation and activation of ATM after DNA damage, facilitating downstream signaling cascades .

• DNA-PK Regulation: BRAT1 binds to DNA-PK and is required for its phosphorylation, which is necessary for non-homologous end joining (NHEJ) repair of double-strand breaks .

• BRCA1 Interaction: Through its association with BRCA1, BRAT1 supports homology-directed repair (HDR) processes .

• Protein Stability: BRAT1 appears to maintain the stability of several PIKK family members, including ATM and DNA-PK, as BRAT1 knockdown results in diminished reaction stability of these proteins .

The table below summarizes BRAT1's key interaction partners in the DDR pathway:

What experimental models are available for studying mouse BRAT1?

Several experimental models have been developed for investigating mouse BRAT1 function:

• Conditional Knockout Mouse Models: The BRAT1^flox/flox/129S6 strain has been established, allowing for tissue-specific deletion of BRAT1 through Cre-lox technology . This model enables the study of BRAT1 function in specific tissues and developmental stages while avoiding the potential embryonic lethality of complete knockout.

• Mouse Embryonic Fibroblasts (MEFs): MEFs derived from BRAT1^flox/flox mice provide a versatile cellular system for studying BRAT1 function. BRAT1-deficient MEFs show reduced mTOR protein levels and impaired serum-induced cell cycle progression .

• Knockdown Cell Lines: Various stable knockdown cell lines, including human HeLa cells with BRAT1 knockdown, have been established to study BRAT1 function in different cellular contexts .

• Neuronal Differentiation Models: Mouse embryonic stem cells with BRAT1 deletion have been utilized to study its role in neuronal differentiation, showing that loss of Brat1 leads to defects in neuronal differentiation assays .

• Recombinant Protein Expression Systems: Though not explicitly detailed in the provided search results, recombinant mouse BRAT1 protein can be expressed using similar systems to those used for other recombinant proteins, such as the carrier-free systems described for other proteins .

How does BRAT1 deficiency affect mTOR signaling and cell cycle progression?

BRAT1 deficiency significantly impacts mTOR signaling and subsequent cell cycle progression through several mechanistic pathways:

• Reduced mTOR Protein Stability: Research has demonstrated that BRAT1 knockdown or deletion results in markedly reduced protein levels of mTOR, particularly evident after serum starvation conditions. The normal restoration of mTOR protein levels following serum reintroduction is suppressed in BRAT1-deficient cells .

• Direct Interaction with mTORC1 Complex: Immunoprecipitation studies have revealed that BRAT1 directly binds to mTOR and Raptor (a component of mTORC1), but not to Akt. This suggests BRAT1 specifically interacts with the mTORC1 complex rather than upstream regulators or mTORC2 components .

• Cell Cycle Regulation: Conditional deletion of BRAT1 in mouse embryonic fibroblasts suppresses serum-induced cell cycle progression, indicating that BRAT1's role in maintaining mTOR stability directly impacts cell proliferation .

• p70 S6 Kinase Effects: While BRAT1 deletion does not significantly alter baseline expression of p70 S6 kinase (a downstream target of mTOR), the protein level of S6K is markedly reduced after serum-starvation in BRAT1-deficient cells compared to controls .

The protein binding relationship can be visualized in this interaction matrix:

These findings suggest that BRAT1 functions as a critical stabilizer of the mTORC1 complex, making it essential for proper cell growth through the PI3K/Akt/mTOR signaling cascade .

What is the mechanistic relationship between BRAT1, INTS11, and INTS9 in neuronal gene activation?

The mechanistic relationship between BRAT1, INTS11, and INTS9 in neuronal gene activation represents a critical pathway for proper neuronal differentiation:

• Distinct Trimeric Complex Formation: BRAT1 interacts with INTS11 and INTS9 subunits of the Integrator complex to form a distinct trimeric complex separate from the core Integrator complex. This specialized complex is specifically involved in activating critical neuronal genes during differentiation .

• Promoter Co-occupancy: BRAT1 and INTS11 co-occupy the promoter regions of REST-regulated neuronal genes. BRAT1 plays a crucial role in recruiting INTS11 to these promoters, suggesting a sequential assembly mechanism .

• Regulation of REST Occupancy: During neuronal differentiation, BRAT1 is required for the removal of the transcriptional repressor REST from neuronal gene promoters. In BRAT1-depleted cells, REST persists at these regulatory regions, preventing the activation of key neuronal genes .

• Mutation Impact: Disease-causing mutations in BRAT1 diminish its association with INTS11/INTS9, providing a mechanistic link between these mutations and neurodegeneration phenotypes. This suggests that the integrity of the BRAT1-INTS11-INTS9 complex is essential for proper neuronal development .

• Differentiation Defects: Loss of BRAT1 in mouse embryonic stem cells leads to defects in neuronal differentiation assays, confirming the functional significance of this complex in vivo .

The relationship between these proteins appears to be sequential and hierarchical: BRAT1 is required to recruit INTS11/INTS9 to specific promoters, which then facilitates the removal of REST and subsequent activation of neuronal genes, ultimately permitting proper neuronal differentiation .

How do disease-causing mutations in BRAT1 alter its molecular interactions and functions?

Disease-causing mutations in BRAT1 significantly disrupt its molecular interactions and cellular functions, providing insight into the pathogenic mechanisms of BRAT1-associated neurodegenerative disorders:

• Disrupted Interaction with INTS11/INTS9: Disease-causing mutations in BRAT1 diminish its association with INTS11 and INTS9 subunits of the Integrator complex. This destabilization affects the 3'-end processing functions of this complex and its ability to activate neuronal genes .

• Impaired Promoter Recruitment: The mutations likely impair BRAT1's ability to recruit INTS11 to the promoters of REST-responsive genes, preventing the displacement of REST from these regulatory regions .

• Persistent REST Occupancy: With compromised BRAT1 function, REST continues to occupy and repress critical neuronal genes, disrupting the gene expression program required for proper neuronal differentiation .

• Neuronal Differentiation Defects: The functional consequence of these molecular disruptions is a significant defect in neuronal differentiation, which explains the neurodegenerative phenotypes observed in patients with BRAT1 mutations .

• Potential Disruption of DDR Functions: While not explicitly detailed in the provided search results, the mutations may also affect BRAT1's interactions with DDR proteins like BRCA1, ATM, and DNA-PK, potentially leading to genomic instability in developing neurons .

The connection between BRAT1 mutations and neurodegenerative diseases highlights the critical importance of proper BRAT1 function in neuronal development and suggests that therapeutic strategies targeting the REST pathway might be beneficial for patients with BRAT1 mutations .

What methodologies are most effective for studying BRAT1's role in DNA damage repair?

Studying BRAT1's role in DNA damage repair requires a comprehensive toolbox of methodologies to assess its multiple functions. The following approaches have proven effective:

• Genetic Manipulation Models:

  • Conditional knockout systems (such as BRAT1^flox/flox/129S6) allow for precise temporal control of BRAT1 deletion

  • RNA interference techniques can be used for transient knockdown studies to assess acute effects of BRAT1 reduction

  • CRISPR/Cas9-based gene editing enables the introduction of specific mutations to mimic disease states

• Homology-Directed Repair (HDR) Assays:

  • Reporter assays utilizing fluorescent proteins can measure HDR efficiency in control versus BRAT1-depleted cells

  • Combined inhibition of ATM in BRAT1-mutant backgrounds can reveal synthetic interactions, as demonstrated in BRCA1 BRCT mutant models

• Protein-Protein Interaction Analysis:

  • Co-immunoprecipitation using full-length and fragment constructs of BRAT1 has successfully identified interactions with ATM, DNA-PK, and mTOR/Raptor

  • Proximity ligation assays can detect endogenous protein interactions in situ

  • Domain mapping using truncated constructs helps identify specific interaction regions, as shown with the C-terminal fragments (#5 and #6) binding strongly to ATM and DNA-PK

• Phosphorylation Analysis:

  • Phospho-specific antibodies against ATM and DNA-PK substrates can assess the impact of BRAT1 depletion on DDR signaling

  • Western blotting with phospho-specific antibodies after DNA damage induction (e.g., with ionizing radiation or chemical agents)

• Cell Cycle and Apoptosis Assessment:

  • Flow cytometry analysis of cell cycle distribution in BRAT1-deficient versus control cells after serum starvation and release

  • Apoptosis assays (Annexin V/PI staining, caspase activation) to quantify the constitutive apoptosis reported in BRAT1 knockdown cells

These methodologies, when combined, provide a comprehensive assessment of BRAT1's multifaceted roles in DNA damage repair, cell cycle regulation, and apoptosis control.

How can researchers effectively assess BRAT1's impact on neuronal differentiation?

Researchers can implement several specialized methodologies to assess BRAT1's impact on neuronal differentiation:

• Neuronal Differentiation Assays:

  • Mouse embryonic stem cell differentiation protocols that direct cells toward neuronal lineages can demonstrate the functional requirement for BRAT1

  • NT2 cell differentiation into astrocytes and neuronal cells provides another model system where BRAT1's role has been established

  • Morphological assessment of neurite outgrowth and quantification of neuronal markers throughout differentiation

• REST Occupancy Analysis:

  • Chromatin immunoprecipitation (ChIP) assays to measure REST binding at key neuronal gene promoters in control versus BRAT1-depleted cells

  • Sequential ChIP (re-ChIP) to examine co-occupancy of BRAT1, INTS11, and REST at target promoters

  • ChIP-seq to identify genome-wide binding patterns and changes upon BRAT1 depletion or mutation

• Gene Expression Profiling:

  • RNA-seq analysis comparing wild-type and BRAT1-deficient cells during neuronal differentiation to identify dysregulated REST-responsive genes

  • RT-qPCR validation of key neuronal marker genes

  • Single-cell RNA-seq to capture heterogeneity in neuronal differentiation processes

• Protein Complex Analysis:

  • Co-immunoprecipitation of BRAT1 with INTS11 and INTS9 to confirm complex formation

  • Mass spectrometry to identify additional components of the complex

  • Gel filtration chromatography to determine complex size and stability

  • Introduction of disease-causing BRAT1 mutations to assess impact on complex formation

• In vivo Models:

  • Conditional knockout of BRAT1 in neural progenitors using Nestin-Cre or similar drivers

  • Assessment of neuroanatomical development and behavioral phenotypes

  • Rescue experiments with wild-type or mutant BRAT1 to establish causality

These approaches collectively provide a comprehensive assessment of how BRAT1 regulates neuronal differentiation through its interactions with the Integrator complex and control of REST occupancy.

What experimental approaches can reveal the relationship between BRAT1 and mTOR signaling?

To thoroughly investigate the relationship between BRAT1 and mTOR signaling, researchers should consider the following experimental approaches:

• Protein Stability and Expression Analysis:

  • Serum starvation and reintroduction experiments in control versus BRAT1-deficient cells to monitor mTOR and S6K protein levels over time

  • Cycloheximide chase assays to determine mTOR protein half-life in the presence or absence of BRAT1

  • Quantitative RT-PCR to distinguish between transcriptional and post-transcriptional effects on mTOR expression

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation with full-length and truncated BRAT1 constructs to map the specific domains interacting with mTOR and Raptor

  • Proximity ligation assays to visualize and quantify endogenous BRAT1-mTOR interactions in situ

  • In vitro binding assays with purified components to determine if interactions are direct

• mTOR Activity Assessment:

  • Phosphorylation analysis of mTOR targets (e.g., S6K, 4E-BP1) in control and BRAT1-deficient cells under various conditions

  • mTOR kinase assays using immunoprecipitated complexes from cells with or without BRAT1

  • Reporter assays for mTOR-dependent translation

• Cell Cycle and Proliferation Studies:

  • BrdU incorporation assays to measure S-phase entry in BRAT1-deficient cells after serum stimulation

  • Cell count and doubling time measurements under various nutrient conditions

  • Synchronization experiments to pinpoint specific cell cycle phases affected by BRAT1 deficiency

• Rescue Experiments:

  • Expression of constitutively active mTOR in BRAT1-deficient cells to determine if mTOR activation can rescue proliferation defects

  • Introduction of BRAT1 into knockout cells to confirm restoration of mTOR levels and signaling

  • Domain mapping through expression of specific BRAT1 fragments to identify minimal regions required for mTOR stabilization

The experimental approach can be summarized in this sequential workflow:

These approaches would provide comprehensive insights into how BRAT1 regulates mTOR signaling and subsequent cell cycle progression .

What are the implications of synthetic lethality between ATM loss and BRCA1 BRCT mutations for potential cancer therapies?

The synthetic lethality observed between ATM loss and BRCA1 BRCT mutations has significant implications for cancer therapy development:

• Mechanism of Synthetic Lethality: In mice carrying a breast cancer-derived mutation in the BRCA1 C-terminal (BRCT) domain (S1598F), ATM becomes essential for supporting residual levels of homology-directed repair (HDR) necessary to repair DNA double-strand breaks. Loss of ATM in this context leads to synthetic lethality .

• Independence from 53BP1 Status: Unlike the relationship between BRCA1 and 53BP1 (where 53BP1 deletion can rescue HDR defects in BRCA1-mutant cells), the ATM-mediated HDR is not affected by 53BP1 status. This suggests ATM has a role in HDR independent of the BRCA1-53BP1 antagonism .

• Therapeutic Targeting Potential: These findings suggest that ATM kinase inhibitors could potentially be used in combination with PARP inhibitor therapy for certain BRCA1-deficient tumors, particularly those with specific BRCT domain mutations .

• Precision Medicine Approach: The synthetic lethality relationship suggests a potential biomarker-guided approach where patients with specific BRCA1 BRCT mutations might particularly benefit from ATM inhibition strategies .

• Dual Targeting Strategy: The data imply that simultaneous inhibition of both HDR (through BRCA1 mutation) and ATM pathways could create a therapeutic vulnerability that cancer cells cannot overcome, leading to selective cancer cell death .

The potential therapeutic benefits can be summarized in this comparative table:

Understanding the relationship between BRAT1, ATM, and BRCA1 could further refine these therapeutic approaches, as BRAT1 is known to interact with both ATM and BRCA1 in DNA damage response pathways .

How might understanding BRAT1's role in neurodegeneration inform therapeutic strategies?

Understanding BRAT1's role in neurodegeneration provides several promising avenues for therapeutic intervention:

• REST Pathway Modulation: Since BRAT1 deficiency leads to persistent REST occupancy at neuronal gene promoters, therapies targeting REST repression may bypass the need for functional BRAT1. Small molecules or peptides that inhibit REST binding or promote its degradation could potentially restore neuronal gene expression in patients with BRAT1 mutations .

• INTS11/INTS9 Complex Enhancement: As BRAT1 mutations diminish its association with INTS11 and INTS9, strategies to stabilize this interaction or directly enhance INTS11/INTS9 recruitment to neuronal gene promoters could compensate for BRAT1 dysfunction .

• Neuronal Gene Expression Restoration: Gene therapy approaches delivering key neuronal genes that are normally repressed in BRAT1-deficient conditions could potentially bypass the transcriptional block caused by persistent REST occupation .

• Stem Cell Therapy: Since BRAT1 is essential for proper neuronal differentiation, transplantation of healthy neuronal progenitors or neurons derived from stem cells could potentially replace degenerated neurons in affected patients .

• BRAT1 Stabilization or Delivery: For mutations that affect BRAT1 stability rather than function, small molecules that stabilize the mutant protein might preserve residual activity. Alternatively, viral vector-mediated delivery of functional BRAT1 to affected tissues could restore normal neuronal development and function .

The table below summarizes potential therapeutic approaches based on different aspects of BRAT1 dysfunction:

These approaches represent promising directions for addressing BRAT1-associated neurodegenerative diseases, though each would require extensive preclinical development and validation .

What are the optimal conditions for expressing and purifying recombinant mouse BRAT1?

While the search results don't provide specific details for BRAT1 expression and purification, we can derive optimal conditions based on comparable recombinant protein production systems:

• Expression Systems:

  • Mammalian expression systems (HEK293, CHO cells) would be preferred for BRAT1 due to its multiple post-translational modifications and interactions with other mammalian proteins

  • Baculovirus-insect cell expression systems represent an alternative that balances yield with proper folding

  • Bacterial expression may be suitable for specific domains but likely problematic for full-length BRAT1 due to its size and complexity

• Purification Tags and Strategies:

  • Addition of a C-terminal tag is recommended to avoid interference with N-terminal protein interactions

  • His6 or GST tags facilitate purification while allowing for tag removal through precision protease cleavage sites

  • The GST tag has been successfully used for BRAT1 fragment pulldown experiments

  • Multi-step purification likely necessary: affinity chromatography followed by ion exchange and size exclusion

• Buffer Conditions:

  • Based on comparable proteins, PBS-based buffers with stabilizing agents would be appropriate

  • Carrier-free formulations (without BSA) should be considered for applications where BSA might interfere

  • Lyophilization from a 0.2 μm filtered solution in PBS can enhance stability for long-term storage

• Storage and Stability:

  • Frozen storage at -80°C in single-use aliquots to avoid freeze-thaw cycles

  • Addition of glycerol (10-15%) to prevent damage during freezing

  • Reconstitution at approximately 100 μg/mL in sterile PBS for immediate use

• Quality Control Assessments:

  • SDS-PAGE and Western blotting to confirm size and immunoreactivity

  • Mass spectrometry to verify identity and assess post-translational modifications

  • Functional assays to confirm BRAT1 activity, such as binding to known partners (BRCA1, ATM, mTOR)

Recombinant BRAT1 production would benefit from approaches similar to those used for other complex proteins, with careful attention to preserving functional interactions with its numerous binding partners .

What controls and validation steps are essential when studying BRAT1 in experimental systems?

When investigating BRAT1 function in experimental systems, several critical controls and validation steps must be implemented to ensure reliable and reproducible results:

• Genetic Manipulation Validation:

  • Confirmation of BRAT1 knockdown/knockout efficiency by both RT-qPCR and Western blot analysis

  • Rescue experiments with wild-type BRAT1 to confirm phenotype specificity

  • Use of multiple independent shRNA/siRNA sequences or CRISPR guide RNAs to rule out off-target effects

• Protein Interaction Controls:

  • Inclusion of appropriate negative controls in co-immunoprecipitation experiments (e.g., IgG controls, irrelevant proteins)

  • Reciprocal co-immunoprecipitation to confirm interactions

  • Domain mapping using truncated BRAT1 constructs, as demonstrated in the BRAT1-mTOR interaction studies

  • Competition assays with excess untagged protein to confirm binding specificity

• Functional Assay Validation:

  • Positive and negative controls for DNA damage response assays (e.g., ATM inhibitors as positive controls)

  • Time-course experiments to capture dynamic processes like serum-induced mTOR regulation

  • Dose-response relationships to establish specificity

  • Multiple complementary assays measuring the same biological outcome

• Cell Model Considerations:

  • Use of multiple cell lines to ensure findings are not cell-type specific

  • Careful selection of control cell lines (empty vector controls, wild-type isogenic lines)

  • Consideration of cell passage number and culture conditions

  • Authentication of cell lines through STR profiling

• In Vivo Model Validation:

  • Thorough characterization of conditional knockout efficiency in target tissues

  • Use of appropriate Cre driver lines for tissue-specific studies

  • Littermate controls to minimize genetic background effects

  • Phenotypic rescue through transgenic expression of wild-type BRAT1

These validation steps ensure that experimental observations truly reflect BRAT1 biology rather than technical artifacts or off-target effects, providing a solid foundation for advancing our understanding of this multifunctional protein .

What are the most promising unresolved questions about BRAT1 function?

Several critical unresolved questions about BRAT1 function represent promising areas for future research:

• Integration of Multiple BRAT1 Functions:

  • How are BRAT1's roles in DNA damage response, neuronal differentiation, and mTOR signaling interconnected?

  • Does BRAT1 serve as a central coordinator of these pathways, or does it have independent functions in each context?

  • What determines the pathway specificity of BRAT1 in different cellular contexts?

• Structure-Function Relationships:

  • What is the three-dimensional structure of BRAT1, and how does it facilitate interactions with multiple partners?

  • Which specific domains mediate interactions with BRCA1, ATM, mTOR, and the INTS11/INTS9 complex?

  • How do disease-causing mutations alter BRAT1's structure and subsequent functions?

• Regulation of BRAT1 Activity:

  • How is BRAT1 itself regulated at the transcriptional, post-transcriptional, and post-translational levels?

  • Does BRAT1 undergo modifications (phosphorylation, ubiquitination) that alter its activity or localization?

  • What signaling pathways control BRAT1 function in different cellular contexts?

• Developmental Roles:

  • Beyond neuronal differentiation, what are BRAT1's roles in other developmental processes?

  • How does embryonic expression of BRAT1 contribute to normal development and organogenesis?

  • What are the tissue-specific requirements for BRAT1 function?

• Therapeutic Targeting Potential:

  • Can BRAT1 itself be directly targeted for therapeutic benefit in cancer or neurodegenerative disease?

  • Are there synthetic lethal interactions involving BRAT1 that could be exploited for cancer therapy?

  • How might restoration of BRAT1 function ameliorate neurodevelopmental disorders?

These unresolved questions highlight the need for continued investigation into this multifunctional protein, with potential implications for both basic science understanding and therapeutic development .

How might emerging technologies advance our understanding of BRAT1 biology?

Emerging technologies offer powerful new approaches to address the complex biology of BRAT1:

• Cryo-Electron Microscopy and Structural Biology:

  • Determination of the BRAT1 protein structure alone and in complex with its binding partners

  • Visualization of conformational changes upon binding to different partners

  • Structure-guided drug design targeting BRAT1 or its interaction interfaces

• CRISPR-Based Technologies:

  • Base editing and prime editing for introducing precise disease-associated mutations

  • CRISPR activation/inhibition systems to modulate BRAT1 expression without genetic deletion

  • CRISPR screens to identify synthetic lethal interactions with BRAT1 deficiency

  • In vivo CRISPR editing in specific neuronal populations

• Advanced Imaging Technologies:

  • Super-resolution microscopy to visualize BRAT1 localization and dynamics at high precision

  • Live-cell imaging of fluorescently tagged BRAT1 to track its movement during DNA damage or neuronal differentiation

  • Intravital imaging to monitor BRAT1 function in developing tissues

• Single-Cell Multi-Omics:

  • Single-cell RNA-seq to capture heterogeneity in cellular responses to BRAT1 deficiency

  • Single-cell ATAC-seq to map chromatin accessibility changes at REST-regulated genes

  • Integrated multi-omics to correlate transcriptional, epigenetic, and proteomic changes

• Organoid and iPSC Technologies:

  • Brain organoids from patient-derived iPSCs with BRAT1 mutations

  • CRISPR-corrected isogenic controls for precise attribution of phenotypes

  • High-throughput drug screening in disease-relevant organoid systems

• Protein Interaction Mapping Technologies:

  • BioID or APEX proximity labeling to map the complete BRAT1 interactome

  • Hydrogen-deuterium exchange mass spectrometry to map binding interfaces

  • Cross-linking mass spectrometry to capture transient protein interactions