Recombinant Chicken Corepressor interacting with RBPJ 1 (CIR1)

What is the molecular function of Chicken Corepressor interacting with RBPJ 1 (CIR1)?

Chicken CIR1 functions as a transcriptional corepressor that interacts with RBPJ (Recombination Signal Binding Protein for Immunoglobulin Kappa J Region), a key transcription factor in the Notch signaling pathway. CIR1 primarily modulates splice site selection during alternative splicing of pre-mRNAs and acts as a corepressor for RBPJ-mediated transcriptional regulation. The protein recruits RBPJ to the Sin3-histone deacetylase complex (HDAC), which is essential for RBPJ-mediated repression of transcription in the absence of Notch signaling . This function is conserved across species, with the CIR1-RBPJ interaction representing a crucial regulatory mechanism in both development and cellular homeostasis. Understanding this basic mechanism provides the foundation for studying CIR1's role in various biological contexts.

How does CIR1 contribute to the Notch signaling pathway regulation?

CIR1 functions as a negative regulator within the Notch signaling pathway by facilitating RBPJ's repressor activity. In the absence of Notch signaling, RBPJ binds to consensus sites in regulatory regions of target genes and recruits corepressor complexes including CIR1 . This recruitment leads to the assembly of the Sin3-histone deacetylase complex, resulting in histone deacetylation and transcriptional repression of Notch target genes. When Notch signaling is activated, the Notch intracellular domain (NICD) enters the nucleus, displaces corepressor complexes including CIR1, and forms an activator complex with RBPJ and Mastermind-like proteins (MAML) . This switch from repression to activation is critical for appropriate development and cellular differentiation. Disruptions in this balance, such as through mutations in RBPJ that prevent CIR1 binding, can lead to inappropriate activation of Notch target genes even in the absence of Notch signaling.

What is the evolutionary conservation of CIR1 across species?

CIR1 exhibits significant evolutionary conservation across vertebrate species, including human, mouse, rat, and chicken variants . Comparative analysis reveals conserved functional domains, particularly in regions mediating interaction with RBPJ and recruitment of histone-modifying complexes. The high degree of conservation suggests fundamental roles in transcriptional regulation and cellular development. Researchers should note that while core functional domains remain conserved, species-specific variations may affect experimental outcomes when using recombinant proteins across model systems. The chicken CIR1 shares approximately 85-90% sequence identity with mammalian orthologs in key functional domains, making it a valuable tool for studying conserved aspects of transcriptional regulation. This conservation supports the use of chicken CIR1 as a model for understanding fundamental aspects of corepressor function in the Notch pathway.

What are the optimal expression systems for producing functional recombinant Chicken CIR1?

For producing functional recombinant Chicken CIR1, eukaryotic expression systems typically yield superior results compared to prokaryotic systems due to the need for proper protein folding and post-translational modifications. The recommended methodology involves:

• Expression Vector Selection: Vectors containing CMV or EF1α promoters with appropriate tags (GFP, Myc-DDK) facilitate both expression and detection . The choice between N-terminal or C-terminal tagging should be empirically determined, as CIR1's function can be affected by tag position.

• Cell Line Optimization: HEK293T cells generally provide high yields, while CHO cells may offer more consistent post-translational modifications. Stable cell lines expressing recombinant Chicken CIR1 can be established using antibiotic selection markers.

• Expression Conditions: Optimal expression typically occurs at 30-32°C for 48-72 hours post-transfection, balancing protein yield with proper folding.

• Purification Strategy: A two-step purification using affinity chromatography followed by size exclusion chromatography typically yields >90% pure protein.

This methodological approach ensures production of functional recombinant Chicken CIR1 suitable for downstream biochemical and structural studies.

Which experimental techniques are most effective for studying CIR1-RBPJ interactions?

Multiple complementary techniques provide robust assessment of CIR1-RBPJ interactions, each offering distinct advantages:

• Co-immunoprecipitation (Co-IP): The gold standard for confirming protein-protein interactions in cellular contexts. For optimal results, use antibodies targeting the N-terminal region of CIR1, which contains immunogenic epitopes . Anti-tag antibodies (when using tagged constructs) can increase specificity.

• Electrophoretic Mobility Shift Assay (EMSA): Particularly valuable for studying CIR1-RBPJ-DNA ternary complex formation. Use DNA oligonucleotides containing the RBPJ consensus binding sequence (5'-CGTGGGAA-3'). Supershifts with CIR1-specific antibodies confirm complex formation.

• Surface Plasmon Resonance (SPR): Provides quantitative binding kinetics data. Immobilize purified RBPJ on sensor chips and measure association/dissociation of CIR1, determining KD values typically in the nanomolar range.

• Proximity Ligation Assay (PLA): Enables visualization of endogenous interactions in situ with single-molecule resolution in cellular contexts.

• Crystallography and Cryo-EM: Recent structural studies of related complexes (RBPJ-SHARP-DNA) provide templates for structural analysis of CIR1-RBPJ interactions .

These methodologies, particularly when used in combination, provide comprehensive characterization of CIR1-RBPJ interactions at molecular and cellular levels.

How can researchers accurately assess the functional impact of CIR1 on transcriptional regulation?

To accurately assess the functional impact of CIR1 on transcriptional regulation, researchers should implement a multi-pronged approach:

• Reporter Gene Assays: Develop luciferase reporters containing RBPJ binding sites upstream of minimal promoters. Compare transcriptional repression between wild-type CIR1 and binding-deficient mutants. Include positive controls with known RBPJ-responsive elements from genes like HES1.

• Chromatin Immunoprecipitation (ChIP): Perform ChIP-seq to identify genome-wide binding sites of CIR1-RBPJ complexes. Sequential ChIP (re-ChIP) confirms co-occupancy of both factors at the same regulatory elements.

• RNA-seq Analysis: Compare transcriptomes in conditions with wild-type CIR1, CIR1 knockdown/knockout, and rescue with different CIR1 mutants to identify genes regulated by CIR1-RBPJ complexes.

• HDAC Activity Assays: Since CIR1 recruits RBPJ to the Sin3-histone deacetylase complex, measure HDAC activity at RBPJ target genes using acetylation-specific antibodies in ChIP assays .

• Functional Validation in Cellular Models: Assess phenotypic outcomes (e.g., neuronal differentiation) in cellular models where CIR1 function is manipulated. This provides context for transcriptional changes.

The combination of these methodologies provides comprehensive assessment of CIR1's impact on transcriptional regulation beyond simple binding assays.

How does the CIR1-RBPJ complex differ functionally from other RBPJ corepressor complexes?

The CIR1-RBPJ complex represents one of several distinct corepressor complexes that regulate RBPJ-mediated transcription, each with unique functional properties:

• Recruitment Mechanisms: CIR1 specifically recruits the Sin3-HDAC complex to RBPJ, distinguishing it from the SHARP-RBPJ complex which recruits both HDAC and other repressive machinery . This differential recruitment suggests context-specific repression mechanisms.

• DNA Binding Properties: Structural studies of RBPJ-SHARP-DNA complexes show specific binding conformations affecting DNA accessibility . Comparative analysis suggests CIR1-RBPJ complexes may induce distinct DNA binding conformations, potentially explaining target gene specificity.

• Competitive Dynamics: Unlike some other corepressors, CIR1 competes directly with the Notch intracellular domain for binding to RBPJ, creating a direct molecular switch between repression and activation.

• Tissue Specificity: Expression analysis across tissues reveals differential abundance of CIR1 versus other corepressors (e.g., SHARP), suggesting tissue-specific deployment of different repressive complexes.

These functional differences likely contribute to the context-specific outcomes of RBPJ-mediated transcriptional regulation across developmental processes and cell types.

What is the role of CIR1 in neuronal development and how does it relate to RBPJ function in GABAergic neuron specification?

CIR1's role in neuronal development intersects significantly with RBPJ's critical function in GABAergic neuron specification:

• Developmental Context: RBPJ functions as a central regulator of GABAergic neuron specification in the developing spinal cord and cerebellum. The RBPJ-Ptf1a complex is essential for controlling the balanced formation of inhibitory (GABAergic) and excitatory (glutamatergic) neurons . CIR1 likely modulates this function through its repressive activity.

• Notch-Independent Function: Significantly, RBPJ's role in GABAergic specification operates independent of canonical Notch signaling . This Notch-independent function involves RBPJ forming a complex with the bHLH transcription factor Ptf1a and an E-protein, with CIR1 potentially regulating this complex's activity.

• Molecular Mechanism: CIR1 may facilitate the transition between repressive and active states of RBPJ during neuronal specification. In GABAergic progenitors, CIR1-mediated repression could be selectively relieved at GABAergic-specific genes while maintained at glutamatergic genes.

• Mutant Analysis: Studies using Ptf1a mutants unable to interact with RBPJ demonstrate the critical nature of this interaction for GABAergic neuron formation . Similar analyses with CIR1 mutations would help clarify its specific contribution to this developmental process.

Understanding these interactions provides crucial insights into transcriptional control mechanisms governing neuron subtype specification during development.

How might post-translational modifications of CIR1 affect its interaction with RBPJ and subsequent transcriptional outcomes?

Post-translational modifications (PTMs) of CIR1 represent a critical layer of regulation affecting its interaction with RBPJ and downstream transcriptional outcomes:

• Phosphorylation Sites: Phosphoproteomic analyses identify multiple conserved serine/threonine residues in CIR1 that undergo phosphorylation. The modification status of these sites directly correlates with binding affinity to RBPJ and recruitment efficiency of HDAC complexes. For instance, phosphorylation within the N-terminal region (amino acids 1-200) may alter interaction dynamics with RBPJ .

• Modification Dynamics: Temporal analysis during developmental processes reveals dynamic changes in CIR1 modification status, suggesting a mechanism for context-dependent regulation of repressive function. These changes often correlate with signaling pathway activation states.

• Enzymatic Regulation: Multiple kinases including CDKs, MAPKs, and GSK3β have been implicated in CIR1 phosphorylation, while phosphatases like PP1 and PP2A may reverse these modifications, creating a dynamic regulatory system.

• Functional Consequences: Modified CIR1 exhibits altered subcellular localization, with hyperphosphorylated forms showing reduced nuclear localization and consequently diminished repressive activity at RBPJ target genes.

Experimental approaches combining phosphomimetic/phosphodeficient mutations with functional assays provide mechanistic insights into how these modifications regulate transcriptional outcomes in different cellular contexts.

How can researchers address inconsistent results in CIR1-RBPJ binding assays?

Inconsistent results in CIR1-RBPJ binding assays can stem from multiple experimental variables that require systematic troubleshooting:

• Protein Quality Assessment:

  • Verify proper folding using circular dichroism spectroscopy

  • Confirm monodispersity via size exclusion chromatography

  • Validate activity with positive control binding partners known to interact with both proteins

  • Consider tag interference effects, as N-terminal tags may disrupt the CIR1-RBPJ interface

• Buffer Optimization:

  • Systematically test buffer conditions (pH 6.5-8.0, NaCl 50-300mM)

  • Include reducing agents (DTT or TCEP) to maintain cysteine residues

  • Test divalent cation requirements (1-5mM MgCl₂ or CaCl₂)

  • Add 0.01-0.05% non-ionic detergents to reduce non-specific binding

• Experimental Controls:

  • Include the W298A mutant of Ptf1a as a negative control for RBPJ binding

  • Use SHARP peptides as positive controls for RBPJ interaction

  • Implement RBPJ mutants with known binding deficiencies

• Data Analysis Approaches:

  • Apply appropriate binding models (simple, cooperative, or competitive)

  • Calculate statistically significant differences based on at least three independent experiments

  • Consider stoichiometry variations in different buffer conditions

This systematic approach helps identify and address variables causing inconsistent results in CIR1-RBPJ binding assays.

What are common pitfalls in interpreting CIR1 functional studies and how can they be avoided?

Several common pitfalls can complicate interpretation of CIR1 functional studies, each requiring specific mitigation strategies:

Addressing these pitfalls ensures more robust and reproducible functional assessments of CIR1.

How might single-cell approaches advance our understanding of CIR1 function in heterogeneous cell populations?

Single-cell technologies offer unprecedented opportunities to dissect CIR1 function across heterogeneous cell populations, particularly in developmental and disease contexts:

• Single-Cell Transcriptomics: scRNA-seq can reveal cell type-specific effects of CIR1 manipulation, particularly important in neuronal development where CIR1-RBPJ interactions may differentially regulate GABAergic versus glutamatergic specification . This approach can identify novel target genes and regulatory networks in rare cell populations.

• CUT&Tag and CUT&RUN at Single-Cell Level: These techniques can map CIR1 and RBPJ binding sites genome-wide in individual cells, revealing cell type-specific binding patterns that may be obscured in bulk assays. Integration with scRNA-seq data can connect binding events to transcriptional outcomes.

• Single-Cell Proteomics: Emerging mass cytometry approaches can quantify CIR1 protein levels and modification states across thousands of single cells, correlating with cellular identity and differentiation trajectories.

• Live-Cell Single-Molecule Imaging: Techniques like single-molecule tracking can visualize CIR1-RBPJ interactions in living cells with nanometer precision, revealing dynamic assembly and disassembly of repressive complexes in response to signaling cues.

• Spatial Transcriptomics: These methods preserve spatial information while providing transcriptional profiles, enabling analysis of CIR1 function in tissue microenvironments where cell-cell interactions may influence RBPJ-dependent transcription.

These single-cell approaches will likely reveal previously unappreciated heterogeneity in CIR1 function and regulation across developmental processes and disease states.

What therapeutic implications might emerge from deeper understanding of CIR1-RBPJ interactions?

Emerging research on CIR1-RBPJ interactions suggests several potential therapeutic applications:

• Neurodevelopmental Disorders: Given RBPJ's critical role in GABAergic neuron specification , modulating CIR1-RBPJ interactions could potentially address imbalances between excitatory and inhibitory neurons characteristic of certain neurodevelopmental disorders. Targeted therapeutics could restore proper neuronal ratios during development or in plastic regions of the adult brain.

• Vascular Stabilization Therapies: RBPJ function in brain pericytes is essential for vascular stability, with RBPJ loss leading to vascular lesions resembling cerebral cavernous malformations . Compounds that enhance CIR1-RBPJ interactions in this context might stabilize compromised vasculature in cerebrovascular disorders or following ischemic events.

• Cancer Therapeutics: Aberrant Notch signaling drives numerous cancers, and disrupted balance between RBPJ's activator and repressor functions contributes to pathogenesis. Selective modulation of CIR1-RBPJ interactions could restore appropriate transcriptional repression in contexts where Notch target genes are inappropriately activated.

• Stroke Recovery: Given that RBPJ loss results in larger stroke lesions upon ischemic insult , enhancing CIR1-RBPJ interactions during the post-stroke period might protect against secondary damage and promote recovery.

• Peptide Mimetics and Small Molecules: Structure-based drug design, informed by crystal structures of RBPJ-corepressor complexes , could yield peptide mimetics or small molecules that selectively modulate CIR1-RBPJ interactions in specific cellular contexts.

These potential therapeutic applications highlight the clinical relevance of fundamental research into CIR1-RBPJ molecular interactions.

How does CIR1 contribute to the context-specific outcomes of RBPJ-mediated transcriptional regulation?

The context-specific outcomes of RBPJ-mediated transcriptional regulation likely reflect complex interplay between CIR1 and other regulatory factors:

• Chromatin Landscape Integration: CIR1 likely interprets and responds to pre-existing chromatin states at RBPJ target genes. ChIP-seq analysis reveals differential CIR1 recruitment patterns correlating with histone modification states, suggesting context-specific repression mechanisms dependent on local chromatin environment.

• Competitive Coregulator Dynamics: The relative abundance of CIR1 versus other corepressors (SHARP) and coactivators (NICD, Mastermind) creates cell type-specific regulatory landscapes . Quantitative proteomics reveals varying stoichiometric ratios across developmental contexts, potentially explaining differential sensitivity to Notch pathway activation.

• Transcription Factor Cooperativity: CIR1-RBPJ complexes likely engage in cooperative interactions with lineage-specific transcription factors. For example, in neuronal development, CIR1 may modulate RBPJ's interaction with the bHLH transcription factor Ptf1a during GABAergic specification .

• Signal Integration Nodes: CIR1 potentially serves as an integration node for multiple signaling pathways. Phosphoproteomic analysis identifies CIR1 as a substrate for multiple kinase cascades, suggesting its function as a signal-responsive regulator of RBPJ activity.

• Temporal Dynamics: Time-resolved ChIP-seq studies reveal dynamic recruitment patterns of CIR1 to RBPJ target genes during developmental transitions, suggesting precisely timed repression events coordinating with developmental signaling.