Recombinant Mouse CDKN2A-interacting protein (Cdkn2aip)

What is CDKN2A-interacting protein (Cdkn2aip) and what cellular functions does it perform?

CDKN2AIP is a member of the RNA-binding protein family with critical roles in cellular processes including DNA damage response, cell cycle regulation, and cellular differentiation. Originally identified as an ARF-binding protein in the p53 pathway, CDKN2AIP has been shown to interact directly with p53 and facilitate its activation independent of ARF . As a multifunctional protein, CDKN2AIP is highly expressed in the testis but is also present in multiple other tissues .

The protein is involved in:

• Stem cell pluripotency and somatic differentiation pathways

• Spermatogonial self-renewal through Wnt-signaling pathway activation

• DNA damage response mechanisms via the ATR/CHK1 pathway

• Regulation of cell proliferation through p53-HDM2-p21 pathway interaction

Methodologically, when studying CDKN2AIP's cellular functions, researchers should employ a combination of co-immunoprecipitation, mass spectrometry analysis, and cellular localization studies to comprehensively characterize its interactome and functional domains.

How should I design knockout models to study Cdkn2aip function?

Based on published methodologies, effective CDKN2AIP knockout models can be generated using CRISPR/Cas9 genome editing. The following approach has been successfully employed:

• Design sgRNAs targeting the Cdkn2aip locus (typically creating a large deletion)

• Co-inject sgRNA and Cas9 mRNA into fertilized eggs of C57BL/6 mice

• Genotype founders by PCR followed by DNA sequencing analysis

• Establish breeding colonies through intercrossing of heterozygous mice

Mouse models with a 3687-base deletion have been generated and successfully validated. Importantly, intercrossing of Cdkn2aip heterozygous mice yielded healthy offspring at Mendelian ratios, indicating no embryonic lethality .

For phenotypic analysis, researchers should prepare for:

• Histological examination of tissues (particularly testes and epididymides)

• Fertility assessments in both heterozygous and homozygous animals

• Age-dependent phenotypic progression analysis

• Molecular characterization of cell cycle parameters

What are the established protein interactions of Cdkn2aip?

CDKN2AIP has several well-characterized protein-protein interactions that contribute to its cellular functions:

• p53 interaction: CDKN2AIP can directly bind to p53 tumor suppressor protein, facilitating its activation and contributing to cell cycle arrest and apoptosis pathways .

• ARF binding: As suggested by its name (CDKN2A-interacting protein), it interacts with the ARF protein (p14ARF in humans, p19ARF in mice) encoded by the CDKN2A locus . CDKN2A generates several transcript variants including ARF, which functions as a stabilizer of p53 by sequestering MDM2 .

• Cell cycle regulatory proteins: Through its association with the p53 pathway, CDKN2AIP indirectly affects interactions with CDK4 and CDK6, which normally form complexes with D-type G1 cyclins to phosphorylate pRb and control cell cycle progression .

For studying these interactions, co-immunoprecipitation followed by mass spectrometry (IP-MS) has proven to be an effective methodology, using testis tissue from 8-week-old male mice .

How does Cdkn2aip contribute to spermatogenesis and male fertility?

CDKN2AIP plays multiple critical roles in spermatogenesis and male fertility as demonstrated by recent research:

• Expression pattern: CDKN2AIP is expressed in spermatocytes and spermatids, suggesting important functions throughout multiple stages of sperm development .

• Spermiogenesis involvement: The protein participates directly in spermiogenesis, with knockout models showing multiple sperm head defects .

• Age-dependent germ cell loss: Cdkn2aip-/- mice exhibit progressive loss of germ cells with age, potentially resulting from:

  • Protamine replacement failure

  • Impaired SUN1 expression (a nuclear envelope protein essential for proper chromosome dynamics)

• Meiotic functions: Loss of Cdkn2aip expression results in:

  • Synapsis failure in 19% of spermatocytes

  • Increased apoptosis due to damaged DNA double-strand break repair

  • Impaired crossover formation during meiosis

• Spermatogonial dynamics: CDKN2AIP is essential for spermatogonial self-renewal and proliferation, potentially through its role in activating the Wnt-signaling pathway .

Methodologically, researchers should employ histological analysis using Bouin's fixative solution and H&E staining of 5μm testis and epididymis sections to characterize spermatogenic defects in mouse models .

What are the consequences of Cdkn2aip knockdown in cellular systems?

Knockdown of CDKN2AIP in cellular systems leads to multiple significant cellular abnormalities:

• Cell cycle disruption: In vitro studies demonstrate that CDKN2AIP knockdown results in:

  • Extended S phase

  • Cell cycle checkpoint activation

  • Mitotic catastrophe

• Genomic instability: Depletion of CDKN2AIP leads to:

  • Increased DNA damage

  • Aneuploidy

  • Chromosomal aberrations

• Cellular fate changes: Knockdown cells show:

  • Increased apoptosis via the ATR/CHK1 pathway

  • Loss of normal differentiation capacity

  • Altered cellular morphology

• Molecular pathway disruption: Reduced CDKN2AIP affects:

  • p53 activation pathways

  • DNA damage response signaling

  • Cell cycle checkpoint mechanisms

When designing knockdown experiments, researchers should employ multiple siRNA sequences or shRNA constructs to minimize off-target effects, and should include rescue experiments with recombinant protein to confirm specificity of observed phenotypes.

What signaling pathways does Cdkn2aip participate in?

CDKN2AIP participates in several signaling pathways that regulate critical cellular processes:

• p53-HDM2-p21 pathway: Overexpression of CDKN2AIP impairs cell proliferation and results in senescence through activation of this pathway .

• ATR/CHK1 pathway: CDKN2AIP depletion triggers DNA damage responses through this pathway, ultimately leading to apoptosis .

• Wnt-signaling pathway: CDKN2AIP appears to be essential for spermatogonial self-renewal and proliferation through activation of Wnt signaling .

• CDKN2A/ARF pathway: As a binding partner of ARF, CDKN2AIP participates in the CDKN2A tumor suppressor network. The CDKN2A locus generates multiple transcript variants including those encoding inhibitors of CDK4 kinase and ARF, which functions as a stabilizer of p53 by sequestering MDM2 .

• Cell cycle control pathways: Through its connections to p53 and the CDKN2A/ARF network, CDKN2AIP influences the CDK4/6-cyclin D-pRb axis that regulates G1 progression .

For interrogating these pathways, researchers should utilize pathway reporter assays, phosphorylation-specific antibodies, and genetic epistasis experiments to determine the precise positioning of CDKN2AIP within these signaling cascades.

How does the interaction between Cdkn2aip and p53 affect cellular responses to DNA damage?

The CDKN2AIP-p53 interaction plays a sophisticated role in DNA damage response:

• Direct p53 activation: CDKN2AIP can interact with p53 directly and facilitate its activation independent of ARF, providing an alternative pathway for p53 stabilization following DNA damage .

• Checkpoint regulation: Through p53 activation, CDKN2AIP contributes to G1 and G2/M checkpoint activation after DNA damage, allowing time for repair mechanisms to function.

• Apoptotic threshold modulation: CDKN2AIP appears to modulate the threshold for p53-dependent apoptosis, as knockdown of CDKN2AIP results in increased apoptosis via the ATR/CHK1 pathway .

• Genomic stability maintenance: CDKN2AIP knockdown leads to aneuploidy and DNA damage, suggesting a role in maintaining genomic stability through p53-dependent and potentially p53-independent mechanisms .

• DSB repair influence: The increased DNA double-strand break repair defects observed in Cdkn2aip-/- mice suggest a direct role in DNA repair processes, potentially through p53-regulated repair mechanisms .

Research methodologies should include:

• Comet assays to assess DNA damage levels

• γH2AX foci quantification as markers of DNA double-strand breaks

• Chromatin immunoprecipitation to identify p53 binding at target genes

• Cell cycle analysis following various DNA damaging agents in the presence/absence of CDKN2AIP

What is the molecular basis for Cdkn2aip's role in meiotic processes and chromosome dynamics?

The molecular mechanisms underlying CDKN2AIP's functions in meiosis include:

• Synapsis regulation: Loss of Cdkn2aip results in synapsis failure in 19% of spermatocytes, suggesting a role in homologous chromosome pairing and synapsis during meiotic prophase I .

• DSB repair facilitation: CDKN2AIP appears to be involved in DNA double-strand break repair during meiotic recombination, as its absence leads to damaged DSB repair .

• Crossover formation: Impaired crossover formation in Cdkn2aip-/- mice indicates a role in regulating meiotic recombination events critical for proper chromosome segregation .

• Nuclear envelope protein regulation: The impaired SUN1 expression observed in knockout mice suggests CDKN2AIP may regulate components of the nuclear envelope essential for chromosome movement and dynamics during meiosis .

• Chromatin structure maintenance: The protamine replacement failures in Cdkn2aip-/- mice point to a potential role in chromatin reorganization during spermiogenesis .

Advanced experimental approaches should include:

• Immunofluorescence analysis of meiotic chromosome spreads

• Analysis of meiotic recombination markers (MLH1, DMC1)

• Super-resolution microscopy to visualize chromosome dynamics

• Chromatin immunoprecipitation sequencing (ChIP-seq) to identify CDKN2AIP binding sites

• RNA-binding protein immunoprecipitation (RIP) to identify RNA targets

What are the structural and functional relationships between CDKN2A and Cdkn2aip?

The complex structural and functional relationships between CDKN2A and CDKN2AIP involve:

• Alternative reading frames: The CDKN2A locus generates several transcript variants through alternative splicing:

  • p16INK4a, which functions as a CDK4/CDK6 inhibitor

  • ARF (p14ARF in humans, p19ARF in mice), which interacts with CDKN2AIP

• Functional pathways:

  • p16INK4a inhibits CDK4 and CDK6, preventing their interaction with cyclins D and phosphorylation of retinoblastoma protein

  • ARF stabilizes p53 by sequestering MDM2 (an E3 ubiquitin-protein ligase responsible for p53 degradation)

  • CDKN2AIP binds to ARF and can also interact directly with p53

• Tumor suppression mechanisms:

  • CDKN2A is frequently mutated or deleted in various tumors and is a known tumor suppressor

  • CDKN2AIP expression in testis appears to be associated with testicular tumor suppression

  • Both proteins participate in p53 pathway regulation, contributing to tumor suppression

• Genomic structure:

  • In mice, Cdkn2a is located on chromosome 4 between Ifna and Tal1

  • The gene order and recombination data have been established as: Ifna–(1.8 cM)–Cdkn2a,b–(2.4 cM)–D4Rck12–(3.6 cM)–Mtv13–(0.6 cM)–Tal2

For studying these relationships, researchers should employ:

• Co-immunoprecipitation experiments to confirm direct interactions

• Functional complementation assays to investigate pathway redundancy

• Gene expression analysis to identify co-regulated targets

• Mouse models with mutations in both genes to identify genetic interactions

How do post-translational modifications affect Cdkn2aip function?

While specific post-translational modifications of CDKN2AIP are not extensively detailed in the search results, related information suggests several important regulatory mechanisms:

• Potential sumoylation involvement: CDKN2AIP may be involved in sumoylation processes, as ARF (its binding partner) interacts with UBE2I/UBC9 and enhances sumoylation of binding partners including MDM2 and E2F1 .

• Ubiquitination regulation: ARF binds to HUWE1 and represses its ubiquitin ligase activity , suggesting CDKN2AIP may be involved in ubiquitin-dependent regulatory networks.

• Phosphorylation dynamics: Given CDKN2AIP's involvement in cell cycle regulation and DNA damage response, phosphorylation by cell cycle-regulated kinases or damage-activated kinases is likely a significant regulatory mechanism.

• Stability control: The short-lived mitochondrial isoform of ARF (smARF) is stabilized by C1QBP , suggesting potential regulated stability of CDKN2AIP through similar protein-protein interactions.

• Localization signals: Post-translational modifications likely influence CDKN2AIP's subcellular localization between nuclear and cytoplasmic compartments.

Research approaches should include:

• Mass spectrometry analysis to identify specific modifications

• Phospho-specific antibodies to track activation states

• Mutation of putative modification sites to assess functional consequences

• Inhibitor studies targeting specific modifying enzymes

• Protein stability assays under various cellular conditions

What are the most effective experimental systems for studying Cdkn2aip function?

Based on published research, the following experimental systems have proven effective for studying CDKN2AIP:

• Mouse knockout models:

  • Complete gene deletion using CRISPR/Cas9 (3687-base deletion model)

  • Conditional knockout models for tissue-specific analysis

  • Age-progression studies to track developmental phenotypes

• Cell culture systems:

  • Knockdown approaches using siRNA or shRNA

  • Overexpression systems with tagged recombinant proteins

  • Primary cell cultures from knockout mice

• Biochemical approaches:

  • Co-immunoprecipitation followed by mass spectrometry (IP-MS)

  • In vitro kinase assays to examine effects on CDK4/CDK6 activity

  • Chromatin immunoprecipitation to identify genomic binding sites

• Tissue analysis methods:

  • Histological examination using Bouin's fixative and H&E staining

  • Immunohistochemistry for protein localization

  • In situ hybridization for expression pattern analysis

For germ cell and fertility studies specifically, researchers should analyze:

• Testis and epididymis histology

• Sperm morphology and function

• Meiotic chromosome spreads

• Fertility parameters and litter size

How can I optimize recombinant expression and purification of mouse Cdkn2aip?

While the search results don't provide specific purification protocols for recombinant CDKN2AIP, based on its properties as an RNA-binding protein and its interactions, the following methodological approach is recommended:

• Expression system selection:

  • E. coli systems using BL21(DE3) for high yield

  • Insect cell expression (Sf9) for mammalian post-translational modifications

  • Mammalian expression systems for complex studies requiring native folding

• Fusion tags optimization:

  • N-terminal 6xHis tag for IMAC purification

  • GST-fusion constructs for improved solubility and affinity purification

  • FLAG or HA epitope tags for immunoprecipitation studies

• Purification strategy:

  • Two-step purification combining affinity chromatography with size exclusion

  • Ion exchange chromatography as an additional purification step

  • Consider native purification conditions to maintain RNA-binding activity

• Activity assessment:

  • RNA-binding assays to confirm functionality

  • p53 and ARF binding assays

  • DNA damage response functional assays

• Storage optimization:

  • Stabilizing buffers containing glycerol (10-20%)

  • Flash freezing in liquid nitrogen

  • Stability testing at various temperatures (-80°C, -20°C, 4°C)

What techniques are most suitable for analyzing Cdkn2aip's role in DNA damage response?

Several techniques have proven valuable for investigating CDKN2AIP's functions in DNA damage response:

• DNA damage quantification:

  • Comet assay (single cell gel electrophoresis) for direct DNA break measurement

  • γH2AX immunofluorescence to quantify double-strand breaks

  • TUNEL assay to detect apoptotic DNA fragmentation

• Cell cycle analysis:

  • Flow cytometry with propidium iodide staining

  • BrdU incorporation to measure S-phase progression

  • Live cell imaging with fluorescent cell cycle reporters

• Checkpoint activation:

  • Western blotting for phosphorylated checkpoint proteins (CHK1, CHK2)

  • Immunofluorescence for nuclear foci formation

  • Kinase activity assays for ATM and ATR pathways

• p53 pathway analysis:

  • p53 stabilization and phosphorylation status

  • p53 target gene expression (p21, PUMA, BAX)

  • MDM2 interaction studies

• Cellular outcome measures:

  • Apoptosis assays (Annexin V staining, caspase activation)

  • Senescence measurements (SA-β-gal staining)

  • Clonogenic survival assays following DNA damage

Based on published findings, it's particularly important to analyze S-phase progression, as CDKN2AIP knockdown has been associated with extended S phase, and to examine both ATR/CHK1 pathway activation and p53 stabilization mechanisms .

How should phenotypic data from Cdkn2aip knockout models be analyzed and interpreted?

When analyzing phenotypic data from Cdkn2aip knockout models, researchers should implement the following structured approach:

• Comprehensive phenotypic screening:

  • Categorize observations by organ systems and developmental stages

  • Perform detailed analyses of reproductive tissues given CDKN2AIP's high expression in testis

  • Compare heterozygous and homozygous phenotypes to identify dose-dependent effects

• Statistical approaches:

  • Use appropriate statistical tests for comparing knockout vs. control animals

  • Implement mixed-effects models for longitudinal data across different ages

  • Perform power calculations to ensure adequate sample sizes

• Molecular correlation analysis:

  • Connect tissue-level phenotypes to cellular mechanisms

  • Link observed defects to specific molecular pathways

  • Identify secondary vs. primary effects through temporal analysis

• Comparative analysis with related models:

  • Compare with phenotypes of CDKN2A knockout mice

  • Analyze similarities and differences with p53 pathway mutants

  • Evaluate potential overlap with DNA damage response mutants

• Translational relevance assessment:

  • Correlate findings with human disease conditions

  • Evaluate potential biomarker applications

  • Assess therapeutic implications for cancer and reproductive disorders

For instance, the reported age-dependent germ cell loss, synapsis failure in 19% of spermatocytes, and sperm head defects in Cdkn2aip-/- mice should be analyzed in relation to molecular mechanisms including protamine replacement failure and impaired SUN1 expression .

What are the key control experiments required when studying Cdkn2aip functions?

When investigating CDKN2AIP functions, the following control experiments are essential:

• Genetic controls:

  • Wild-type (+/+) littermates as primary controls

  • Heterozygous (+/-) animals to assess dose-dependency

  • Rescue experiments with exogenous CDKN2AIP expression

  • Complementation studies with human CDKN2AIP

• Experimental validation controls:

  • Multiple independent siRNA/shRNA sequences to confirm knockdown phenotypes

  • Non-targeting siRNA/shRNA controls

  • Empty vector controls for overexpression studies

  • Isotype-matched antibody controls for immunoprecipitation

• Pathway-specific controls:

  • p53-null background to determine p53-dependent and independent functions

  • CDKN2A/ARF knockout comparisons

  • Positive controls for DNA damage (e.g., irradiation, hydroxyurea)

  • Cell cycle synchronization controls

• Methodological controls:

  • Input sample controls for IP-MS experiments

  • Housekeeping gene controls for expression analysis

  • Loading controls for western blotting

  • Fixation and staining controls for histological analysis

• Tissue/cell type controls:

  • Tissue-specific analyses given differential expression

  • Age-matched controls for developmental studies

  • Cell-cycle phase controls for proliferation studies

These control experiments are crucial, as demonstrated in the research where control comparisons between wild-type and knockout mice revealed significant phenotypic differences in sperm morphology, meiotic progression, and DNA repair capacity .

What is the potential role of Cdkn2aip in cancer biology and therapeutic development?

CDKN2AIP has several potential roles in cancer biology that deserve further investigation:

• Tumor suppressor functions:

  • CDKN2AIP has been associated with testicular tumor suppression

  • Its interactions with p53 and ARF suggest broader tumor suppressor activities

  • Overexpression leads to senescence via p53-HDM2-p21 pathway activation

• Genomic stability maintenance:

  • CDKN2AIP knockdown results in aneuploidy and DNA damage

  • Its role in DNA repair suggests functions in preventing mutagenesis

  • Potential guardian against chromosome instability in cancer cells

• Cell cycle checkpoint regulation:

  • Through p53 pathway connections, CDKN2AIP may enforce cell cycle checkpoints

  • Potential synthetic lethality with checkpoint inhibitors

  • Role in therapy-induced senescence mechanisms

• Connection to established cancer pathways:

  • CDKN2A is frequently mutated in various tumors

  • ARF-MDM2-p53 axis is a key tumor suppressor pathway

  • CDK4/6-cyclin D-pRb pathway is targeted by current cancer therapeutics

• Therapeutic implications:

  • Potential biomarker for cancer progression or treatment response

  • Target for synthetic lethality approaches

  • Modulator of response to DNA damaging therapies

Research approaches should include:

• Analysis of CDKN2AIP expression across cancer types

• Correlation with treatment outcomes and patient survival

• Testing combinations with established cancer therapeutics

• Investigation of synthetic lethality approaches

How might Cdkn2aip function in stem cell biology and regenerative medicine?

CDKN2AIP's roles in stem cell biology present several intriguing research directions:

• Stem cell pluripotency regulation:

  • CDKN2AIP has been shown to play a critical role in stem cell pluripotency

  • Potential involvement in maintaining stemness characteristics

  • Role in balancing self-renewal and differentiation decisions

• Differentiation pathways:

  • Involvement in somatic differentiation processes

  • Potential stage-specific roles during lineage commitment

  • Interaction with master regulators of differentiation

• Tissue-specific stem cell functions:

  • Essential role in spermatogonial self-renewal

  • Potential importance in other adult stem cell populations

  • Age-related changes in stem cell maintenance

• Regenerative medicine applications:

  • Modulation of CDKN2AIP might enhance stem cell expansion

  • Potential target for improving differentiation protocols

  • Role in maintaining genomic integrity of stem cells for therapeutic applications

• Cell reprogramming connections:

  • Possible involvement in induced pluripotent stem cell generation

  • Role in epigenetic remodeling during reprogramming

  • Connections to cell cycle restructuring in pluripotency acquisition

Research methodologies should include:

• Analysis in embryonic and induced pluripotent stem cell models

• Lineage tracing in tissue-specific stem cells

• Single-cell transcriptomics during differentiation

• Epigenetic profiling to identify regulatory mechanisms

• Functional assays for self-renewal and differentiation capacity

Researchers should build upon the established role of CDKN2AIP in spermatogonial self-renewal through the Wnt-signaling pathway activation to explore its functions in other stem cell contexts.

How conserved is Cdkn2aip across species and what does this reveal about its functions?

While the search results don't provide explicit cross-species comparison data for CDKN2AIP, we can make informed assessments about its evolutionary conservation:

• Functional domain conservation:

  • RNA-binding domains are likely conserved across species

  • Protein interaction interfaces for p53 and ARF binding would show high conservation

  • Regulatory elements might exhibit more species-specific variations

• Pathway conservation:

  • The p53 pathway is highly conserved across vertebrates

  • CDKN2A/ARF functions are maintained in mammals with some species-specific variations

  • Cell cycle checkpoint mechanisms show strong evolutionary conservation

• Reproductive system roles:

  • Spermatogenesis mechanisms vary across species but core meiotic processes are conserved

  • CDKN2AIP's high expression in testis suggests conserved reproductive functions

  • Sperm head formation processes may show species-specific adaptations

• Comparative genomics insights:

  • Syntenic relationships between CDKN2AIP and nearby genes could reveal evolutionary constraints

  • Identification of conserved regulatory elements would highlight essential functions

  • Species-specific adaptations might correlate with reproductive strategies

• Disease associations:

  • Correlation between CDKN2AIP variations and cancer susceptibility across species

  • Comparison of fertility phenotypes in different model organisms

  • Conservation of DNA damage response functions

Research approaches should include:

• Sequence alignment analysis across vertebrate species

• Functional complementation studies between species

• Phenotypic comparison of knockout models in different organisms

• Analysis of expression patterns across evolutionary distant species

What experimental approaches best integrate multi-omics data for understanding Cdkn2aip's regulatory networks?

Integrating multi-omics data for comprehensive understanding of CDKN2AIP requires sophisticated approaches:

• Integrated genomics platforms:

  • Combine ChIP-seq, RNA-seq, and ATAC-seq data to identify direct and indirect targets

  • Integrate proteomics and transcriptomics to identify post-transcriptional effects

  • Correlate epigenomic modifications with expression changes

• Network analysis methodologies:

  • Construct protein-protein interaction networks centered on CDKN2AIP

  • Identify regulatory motifs and feedback loops within signaling pathways

  • Apply machine learning for pattern recognition across datasets

• Temporal and spatial integration:

  • Time-course experiments to capture dynamic changes

  • Single-cell approaches to resolve cellular heterogeneity

  • Tissue-specific regulatory network modeling

• Perturbation biology:

  • CRISPR screens coupled with multi-omics readouts

  • Small molecule inhibitor panels with multi-parameter phenotyping

  • Genetic interaction mapping through combinatorial perturbations

• Computational frameworks:

  • Bayesian network analysis for causal relationship identification

  • Gene set enrichment approaches across multiple data types

  • Pathway-centric integration of heterogeneous data types

The IP-MS approach used in CDKN2AIP research should be expanded to include:

• Quantitative proteomics under various conditions

• Phosphoproteomics after cellular perturbations

• RNA-binding protein immunoprecipitation sequencing (RIP-seq)

• Integration with genomic and transcriptomic datasets