SPIN1 Human

What is the basic structure of human SPIN1 protein?

SPIN1 is a 262 amino acid protein with a molecular mass of approximately 32.0kDa. The protein structure consists of an N-terminal intrinsically disordered region (IDR) and three tudor-like domains, with each domain containing approximately 50 amino acids . This structural arrangement enables SPIN1 to function as a "reader" module that recognizes specific histone methylation patterns. The protein can be produced as a recombinant for research purposes, typically as a single polypeptide chain with additional tags such as a 23 amino acid His-tag at the N-terminus for purification purposes .

What histone modifications does SPIN1 recognize and how does this affect its function?

SPIN1 functions as a multifunctional histone reader with remarkable specificity for several key histone modifications. Structural studies have revealed that the second Tudor-like domain of SPIN1 recognizes H3K4me3 (histone H3 trimethylated at lysine 4) or H4K20me3, while the first Tudor module recognizes H3R8me2a or H3K9me3 . This indicates SPIN1's versatility in recognizing multiple histone modifications including H3K4me3, H4K20me3, H3"K4me3-R8me2a" and H3K4me3-K9me3 . The binding of SPIN1 to H3K4me3 occurs with high affinity and this association is enhanced by the presence of asymmetrically dimethylated arginine 8 of histone H3 . This histone code reading capability enables SPIN1 to function as a transcriptional coactivator, promoting the binding of various factors in promoter regions and facilitating gene transcription, including rDNA, IL1B, and BST2 .

How does SPIN1 contribute to normal cellular functions?

SPIN1 plays essential roles in several fundamental cellular processes:

• Transcriptional Regulation: SPIN1 acts as a transcriptional coactivator by binding to H3K4me3, promoting its binding in promoter regions of genes, and facilitating transcription of various genes including rDNA .

• Cell Cycle Regulation: Studies show SPIN1 affects cell cycle progression, with knockdown resulting in reduction in S-phase and increase in G1 phase in HeLa and HEK293T cells .

• DNA Damage Response: SPIN1 is recruited to DNA lesions through its N-terminal disordered region that binds to Poly-ADP-ribose (PAR) and facilitates homologous recombination (HR)-mediated DNA damage repair .

• Embryonic Development: SPIN1 serves critical functions in embryonic development, with evidence showing its importance in skeletal muscle development in mice .

The various functional roles of SPIN1 highlight its significance in maintaining normal cellular homeostasis and developmental processes.

How is SPIN1 recruited to DNA damage sites and what is its role in DNA repair?

SPIN1 is rapidly recruited to DNA damage sites through a specific mechanism involving direct binding to Poly-ADP-ribose (PAR) through its N-terminal disordered region . Upon recruitment to DNA lesions, SPIN1 facilitates homologous recombination (HR)-mediated DNA damage repair through several coordinated actions:

• It promotes H3K9me3 accumulation at DNA damage sites

• It enhances the interaction between H3K9me3 and Tip60

• This interaction promotes the activation of ATM (Ataxia Telangiectasia Mutated) kinase

• The activated pathway ultimately enhances HR repair efficiency

Experimental evidence demonstrates that knockdown of SPIN1 specifically reduces the GFP signaling associated with HR repair pathway, while having no significant effect on non-homologous end joining (NHEJ) repair pathway . Further supporting this specificity, SPIN1 depletion results in reduced numbers of RAD51 and BRCA1 foci (HR repair factors) following ionizing radiation, while the number of 53BP1 foci (NHEJ factor) remains unchanged .

What experimental approaches can be used to study SPIN1's role in DNA damage repair?

Researchers investigating SPIN1's function in DNA damage repair can employ several methodological approaches:

• GFP Reporter Assays: These can be used to quantitatively assess HR and NHEJ repair pathway efficiency following SPIN1 knockdown or overexpression .

• Immunofluorescence Staining: This approach allows quantification of repair foci (RAD51, BRCA1, 53BP1, γH2AX) following DNA damage induction in SPIN1-depleted or overexpressing cells .

• Cell Cycle Analysis: Flow cytometry can be used to determine SPIN1's impact on cell cycle progression, which may indirectly affect DNA repair pathway choice .

• Chromatin Immunoprecipitation (ChIP): This technique can assess SPIN1 recruitment to damage sites and examine histone modification changes (particularly H3K9me3) at these sites.

• Co-Immunoprecipitation: This method can validate protein-protein interactions, such as between SPIN1 and repair factors or between H3K9me3 and Tip60 .

• PAR-Binding Assays: These can confirm SPIN1's direct interaction with PAR at DNA damage sites .

These methodologies, used in combination, provide comprehensive insights into SPIN1's mechanistic role in DNA repair processes.

How does SPIN1 contribute to cancer development and progression?

SPIN1 is highly expressed in various types of cancers compared to normal tissues, as demonstrated by analysis using the GEPIA (Gene Expression Profiling Interactive Analysis) web server . The protein's contribution to cancer biology is multifaceted:

• Transcriptional Dysregulation: As a transcriptional coactivator, SPIN1 overexpression can alter expression patterns of genes involved in proliferation and survival.

• Cell Cycle Effects: SPIN1 influences cell cycle progression, with studies showing its impact on transformation of cell lines and tumor formation in nude mice .

• Apoptosis Regulation: Research indicates SPIN1 affects apoptotic pathways, potentially allowing cancer cells to evade programmed cell death .

• DNA Repair Enhancement: By promoting efficient HR-mediated repair, SPIN1 may help cancer cells survive DNA damage that would otherwise lead to cell death .

• Chemoresistance Promotion: SPIN1 increases resistance to DNA-damaging chemotherapeutic agents like Cisplatin and PARP inhibitors like Olaparib both in vitro and in vivo .

This collective evidence positions SPIN1 as a potential tumor promoter and therapeutic target in cancer treatment strategies.

What is the relationship between SPIN1 expression and chemoresistance?

SPIN1 significantly contributes to cancer chemoresistance through multiple mechanisms:

• Enhanced DNA Damage Repair: SPIN1 increases the efficiency of HR-mediated DNA repair, potentially allowing cancer cells to survive DNA damage induced by chemotherapeutic agents .

• Drug Resistance Modulation: Experimental evidence shows that knockdown of SPIN1 increases the sensitivity of cancer cells to Cisplatin and Olaparib, while reintroducing SPIN1 restores resistance to these drugs .

• In Vivo Confirmation: Xenograft mouse models demonstrate that SPIN1 depletion results in significant increase in sensitivity to Cisplatin and Olaparib, as evidenced by decreased tumor growth rate, weight, and size .

Interestingly, while SPIN1's recruitment to DNA damage sites following Olaparib treatment was not observed, its reintroduction still restored resistance to this PARP inhibitor, suggesting alternative mechanisms through which SPIN1 contributes to cell survival in the presence of certain chemotherapeutic agents . This complex relationship between SPIN1 expression and chemoresistance suggests that targeting SPIN1 could be a potential strategy to enhance the sensitivity of cancer cells to DNA damage-inducing anticancer drugs.

What methodological approaches can be used to target SPIN1 in cancer therapy research?

Researchers investigating SPIN1 as a therapeutic target in cancer can employ several approaches:

• RNA Interference (RNAi): siRNA or shRNA targeting SPIN1 can be used to assess the effects of SPIN1 knockdown on cancer cell sensitivity to chemotherapy .

• CRISPR-Cas9 Gene Editing: This can be used to create SPIN1 knockout models to study long-term effects of SPIN1 ablation.

• Small Molecule Inhibitors: Development or screening of compounds that interfere with SPIN1's histone binding domains, particularly targeting its interactions with H3K4me3 or H3K9me3.

• Combination Therapy Assessment: Testing SPIN1 inhibition in combination with DNA-damaging agents (Cisplatin) or PARP inhibitors (Olaparib) to overcome chemoresistance .

• Xenograft Models: In vivo models can validate the effects of SPIN1 targeting on tumor growth and chemosensitivity, as demonstrated in previous research .

• Patient-Derived Xenografts (PDX): These more clinically relevant models can assess the variability of response to SPIN1 targeting across different patient samples.

• Biomarker Development: Developing methods to assess SPIN1 expression or activity as a potential predictor of chemotherapy response.

These methodological approaches provide a framework for translational research aimed at exploiting SPIN1's role in cancer for therapeutic benefit.

What is known about SPIN1's role in skeletal muscle development?

SPIN1 plays a critical role in skeletal muscle (SkM) development, as evidenced by studies using conditional knockout mice. Research has shown that:

• Phenotypic Impact: Mice with ablation of SPIN1 in myoblast precursors (Spin1 M5 mice using Myf5-Cre deleter strain) exhibit severe developmental defects, with most dying shortly after birth due to sarcomere disorganization and necrosis .

• Survival Defects: Surviving Spin1 M5 mice display growth retardation and prominent defects in specific muscles, particularly the soleus, tibialis anterior, and diaphragm muscle .

• Molecular Basis: Transcriptome analyses at different embryonic stages (E15.5, E16.5) and at three weeks of age revealed aberrant fetal myogenesis and identified deregulated SkM functional networks .

• Direct Target Genes: Genome-wide chromatin occupancy studies in primary myoblasts revealed direct Spin1 target genes, suggesting that deregulated basic helix-loop-helix transcription factor networks account for developmental defects in Spin1 M5 fetuses .

• Pathological Mechanisms: Correlating histological and transcriptome analyses demonstrated that aberrant expression of titin-associated proteins, abnormal glycogen metabolism, and neuromuscular junction defects contribute to SkM pathology in Spin1 M5 mice .

This research established SPIN1 as the first example of a histone code reader controlling SkM development in mice, suggesting potential implications for human skeletal muscle diseases .

What experimental models are most effective for studying SPIN1's developmental functions?

Several experimental models have proven valuable for investigating SPIN1's role in development:

• Conditional Knockout Mice: The Spin1 M5 mouse model using the Myf5-Cre deleter strain has been particularly informative for studying skeletal muscle development, allowing tissue-specific ablation of SPIN1 in myoblast precursors .

• Embryonic Stage Analysis: Examining embryos at different developmental stages (e.g., E15.5, E16.5) provides insights into the temporal requirements for SPIN1 during development .

• Primary Cell Cultures: Primary myoblasts isolated from model organisms allow for detailed molecular analyses, including chromatin occupancy studies to identify direct target genes .

• Transcriptome Analysis: RNA sequencing at different developmental stages reveals the dynamic impact of SPIN1 on gene expression networks during development .

• Histological Examination: Sarcomere organization and tissue architecture can be assessed through histological methods to characterize developmental defects .

• Functional Assays: For skeletal muscle development, functional assessments of muscle strength and coordination in surviving mutant animals provide physiological context to molecular findings.

• Genome-wide Chromatin Occupancy: ChIP-seq analysis reveals direct binding sites of SPIN1 across the genome, connecting its histone reader function to specific target genes .

These complementary approaches provide a comprehensive understanding of SPIN1's developmental functions and the consequences of its disruption.

What are the optimal methods for producing and purifying recombinant SPIN1 protein for research?

Recombinant SPIN1 production requires several technical considerations to ensure high-quality protein for research applications:

• Expression System: E. coli has been successfully used as an expression system for human SPIN1 recombinant protein . The protein is typically expressed as a single polypeptide chain containing 285 amino acids (including the 262 amino acids of the native protein plus additional tag sequences) .

• Affinity Tags: A 23 amino acid His-tag at the N-terminus is commonly used to facilitate purification . The resulting protein has a molecular mass of approximately 32.0kDa .

• Purification Techniques: Proprietary chromatographic techniques are employed for purification , likely including immobilized metal affinity chromatography (IMAC) for His-tagged proteins followed by size exclusion chromatography to enhance purity.

• Quality Control: Purity assessment by SDS-PAGE is essential, with optimal preparations achieving greater than 90% purity . Additional quality control may include mass spectrometry confirmation and functional binding assays.

• Storage Conditions: The purified protein is typically maintained as a sterile filtered colorless solution , with specific buffer compositions designed to maintain stability.

• Shipping Considerations: Recombinant SPIN1 requires shipping with ice packs to maintain stability during transport .

These methodological considerations ensure the production of high-quality recombinant SPIN1 protein suitable for downstream applications such as structural studies, binding assays, and enzymatic characterizations.

How can researchers effectively detect and quantify SPIN1 in experimental samples?

Effective detection and quantification of SPIN1 in experimental samples can be achieved through several complementary methods:

• Western Blotting: Polyclonal antibodies against SPIN1 have been validated for Western blot applications, allowing detection of both recombinant and endogenous SPIN1 protein . This technique enables semi-quantitative assessment of SPIN1 expression levels across different experimental conditions.

• Immunohistochemistry (IHC): SPIN1 antibodies have been validated for IHC applications on formalin-fixed paraffin-embedded tissues, including kidney tissue samples . This allows visualization of SPIN1 protein localization within tissue contexts.

• Immunocytochemistry (ICC): For cellular localization studies, validated antibodies can be used for ICC applications to determine SPIN1's subcellular distribution in various cell types and conditions .

• Immunoprecipitation (IP): SPIN1 antibodies have been validated for IP applications, allowing isolation of SPIN1 protein complexes for interaction studies .

• Quantitative PCR (qPCR): For mRNA level quantification, qPCR assays targeting SPIN1 transcript provide a sensitive method to assess expression changes.

• Flow Cytometry: For single-cell analysis, particularly in heterogeneous populations, flow cytometry using fluorescently labeled SPIN1 antibodies can be employed.

• Mass Spectrometry: For absolute quantification and post-translational modification analysis, mass spectrometry approaches offer high precision detection of SPIN1 protein.

When selecting detection methods, researchers should consider the specific experimental questions, sample types, and required sensitivity levels to choose the most appropriate technical approach.

What are the key considerations for designing siRNA knockdown experiments targeting SPIN1?

Designing effective siRNA knockdown experiments for SPIN1 requires attention to several methodological considerations:

• siRNA Design: Multiple siRNA sequences targeting different regions of SPIN1 mRNA should be designed and tested to identify those with highest knockdown efficiency while minimizing off-target effects. Previous studies have successfully used siRNA approaches for SPIN1 knockdown .

• Controls: Proper experimental controls are essential, including:

  • Non-targeting siRNA control (siNC) with similar chemical modifications

  • Positive control targeting a housekeeping gene

  • Untransfected control to assess transfection toxicity

• Validation of Knockdown: Knockdown efficiency should be verified at both:

  • mRNA level by qRT-PCR

  • Protein level by Western blot

• Phenotypic Rescue: To confirm specificity, rescue experiments should be performed by reintroducing siRNA-resistant SPIN1 expression constructs. This approach has been successfully used in previous studies to validate SPIN1-specific effects .

• Temporal Considerations: The timing of analyses after knockdown is critical, particularly when studying:

  • Cell cycle effects, as SPIN1 knockdown has been shown to result in S-phase reduction and G1 phase increase

  • DNA damage response, which may require specific timing of damage induction after knockdown

• Cell Type Selection: Different cell lines may exhibit varying transfection efficiencies and SPIN1 dependency. Previous studies have successfully employed SPIN1 knockdown in HeLa, HEK293T, U2OS, and SGC7901 cell lines .

• Functional Readouts: Appropriate assays should be selected based on the SPIN1 functions being investigated, such as:

  • GFP reporter assays for HR repair pathway assessment

  • Immunofluorescence staining for DNA repair foci quantification

  • Cell viability assays for chemosensitivity assessment

Careful attention to these methodological considerations will enhance the reliability and interpretability of SPIN1 knockdown experiment results.

How should researchers interpret contradictory findings about SPIN1 function across different experimental systems?

When interpreting contradictory findings about SPIN1 function across different experimental systems, researchers should consider several contextual factors:

• Cell Type Specificity: SPIN1 functions may vary significantly between cell types due to:

  • Different expression levels of co-factors or interaction partners

  • Varying chromatin landscapes affecting histone modification patterns

  • Cell-type specific signaling pathways that modulate SPIN1 activity

  • Different cell cycle characteristics, as SPIN1 has established effects on cell cycle progression

• Experimental Conditions: Variations in experimental conditions can lead to contradictory results:

  • Acute versus chronic SPIN1 depletion may reveal different aspects of its function

  • Stress conditions (e.g., DNA damage) may activate specific SPIN1 functions not observed under normal conditions

  • Different methods of inducing DNA damage (IR vs. chemical agents) may engage SPIN1 differently

• Methodological Differences: Technical approaches can influence outcomes:

  • Complete knockout versus partial knockdown may reveal different phenotypes

  • Overexpression studies may identify functions not observed in loss-of-function studies

  • Different tagged versions of SPIN1 may affect its localization or function

• Functional Redundancy: Other proteins with similar functions may compensate for SPIN1 loss in certain contexts but not others.

• Multiple Molecular Functions: As SPIN1 recognizes multiple histone modifications and has diverse cellular roles, different experimental systems may emphasize different aspects of its multifunctional nature .

For example, while SPIN1 was not recruited to DNA damage sites following Olaparib treatment, its reintroduction still restored resistance to this drug, suggesting alternative mechanisms through which SPIN1 contributes to cell survival in different contexts . Researchers should carefully consider these factors when designing experiments and interpreting apparently contradictory findings.

What bioinformatic approaches are most valuable for analyzing SPIN1's genomic binding patterns and target genes?

Several bioinformatic approaches are particularly valuable for analyzing SPIN1's genomic binding patterns and target genes:

• ChIP-Seq Analysis Pipeline:

  • Peak calling algorithms (e.g., MACS2) to identify genomic regions with significant SPIN1 binding

  • Motif discovery tools to identify DNA sequences associated with SPIN1 binding sites

  • Genomic annotation to determine distribution of binding sites relative to genes (promoters, enhancers, gene bodies)

  • Integration with histone modification data, particularly H3K4me3 and H3K9me3, which are recognized by SPIN1

• Multi-omics Integration:

  • Correlation of SPIN1 binding sites with transcriptome data to identify direct target genes

  • Integration with chromatin accessibility data (ATAC-seq) to examine relationship between SPIN1 binding and chromatin state

  • Overlay with other transcription factor binding data to identify potential co-regulatory networks

• Pathway and Network Analysis:

  • Gene Ontology (GO) enrichment analysis of SPIN1 target genes to identify overrepresented biological processes

  • Pathway analysis using KEGG, Reactome, or similar databases to place target genes in functional contexts

  • Protein-protein interaction network analysis to identify functional clusters among target genes

• Comparative Genomics:

  • Cross-species analysis of SPIN1 binding patterns to identify evolutionarily conserved targets

  • Comparison of binding patterns across different cell types or conditions to identify context-specific functions

• Machine Learning Approaches:

  • Predictive modeling of SPIN1 binding based on DNA sequence and epigenetic features

  • Classification of SPIN1 target genes based on response patterns and functional outcomes

These bioinformatic approaches have proven valuable in identifying direct SPIN1 target genes and elucidating how deregulated basic helix-loop-helix transcription factor networks account for developmental defects in SPIN1-deficient models .

What is the potential of SPIN1 as a therapeutic target in cancer treatment?

SPIN1 shows considerable promise as a therapeutic target in cancer treatment based on several lines of evidence:

• Differential Expression: SPIN1 is highly expressed in various types of cancers compared to normal tissues, as demonstrated by analysis using the GEPIA web server . This differential expression provides a potential therapeutic window.

• Chemosensitization Effects: Knockdown of SPIN1 increases cancer cell sensitivity to both:

  • Cisplatin (DNA crosslinking agent)

  • Olaparib (PARP inhibitor)

• In Vivo Validation: Xenograft mouse models confirm that SPIN1 depletion enhances tumor chemosensitivity, with remarkable decreases in tumor growth rate, weight, and size following treatment with Cisplatin and Olaparib .

• Radiotherapy Enhancement: Previous studies have shown that targeting SPIN1 can enhance the radiosensitivity of non-small cell lung cancer (NSCLC) cells and improve radiotherapy efficacy .

• Mechanistic Rationale: SPIN1's role in promoting efficient HR-mediated DNA repair provides a clear mechanistic rationale for targeting it in combination with DNA-damaging therapies .

• Druggable Domains: SPIN1's tudor domains, which recognize specific histone modifications, represent potentially druggable protein-protein interaction surfaces.

Given these findings, developing SPIN1 inhibitors could be a promising strategy to overcome chemoresistance in cancer treatment, particularly when combined with DNA-damaging agents or PARP inhibitors. The synthetic lethality concept that underlies PARP inhibitor efficacy in BRCA-deficient tumors might be extended through SPIN1 inhibition to a broader range of cancers.

How might SPIN1 dysfunction contribute to skeletal muscle diseases in humans?

Based on developmental studies in mice, SPIN1 dysfunction may contribute to skeletal muscle diseases in humans through several potential mechanisms:

• Sarcomere Organization Defects: Spin1 M5 mice with ablation of SPIN1 in myoblast precursors display severe sarcomere disorganization , suggesting SPIN1 dysfunction could contribute to human myopathies characterized by sarcomeric abnormalities.

• Muscle-Specific Pathology: The most prominent defects in surviving Spin1 M5 mice occur in soleus, tibialis anterior, and diaphragm muscle , indicating potential muscle-type specific vulnerability that could be relevant to certain human myopathies.

• Aberrant Myogenesis: Transcriptome analyses revealed that SPIN1 deficiency leads to aberrant fetal myogenesis , suggesting potential involvement in congenital myopathies resulting from developmental defects.

• Dysregulated Transcription Factor Networks: SPIN1 deficiency disrupts basic helix-loop-helix transcription factor networks during development , which may be relevant to human muscular disorders associated with transcriptional dysregulation.

• Titin-Associated Protein Abnormalities: Correlation of histological and transcriptome analyses showed aberrant expression of titin-associated proteins in SPIN1-deficient mice , potentially linking SPIN1 dysfunction to titinopathies in humans.

• Glycogen Metabolism Defects: SPIN1 deficiency leads to abnormal glycogen metabolism , which could contribute to glycogen storage myopathies in humans.

• Neuromuscular Junction Defects: SPIN1-deficient mice exhibit neuromuscular junction defects , suggesting potential relevance to human disorders involving defective neuromuscular transmission.

These findings position SPIN1 as a potential player in human skeletal muscle diseases, particularly those involving developmental defects, sarcomere disorganization, or neuromuscular junction abnormalities. Further research in human patients is needed to establish direct links between SPIN1 dysfunction and specific myopathies.