SUB1 Human

What is the SUB1 gene and what protein does it encode?

SUB1 (SUB1 Regulator of Transcription) is a protein-coding gene located on human chromosome 5. It encodes the Activated RNA polymerase II transcriptional coactivator p15, also known as positive cofactor 4 (PC4) or SUB1 homolog . The human SUB1 gene is named after an orthologous gene in yeast. The protein functions as a general transcriptional coactivator that mediates interactions between upstream activators and the general transcriptional machinery .

The protein encoded by SUB1 has several key molecular functions:

• RNA binding capability

• Single-stranded DNA binding

• Transcriptional coactivator activity

• Identical protein binding

To study SUB1 function, researchers should be aware of its multiple aliases which may appear in scientific literature:

• P15

• PC4

• p14

• TCP4

• SUB1 homolog

• SUB1 regulator of transcription

What are the primary cellular functions of SUB1 protein?

SUB1 plays diverse roles in cellular function, primarily centered around transcriptional regulation and DNA maintenance:

• Transcriptional regulation: SUB1 functions as a general coactivator that stabilizes the multiprotein transcription complex, cooperating with TAFs (TBP-associated factors) to mediate interactions between upstream activators and the general transcription machinery .

• DNA protection: SUB1 is induced by oxidative stress and coordinates cellular responses to DNA strand breaks arising from oxidative damage . This protective function involves:

  • Recognition of damaged DNA sites

  • Recruitment of repair machinery

  • Facilitation of repair processes

• Chromatin organization: SUB1 interacts with various transcription factors and can influence chromatin structure, affecting gene accessibility.

• Gene expression modulation: Research has shown that SUB1 can bind to the promoters of several genes, including oncogenes like PLK1, BUB1B, and C-MYC, directly influencing their expression levels .

Interestingly, yeast SUB1 shares structural and functional similarities with human alpha-synuclein, a protein implicated in Parkinson's disease, particularly in DNA repair mechanisms including double-strand break repair .

How is SUB1 implicated in cancer progression?

SUB1 has been significantly implicated in cancer development and progression, particularly in prostate cancer. Research evidence demonstrates:

• Elevated expression: SUB1 shows increased expression in prostate cancer cell lines and tissues compared to normal controls .

• Functional significance: Knockdown studies reveal SUB1's critical role in:

  • Cancer cell proliferation

  • Cell migration

  • Invasion capacity

  • Colony formation

• Oncogenic regulation: SUB1 directly binds to the promoters of several oncogenes, functioning as a transcriptional activator for:

  • PLK1 (Polo-like kinase 1)

  • BUB1B (Budding Uninhibited by Benzimidazoles 1 Homolog Beta)

  • C-MYC

• Tumor suppressor downregulation: SUB1 has been shown to downregulate CDKN1B (p27), a cell cycle inhibitor and tumor suppressor .

• In vivo evidence: Both chorioallantoic membrane (CAM) assays and murine xenograft models demonstrate that SUB1 inhibition significantly reduces tumor growth and metastasis .

• Other cancer associations: Beyond prostate cancer, SUB1 has been linked to:

  • Lung adenocarcinoma (correlated with lymphangiogenesis and lymphatic metastasis)

  • Non-small-cell lung cancer

  • Astrocytoma

The cumulative evidence positions SUB1 as an important contributor to oncogenic processes across multiple cancer types.

What is the relationship between microRNA-101 and SUB1 in cancer?

The relationship between microRNA-101 (miR-101) and SUB1 represents an important regulatory mechanism in cancer progression:

• Negative regulation: miR-101, a tumor suppressor microRNA, directly targets the 3'-UTR of SUB1, causing reduction in SUB1 protein levels .

• Validation evidence: Research has confirmed this regulatory relationship through:

  • Co-transfection experiments with miR-101 and pMir-REPORT-SUB1 3′-UTR plasmids

  • Demonstration of reduced SUB1 protein levels specifically in miR-101-treated cells, while other miRs (miR-23a, -23b, -30a, -30b, -124, -122) showed no effect

• Cancer implications: miR-101 is frequently downregulated in prostate cancer, which consequently leads to:

  • Increased SUB1 expression

  • Subsequent activation of downstream oncogenes

  • Enhanced cancer cell proliferation, invasion, and metastasis

• Therapeutic potential: This regulatory axis suggests potential therapeutic approaches:

  • miR-101 replacement therapy

  • Targeting SUB1 directly

  • Inhibiting downstream effectors of SUB1 (e.g., PLK1)

The miR-101/SUB1 axis represents a critical regulatory mechanism that, when dysregulated, contributes significantly to prostate cancer progression, making it an attractive target for therapeutic intervention.

What methodologies are most effective for studying SUB1 function?

Research on SUB1 employs diverse experimental approaches depending on the specific aspects being investigated:

• Expression analysis:

  • Quantitative PCR (qPCR) for mRNA expression quantification

  • Western blotting for protein level assessment

  • Immunohistochemistry for tissue localization and expression patterns

• Functional manipulation:

  • RNA interference (siRNA and shRNA) for transient and stable knockdown

  • CRISPR-Cas9 gene editing for knockout studies

  • Lentiviral-mediated overexpression for gain-of-function experiments

• Protein-DNA interaction studies:

  • Chromatin immunoprecipitation (ChIP) to identify SUB1 binding sites on target gene promoters

  • Electrophoretic mobility shift assays (EMSA) for DNA binding characterization

• Cellular phenotype assessment:

  • Proliferation assays (e.g., colony formation assays)

  • Migration assays (e.g., wound healing assay)

  • Invasion assays (e.g., Boyden chamber Matrigel invasion assay)

• In vivo models:

  • Chorioallantoic membrane (CAM) assays

  • Murine xenograft models

  • Transgenic mouse models

Researchers should select methods appropriate to their specific research questions while ensuring proper controls and validation approaches.

How can researchers effectively modulate SUB1 expression for functional studies?

Effective modulation of SUB1 expression is crucial for investigating its functional roles. Several approaches have proven successful:

• Knockdown strategies:

  • Transient siRNA transfection: Provides short-term reduction in SUB1 levels

    • Typically using pools of 3-4 siRNAs targeting different regions

    • Effective for acute functional assays (48-72 hours)

  • Stable shRNA expression: Enables long-term studies

    • Lentiviral delivery systems allow integration and sustained expression

    • Multiple shRNA constructs should be tested to confirm specificity

• Overexpression approaches:

  • Lentiviral expression systems: Ideal for stable expression

    • Myc-DDK-tagged SUB1 constructs have been successfully employed

    • Can include inducible promoters for temporal control

  • Transient plasmid transfection: Suitable for short-term studies

• MicroRNA modulation:

  • miR-101 precursor transfection reduces SUB1 levels

  • miR-101 inhibitors can increase SUB1 expression

• Pharmacological interventions:

  • While specific SUB1 inhibitors are not well established, downstream effector inhibition can be used

  • For example, PLK1 inhibitor (volasertib/BI6727) can mitigate SUB1 overexpression effects

Researchers should confirm modulation efficiency through both mRNA (qPCR) and protein (Western blot) level assessments.

What are the key protein-protein interactions of SUB1 and their functional significance?

SUB1 engages in numerous protein-protein interactions that mediate its biological functions:

• Transcriptional machinery interactions:

  • SUB1 interacts with TAFs (TBP-associated factors) to stabilize the transcription complex

  • Functions cooperatively with general transcription factors

• Activator domain interactions:

  • SUB1 has been shown to interact with distinct domains of various activators including:

    • VP16

    • GAL4

    • AP2

    • HIV-TAT

    • p53

    • SMYD3

  • These interactions are critical for SUB1's role in transcriptional modulation

• RNA processing factors:

  • SUB1 interacts with CSTF2 (cleavage stimulation factor), suggesting a role in RNA processing

• DNA repair complexes:

  • SUB1's involvement in DNA repair implies interactions with repair machinery components

• Cancer-specific interactions:

  • In lung adenocarcinoma, SUB1 expression correlates with VEGF-C, VEGF-D, and VEGFR-3 levels, suggesting potential functional interactions related to lymphangiogenesis and metastasis

The diverse interaction network of SUB1 underscores its multifunctional nature, acting as a hub protein that influences multiple cellular processes through protein-protein interactions.

How does SUB1 contribute to DNA damage response and repair?

SUB1 plays a significant role in DNA damage response (DDR) and repair mechanisms:

The dual role of SUB1 in transcription and DNA repair highlights its importance in maintaining cellular homeostasis, with implications for both normal physiology and disease states when dysregulated.

What are the critical methodological considerations when investigating SUB1's role in cancer?

When investigating SUB1's role in cancer, researchers should consider several critical methodological aspects:

• Model selection:

  • Cell line considerations:

    • Use multiple cell lines to account for heterogeneity

    • Include both hormone-responsive (e.g., LnCaP, VCaP) and hormone-independent (e.g., DU145, PC3) prostate cancer cell lines for comprehensive analysis

    • Include appropriate non-cancerous controls (e.g., RWPE cells for prostate studies)

  • In vivo models:

    • Xenograft models in immunocompromised mice

    • Chorioallantoic membrane (CAM) assays for tumor growth and metastasis

    • Consider genetically engineered mouse models for more physiologically relevant contexts

• Expression modulation approaches:

  • Implement both gain- and loss-of-function experiments

  • Validate knockdown/overexpression at both mRNA and protein levels

  • Consider rescue experiments to confirm specificity of observed phenotypes

• Functional assays:

  • Implement comprehensive phenotypic assessment:

    • Proliferation (short-term and long-term)

    • Migration (wound healing assays)

    • Invasion (Matrigel invasion assays)

    • Colony formation

• Molecular mechanism investigation:

  • Examine transcriptional targets using ChIP followed by sequencing (ChIP-seq)

  • Validate direct binding to promoters of interest

  • Assess effects on downstream pathway activation

• Therapeutic relevance:

  • Test combinations of SUB1 modulation with standard therapies

  • Investigate downstream effector inhibition (e.g., PLK1 inhibition) as alternative approaches

These methodological considerations ensure robust, reproducible results that can advance our understanding of SUB1's role in cancer biology and potentially inform therapeutic strategies.

How can researchers investigate potential contradictions in SUB1 functional data?

Investigating contradictions in SUB1 functional data requires systematic approaches to resolve discrepancies:

• Context-dependent function analysis:

  • Cell type specificity:

    • Compare results across multiple cell types and lineages

    • Determine if contradictions are cell-type specific

  • Microenvironment influence:

    • Test functionality under different conditions (normoxia vs. hypoxia)

    • Examine effects of growth factors and cytokines on SUB1 function

• Isoform and post-translational modification assessment:

  • Alternative splicing:

    • Determine if contradictory data relates to different SUB1 isoforms

    • Use isoform-specific detection methods

  • Post-translational modifications:

    • Investigate how phosphorylation, acetylation, or other modifications affect function

    • Use modification-specific antibodies or mass spectrometry approaches

• Methodological reconciliation:

  • Technical approaches:

    • Compare results from different knockdown methods (siRNA vs. shRNA vs. CRISPR)

    • Assess timing differences (acute vs. chronic modulation)

  • Data validation:

    • Use multiple experimental approaches to confirm findings

    • Implement rescue experiments to verify specificity

• Pathway interaction analysis:

  • Compensatory mechanisms:

    • Investigate potential redundant systems that may activate upon SUB1 modulation

    • Perform time-course studies to identify adaptive responses

  • Feedback loops:

    • Determine if contradictions result from feedback regulation

    • Map complete network interactions

When faced with contradictory data, researchers should systematically explore these dimensions to identify the source of discrepancy and develop a more nuanced understanding of SUB1's context-dependent functions.

What are the most promising therapeutic approaches targeting SUB1 in cancer?

Several promising therapeutic approaches targeting SUB1 in cancer warrant further investigation:

• Direct SUB1 inhibition strategies:

  • Small molecule inhibitors: Development of compounds targeting SUB1's DNA-binding domain or protein-protein interaction interfaces

  • Peptide-based inhibitors: Design of peptides that disrupt critical SUB1 interactions with transcriptional machinery

• MicroRNA-based approaches:

  • miR-101 replacement therapy: Since miR-101 negatively regulates SUB1, its delivery to cancer cells could reduce SUB1 expression

  • Combined miRNA approaches: Targeting SUB1 with multiple regulatory miRNAs for enhanced effect

• Downstream effector inhibition:

  • PLK1 inhibitors: Compounds like volasertib (BI6727) can mitigate the oncogenic effects of SUB1 overexpression

  • BUB1B and C-MYC targeting: Inhibitors of these downstream targets may be effective in SUB1-overexpressing tumors

• Combination therapeutic approaches:

  • SUB1 inhibition with standard chemotherapy: May enhance sensitivity to conventional treatments

  • Synthetic lethality: Identifying genes that, when inhibited along with SUB1, cause selective cancer cell death

• Gene therapy approaches:

  • CRISPR-based gene editing: For targeted disruption of SUB1 in cancer cells

  • Antisense oligonucleotides: To reduce SUB1 expression through complementary binding to mRNA

Each approach has distinct advantages and challenges, with downstream effector inhibition (particularly PLK1 inhibition) currently showing the most immediate translational potential based on existing research .

What emerging technologies could advance SUB1 research?

Emerging technologies offer exciting opportunities to advance SUB1 research across multiple dimensions:

• Single-cell technologies:

  • Single-cell RNA sequencing: To understand SUB1 expression heterogeneity within tumors

  • Single-cell proteomics: For protein-level analysis of SUB1 and its interactors at individual cell resolution

  • Spatial transcriptomics: To map SUB1 expression patterns within tumor microenvironments

• Advanced genome editing:

  • Prime editing: For precise modification of SUB1 regulatory elements

  • Base editing: To introduce specific mutations for structure-function studies

  • CRISPRi/CRISPRa: For reversible modulation of SUB1 expression without genomic alteration

• Protein interaction and structural biology tools:

  • Proximity labeling (BioID, APEX): To map the complete SUB1 interactome in living cells

  • Cryo-electron microscopy: For high-resolution structural studies of SUB1 complexes

  • AlphaFold2 and other AI protein structure prediction: To model SUB1 interactions

• Patient-derived models:

  • Organoids: Three-dimensional culture systems that better recapitulate tumor heterogeneity

  • Patient-derived xenografts: For more clinically relevant in vivo models

  • Humanized mouse models: To study SUB1 function in the context of human immune interactions

• Computational and systems biology approaches:

  • Network analysis: To position SUB1 within larger regulatory networks

  • Multi-omics integration: Combining genomics, transcriptomics, proteomics, and metabolomics data

  • Machine learning: For prediction of SUB1 regulatory relationships and therapeutic vulnerabilities

These emerging technologies will enable researchers to develop a more comprehensive understanding of SUB1 biology, potentially revealing new therapeutic opportunities and biological insights.

How is SUB1 expression regulated in normal and disease states?

SUB1 expression is regulated through multiple mechanisms that differ between normal and disease states:

• Transcriptional regulation:

  • Normal contexts: Standard promoter-driven expression with tissue-specific factors

  • Disease states: Potential dysregulation through altered transcription factor activity or chromatin modifications

  • Stress response: SUB1 is induced by oxidative stress, suggesting stress-responsive elements in its promoter

• Post-transcriptional regulation:

  • MicroRNA control:

    • miR-101 directly targets SUB1 mRNA at its 3'-UTR

    • Downregulation of miR-101 in cancer leads to increased SUB1 expression

  • mRNA stability: Potential regulation through RNA-binding proteins that affect transcript half-life

• Post-translational modifications:

  • Phosphorylation, acetylation, and other modifications likely regulate SUB1 activity, stability, and interactions

  • These modifications may be dysregulated in disease contexts

• Protein stability regulation:

  • Ubiquitin-proteasome pathway likely controls SUB1 protein levels

  • Alterations in this pathway could contribute to SUB1 overexpression in cancer

• Disease-specific mechanisms:

  • Cancer:

    • Loss of miR-101 expression

    • Potential genomic amplification

    • Altered promoter methylation status

  • Other diseases: Different regulatory mechanisms may apply in non-cancer pathologies

Understanding these regulatory mechanisms provides potential intervention points for therapeutic strategies targeting SUB1 expression in disease contexts.

What are the tissue-specific expression patterns of SUB1 in humans?

SUB1 demonstrates distinct tissue-specific expression patterns in humans, which has implications for both normal physiology and disease:

This tissue-specific expression profile suggests context-dependent functions for SUB1 and may explain why its dysregulation contributes to specific disease states in particular tissues.

How conserved is SUB1 across species and what does this suggest about its function?

The evolutionary conservation of SUB1 provides important insights into its fundamental biological functions:

• Cross-species conservation:

  • SUB1 is evolutionarily conserved from yeast to humans, suggesting ancient and fundamental cellular functions

  • The human SUB1 gene is named after its orthologous gene in yeast, emphasizing this evolutionary relationship

• Functional conservation:

  • DNA binding: The DNA-binding capability is preserved across species

  • Transcriptional regulation: The role in transcriptional processes appears to be an ancient function

  • DNA repair: Involvement in DNA damage response pathways is observed in both yeast and humans

• Structural similarities with other proteins:

  • Yeast SUB1 shares structural and functional similarities with human alpha-synuclein, particularly in DNA repair functions

  • This suggests potential evolutionary convergence or divergence of similar functional domains

• Species-specific adaptations:

  • Despite conservation, species-specific roles have evolved

  • In yeast, SUB1 plays a negative role during starvation-induced sporulation , which may represent a specialized function

• Orthology relationships:

  • SUB1 has established orthologs documented in:

    • HomoloGene database (ID: 38218)

    • OMA orthology database

    • MGI database (ID: 104811) for mouse ortholog

The high degree of conservation across diverse species emphasizes SUB1's fundamental importance in cellular processes, particularly in transcriptional regulation and DNA repair, while species-specific adaptations highlight its evolutionary plasticity to serve specialized functions in different organisms.

What is the potential of SUB1 as a diagnostic or prognostic biomarker?

SUB1's involvement in cancer progression suggests significant potential as a diagnostic and prognostic biomarker:

• Diagnostic applications:

  • Expression level analysis:

    • Elevated SUB1 expression has been observed in multiple cancer types including prostate cancer, lung adenocarcinoma, non-small-cell lung cancer, and astrocytoma

    • This differential expression could serve as a diagnostic indicator

  • Tissue specificity:

    • Cancer-specific expression patterns may help distinguish malignant from benign conditions

• Prognostic value:

  • Correlation with aggressiveness:

    • SUB1 expression levels may correlate with cancer aggressiveness and clinical outcomes

    • Functional studies showing its role in proliferation, invasion, and metastasis support this potential

  • Predictive capabilities:

    • May predict response to specific therapies, particularly those targeting downstream effectors like PLK1

• Biomarker development considerations:

  • Assay development:

    • Immunohistochemistry for tissue samples

    • qPCR for mRNA quantification

    • Potential liquid biopsy applications (circulating tumor DNA or exosomes)

  • Clinical validation requirements:

    • Large cohort studies with clinical outcome data

    • Multivariate analysis with established prognostic factors

• Combinatorial biomarker approaches:

  • SUB1 with downstream targets:

    • Combined assessment of SUB1 with PLK1, BUB1B, or C-MYC may enhance prognostic value

  • SUB1 with miR-101:

    • The ratio of miR-101 to SUB1 expression might provide additional prognostic information

While current research establishes SUB1's potential as a biomarker, comprehensive clinical validation studies are needed before implementation in diagnostic or prognostic protocols.

What resources and databases are available for SUB1 research?

A comprehensive array of resources and databases is available to support SUB1 research:

• Genomic and genetic databases:

  • NCBI Gene: ID 10923

  • Ensembl: ENSG00000113387

  • OMIM: 600503 (Online Mendelian Inheritance in Man)

  • HGNC: 19985 (HUGO Gene Nomenclature Committee)

• Protein databases and resources:

  • UniProtKB/Swiss-Prot: P53999

  • PDB: Protein Data Bank entries for structural information

  • PDBe-KB: Consolidated structural and functional information

• Expression and functional data:

  • Gene Expression Omnibus (GEO): Repository for expression data

  • The Cancer Genome Atlas (TCGA): Cancer-specific expression and mutation data

  • GeneCards: Integrated database of human genes

  • Allen Brain Atlas: Brain-specific expression data

• Orthology resources:

  • HomoloGene: ID 38218 for evolutionary conservation data

  • OMA: Orthology matrix for SUB1 across species

  • MGI: ID 104811 for mouse ortholog information

• Functional annotation resources:

  • Gene Ontology (GO): Functional annotations including:

    • RNA binding

    • Single-stranded DNA binding

    • Transcription coactivator activity

  • Reactome: Pathway database

  • KEGG: Metabolic and signaling pathway data

• Research tools and reagents:

  • Commercial antibodies for various applications

  • Verified siRNA/shRNA constructs

  • Expression vectors

  • CRISPR guide RNA designs

Researchers should utilize these complementary resources to gain comprehensive insights into SUB1 structure, function, expression, and evolution, facilitating more effective experimental design and interpretation.

What are the best experimental models for studying SUB1 function?

Selecting appropriate experimental models is crucial for investigating SUB1 function effectively:

• Cell line models:

  • Cancer cell lines:

    • Prostate cancer: DU145, PC3 (hormone-independent), LnCaP, VCaP (hormone-responsive)

    • Lung cancer: A549, H1299

    • Other cancer types: Context-dependent selection based on research question

  • Non-cancerous controls:

    • Prostate: RWPE cells (normal prostate epithelial cells)

    • Other tissues: Primary cells or immortalized normal cell lines

  • Advantages: Easy manipulation, consistent results, well-characterized

  • Limitations: May not fully recapitulate in vivo complexity

• Patient-derived models:

  • Patient-derived xenografts (PDX)

  • Organoids

  • Primary cell cultures

  • Advantages: Better representation of tumor heterogeneity and patient-specific features

  • Limitations: More challenging to establish and manipulate

• Animal models:

  • Mouse models:

    • Xenograft models (as used in SUB1 studies)

    • Genetically engineered models

    • Conditional knockout/knockin models

  • Other models:

    • Chorioallantoic membrane (CAM) assay (used in SUB1 studies)

    • Zebrafish models for specific applications

  • Advantages: In vivo context, systemic effects

  • Limitations: Species differences, ethical considerations, cost

• Yeast models:

  • Given the conservation between yeast and human SUB1, Saccharomyces cerevisiae provides a valuable model system

  • Advantages: Genetic tractability, rapid generation time

  • Limitations: Differences in cellular complexity