Recombinant Human Suppressor of tumorigenicity 7 protein-like (ST7L)

What is ST7L and how does it relate to the ST7 gene family?

ST7L (Suppressor of Tumorigenicity 7-Like, also known as ST7R) is a novel gene homologous to the tumor suppressor gene ST7. It encodes a 575-amino-acid polypeptide that contains a leucine zipper domain and three tyrosine-phosphorylation sites. ST7L shares 72.1% total-amino-acid identity with human ST7 and 56.8% total-amino-acid identity with Drosophila CG3634 . The gene was identified through bioinformatics approaches and subsequently isolated using cDNA-PCR methods. The relationship between ST7L and ST7 suggests a potential evolutionary duplication event, with both genes maintaining similar functional domains despite their location on different chromosomes. Understanding this relationship provides valuable context for researchers studying tumor suppressor gene families and their evolutionary conservation.

What is the genomic location and structure of the ST7L gene?

The ST7L gene is located on human chromosome 1p13 and consists of at least 15 exons . Its genomic organization is notable for its proximity to the WNT2B gene, as they are clustered in a tail-to-tail arrangement with an interval of less than 5.0-kb. This arrangement mirrors the clustering of ST7 and WNT2 genes on chromosome 7q31, suggesting that these gene clusters originated from duplication of an ancestral gene cluster . The gene produces four distinct isoforms through alternative splicing mechanisms, allowing for potential functional diversity of the resulting proteins. This genomic arrangement may have implications for coordinated regulation of these genes in cellular processes and potentially in tumor suppression pathways.

What are the key structural domains and motifs in the ST7L protein?

The ST7L protein contains several noteworthy structural features that distinguish it from other proteins, including:

• A leucine zipper domain, which is unique to ST7L and not found in ST7

• Three tyrosine-phosphorylation sites, with Tyr268 and Tyr441 being conserved across ST7L, ST7, and Drosophila CG3634

• Three conserved domains designated as S7H1, S7H2, and S7H3, which are shared among ST7L, ST7, and CG3634

These structural elements provide important clues about the protein's potential functions. The leucine zipper domain suggests capability for protein-protein interactions or DNA binding, while the conserved tyrosine-phosphorylation sites indicate possible regulation through phosphorylation events. The high degree of conservation in specific domains across species emphasizes their functional importance and suggests evolutionary pressure to maintain these regions intact.

What are the most effective methods for recombinant expression of ST7L protein?

When expressing recombinant ST7L protein, researchers should consider a systematic experimental design approach. Begin by optimizing codon usage for the expression system of choice, as this can significantly impact protein yield. For mammalian expression, HEK293 or CHO cells often provide proper post-translational modifications, while E. coli systems may be suitable if glycosylation is not critical.

A typical expression protocol includes:

• Gene synthesis or PCR amplification from cDNA with appropriate restriction sites

• Cloning into an expression vector with a suitable tag (His, GST, or FLAG)

• Transfection/transformation into the chosen expression system

• Expression optimization by varying temperature, induction time, and media composition

• Protein extraction using methods compatible with downstream applications

For complex proteins like ST7L with multiple domains, it may be beneficial to express individual domains separately if full-length expression proves challenging. Additionally, co-expression with chaperone proteins may improve folding efficiency. The experimental design should include appropriate controls and multiple replication to ensure statistical validity of the results .

How can researchers effectively design experiments to study ST7L functionality?

When designing experiments to study ST7L functionality, researchers should employ a true experimental research design with proper controls and variable manipulation. Begin by clearly defining your research questions and hypotheses regarding ST7L function. For example, a null hypothesis might state: "ST7L knockdown has no effect on cell proliferation in cancer cell lines."

The experimental design should include:

• Independent Variables: Manipulation of ST7L expression (overexpression, knockdown, mutation of specific domains)

• Dependent Variables: Measurable outcomes such as cell proliferation, migration, gene expression profiles, or protein interactions

• Control of Extraneous Variables: Use of proper control groups, randomization, and standardized conditions

For ST7L functional studies, consider employing both gain-of-function (overexpression) and loss-of-function (siRNA knockdown or CRISPR/Cas9 knockout) approaches in relevant cell lines. Time-course experiments are recommended to capture dynamic processes. Additionally, rescue experiments where wild-type ST7L is reintroduced after knockdown provide powerful evidence for function specificity. The leucine zipper domain unique to ST7L suggests potential DNA binding or protein interaction capabilities, which could be investigated using chromatin immunoprecipitation or co-immunoprecipitation methods respectively .

What cell models are most appropriate for studying ST7L in tumor suppression research?

Selecting appropriate cell models for ST7L tumor suppression research requires consideration of the tissue context and potential pathway interactions. Based on the genomic proximity of ST7L to WNT2B and its homology to the tumor suppressor ST7, cell lines with active WNT signaling pathways may be particularly informative .

Consider the following approach:

• Primary Screening: Test ST7L expression levels across a panel of normal and cancer cell lines from multiple tissue origins using qRT-PCR and western blotting

• Selection Criteria:

  • Cell lines with varied endogenous ST7L expression

  • Cell lines where WNT signaling status is characterized

  • Models representing tissues where chromosome 1p13 alterations are documented

• Experimental Models:

  • 2D cell culture for basic mechanistic studies

  • 3D organoids for more physiologically relevant contexts

  • Xenograft models for in vivo validation

When designing your experimental panels, include both cell lines with intact tumor suppressor pathways and those with specific defects. This comparative approach allows identification of synthetic lethal interactions and context-dependent functions. Additionally, isogenic cell line pairs differing only in ST7L status provide powerful tools for attributing observed phenotypes specifically to ST7L function .

How does ST7L potentially interact with the WNT signaling pathway?

The genomic arrangement of ST7L and WNT2B in a tail-to-tail cluster on chromosome 1p13 mirrors the arrangement of ST7 and WNT2 on chromosome 7q31, suggesting potential functional relationships between these genes . This genomic organization raises intriguing questions about ST7L's role in WNT signaling.

When investigating ST7L-WNT pathway interactions, consider these methodological approaches:

• Pathway Reporter Assays: Utilize TCF/LEF luciferase reporters to measure canonical WNT pathway activity in the presence of normal or altered ST7L expression

• Protein Interaction Studies: Perform co-immunoprecipitation experiments to determine if ST7L directly interacts with WNT pathway components

• Gene Expression Analysis: Conduct RNA-seq following ST7L modulation to identify changes in WNT target gene expression

• Phosphorylation Status: Assess β-catenin phosphorylation levels in response to ST7L manipulation

Given that WNT2 and WNT2B isoform 2 (WNT2B2) are positive regulators of the WNT-β-catenin-TCF signaling pathway , researchers should specifically investigate whether ST7L functions as a tumor suppressor by modulating this pathway. The leucine zipper domain unique to ST7L may enable protein-protein interactions or transcriptional regulation functions that affect WNT signaling output. A systematic approach comparing the effects of wild-type ST7L versus domain-specific mutants will help delineate the precise mechanisms of these interactions.

What are the implications of ST7L's tyrosine phosphorylation sites for its function?

ST7L contains three tyrosine phosphorylation sites, with Tyr268 and Tyr441 conserved across ST7L, ST7, and Drosophila CG3634 . This high level of evolutionary conservation strongly suggests functional importance of these sites. When investigating the role of these phosphorylation sites, researchers should employ the following methodological approaches:

• Site-Directed Mutagenesis: Generate phospho-null (Y→F) and phospho-mimetic (Y→E or Y→D) mutants for each site

• Phosphorylation Detection: Use phospho-specific antibodies or mass spectrometry to determine basal phosphorylation status and stimulus-induced changes

• Kinase Identification: Perform in vitro kinase assays with candidate kinases and utilize kinase inhibitors in cellular systems

• Functional Comparisons: Assess how phosphorylation site mutants differ from wild-type ST7L in cellular assays of proliferation, migration, and pathway activation

Experimental designs should include appropriate controls and statistical analyses to validate findings. For instance, experiments testing phosphorylation-dependent functions should include both phospho-null and phospho-mimetic variants alongside wild-type protein to distinguish between structural and phosphorylation-specific effects .

The functional consequences of phosphorylation may include altered protein stability, subcellular localization, protein-protein interactions, or enzymatic activity. Systematic characterization of these parameters in wild-type versus mutant ST7L will provide mechanistic insights into how post-translational modifications regulate this potential tumor suppressor.

How do the four ST7L isoforms differ in structure and potential function?

The ST7L gene produces four distinct isoforms through alternative splicing , which may exhibit isoform-specific functions in different cellular contexts. When investigating these isoforms, researchers should employ a systematic comparative approach:

• Structural Analysis:

  • Perform computational domain analysis to identify differences in functional motifs

  • Determine which domains are present/absent in each isoform

  • Assess conservation of key features (leucine zipper, phosphorylation sites) across isoforms

• Expression Profiling:

  • Quantify isoform-specific expression across tissues and cell lines using RT-PCR with isoform-specific primers

  • Analyze public RNA-seq datasets to identify tissue-specific or disease-associated expression patterns

• Functional Characterization:

  • Express each isoform individually in appropriate cell models

  • Compare effects on cellular phenotypes (proliferation, migration, differentiation)

  • Assess molecular interactions specific to each isoform

• Localization Studies:

  • Determine subcellular localization of each isoform using fluorescent tagging

  • Investigate whether localization changes in response to cellular stimuli

A well-designed experimental approach would involve expressing each isoform individually in a cell line with minimal endogenous ST7L expression, followed by comprehensive phenotypic and molecular characterization. This controlled system allows direct comparison of isoform-specific effects while minimizing confounding variables .

What are the optimal approaches for studying ST7L in cancer genomics databases?

When investigating ST7L in cancer genomics databases, researchers should implement a comprehensive multi-database strategy focusing on both genomic alterations and expression patterns. The chromosome 1p13 region where ST7L resides may be subject to copy number alterations or structural variations in certain cancer types.

A methodical approach includes:

• Database Selection and Integration:

  • Primary databases: TCGA, ICGC, cBioPortal, COSMIC

  • Specialized databases: Cancer Cell Line Encyclopedia, Cancer Dependency Map

  • Expression databases: GTEx, CCLE, Human Protein Atlas

• Analysis Parameters:

  • Copy number variations affecting the ST7L locus

  • Mutational patterns (missense, nonsense, frameshift)

  • Expression correlation with clinical outcomes

  • Co-expression patterns with WNT2B and pathway components

• Comparative Analysis:

  • Compare ST7L alterations with those of ST7 to identify shared or distinct patterns

  • Analyze chromosome 1p13 and 7q31 regions for coordinated alterations

• Validation Approaches:

  • Select representative cell lines based on database findings for experimental validation

  • Design experiments to test hypotheses generated from in silico analysis

This bioinformatic approach should be structured as a true experimental design with clearly defined hypotheses and appropriate statistical analyses . For example, researchers might hypothesize that ST7L expression correlates with WNT pathway gene expression signatures in specific cancer types. Such hypotheses can be tested through correlation analysis in public datasets before moving to experimental validation.

How can CRISPR/Cas9 technology be optimized for studying ST7L function?

CRISPR/Cas9 technology offers powerful approaches for investigating ST7L function through precise genetic manipulation. When designing CRISPR experiments for ST7L research, consider the following methodological framework:

• Guide RNA Design:

  • Target conserved exons present in all four splice variants for complete knockout

  • For isoform-specific studies, design gRNAs targeting unique exons

  • Include controls for off-target effects using multiple guide RNA strategies

• Modification Strategies:

  • Complete knockout: Use paired gRNAs to create frameshift deletions

  • Domain-specific studies: Create precise edits to modify the leucine zipper domain or phosphorylation sites

  • Endogenous tagging: Knock-in fluorescent or affinity tags for localization and interaction studies

• Validation Protocols:

  • Genomic verification: PCR and sequencing of target region

  • Transcript analysis: RT-PCR and RNA-seq to confirm altered expression

  • Protein verification: Western blot to confirm protein loss or modification

• Phenotypic Assays:

  • Proliferation, migration, and invasion assays

  • Pathway reporter assays focusing on WNT signaling

  • Gene expression profiling to identify downstream effects

A particularly powerful approach is to create isogenic cell line pairs differing only in ST7L status, allowing direct attribution of observed phenotypes to ST7L function. For studying the physiological relevance of specific domains, consider using homology-directed repair to introduce point mutations affecting the leucine zipper domain or phosphorylation sites .

What are the best practices for developing antibodies against ST7L for research applications?

Developing specific antibodies against ST7L requires careful antigen design and validation strategies to ensure specificity, particularly given its homology to ST7. The following methodological approach is recommended:

• Antigen Design Strategy:

  • Identify unique epitopes in ST7L not present in ST7 (approximately 28% of residues differ)

  • Consider multiple antigens: full-length protein, unique peptide sequences, and domain-specific antigens

  • For phospho-specific antibodies, design peptides containing phosphorylated Tyr268 or Tyr441

• Production Approaches:

  • Monoclonal antibodies: Provide high specificity but recognize single epitopes

  • Polyclonal antibodies: Recognize multiple epitopes but may have cross-reactivity

  • Recombinant antibodies: Allow for reproducible production and engineering

• Validation Protocol:

  Validation Method	Purpose	Acceptance Criteria
  Western blot	Confirm specificity	Single band at expected MW; absence in knockout cells
  Immunoprecipitation	Verify native protein recognition	Enrichment of target protein
  Immunofluorescence	Assess subcellular localization	Expected localization pattern; absence in knockout cells
  Peptide competition	Confirm epitope specificity	Signal reduction with specific peptide
  Cross-reactivity test	Ensure no ST7 recognition	No signal with purified ST7 protein

• Application Optimization:

  • Determine optimal working conditions for each application

  • Create standard operating procedures for reproducibility

  • Validate across multiple cell types expressing different ST7L isoforms

A rigorous validation approach is essential, particularly testing antibodies in both ST7L-expressing and ST7L-knockout cells to confirm specificity. For phospho-specific antibodies, additional validation using phosphatase treatment and kinase activators/inhibitors is required to confirm phosphorylation specificity .

How does ST7L compare evolutionarily with ST7 and other related proteins?

Evolutionary analysis of ST7L reveals important insights into functional conservation and specialization. ST7L shares 72.1% total amino acid identity with human ST7 and 56.8% with Drosophila CG3634, suggesting a common ancestral origin . When conducting evolutionary analyses of ST7L, researchers should employ these methodological approaches:

• Sequence-Based Phylogenetic Analysis:

  • Construct multiple sequence alignments using MUSCLE or CLUSTAL algorithms

  • Generate phylogenetic trees using maximum likelihood or Bayesian methods

  • Calculate evolutionary rates to identify rapidly evolving or conserved regions

• Domain Conservation Assessment:

  • Compare conservation of the three shared domains (S7H1, S7H2, and S7H3)

  • Analyze the unique leucine zipper domain in ST7L to determine when this feature emerged

  • Evaluate conservation of phosphorylation sites across species

• Genomic Synteny Analysis:

  • Examine the ST7L-WNT2B and ST7-WNT2 genomic arrangements across species

  • Identify when the proposed ancestral gene duplication event likely occurred

  • Map chromosomal rearrangements that may have influenced gene function

• Functional Divergence Assessment:

  • Compare expression patterns of ST7L and ST7 across tissues and developmental stages

  • Evaluate differences in protein interaction networks

  • Analyze selection pressure on different domains using dN/dS ratios

This evolutionary perspective provides crucial context for understanding ST7L function. The high conservation of certain domains suggests core functional roles, while divergent features like the leucine zipper domain may represent functional specialization. The tail-to-tail clustering with WNT family genes across evolutionary time emphasizes the potential importance of coordinated regulation between these gene pairs .

What techniques are most effective for comparing the functional differences between ST7 and ST7L?

To rigorously compare the functional differences between ST7 and ST7L, researchers should implement parallel experimental designs that systematically assess various aspects of protein function. The 72.1% amino acid identity between these proteins suggests both shared and distinct functions .

A comprehensive comparative approach includes:

• Expression Pattern Comparison:

  • Analyze tissue-specific expression using qRT-PCR and western blotting

  • Examine subcellular localization patterns through immunofluorescence

  • Investigate expression changes during development or disease progression

• Functional Substitution Experiments:

  • Conduct rescue experiments in knockout/knockdown systems

  • Determine whether ST7L can functionally compensate for ST7 loss and vice versa

  • Create chimeric proteins exchanging specific domains to identify functional regions

• Protein Interaction Network Mapping:

  • Perform parallel co-immunoprecipitation studies followed by mass spectrometry

  • Conduct yeast two-hybrid screenings for both proteins

  • Compare interaction partners using proximity labeling techniques (BioID, APEX)

• Transcriptional Impact Assessment:

  • Compare gene expression changes following ST7 or ST7L modulation using RNA-seq

  • Identify shared and distinct transcriptional targets

  • Conduct chromatin immunoprecipitation if DNA binding is suspected

A well-designed experimental approach would use isogenic cell lines with individual or combined knockouts of ST7 and ST7L, followed by rescue with either wild-type protein or domain swaps between the two. This strategy allows precise delineation of shared functions versus unique roles of each protein, with particular attention to the leucine zipper domain unique to ST7L .

How might the genomic clustering of ST7L-WNT2B and ST7-WNT2 influence their coordinated functions?

The tail-to-tail genomic arrangement of ST7L-WNT2B and ST7-WNT2 gene pairs, with intervals of less than 5.0 kb, suggests potential coordinated regulation or functional relationships . This genomic architecture may influence gene expression, chromatin organization, and functional interactions. When investigating these relationships, researchers should consider these methodological approaches:

• Coordinated Expression Analysis:

  • Quantify expression correlation between gene pairs across tissues and conditions

  • Investigate shared regulatory elements using chromatin accessibility assays

  • Determine whether the genes respond similarly to cellular stimuli

• Chromatin Structure Investigation:

  • Employ Chromosome Conformation Capture (3C, 4C, Hi-C) to identify physical interactions

  • Map enhancer-promoter interactions that might influence both genes

  • Investigate insulator elements that could independently regulate each gene

• Functional Interaction Assessment:

  • Determine whether ST7L modulates WNT2B signaling output

  • Compare with ST7's influence on WNT2 signaling

  • Investigate whether gene pairs show synthetic phenotypes when co-modified

• Evolutionary Conservation Analysis:

  • Examine whether the tail-to-tail arrangement is conserved across species

  • Identify species where gene duplication or divergence occurred

  • Assess selective pressure maintaining this genomic arrangement

A particularly informative experimental design would utilize CRISPR/Cas9 to modify the genomic distance or orientation between gene pairs and assess the impact on expression and function. Complementary approaches could include enhancer deletion studies and forced expression of one gene to determine effects on its partner .