POU5F1 Human, PolyR

What is the structure and function of POU5F1?

POU5F1 (also known as Oct4, Oct-3, Oct-4, OTF-3, OTF-4) is a homeodomain transcription factor belonging to the POU family. The human recombinant protein is a single, non-glycosylated polypeptide chain containing 360 amino acids with a molecular mass of 38.6kDa. Structurally, human POU5F1 contains a 75aa POU-specific (POUS) domain and a 60aa POU-Homeo-(POUH) domain connected by a linker region .

The protein functions as a critical transcription factor that specifically interacts with the Octamer motif ATGCAAAT. Its significance lies in its role in maintaining self-renewal and pluripotency of embryonic stem (ES) cells. POU5F1 possesses two distinct DNA binding domains that independently bind half-sites of the canonical octamer motif, giving it flexibility to bind with distinct DNA motifs by forming heterodimers with other transcription factors or homodimers in various conformations .

How does POU5F1 regulate pluripotency at the molecular level?

POU5F1 regulates pluripotency through multiple molecular mechanisms:

• Transcriptional regulation: POU5F1 regulates numerous target genes essential for maintaining the undifferentiated state of stem cells.

• Cooperative action: It works jointly with other transcription factors, particularly Sox2 and Nanog, forming a core regulatory network that sustains stem cell potency and self-renewal .

• Domain-specific interactions: The two proline-rich domains in the N-terminal and C-terminal regions are vital for POU5F1 transactivation, enabling precise control of target gene expression .

• Enhancer regulation: The expression of POU5F1 itself is regulated through distal and proximal enhancers, allowing for context-specific control of pluripotency networks .

Research indicates that disruption of POU5F1 expression leads to loss of pluripotency and differentiation of stem cells, highlighting its essential role in stem cell biology.

What are the optimal storage and handling conditions for POU5F1 Human, PolyR recombinant protein?

For optimal results when working with POU5F1 Human, PolyR recombinant protein, follow these evidence-based handling protocols:

Storage conditions:

• Short-term storage (2-4 weeks): Store at 4°C if the entire vial will be used within this period

• Long-term storage: Store frozen at -20°C

• Avoid multiple freeze-thaw cycles to maintain protein integrity and activity

Formulation details:

• The protein is supplied as a 0.2μm filtered solution in PBS, pH 7.4 with 5% glycerol

• This formulation provides stability while maintaining biological activity

Quality control parameters:

• Purity: >95.0% as determined by SEC-HPLC and SDS-PAGE analysis

• N-terminal sequence verification: The first five amino acids are Met-Ala-Gly-His-Leu

When designing experiments, consider the C-terminal poly-arginine tag, which may influence protein behavior in certain experimental contexts, particularly in protein-protein interaction studies.

How can POU5F1 Human, PolyR be effectively used in stem cell differentiation studies?

When implementing POU5F1 Human, PolyR in stem cell differentiation studies, consider this methodological framework:

• Baseline expression mapping: Before experimental manipulation, determine endogenous POU5F1 expression levels in your stem cell system. This provides a reference point for interpreting results after recombinant protein introduction.

• Dosage optimization: Titrate the recombinant protein concentration (typically starting with 10-100 ng/mL) to determine the optimal concentration for your specific cell type.

• Temporal dynamics assessment: Monitor POU5F1 levels throughout differentiation processes. Research shows that Pcbp1 is essential for timely Oct4 downregulation upon differentiation signals, and residual Oct4 expression leads to primitive endoderm specification .

• Integration with differentiation protocols: When using all-trans retinoic acid (RA) for differentiation, note that cells typically show complete pluripotency shutdown after three days, evidenced by loss of Oct4 and Nanog expression .

• Interaction studies: Design experiments to examine POU5F1 interactions with other pluripotency factors like Sox2 and Nanog, as these cooperative interactions are critical for maintaining stem cell states .

This approach allows for systematic evaluation of POU5F1's role in maintaining pluripotency and regulating differentiation pathways.

How can chromatin immunoprecipitation (ChIP) experiments be optimized when studying POU5F1 binding sites?

When conducting ChIP experiments to study POU5F1 binding sites, implement these advanced protocol optimizations:

• Fixation optimization: Due to POU5F1's dynamic binding properties, test crosslinking times (typically 10-15 minutes with 1% formaldehyde) to preserve authentic protein-DNA interactions without over-fixation.

• Sonication parameters: Optimize sonication to generate DNA fragments of 200-500 bp, which is ideal for detecting specific octamer motif binding sites (ATGCAAAT) that POU5F1 preferentially targets .

• Antibody selection: For recombinant POU5F1 Human, PolyR, use antibodies that recognize the native protein structure rather than the poly-arginine tag to avoid artificial binding patterns.

• Control selection: Include the following controls:

  • Input chromatin (pre-immunoprecipitation)

  • IgG negative control

  • Positive control regions (known POU5F1 binding sites)

  • Negative control regions (genomic regions without octamer motifs)

• Enhanced detection of enhancer regions: Research has shown that Pcbp1 occupies poly(C)-sites of the POU5F1 enhancers in embryonic stem cells. Design your primers to cover both the 2A and 1A sites of the POU5F1 gene to capture these regulatory interactions .

• Cross-validation: Validate ChIP-qPCR findings with ChIP-seq when possible to generate genome-wide binding profiles.

This methodological approach allows for precise mapping of POU5F1 binding sites and helps elucidate its role in transcriptional regulation networks.

What are the methodological considerations when investigating POU5F1's role in cancer progression models?

When investigating POU5F1's role in cancer progression, implement these evidence-based methodological approaches:

• Expression profiling stratification: Quantify POU5F1 expression across different cancer stages and correlate with clinical outcomes. Research has shown that POU5F1 gene expression in colorectal cancer serves as a novel prognostic marker .

• Functional analysis in cancer stem cells (CSCs):

  • Design experiments to test the cancer stem cell hypothesis that reactivation of early embryonic stem cell genes like POU5F1 in somatic stem cells may lead to transformation into CSCs

  • This approach helps investigate cancer initiation, promotion, and progression mechanisms

• Genetic polymorphism analysis: Consider incorporating genotyping for POU5F1 polymorphisms in your cancer models. Studies have identified that variants of rs887468 and rs3130457 are significantly associated with increased lung cancer risk .

• Interaction studies with oncogenic pathways:

  • Investigate POU5F1's interaction with the AKT-Onzin axis in lung adenocarcinoma to understand radiation resistance mechanisms

  • Design experiments to evaluate how POU5F1 and Nanog co-expression enhances drug resistance and promotes epithelial-mesenchymal transition in lung adenocarcinoma

• Knockdown/overexpression approach: Implement both knockdown (using siRNA or CRISPR) and overexpression (using recombinant POU5F1 Human, PolyR) strategies to investigate how POU5F1 modulates tumorigenic and metastatic abilities .

These approaches provide a comprehensive framework for investigating POU5F1's multifaceted roles in cancer biology.

How should researchers address inconsistent results in POU5F1 expression studies?

When encountering variability in POU5F1 expression studies, implement this systematic troubleshooting approach:

• Validate protein quality: Confirm recombinant POU5F1 Human, PolyR integrity using:

  • SDS-PAGE analysis to verify the expected 38.6kDa molecular weight

  • SEC-HPLC to ensure >95% purity as specified in product documentation

• Evaluate experimental conditions:

  • Culture medium composition: POU5F1 expression is sensitive to serum factors and LIF (leukemia inhibitory factor)

  • Cell density: Overgrowth can affect expression patterns

  • Passage number: Higher passages may show altered expression profiles

• Control for post-translational modifications: POU5F1 undergoes various post-translational modifications that affect its activity. Standardize protocols to account for these modifications when comparing across experiments .

• Address technical variability:

  • Normalize data using multiple reference genes when performing qPCR

  • Use consistent antibody lots for immunodetection methods

  • Include positive controls (embryonic stem cells) and negative controls (fully differentiated cells)

• Context-dependent regulation: Be aware that POU5F1 expression is regulated differently depending on cell type and developmental context. For example, in zebrafish development, both protein expression and post-translational modifications vary significantly during early developmental stages .

By systematically addressing these factors, researchers can improve reproducibility and interpretation of POU5F1 expression studies.

What are the key considerations when interpreting POU5F1 binding data in different cellular contexts?

When interpreting POU5F1 binding data across different cellular contexts, consider these methodological principles:

• Context-specific binding partners: POU5F1 binding patterns vary based on available cofactors in different cell types:

  • In embryonic stem cells, POU5F1 frequently partners with Sox2

  • In cancer cells, alternative binding partners may emerge, altering target gene selection

• Chromatin accessibility variations: Different cell types exhibit unique chromatin landscapes:

  • Use ATAC-seq or DNase-seq data to correlate POU5F1 binding with accessible chromatin regions

  • Note that rs887468 and rs3130457 fall into DNase I peaks, suggesting these regions are potential genetic regulatory loci that correlate with binding sites of sequence-specific DNA-binding proteins

• Differential enhancer utilization:

  • POU5F1 expression is regulated through different enhancers depending on context

  • When analyzing binding in stem cells versus differentiated cells, note that both distal and proximal enhancers of POU5F1 are active in the presence of serum and LIF (SL condition)

• Binding versus functional impact:

  • Distinguish between mere occupancy and functional regulation

  • Integrate binding data with expression data to determine which binding events lead to transcriptional changes

• Technical biases in detection methods:

  • ChIP-seq may favor high-affinity binding sites

  • Different antibodies may detect distinct POU5F1 conformations or modified forms

This framework helps researchers accurately interpret POU5F1 binding data across diverse experimental systems and avoid context-dependent misinterpretations.

How might POU5F1 polymorphisms contribute to personalized cancer risk assessment and treatment?

Recent research suggests a promising role for POU5F1 polymorphisms in personalized oncology, with important methodological considerations for implementation:

• Genetic risk stratification:

  • Studies have identified that variant alleles of rs887468 and rs3130457 in POU5F1 are significantly associated with increased lung cancer risk (OR = 1.29)

  • These findings suggest potential utility in genetic screening panels for cancer risk assessment

• Interaction with environmental factors:

  • Research has detected significant interactions between rs887468 genotypes and smoking status on lung cancer risk (P = 0.017)

  • This gene-environment interaction model could be integrated into comprehensive risk assessment tools

• Allele-dosage effects:

  • Combined analysis shows a significant allele-dosage association between the number of risk alleles and increased lung cancer risk (P trend < 0.001)

  • This dose-response relationship provides a quantitative framework for risk assessment

• Tissue-specific expression patterns:

  • POU5F1 shows differential expression across tissue types, with notable expression in reproductive tissues and certain brain regions

  • This tissue specificity should inform targeted therapeutic approaches

• Potential therapeutic targeting:

  • POU5F1's role in cancer stem cell maintenance suggests it could be a viable therapeutic target

  • Knockdown of POU5F1 has been shown to impede tumorigenic and metastatic ability and reverse the epithelial-mesenchymal transition process in lung adenocarcinoma

These findings point toward a future where POU5F1 genotyping could become part of personalized cancer risk assessment and treatment selection protocols, particularly for lung and colorectal cancers.

What are the emerging techniques for studying POU5F1's role in the dynamic regulation of pluripotency networks?

Cutting-edge methodological approaches for investigating POU5F1's role in pluripotency regulation include:

• Single-cell multi-omics integration:

  • Combining single-cell RNA-seq with single-cell ATAC-seq or ChIP-seq to correlate POU5F1 binding with transcriptional outcomes at the individual cell level

  • This approach reveals heterogeneity in pluripotency states that bulk analysis might miss

• Live-cell imaging of POU5F1 dynamics:

  • Using fluorescently tagged POU5F1 to monitor real-time protein dynamics during differentiation

  • This technique has revealed that Pcbp1 is essential for timely Oct4 downregulation upon differentiation signals

• CRISPR-based functional genomics:

  • Employing CRISPR activation/inhibition systems to modulate POU5F1 expression or target its regulatory elements

  • Genetic tools for fate mapping of Oct4 (Pou5f1)-expressing cells provide temporal control for lineage tracing experiments

• Structural biology approaches:

  • Cryo-EM and X-ray crystallography studies of POU5F1 complexes with DNA and partner proteins

  • These methods provide atomic-level insights into how POU5F1's POUS and POUH domains interact with their targets

• Computational modeling of pluripotency networks:

  • Using machine learning algorithms to predict POU5F1 binding sites and regulatory networks

  • These models can integrate multiple data types to predict cell state transitions