EREG Human, His

How does EREG expression differ between humans and other species?

EREG demonstrates remarkable species-specific expression patterns that have significant research implications:

• EREG is expressed in the developing neocortex of humans and gorillas but is undetectable in mouse neocortex

• In human fetal neocortex (12-19 gestation weeks), EREG is expressed at higher levels in radial glia (both apical and basal) than in neurons

• The mouse Ereg gene locus shows repressive H3K27me3 modifications and lacks active chromatin marks, while the human EREG locus exhibits active H3K27ac marks in the fetal neocortex

• RT-qPCR analysis confirms this inter-species difference in EREG mRNA expression

These expression differences suggest that EREG may play an important role in primate-specific aspects of brain development, making it a crucial target for comparative developmental neurobiology research .

What technical considerations affect EREG functionality in experimental systems?

When working with recombinant EREG Human, His protein, several factors can influence its experimental performance:

• Protein conformation: Commercial EREG is often available in both native and denatured forms, with the latter primarily suitable for antibody production and immunoassays rather than functional studies

• Storage conditions: Repeated freeze-thaw cycles can diminish biological activity, necessitating single-use aliquots

• Buffer composition: The presence of carrier proteins (e.g., BSA) can prevent non-specific binding to surfaces and improve stability

• Concentration effects: EREG demonstrates dose-dependent effects, typically requiring 1-100 ng/mL for biological responses

• Receptor expression: The magnitude of cellular responses correlates with EGFR/ERBB4 receptor abundance on target cells

The purity of the recombinant protein is also critical, with most research applications requiring >80% purity as typically confirmed by SDS-PAGE analysis .

How does EREG contribute to neocortical development in primates?

EREG plays a significant role in primate-specific aspects of brain development:

• EREG supports proliferation of basal progenitor cells in the developing neocortex of primates

• Addition of EPIREGULIN to mouse neocortex (which naturally lacks Ereg expression) increases proliferation of basal progenitor cells

• EREG ablation in human cortical organoids reduces proliferation in the subventricular zone

• Treatment with EPIREGULIN promotes a further increase in proliferation of gorilla but not human basal progenitor cells, suggesting species-tailored sensitivity

• EPIREGULIN competes with EGF to promote proliferation, and inhibition of the EGF receptor abrogates the EPIREGULIN-mediated increase in basal progenitor cells

These findings suggest that species-specific regulation of EPIREGULIN expression may contribute to the increased neocortex size of primates by providing a tunable pro-proliferative signal to basal progenitor cells in the subventricular zone .

What methodological approaches can be used to study EREG in brain development?

Researchers investigating EREG's role in neurological development can employ several experimental approaches:

• Cerebral organoid models: Human or non-human primate cerebral organoids can be treated with recombinant EREG to study effects on progenitor proliferation

• CRISPR/Cas9 gene editing: EREG can be ablated in human cortical organoids to study loss-of-function effects on neurodevelopment

• Cross-species comparative studies: Adding EREG to mouse neocortical cultures (where endogenous Ereg is absent) enables examination of evolutionary differences in receptor responses

• Pharmacological inhibition: Combining EREG treatment with EGFR inhibitors confirms pathway specificity

• Epigenetic analysis: Examining histone modifications (H3K27ac, H3K27me3, H3K4me3) at the EREG locus provides insights into expression regulation

When designing these experiments, researchers should consider developmental timing, concentration-dependent effects, and the need for appropriate controls including other growth factors to establish EREG-specific effects.

What explains the differential expression of EREG between human and mouse neocortex?

The species-specific expression of EREG is regulated by complex epigenetic and genomic mechanisms:

• Chromatin state: In mouse neocortex, the Ereg locus is marked by repressive H3K27me3 and lacks active modifications like H3K4me3 and H3K27ac. In contrast, the human EREG locus shows enrichment in active H3K27ac marks

• Cis-regulatory elements (CREs): ATAC-seq and H3K27ac ChIP-seq data of fetal human neocortex identified 11 regions of open chromatin within 100 kb of the EREG gene that may represent putative active enhancers

• Genomic conservation: Of the 11 putative EREG CREs, only one shows even a small ATAC-seq peak in mouse neocortex, while the orthologous regions in mouse lack H3K27ac or ATAC-seq peaks

• Sequence divergence: While most putative CREs are at least partially conserved in the mouse, they show higher sequence divergence compared to primate species

Interestingly, experimental reduction of repressive H3K27me3 at the mouse Ereg locus using CRISPR/Cas9-based epigenome editing did not result in upregulation of Ereg gene expression, suggesting the mouse locus is in a fully repressed rather than poised state .

How is EREG being targeted in colorectal cancer research?

EREG has emerged as a promising target in cancer research, particularly for colorectal cancer (CRC), with several strategic approaches:

• Antibody-Drug Conjugates (ADCs): EREG-targeting ADCs show efficacy in both RAS wildtype and mutant colorectal cancer models

• Monoclonal antibodies: High-specificity antibodies like H231 can bind EREG with Kd values as low as 0.01 μg/ml (0.1 nmol/L)

• Expression targeting: CRISPR-Cas9-based knockout of EREG can help evaluate its role in cancer cell proliferation and survival

• Pathway inhibition: Combined targeting of EREG and downstream EGFR signaling may enhance therapeutic efficacy

The development of EREG-targeting strategies is particularly significant because EREG is highly expressed in both RAS wildtype and mutant CRC with minimal expression in normal tissues, making it an attractive target for therapeutic development .

What are the mechanisms and efficacy of EREG-targeting antibody-drug conjugates?

EREG-targeting antibody-drug conjugates (ADCs) represent a sophisticated approach to cancer therapy with several key components:

• Target selection: EREG's high expression in colorectal cancers with minimal normal tissue expression provides tumor specificity

• Antibody development: Monoclonal antibodies like H231 are developed with high specificity and affinity for human and mouse EREG

• Internalization properties: Selected antibodies must internalize to lysosomes following EREG binding

• Conjugation chemistry: Antibodies are conjugated to cytotoxic payloads like duocarmycin DM via cleavable dipeptide or tripeptide chemical linkers

• Payload mechanism: Upon internalization, lysosomal enzymes cleave the linkers, releasing the DNA-alkylating payload

Preclinical studies demonstrate that EREG ADCs are well-tolerated, neutralize EGFR pathway activity, cause significant tumor growth inhibition or regression, and increase survival in various CRC models .

What advantages do EREG-targeted therapies offer compared to direct EGFR targeting?

EREG-targeted therapies present several distinct advantages over conventional EGFR-targeted approaches:

• RAS-independence: While the efficacy of clinically approved anti-EGFR mAbs is largely limited by RAS mutational status, EREG ADCs show promise for both RAS mutant and wildtype patients

• Tumor specificity: EREG's restricted expression pattern (high in tumors, low in normal tissues) potentially reduces off-target effects compared to ubiquitous EGFR targeting

• Novel mechanism: EREG-targeted ADCs combine EGFR pathway neutralization with direct cytotoxic effects through payload delivery

• Resistance bypass: EREG targeting may overcome resistance mechanisms that develop against direct EGFR inhibition

These advantages suggest that EREG-targeted approaches could expand therapeutic options for colorectal cancer patients, particularly those with RAS mutations who derive limited benefit from current EGFR-targeted therapies .

What techniques are optimal for validating EREG-binding antibodies?

Comprehensive validation of EREG-specific antibodies requires multiple complementary approaches:

• Binding affinity determination: Surface plasmon resonance or biolayer interferometry to quantify Kd values (e.g., H231 antibody demonstrated Kd of 0.01 μg/ml or 0.1 nmol/L)

• Cell-based binding assays: Testing antibody binding using cells with confirmed EREG expression versus control cells

• Cross-reactivity testing: Evaluating binding to related EGF family members to confirm specificity

• Internalization assessment: Confocal microscopy or flow cytometry to confirm antibody internalization (critical for ADC development)

• Functional neutralization: Testing the antibody's ability to block EREG-mediated EGFR activation

• Epitope mapping: Determining the specific binding region to ensure it doesn't interfere with intended applications

For example, the development of the H231 mAb included validation using 293T cells (which lack endogenous EREG) transfected with EREG expression constructs versus vector controls to confirm binding specificity .

What are the recommended approaches for studying EREG-receptor interactions?

Several methodological approaches are valuable for investigating EREG interactions with its receptors:

• Binding kinetics: Surface plasmon resonance to measure association and dissociation rates

• Receptor activation: Western blotting or ELISA to quantify EGFR and ERBB4 phosphorylation after EREG treatment

• Downstream signaling: Monitoring activation of pathways including MAPK/ERK and PI3K/AKT

• Competitive binding: Assessing EREG competition with other EGFR ligands like EGF

• Structural studies: X-ray crystallography or cryo-EM of EREG-receptor complexes

• Crosslinking: Chemical crosslinkers to stabilize complexes for co-immunoprecipitation

When designing these experiments, it's important to include appropriate controls such as other EGFR ligands for comparison and receptor-blocking antibodies to confirm specificity. Time-course studies (5-60 minutes post-treatment) are essential to capture transient signaling events.

What experimental design is required for EREG knockout or overexpression studies?

For rigorous EREG genetic manipulation studies, several methodological considerations are critical:

• CRISPR-Cas9 knockout: Design guide RNAs targeting early exons (e.g., the sgRNA sequence 5'-GACAGAAGACAATCCACGTG-3' has been validated for EREG knockout)

• Validation of knockout: Confirm EREG deletion at both genomic (PCR, sequencing) and protein levels (Western blot, immunofluorescence)

• Overexpression systems: Clone full-length EREG or specific domains into appropriate expression vectors with tags for detection

• Expression verification: Use anti-tag antibodies (e.g., anti-myc) to confirm expression in transfected cells

• Selection strategies: Establish stable lines using appropriate antibiotics for long-term studies

• Controls: Include both vector-only controls and rescue experiments to confirm phenotype specificity

For EREG overexpression, researchers have successfully used the pIRESpuro3 vector with EREG fused to the CD8 signal peptide sequence (MALPVTALLLPLALLLHAA) followed by a Myc-tag at the N-terminus .

What are common technical challenges when working with recombinant EREG?

Researchers working with EREG Human, His protein frequently encounter several technical issues:

• Protein aggregation: His-tagged EREG may form aggregates that reduce activity

  • Solution: Add carrier proteins (0.1% BSA) and avoid vigorous vortexing

• Inconsistent cellular responses: Variability in EREG-induced signaling

  • Solution: Verify receptor expression levels and standardize cell density

• Batch variation: Different preparations showing varying potency

  • Solution: Perform functional validation of each batch using standard assays

• Signal-to-noise ratio: High background in phosphorylation assays

  • Solution: Optimize serum starvation conditions (16-24 hours) before EREG treatment

• Species cross-reactivity: Human EREG may have different potency on cells from different species

  • Solution: Validate activity using species-matched positive controls

For cellular experiments, researchers should carefully control cell confluency, passage number, and starvation conditions, as these factors can significantly influence EREG responsiveness.

How can experimental variability be minimized in EREG functional studies?

To ensure reproducible results in EREG functional studies, implement these methodological controls:

• Standardized protocols: Develop detailed SOPs for all experimental procedures

• Positive controls: Include established EGFR ligands (EGF, TGF-α) in parallel with EREG

• Dose-response analysis: Test multiple EREG concentrations (typically 0.1-100 ng/mL)

• Biological replicates: Perform experiments across multiple cell passages or donors

• Technical replicates: Include at least triplicate measurements within each experiment

• Receptor quantification: Regularly verify EGFR/ERBB4 expression levels on target cells

• Time-course studies: Examine responses at multiple time points to capture both immediate and delayed effects

• Reagent quality control: Use the same lot of recombinant EREG when possible, or validate new lots

Documentation of all experimental parameters, including cell source, passage number, culture conditions, and reagent details, is essential for troubleshooting inconsistencies and ensuring reproducibility.

What controls are essential for validating EREG-specific effects?

Robust experimental design for EREG studies requires comprehensive controls:

• Receptor dependency: Include EGFR/ERBB4 inhibitors (e.g., erlotinib) to confirm receptor-mediated effects

• Specificity controls: Test other EGF family ligands to distinguish EREG-specific versus general EGFR effects

• Genetic validation: Compare effects in EREG-knockout versus wildtype cells

• Functional neutralization: Use EREG-neutralizing antibodies to block activity

• Concentration controls: Demonstrate dose-dependent responses to establish specificity

• Vehicle controls: Include buffer-only treatments with identical carrier proteins and solvents

• Sequential treatment: Pre-treat with EGFR blockers before EREG to confirm receptor dependency

For developmental studies using organoids, additional controls should include treatment with other growth factors and comparison of effects across species (human vs. gorilla vs. mouse) to highlight species-specific responses .