Recombinant Dog Steroid hormone receptor ERR1 (ESRRA)

What is the structure and function of canine ESRRA?

ESRRA (Estrogen-related receptor alpha) is a NR3 Steroid Receptor that was isolated based on sequence similarity in its DNA-binding domain to estrogen receptor alpha (ER alpha). Structurally, ESRRA binds as a monodimer to the extended half-site TNAAGGTCA and as a homodimer to the estrogen response element (ERE). It functions as a constitutive activator of the estrogen response element and the palindromic thyroid hormone response element (TRE) but not of the glucocorticoid response element (GRE) .

Functionally, ESRRA regulates the promoters of several genes including lactoferrin, medium-chain acyl CoA dehydrogenase, osteopontin, and thyroid receptor alpha. The receptor may affect cellular energy balance and bone formation. Recent studies have identified Phe-329 as the residue responsible for the constitutive activity of ESRRA .

How does canine ESRRA differ from human ESRRA in terms of sequence homology and function?

While the search results don't provide specific information about sequence differences between canine and human ESRRA, research suggests conservation of key functional domains across species. When working with recombinant canine ESRRA, researchers should consider:

• DNA binding domain conservation

• Ligand binding pocket structure

• Post-translational modification sites

• Species-specific cofactor interactions

Experimental validation of cross-species functional conservation is recommended before extrapolating findings from canine to human models or vice versa.

What are the recommended expression systems for producing recombinant canine ESRRA?

For optimal expression of functional recombinant canine ESRRA, consider these methodological approaches:

• Bacterial expression systems: Suitable for producing protein fragments for structural studies, but may lack post-translational modifications

• Mammalian expression systems: HEK293 or CHO cells are preferred for full-length ESRRA with proper folding and modifications

• Baculovirus expression: Useful for large-scale production with intermediate level of post-translational modifications

Expression optimization should include codon optimization, proper selection of affinity tags (His-tag is commonly used), and validation of protein functionality through DNA binding assays .

How can ESRRA knockout models be used to study adipogenesis and bone formation?

ESRRA knockout models provide valuable insights into the receptor's role in adipogenesis and bone formation. Research indicates that ERRalpha null mice exhibit altered regulation of genes involved in adipogenesis . When designing knockout studies:

• Consider both global and tissue-specific knockout approaches

• Evaluate phenotypic changes in bone marrow adipocytes (BMAds) and white adipose tissue (WAT)

• Monitor changes in adipogenic marker genes such as Pparg, Cebpa, and Fabp4, which are down-regulated upon ESRRA abrogation

• Assess bone formation parameters including osteoblast activity and mineralization

Recent findings demonstrate that ESRRA ablation in BMAds leads to altered expression of secreted factors, particularly a significant increase in SPP1 (osteopontin) expression . This suggests a potential regulatory role in the balance between adipogenesis and osteogenesis.

What are the methodological considerations for studying ESRRA-mediated transcriptional regulation?

When investigating ESRRA-mediated transcriptional regulation:

• Promoter binding analysis: Use chromatin immunoprecipitation (ChIP) to confirm direct binding to target gene promoters. ESRRA binds to specific DNA sequences, including extended half-site TNAAGGTCA and ERE elements .

• Reporter assays: Employ luciferase reporter constructs containing potential ERRE (ESRRA response elements) to quantify transcriptional activity.

• Mutational analysis: Generate point mutations in potential binding sites to confirm functional relevance, similar to studies that identified Phe-329 as critical for constitutive activity .

• Coregulator interactions: Investigate interactions with coactivators and corepressors that may modulate ESRRA activity in different cellular contexts.

Research has shown that ESRRA positively regulates Leptin transcription by binding directly to its promoter, while negatively regulating SPP1 expression by interfering with E2/ESR1 signaling . These opposing regulatory mechanisms highlight the complexity of ESRRA-mediated transcriptional control.

What approaches can be used to study cross-talk between ESRRA and other nuclear receptors in dogs?

To investigate nuclear receptor cross-talk involving ESRRA:

• Co-immunoprecipitation (Co-IP): Detect physical interactions between ESRRA and other nuclear receptors

• Sequential ChIP (ChIP-reChIP): Determine co-occupancy of regulatory regions by multiple transcription factors

• Transcriptome analysis: Compare gene expression profiles in the presence of ESRRA alone versus ESRRA with other nuclear receptors

• Competition assays: Assess competition for DNA binding sites or coregulators

The relationship between ESRRA and ESR1 (estrogen receptor alpha) is particularly interesting, as evidence suggests ESRRA can interfere with E2/ESR1 signaling to regulate SPP1 expression . This interference mechanism represents an important aspect of nuclear receptor cross-talk that may influence various physiological processes.

How does ESRRA regulate SPP1 (osteopontin) expression in adipocytes?

ESRRA functions as a negative regulator of SPP1 expression in adipocytes. The molecular mechanism involves:

• ESRRA interference with E2/ESR1 signaling pathways

• ESRRA ablation in bone marrow adipocytes (BMAds) leads to dramatically enhanced SPP1 expression

• ESRRA knockdown increases SPP1 expression in white adipose tissue (gWAT) adipocytes

Research using qRT-PCR profiling and western blot analysis confirmed that SPP1 expression is significantly enhanced by ESRRA abrogation in fully differentiated BMAds . This regulatory mechanism appears to be consistent across different adipose tissue types, suggesting a conserved role for ESRRA in controlling SPP1 expression.

Comparative studies in ovariectomized (OVX) mice showed that circulating SPP1 was significantly decreased in OVX mice but was partially rescued in EsrraAKO OVX mice (adipocyte-specific ESRRA knockout) . This further supports the negative regulatory role of ESRRA on SPP1 expression.

What is the relationship between ESRRA and LEPTIN expression in adipocytes?

ESRRA positively regulates LEPTIN expression through direct transcriptional control:

• ESRRA deficient mice display declined levels of circulating LEPTIN

• LEPTIN expression is repressed in both white adipose tissue (WAT) adipocytes and bone marrow adipocytes (BMAds) in ESRRA knockout models

• ESRRA directly binds to the Leptin promoter through putative ESRRA response elements (ERREs)

Experimental evidence has identified four putative ERREs (S1-S4) on the Leptin promoter that serve as binding sites for ESRRA . This direct binding mechanism explains how ESRRA positively regulates Leptin transcription.

This regulatory relationship has significant implications for energy metabolism and adipocyte function, as LEPTIN is a critical hormone in regulating energy homeostasis and fat storage.

How can the constitutive activity of ESRRA be modulated experimentally?

ESRRA is known for its constitutive (ligand-independent) activity, with Phe-329 being identified as the residue responsible for this constitutive activity . Researchers can experimentally modulate this activity through:

• Site-directed mutagenesis: Mutating Phe-329 can alter the constitutive activity of ESRRA

• Synthetic ligands: Using inverse agonists that can bind to ESRRA and reduce its constitutive activity

• Coregulator manipulation: Overexpressing or knocking down specific coactivators or corepressors

• Post-translational modifications: Inducing or inhibiting phosphorylation, acetylation, or SUMOylation that may affect ESRRA activity

Understanding and manipulating the constitutive activity of ESRRA is particularly important when studying its role in physiological and pathological conditions where its activity may be dysregulated.

What is the role of ESRRA in canine metabolic disorders?

While the search results don't provide specific information about ESRRA in canine metabolic disorders, its established role in regulating energy metabolism suggests potential involvement. Experimental approaches to investigate this include:

• Comparing ESRRA expression and activity in healthy dogs versus those with metabolic disorders

• Correlating ESRRA expression with metabolic parameters such as insulin sensitivity and lipid profiles

• Examining the effects of ESRRA modulation on metabolic outcomes in canine models

ESRRA's regulation of Leptin expression is particularly relevant to metabolic disorders, as Leptin plays a central role in energy homeostasis. Additionally, ESRRA's effects on adipogenesis suggest it could influence obesity development and metabolic syndrome in dogs.

How can ESRRA be targeted for therapeutic applications in dogs?

Based on research findings, potential therapeutic strategies targeting ESRRA include:

• Inverse agonists: Developing compounds that reduce ESRRA's constitutive activity

• Tissue-specific modulation: Creating delivery systems that target ESRRA in specific tissues (e.g., adipose tissue)

• Gene therapy approaches: Using viral vectors to modulate ESRRA expression in target tissues

• Indirect targeting: Modulating upstream regulators or downstream effectors of ESRRA signaling

ESRRA has been identified as a potential biomarker for unfavorable clinical outcomes in human breast tumors and may predict sensitivity to hormonal blockade therapy and ErbB2-based therapy such as Herceptin . Similar applications could be explored in canine oncology.

How is ESRRA involved in bone formation and potential applications in canine orthopedics?

ESRRA plays a significant role in bone formation and may have applications in canine orthopedic conditions:

• ESRRA affects cellular energy balance and bone formation

• ESRRA ablation in adipocytes promotes osteogenesis and vascular marrow

• ESRRA regulates SPP1 (osteopontin), which is an important bone matrix protein involved in bone remodeling

The regulatory relationship between ESRRA and SPP1 is particularly relevant for bone health. SPP1 is dramatically enhanced by ESRRA abrogation in adipocytes , suggesting that targeted inhibition of ESRRA in adipose tissue could potentially promote bone formation.

This mechanism could be explored for therapeutic applications in canine orthopedic conditions such as osteoporosis, fracture healing, or osteoarthritis.

What are the optimal conditions for expressing and purifying recombinant canine ESRRA?

For successful expression and purification of recombinant canine ESRRA:

Expression Conditions:

• Temperature: 16-18°C for mammalian cells to enhance proper folding

• Induction parameters: For bacterial systems, optimize IPTG concentration (0.1-1 mM) and induction time (4-16 hours)

• Cell density: Induce at mid-log phase (OD600 of 0.6-0.8) for bacterial systems

Purification Strategy:

• Affinity chromatography using His-tag or GST-tag

• Ion exchange chromatography to remove contaminants

• Size exclusion chromatography for final polishing

• Consider adding protease inhibitors throughout purification

Quality Control Measures:

• SDS-PAGE and Western blot to confirm size and immunoreactivity

• DNA binding assay to verify functional activity (EMSA with ERE sequences)

• Mass spectrometry to confirm protein identity and modifications

What are the key considerations for designing ESRRA-targeted knockout or knockdown experiments?

When designing ESRRA knockout or knockdown experiments:

Knockout Strategies:

• CRISPR-Cas9: Target conserved functional domains; design multiple gRNAs

• Conditional knockout: Use Cre-loxP system for tissue-specific deletion

• Validate knockout efficiency at both mRNA and protein levels

Knockdown Approaches:

• siRNA: Design 3-4 different siRNAs targeting different regions

• shRNA: For stable knockdown, ensure target specificity

• Antisense oligonucleotides: Consider modified backbones for stability

Controls and Validation:

• Include scrambled/non-targeting controls

• Quantify knockdown efficiency by qRT-PCR and Western blot

• Perform rescue experiments with recombinant ESRRA to confirm specificity

Phenotypic Assessment:

• Monitor effects on adipogenic marker genes (Pparg, Cebpa, Fabp4)

• Assess SPP1 and LEPTIN expression changes

• Evaluate adipocyte differentiation and bone formation parameters

What analytical methods are recommended for studying ESRRA-regulated gene expression?

For comprehensive analysis of ESRRA-regulated gene expression:

Transcriptomic Approaches:

• RNA-Seq: For genome-wide expression analysis

• qRT-PCR: For targeted validation of specific genes

• Single-cell RNA-Seq: To assess cell-type-specific effects

Protein-Level Validation:

• Western blot: For key proteins like SPP1 and LEPTIN

• Immunofluorescence: For tissue localization studies

• ELISA: For quantifying secreted factors in culture media or serum

Functional Analyses:

• Luciferase reporter assays: To assess promoter activity

• ChIP-Seq: To identify genome-wide ESRRA binding sites

• 3C/4C/Hi-C: To examine chromatin interactions at ESRRA binding sites

When studying ESRRA's regulation of specific genes like SPP1 and LEPTIN, combine multiple approaches to establish direct regulation versus indirect effects. For example, research has shown that ESRRA knockdown increases SPP1 expression while decreasing LEPTIN expression, demonstrating opposing regulatory mechanisms .