NR1D1 Antibody

What is NR1D1 and what are its primary functions in cellular biology?

NR1D1 (nuclear receptor subfamily 1, group D, member 1), also known as REV-ERBα, EAR1, THRAL, ear-1, and hRev, is a transcriptional repressor that contains one nuclear receptor DNA-binding domain and belongs to the nuclear hormone receptor family . It functions as an integral component of the complex transcription machinery governing circadian rhythmicity, forming a critical negative limb of the circadian clock by directly repressing the expression of core clock components BMAL1, CLOCK, and CRY1 .

Additionally, NR1D1 regulates genes involved in multiple metabolic pathways, including:

• Lipid and bile acid metabolism

• Adipogenesis and adipocyte differentiation

• Gluconeogenesis

• Macrophage inflammatory responses

NR1D1 acts as a receptor for heme, which stimulates its interaction with the NCOR1/HDAC3 corepressor complex, enhancing transcriptional repression . It recognizes two classes of DNA response elements within promoters of target genes and can bind as either monomers or homodimers, depending on the nature of the response element .

What are the molecular characteristics of commercial NR1D1 antibodies?

Commercial NR1D1 antibodies, such as the 14506-1-AP from Proteintech, typically possess the following molecular characteristics:

The antibody may be generated using a specific immunogen, such as NR1D1 fusion protein (Ag5964 in the case of 14506-1-AP) . Different manufacturers may produce various formats including monoclonal and recombinant antibodies with specific applications in mind.

What are the validated applications for NR1D1 antibodies in research?

NR1D1 antibodies have been validated for multiple research applications, with specific dilution recommendations for optimal results:

It is recommended that researchers titrate these antibodies in each testing system to obtain optimal results, as dilution requirements may be sample-dependent .

How should researchers optimize Western blot protocols for NR1D1 detection?

For optimal Western blot detection of NR1D1:

• Sample Preparation:

  • Use fresh samples from validated cellular sources (A549, HepG2, Jurkat, NIH/3T3, Y79, HeLa cells, or mouse skeletal muscle tissue have shown positive results)

  • Employ appropriate lysis buffers containing protease inhibitors

  • Keep samples on ice throughout preparation

• Gel Selection and Transfer:

  • Use 8-10% SDS-PAGE gels to properly resolve the 55-68 kDa bands of NR1D1

  • Ensure complete transfer to PVDF or nitrocellulose membranes

• Antibody Incubation:

  • Block membranes thoroughly with 5% non-fat milk or BSA

  • Start with a 1:2000 dilution of primary antibody and adjust as needed

  • Incubate overnight at 4°C for optimal binding

  • Use appropriate secondary antibodies (typically anti-rabbit HRP conjugates)

• Detection and Validation:

  • Use enhanced chemiluminescence reagents for detection

  • Expected band size is 55-68 kDa (observed) versus 67 kDa (calculated)

  • Validate specificity using positive controls (such as known NR1D1-expressing cell lines)

  • Consider using knockout/knockdown samples as negative controls

• Troubleshooting:

  • If high background occurs, increase the blocking time and washing steps

  • If no signal is detected, check protein loading or consider using a different lysis buffer

What are the best cellular models for studying NR1D1 function?

Based on research literature and validated antibody applications, the following cellular models are recommended for studying NR1D1 function:

Cell Lines with Validated NR1D1 Expression:

• Cancer cell lines: A549, HepG2, Jurkat, Y79, HeLa cells

• Fibroblasts: NIH/3T3 cells (positive for both WB and IF)

• Breast cancer models: Multiple breast cancer cell lines are suitable given NR1D1's role in breast cancer progression

• Synovial cells: Fibroblast-like synoviocytes (FLSs) from rheumatoid arthritis patients

• Adipocytes: Primary adipocytes or differentiated preadipocyte lines for metabolic studies

Tissue Models:

• Mouse skeletal muscle tissue

• Adipose tissue (for studying metabolic regulation)

• Synovial tissue (for studying inflammatory mechanisms)

• Breast cancer tissue (for studying cancer progression and immunity)

Genetic Modification Approaches:

• CRISPR/Cas9-mediated knockout of NR1D1

• Conditional knockout models (e.g., adipocyte-specific deletion using adiponectin-Cre)

• NR1D1 overexpression systems

• Reporter assays to monitor NR1D1 transcriptional repression activity

How does NR1D1 function in the regulation of circadian rhythm at the molecular level?

NR1D1 functions as a critical component in the circadian clock mechanism through several molecular interactions:

• Core Circadian Clock Repression:

  • NR1D1 directly represses the expression of core clock components BMAL1, CLOCK, and CRY1

  • It forms a critical negative limb of the circadian clock machinery

• Transcriptional Regulation Mechanism:

  • NR1D1 recognizes specific DNA response elements in target gene promoters:

    • As a monomer: Binds to response elements with consensus half-site motif 5'-[A/G]GGTCA-3' preceded by A/T-rich 5' sequence (RevRE)

    • As a homodimer: Binds to direct repeats of the core motif spaced by two nucleotides (RevDR-2)

• Heme-Dependent Regulation:

  • Acts as a receptor for heme, which modulates its activity

  • Heme binding stimulates NR1D1's interaction with the NCOR1/HDAC3 corepressor complex

  • This interaction enhances transcriptional repression activity

• Feedback Mechanisms:

  • BMAL1-CLOCK activates Rev-erbα (NR1D1) itself, creating a regulatory feedback loop

  • NR1D1 acts as a potent competitive repressor of ROR alpha (RORA) function

  • It regulates heme levels by repressing the expression of PPARGC1A, a potent inducer of heme synthesis

For research applications, ChIP assays using NR1D1 antibodies can identify direct binding targets and circadian occupancy patterns at promoters of clock-controlled genes. Time-course experiments with synchronized cells can reveal oscillation patterns of NR1D1 binding and repressive activity.

What is the role of NR1D1 in cancer biology, particularly in breast cancer?

NR1D1 plays significant roles in breast cancer biology through multiple mechanisms:

• DNA Damage Response and Repair:

  • NR1D1 inhibits DNA repair in breast cancer cells

  • This inhibition leads to accumulation of cytosolic DNA fragments

  • These fragments activate the cGAS-STING pathway, which is crucial for immune responses

• Antitumor Immune Response Activation:

  • NR1D1 enhances type I interferon (IFN) signaling

  • It promotes the production of type I IFNs and downstream chemokines CCL5 and CXCL10

  • This leads to increased infiltration of CD8+ T cells and natural killer cells into tumors

• Tumor Progression and Metastasis Inhibition:

  • Deletion of Nr1d1 in MMTV-PyMT transgenic mice resulted in increased tumor growth and lung metastasis

  • Loss of Nr1d1 in tumor cells rather than stromal cells played a prominent role in tumor progression

  • NR1D1 suppresses breast cancer progression and lung metastasis

• Pharmacological Activation Effects:

  • The NR1D1 ligand SR9009 enhances type I IFN-mediated antitumor immunity

  • This activation is accompanied by suppression of tumor progression and lung metastasis

These findings suggest that NR1D1 may be a promising therapeutic target for breast cancer. Researchers investigating NR1D1 in cancer settings should consider employing breast cancer models, examining immune cell infiltration, and assessing cGAS-STING pathway activation as key experimental approaches.

How does NR1D1 influence metabolic regulation and adipose tissue function?

NR1D1 serves as a critical regulator of metabolic functions and adipose tissue biology through several mechanisms:

For researchers studying NR1D1 in metabolic contexts, it is important to distinguish between global and tissue-specific effects of NR1D1 manipulation. Conditional knockout models, such as the adiponectin-Cre driver line, provide valuable tools for investigating tissue-specific roles .

What techniques are recommended for investigating NR1D1's transcriptional repression activity?

Researchers investigating NR1D1's transcriptional repression activity should consider the following methodological approaches:

• Chromatin Immunoprecipitation (ChIP) Assays:

  • Use validated NR1D1 antibodies (multiple publications have employed ChIP successfully)

  • Identify direct binding sites of NR1D1 on target gene promoters

  • Analyze binding to known response elements:

    • RevRE (monomeric binding): 5'-[A/G]GGTCA-3' preceded by A/T-rich sequence

    • RevDR-2 (homodimeric binding): Direct repeat of core motif with 2-nucleotide spacing

  • Combine with sequencing (ChIP-seq) for genome-wide binding profile

• Reporter Gene Assays:

  • Construct luciferase reporters containing NR1D1 binding elements

  • Test wild-type vs. mutated binding sites to confirm specificity

  • Assess repression activity with NR1D1 overexpression or knockout

  • Evaluate heme-dependent modulation of repression activity

• Co-Immunoprecipitation (Co-IP):

  • Investigate NR1D1 interaction with NCOR1/HDAC3 corepressor complex

  • Examine how heme binding affects these protein-protein interactions

  • Use IP-grade NR1D1 antibodies for pull-down experiments

• Gene Expression Analysis:

  • Analyze expression of known NR1D1 target genes (BMAL1, CLOCK, CRY1, APOC3, etc.)

  • Use RNA-seq to identify global gene expression changes upon NR1D1 modulation

  • Perform time-course experiments to capture circadian patterns of repression

• Heme Binding and Functional Studies:

  • Assess heme binding to NR1D1 and its impact on repression activity

  • Investigate how heme levels affect NR1D1-mediated transcriptional regulation

  • Examine PPARGC1A regulation by NR1D1 as a feedback mechanism for heme synthesis

How can researchers effectively utilize NR1D1 pharmacological modulators in experimental settings?

NR1D1 pharmacological modulators, particularly the agonist SR9009, have been used effectively in multiple research contexts. Here are methodological guidelines for their application:

• SR9009 Agonist Applications:

  • Cancer Research: SR9009 enhances type I IFN-mediated antitumor immunity and suppresses tumor progression and lung metastasis in breast cancer models

  • Inflammatory Diseases: SR9009 reduces expression of proinflammatory cytokines and matrix metalloproteinases in rheumatoid arthritis models

  • Metabolic Studies: Daily administration of SR9009 reduces fat mass and white adipose tissue (WAT) lipogenic gene expression in mice

• Dosing Considerations:

  • In vitro studies: Typically 1-10 μM concentration range

  • In vivo studies: Usually administered 50-100 mg/kg/day in rodent models

  • Administration route: Intraperitoneal injection is common for animal studies

  • Treatment schedule: Consider circadian timing of administration due to NR1D1's role in circadian rhythm

• Experimental Controls and Validations:

  • Include vehicle controls (DMSO for in vitro, appropriate vehicle for in vivo)

  • Validate target engagement by measuring known NR1D1 target gene expression

  • Consider using Nr1d1 knockout models as negative controls

  • Be aware of potential off-target actions of NR1D1 agonists

• Assessing Pathway Modulation:

  • Monitor MAPK and NF-κB pathway activation, which are blocked by SR9009

  • Measure ROS generation and Nrf2-associated enzyme production

  • Assess effects on immune cell populations, particularly M1 macrophage polarization and osteoclastogenesis

  • Examine metabolic pathway alterations, especially in lipid metabolism contexts

• Experimental Readouts:

  • For inflammatory studies: Measure cytokine production, inflammatory cell infiltration

  • For cancer studies: Assess tumor growth, metastasis, CD8+ T cell and NK cell infiltration

  • For metabolic studies: Evaluate adiposity, glucose tolerance, lipid profiles

What considerations are important when using NR1D1 antibodies for immunofluorescence and immunohistochemistry?

When utilizing NR1D1 antibodies for immunofluorescence (IF) and immunohistochemistry (IHC) applications, researchers should consider these methodological guidelines:

• Sample Preparation:

  • Cell Fixation: 4% paraformaldehyde (10-15 minutes) is typically effective

  • Tissue Fixation: Use freshly prepared 4% paraformaldehyde or formalin-fixed paraffin-embedded (FFPE) sections

  • Antigen Retrieval: Critical for FFPE tissues; citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) heat-induced retrieval is often effective

  • Permeabilization: 0.1-0.3% Triton X-100 for cell membrane permeabilization

• Antibody Selection and Optimization:

  • Dilution Range: Start with 1:50-1:500 for IF/ICC applications

  • Validation: Confirm specificity using positive controls (NIH/3T3 cells have shown positive IF/ICC results)

  • Antibody Format: Consider using antibodies specifically validated for IF/IHC applications (e.g., ab174309 has been validated for IHC-P)

  • Batch Testing: Test each new antibody lot on known positive samples

• Signal Optimization and Controls:

  • Background Reduction: Extended blocking (1-2 hours) with 5-10% normal serum matching secondary antibody host

  • Autofluorescence Quenching: Consider treatments like Sudan Black B for tissues with high autofluorescence

  • Negative Controls: Include secondary-only controls and ideally NR1D1 knockout/knockdown samples

  • Positive Controls: Use tissue or cell lines with known NR1D1 expression

• Subcellular Localization Considerations:

  • NR1D1 is primarily nuclear, so nuclear counterstaining (e.g., DAPI) is essential

  • Consider co-staining with other nuclear markers to confirm localization

  • Evaluate heme-dependent changes in localization and interaction with corepressor complexes

• Tissue-Specific Considerations:

  • Synovial Tissue: Increased NR1D1 expression has been reported in RA patient synovial tissues

  • Breast Cancer Tissue: Consider examining immune cell infiltration alongside NR1D1 expression

  • Adipose Tissue: Important for metabolic studies; evaluate depot-specific expression patterns

  • Circadian Timing: Consider time of sample collection due to potential circadian variations in expression

What are common challenges when detecting NR1D1 protein and how can they be overcome?

Researchers may encounter several challenges when detecting NR1D1 protein. Here are common issues and their solutions:

• Variable Molecular Weight Detection:

  • Challenge: NR1D1 has a calculated molecular weight of 67 kDa but is observed at 55-68 kDa

  • Solution: Use appropriate molecular weight markers spanning 50-70 kDa range

  • Approach: Validate band identity with positive controls and consider post-translational modifications that may affect migration

• Antibody Specificity Issues:

  • Challenge: Cross-reactivity with related nuclear receptors or non-specific binding

  • Solution: Validate antibody specificity using Nr1d1 knockout or knockdown samples

  • Approach: Compare multiple antibodies targeting different epitopes of NR1D1

• Low Signal Intensity:

  • Challenge: Weak detection of endogenous NR1D1

  • Solution: Optimize protein extraction methods (nuclear extraction protocols may improve yield)

  • Approach: Consider signal amplification methods and extended exposure times for Western blot

• Circadian Expression Patterns:

  • Challenge: NR1D1 expression may vary by time of day due to its role in circadian rhythm

  • Solution: Standardize sample collection timing or perform time-course experiments

  • Approach: Document collection time and consider time-dependent changes when interpreting results

• Cell-Type Specific Expression:

  • Challenge: Variable expression across different cell types

  • Solution: Use validated positive control cell lines (A549, HepG2, Jurkat, NIH/3T3, Y79, HeLa)

  • Approach: Consider cell synchronization protocols for circadian studies

How should researchers interpret discrepancies in NR1D1 function between in vitro and in vivo studies?

Interpreting discrepancies between in vitro and in vivo NR1D1 studies requires careful consideration of several factors:

• Adipocyte Differentiation Discrepancies:

  • In vitro evidence: NR1D1 has been implicated in adipocyte differentiation

  • In vivo evidence: Nr1d1-/- mice show pronounced adiposity and adipocyte hypertrophy, even under normal feeding conditions

  • Interpretation approach: Consider cell-autonomous versus systemic effects; examine tissue-specific knockout models to resolve discrepancies

• Pharmacological versus Genetic Manipulation:

  • Pharmacological evidence: NR1D1 agonists reduce fat mass and WAT lipogenic gene expression

  • Genetic evidence: Effects may differ between global and tissue-specific knockouts

  • Interpretation approach: Be aware of potential off-target effects of pharmacological agents; SR9009 has significant off-target actions

• Context-Dependent Functions:

  • Basal versus stimulated conditions: NR1D1 activity may be limited to specific target genes under basal conditions but expand during pathological states

  • Interpretation approach: Compare gene expression changes under various physiological and pathological conditions

• Circadian Timing Considerations:

  • Challenge: Results may vary depending on circadian time of experiments

  • Interpretation approach: Document timing of interventions and measurements; consider time-course experiments

• Integration of Multiple Methodologies:

  • Recommendation: When faced with discrepancies, employ multiple complementary approaches:

    • Combine tissue-specific knockout models with pharmacological studies

    • Validate in vitro findings in primary cells from multiple donors

    • Use transcriptomic profiling alongside targeted gene expression analysis

    • Consider proteomics to assess post-translational regulation

For example, research has shown that global Nr1d1-/- mice exhibit de-repression of lipid synthesis and storage programs, but selective deletion of Nr1d1 in adipocytes does not result in dysregulation of WAT metabolic pathways under basal conditions . This suggests that NR1D1's effects on metabolism may involve inter-tissue communication or developmental programming that isn't captured in cell culture models.

What emerging roles of NR1D1 in disease mechanisms warrant further investigation?

Several emerging roles of NR1D1 in disease mechanisms present promising avenues for future research:

• Cancer Immunotherapy Applications:

  • NR1D1's role in enhancing antitumor immunity via cGAS-STING pathway activation represents a novel therapeutic target

  • Future research should investigate:

    • Combinatorial approaches with immune checkpoint inhibitors

    • Tumor-specific NR1D1 activation strategies

    • Expansion beyond breast cancer to other cancer types

• Inflammatory Disease Modulation:

  • NR1D1 activation decreases proinflammatory cytokines and matrix metalloproteinases in rheumatoid arthritis models

  • Promising areas for investigation include:

    • NR1D1 roles in other autoimmune conditions

    • Mechanisms connecting circadian disruption to inflammatory diseases

    • Development of tissue-specific NR1D1 modulators with reduced off-target effects

• Metabolic Syndrome and Obesity:

  • NR1D1 influences adipose tissue expansion and has been linked to metabolic regulation

  • Key research directions include:

    • Mechanisms behind adipocyte-specific Nr1d1 knockout enhancing adipose tissue expansion yet preventing obesity-related pathology

    • Sex-specific differences in NR1D1 metabolic functions

    • Interaction between diet timing and NR1D1 function

• Circadian Disruption and Chronic Disease:

  • As a key circadian regulator, NR1D1 may mediate effects of circadian disruption on chronic diseases

  • Research should explore:

    • Impact of shift work and jetlag on NR1D1 function

    • Interventions targeting NR1D1 to mitigate circadian disruption effects

    • Personalized timing of therapeutic interventions based on individual circadian profiles

• Neurodegenerative Disorders:

  • Given NR1D1's role in inflammation and circadian regulation, potential connections to neurodegenerative diseases warrant investigation

  • Examine:

    • NR1D1 expression and function in models of Alzheimer's, Parkinson's, and other neurodegenerative conditions

    • Effects of NR1D1 modulation on neuroinflammation and disease progression

How might advanced technologies enhance NR1D1 research methodologies?

Emerging technologies offer significant potential to advance NR1D1 research methodologies:

• Single-Cell Technologies:

  • Single-cell RNA sequencing can reveal cell-type-specific NR1D1 functions and target genes

  • Single-cell ATAC-seq can identify chromatin accessibility changes mediated by NR1D1

  • Implementation strategies:

    • Apply to heterogeneous tissues like tumor microenvironments to assess NR1D1's role in specific cell populations

    • Track temporal dynamics of NR1D1-mediated gene regulation at single-cell resolution

• CRISPR-Based Approaches:

  • CRISPR activation/interference systems allow temporal control of NR1D1 expression

  • CRISPR screens can identify synthetic lethal interactions with NR1D1 in disease contexts

  • Base editing can introduce specific mutations to study structure-function relationships

  • Application examples:

    • Create cell type-specific NR1D1 modulation in complex tissue models

    • Screen for genes that synergize with NR1D1 in controlling tumor immunity

• Spatial Transcriptomics and Proteomics:

  • Spatial profiling can map NR1D1 expression and activity across tissue architectures

  • Particularly valuable for understanding NR1D1's role in heterogeneous tissues like tumors

  • Research applications:

    • Map NR1D1 activity relative to immune cell infiltration in tumor microenvironments

    • Analyze spatial regulation in adipose tissue remodeling during metabolic challenges

• Advanced Imaging Technologies:

  • Live-cell imaging of NR1D1 with fluorescent tags can reveal dynamic regulation

  • FRET/BRET approaches can monitor protein-protein interactions in real-time

  • Super-resolution microscopy can examine subnuclear localization

  • Implementation strategies:

    • Track NR1D1-corepressor complex formation in response to heme or pharmacological modulators

    • Visualize circadian dynamics of NR1D1 nuclear translocation

• Organoid and Tissue-on-Chip Models:

  • 3D organoid systems can better recapitulate in vivo NR1D1 functions

  • Microphysiological systems allow study of inter-tissue communication

  • Research applications:

    • Investigate NR1D1 function in tumor-immune organoids

    • Study metabolic effects in interconnected liver-adipose-muscle platforms

What are the most promising therapeutic approaches targeting NR1D1 in various disease contexts?

Based on current research, several therapeutic approaches targeting NR1D1 show significant promise:

• Cancer Immunotherapy:

  • Approach: NR1D1 agonists (e.g., SR9009) to enhance antitumor immunity

  • Mechanism: Activation promotes DNA damage-induced cytosolic DNA accumulation, activating cGAS-STING pathway and increasing type I IFN production

  • Development considerations:

    • Combination with existing immunotherapies (checkpoint inhibitors)

    • Tumor-targeted delivery approaches

    • Biomarkers to identify patients likely to respond

• Inflammatory Disease Treatment:

  • Approach: NR1D1 agonists to suppress inflammatory pathways

  • Mechanism: NR1D1 activation blocks MAPK and NF-κB pathways, reduces proinflammatory cytokine expression, and inhibits M1 macrophage polarization

  • Applications: Rheumatoid arthritis and potentially other inflammatory conditions

  • Development considerations:

    • Tissue-specific agonist delivery

    • Timing of administration relative to circadian rhythms

    • Monitoring for metabolic side effects

• Metabolic Disorder Interventions:

  • Approach: Tissue-specific NR1D1 modulators

  • Mechanism: NR1D1 regulates lipid metabolism, adipogenesis, and gluconeogenesis

  • Development considerations:

    • Adipocyte-specific targeting strategies

    • Differential approaches based on fed/fasting states

    • Integration with time-restricted feeding approaches

• Circadian Rhythm Stabilization:

  • Approach: Timed administration of NR1D1 modulators

  • Mechanism: NR1D1's central role in circadian clock regulation

  • Applications: Shift work disorder, jetlag, circadian disruption-related conditions

  • Development considerations:

    • Personalized timing based on individual chronotypes

    • Pulsatile release formulations

    • Minimizing disruption to other clock components

• Novel Drug Development Strategies:

  • Selective DNA binding domain modulators: To target specific NR1D1 functions

  • Allosteric modulators: To fine-tune NR1D1 activity beyond simple activation/inhibition

  • Proteolysis-targeting chimeras (PROTACs): For selective NR1D1 degradation

  • Dual-targeting approaches: Combining NR1D1 modulation with complementary pathways

While NR1D1 agonists like SR9009 have shown promise in preclinical models, their development into clinical therapeutics will require addressing potential off-target effects , optimizing pharmacokinetics, and determining appropriate dosing schedules that account for circadian aspects of NR1D1 biology.