DRAP1 Antibody

What is DRAP1 and what is its functional significance in transcriptional regulation?

DRAP1 (DR1-Associated Protein 1, also known as Negative Cofactor 2 Alpha) is a transcriptional corepressor that functions primarily by forming a heterodimeric complex with DR1 (Downregulator of Transcription 1). This complex plays a crucial role in global transcriptional repression by targeting TATA-binding protein (TBP), thereby preventing the association of TFIIB with the TBP-TATA complex .

The functional significance of DRAP1 lies in its ability to dramatically enhance DR1-mediated repression through association via their respective histone-fold motifs . While DR1 alone exhibits some repressor activity, the formation of the DRAP1/DR1 heterodimer substantially increases this repression. The complex has been shown to affect both RNA polymerase II and III transcription, though with selective effects on different gene classes .

Research methodologies to study DRAP1 function typically involve:

• Genetic knockout/knockdown experiments using RNAi

• ChIP assays to detect DRAP1 occupancy at target genes

• Co-immunoprecipitation studies to examine protein-protein interactions

• Transcriptional reporter assays to measure repressive activity

What are the optimal applications and conditions for DRAP1 antibody use in experimental research?

DRAP1 antibodies have been validated for multiple research applications with specific optimal conditions for each method:

Western Blotting (WB):

• Recommended dilution: 1:500-1:1000

• Detected molecular weight: 26-30 kDa (observed) vs. 22 kDa (calculated)

• Positive detection reported in multiple tissues including mouse testis, human heart, human brain, mouse kidney, and rat testis

Immunohistochemistry (IHC):

• Recommended dilution: 1:20-1:200

• Optimal antigen retrieval: TE buffer pH 9.0 (alternative: citrate buffer pH 6.0)

• Positive detection reported in human pancreas cancer tissue

Immunofluorescence (IF)/Immunocytochemistry (ICC):

• Recommended dilution: 1:20-1:200

• Successfully tested in HepG2 cells

Chromatin Immunoprecipitation (ChIP):

• Successfully used to detect DRAP1 occupancy at both RNA polymerase II and III transcribed genes

• Critical control: Include non-specific binding controls (regions upstream of target genes or unrelated loci)

For optimal results across all applications, antibody selection should consider:

• Host species compatibility with experimental system

• Validated reactivity with target species (human, mouse, rat are common)

• Clonality (polyclonal vs. monoclonal) based on experimental needs

• Epitope location relative to protein interaction domains

How should researchers interpret discrepancies between calculated and observed molecular weights for DRAP1 in Western blot analysis?

The discrepancy between the calculated molecular weight of DRAP1 (22 kDa) and the observed molecular weight in Western blot analysis (26-30 kDa) represents a common challenge in protein research. This phenomenon requires methodological consideration:

Explanations for the discrepancy:

• Post-translational modifications: DRAP1 may undergo phosphorylation, ubiquitination, or other modifications that increase its apparent molecular weight

• Structural properties: The amino acid composition and structural features of DRAP1 may cause anomalous migration in SDS-PAGE

• Species variations: Different species may exhibit slightly different migration patterns

Methodological approaches to address this issue:

• Validation experiments:

  • Use multiple antibodies targeting different epitopes of DRAP1

  • Perform knockdown/knockout validation to confirm specificity

  • Include positive controls with recombinant DRAP1 protein

• Technical considerations:

  • Optimize gel percentage to improve resolution in the 20-30 kDa range

  • Consider gradient gels for better separation

  • Adjust running conditions (voltage, time) to enhance resolution

• Confirmatory analyses:

  • Mass spectrometry to confirm protein identity

  • 2D gel electrophoresis to separate modified forms

  • Phosphatase treatment to determine if phosphorylation contributes to the shift

When reporting results, researchers should note both the calculated and observed molecular weights and provide possible explanations for any discrepancies.

What species cross-reactivity can be expected with DRAP1 antibodies and how does this impact experimental design?

DRAP1 antibodies show variable species cross-reactivity depending on the specific antibody and its target epitope. Understanding these patterns is crucial for experimental design:

Reported reactivity patterns:

Impact on experimental design:

• Evolutionary conservation considerations: The high cross-reactivity of some DRAP1 antibodies suggests evolutionary conservation of certain epitopes across species

• Species-specific interactions: Despite protein conservation, the interaction between DRAP1 and DR1 appears to be species-specific , requiring careful consideration in heterologous systems

• Validation requirements: Cross-species applications require thorough validation, especially when the antibody was raised against a human epitope

• Epitope selection for multi-species studies: When designing experiments involving multiple species, select antibodies targeting highly conserved regions

Methodological approach for cross-species studies:

• Perform sequence alignment analysis to identify conserved regions

• Validate antibody specificity in each species before experimental use

• Consider species-specific controls when possible

• When using antibodies in less common species, perform Western blot validation first

What experimental evidence supports the role of DRAP1/DR1 in RNA polymerase III transcription regulation?

The role of the DRAP1/DR1 complex in RNA polymerase III (pol III) transcription regulation is supported by multiple lines of experimental evidence:

ChIP analysis evidence:

• Endogenous Dr1 and DRAP1 have been detected at pol III-transcribed genes in human cells through chromatin immunoprecipitation (ChIP)

• Both proteins were found at tRNA genes, 5S rRNA genes, and U6 snRNA genes with specificity confirmed by the absence of binding at control loci

• Quantitative PCR following ChIP showed highly significant (P < 0.0001) binding to these sites

Functional evidence through gene depletion:

• RNAi-mediated knockdown of Dr1 in HeLa cells resulted in significant upregulation of tRNA expression (both mature tRNAs and pre-tRNAs)

• Three independent RNAi approaches targeting different regions of Dr1 yielded consistent results, confirming specificity

• Interestingly, not all pol III transcripts responded equally: tRNAs and Alu RNA increased, but 5S rRNA, U6 snRNA, and 7SL RNA levels showed no significant response

Protein interaction studies:

• A stable association was detected between endogenous Dr1 and the pol III-specific transcription factor Brf1

• This interaction may recruit Dr1 to pol III templates in vivo, as crosslinking to these sites increases following Brf1 induction

Biochemical evidence:

• Early in vitro studies showed that recombinant Dr1 inhibits tRNA gene transcription, though requiring excess protein

• In reconstituted in vitro transcription systems, the yDr1/Bur6 complex (yeast homolog of Dr1/DRAP1) repressed transcription, and this repression could be overcome by increasing TBP concentration

These findings collectively demonstrate that the physiological functions of human Dr1/DRAP1 include regulation of pol III transcription, though with more selective effects than initially predicted.

How can researchers effectively use DRAP1 antibodies in ChIP experiments to study transcriptional regulation?

Chromatin immunoprecipitation (ChIP) with DRAP1 antibodies has proven valuable for understanding DRAP1's role in transcriptional regulation. The following methodological considerations are essential for successful DRAP1 ChIP experiments:

Antibody selection considerations:

• Validate antibody specificity via Western blot before ChIP application

• Consider using at least two independent antibodies targeting different epitopes to confirm results

• For cross-species studies, ensure the antibody recognizes the target species

Optimized ChIP protocol elements:

• Crosslinking conditions:

  • Standard formaldehyde crosslinking (1% for 10 minutes) has been successful

  • For studying transient interactions, consider shorter crosslinking times

• Sonication parameters:

  • Aim for chromatin fragments of 200-500 bp

  • Verify fragmentation efficiency by gel analysis before immunoprecipitation

• Immunoprecipitation controls:

  • Include IgG negative control

  • Use TBP antibody as a positive control for promoter regions

  • Include input samples (typically 5-10% of starting material)

• Target selection:

  • Include known DRAP1 targets (tRNA genes, 5S rRNA genes) as positive controls

  • Include non-bound regions (e.g., regions upstream of U6 genes, ARPP P0 coding region) as negative controls

  • Consider both pol II and pol III transcribed genes for comprehensive analysis

• Data analysis:

  • Normalize to input samples

  • Compare DRAP1 enrichment to IgG control

  • For relative occupancy comparisons, present as percentage of input or relative to TBP signal

Advanced ChIP applications for DRAP1:

• ChIP-seq for genome-wide DRAP1 binding profile

• Sequential ChIP (re-ChIP) to detect DRAP1-DR1 co-occupancy

• ChIP followed by mass spectrometry to identify DRAP1-associated proteins at chromatin

What methodological approaches can resolve specificity issues in co-immunoprecipitation experiments with DRAP1 antibodies?

Co-immunoprecipitation (co-IP) is critical for studying DRAP1 interactions, but specificity challenges require careful methodological consideration:

Common specificity issues:

• Non-specific antibody binding

• Species cross-reactivity complications

• Buffer conditions affecting protein-protein interactions

• Differential detection of endogenous versus overexpressed proteins

Optimized methodology for DRAP1 co-IP:

• Antibody preparation:

  • Affinity-purify antibodies using recombinant DRAP1 polypeptides

  • Immobilize antibodies (≈1 μg) on protein A-agarose beads

  • Include isotype-matched IgG controls

• Buffer optimization:

  • Successful DRAP1/DR1 co-IP has been performed with buffer containing:

    • 20 mM Hepes–KOH (pH 7.9)

    • 1 mM EDTA

    • 10% (vol/vol) glycerol

    • 0.5% (vol/vol) Nonidet P-40

    • 0.1% (vol/vol) Triton X-100

    • 0.25 M NaCl

  • Multiple washes (at least three) are recommended to reduce background

• Validation approaches:

  • Reciprocal co-IP (using antibodies against both interaction partners)

  • Competitive peptide blocking to confirm specificity

  • Include protein mixture controls without the suspected interaction partner

  • Western blot analysis using antibodies against both proteins

• Addressing species specificity:

  • The interaction between Dr1 and DRAP1(Bur6) is species-specific

  • Human Dr1 failed to immunoprecipitate yeast Bur6

  • Use species-matched components when possible

  • For cross-species studies, consider epitope-tagged constructs

• Detection strategies:

  • SDS-PAGE followed by Western blot

  • Probing with antibodies against the co-precipitated protein

  • Consider stripping and reprobing membranes to detect both interaction partners

These methodological refinements can significantly improve specificity in DRAP1 co-IP experiments and provide reliable data on protein-protein interactions.

What is the current understanding of DRAP1's role in cancer progression, particularly in triple-negative breast cancer?

Recent research has revealed significant insights into DRAP1's role in cancer progression, with particular focus on triple-negative breast cancer (TNBC):

Expression and clinical correlation:

• DRAP1 is upregulated in TNBC compared to other breast cancer subtypes

• Elevated DRAP1 expression correlates with poor recurrence-free survival in TNBC patients

Functional impact in cancer models:

• DRAP1 promotes TNBC proliferation, migration, and invasion in vitro

• DRAP1 enhances tumor growth and metastasis in vivo

Molecular mechanisms:

• mTOR pathway regulation:

  • The DR1/DRAP1 heterodimer complex inhibits expression of CASTOR1 (cytosolic arginine sensor for mTORC1 subunit 1)

  • This inhibition increases activation of mTOR signaling

  • Elevated mTOR activity promotes cancer progression

  • DRAP1-high tumors show increased sensitivity to the mTOR inhibitor everolimus

• DRAP1-DR1 positive feedback loop:

  • DRAP1 enhances DR1 stability by recruiting the deubiquitinase USP7

  • This recruitment inhibits DR1 proteasomal degradation

  • In turn, DR1 directly promotes DRAP1 transcription

  • This bidirectional regulation creates a feed-forward mechanism enhancing TNBC progression

Methodological approaches for studying DRAP1 in cancer:

• Expression analysis:

  • Immunohistochemistry of tumor tissue microarrays

  • Western blot analysis of patient-derived samples

  • RNA-seq data mining from cancer databases

• Functional studies:

  • RNA interference for loss-of-function studies

  • CRISPR/Cas9 knockout models

  • Overexpression systems for gain-of-function analysis

  • Patient-derived xenograft models

• Therapeutic implications:

  • Combination therapy studies with mTOR inhibitors

  • Development of small molecule inhibitors targeting DRAP1/DR1 interaction

  • Biomarker analysis for patient stratification

This evolving understanding suggests that targeting the DRAP1/DR1 complex may represent a novel therapeutic strategy for TNBC treatment .

How do structural and functional differences in DRAP1 across species impact experimental design and data interpretation?

Understanding species-specific differences in DRAP1 is critical for experimental design and interpretation, particularly in comparative studies:

Structural conservation and divergence:

• Sequence alignment shows that human DRAP1 and yeast Bur6 (yDRAP1) are 37% identical (61% similar)

• The highest conservation is found in the histone-fold motif

• These differences may impact epitope recognition by antibodies across species

Functional complementation studies:

Species-specific interaction patterns:

• Immunoprecipitation studies confirm species-specificity of the DR1-DRAP1 interaction:

  • Antibodies against Bur6 successfully immunoprecipitate yeast Dr1

  • Human Dr1 antibodies fail to immunoprecipitate yeast Bur6

  • Human Dr1 antibodies successfully immunoprecipitate human DRAP1

Chromatin association differences:

• In yeast, endogenous Dr1/Bur6 is not detected at tRNA genes under standard growth conditions

• In contrast, human Dr1/DRAP1 is readily detected at tRNA genes in human cells

• This suggests evolutionary divergence in the genomic targeting mechanisms

Methodological implications:

• For heterologous expression studies:

  • Co-express species-matched DR1 and DRAP1 proteins

  • When cross-species studies are necessary, verify protein-protein interactions first

  • Consider creating chimeric proteins to identify interaction domains

• For antibody selection:

  • Use species-specific antibodies when possible

  • For cross-species detection, target the most conserved epitopes

  • Validate antibody specificity in each species independently

• For functional studies:

  • Include species-appropriate controls

  • Consider evolutionary context when interpreting results

  • Be cautious when extrapolating findings across distant species

• For structural biology approaches:

  • Compare protein structures across species to identify conserved interaction surfaces

  • Use molecular modeling to predict species-specific differences

What are the recommended approaches for studying DRAP1-DR1 complex formation and its impact on transcriptional regulation?

Studying the DRAP1-DR1 complex requires integrated methodological approaches spanning biochemical, molecular, and cellular techniques:

1. Biochemical characterization of complex formation:

• Protein purification strategies:

  • Recombinant expression of DRAP1 and DR1 in E. coli or insect cells

  • Affinity tags (His, GST) for purification of individual proteins

  • Co-expression systems for complex isolation

• Interaction analysis methods:

  • Co-immunoprecipitation with antibodies against different epitopes

  • GST pulldown assays using recombinant proteins

  • Size exclusion chromatography to identify complex formation

  • Surface plasmon resonance to measure binding kinetics

  • Isothermal titration calorimetry for thermodynamic parameters

2. Structural characterization approaches:

• Domain mapping:

  • The histone-fold motifs in both proteins mediate their interaction

  • The TBP-binding domain and QA-domain of DR1 are required for transcriptional repression

  • Create deletion constructs to map minimal interaction domains

• Structural analysis techniques:

  • X-ray crystallography of the complex

  • Cryo-EM for larger assemblies including TBP

  • NMR for dynamic interaction studies

  • Hydrogen-deuterium exchange mass spectrometry

3. Transcriptional regulation mechanisms:

• In vitro transcription systems:

  • Reconstituted systems with purified components (TBP, TFIIB, RNAPII/III)

  • Template competition assays

  • Order-of-addition experiments to determine mechanism

• Chromatin association studies:

  • ChIP to identify genomic binding sites

  • ChIP-seq for genome-wide occupancy patterns

  • Sequential ChIP to confirm co-occupancy

  • Chromatin fractionation to study nuclear distribution

4. Functional impact assessment:

• Gene expression analysis:

  • RNAi-mediated depletion of DRAP1 or DR1

  • Overexpression studies

  • qRT-PCR for specific target genes

  • RNA-seq for genome-wide effects

• Reporter assays:

  • Luciferase reporters with promoters of interest

  • Tethering assays using GAL4 fusion proteins

  • In vitro transcription assays with purified components

5. Regulatory mechanism investigation:

• Post-translational modification analysis:

  • Phosphorylation analysis through mass spectrometry

  • Ubiquitination studies (relevant for DR1 stability)

  • Identification of modification sites through mutational analysis

• Protein stability studies:

  • DRAP1 enhances DR1 stability by recruiting USP7 deubiquitinase

  • Cycloheximide chase assays to measure protein half-life

  • Proteasome inhibitor studies

  • Ubiquitination assays