SIN3B Antibody, FITC conjugated

What is SIN3B and what cellular functions does it mediate?

SIN3B (Paired amphipathic helix protein Sin3b) primarily functions as a transcriptional corepressor that mediates gene repression by recruiting histone deacetylase complexes to target gene promoters. It acts by interacting with MXI1 to repress MYC-responsive genes and antagonize MYC oncogenic activities . SIN3B also interacts with MAD-MAX heterodimers by binding to MAD, forming a complex that represses transcription by tethering SIN3B to DNA . Additionally, SIN3B forms a complex with FOXK1 which represses transcription and regulates cell cycle progression, likely by repressing cell cycle inhibitor gene expression . Recent research has revealed that SIN3B can adopt alternative functions under specific conditions, such as activating gene transcription in hepatocellular carcinoma cells .

What are the key specifications of commercially available SIN3B Antibody, FITC conjugated?

The commercially available SIN3B polyclonal antibody with FITC conjugation has the following specifications:

This antibody is designed to recognize human SIN3B protein in research applications, with validated use in ELISA techniques .

How does the SIN3B complex structure contribute to its function?

The human SIN3B complex has a remarkable V-shaped structure with 1:1:1:1 stoichiometry of SIN3B, HDAC, PHF12, and MORF4L1. In this complex, SIN3B wraps around the catalytic HDAC module with its middle domain (MD), HDAC interacting domain (HID), and a loop from the C-terminal domain (CTD) . The structure reveals that SIN3B completely encircles the deacetylase and contacts its allosteric basic patch, thereby stimulating catalysis .

A particularly important structural feature is the SIN3B "Gate loop" that connects MD and HID and inserts into the active site of the HDAC . This loop rearranges to accommodate acetyl-lysine moieties and stabilizes the substrate for specific deacetylation, which is guided by a substrate receptor subunit . This structural arrangement explains how SIN3B can specifically recognize and deacetylate certain histone marks while ignoring others.

What applications is the FITC-conjugated SIN3B antibody suitable for?

• Flow cytometry - The FITC label makes this antibody suitable for flow cytometric analysis of cells expressing SIN3B

• Fluorescence microscopy - Can be used for direct visualization of SIN3B in fixed cells

• Immunohistochemistry on frozen sections - May be applicable with appropriate optimization

• High-content screening - Potential application in automated imaging platforms

It's important to note that while ELISA is the validated application, each additional application would require independent validation by the researcher with appropriate controls.

How does stress-induced Sin3B activation affect p53 target gene regulation?

Under various cellular stress conditions, including DNA damage induced by bleomycin or adriamycin, SIN3B expression is significantly upregulated at both transcript and protein levels . This stress-mediated increase in SIN3B expression is p53-dependent and associated with enhanced interaction between SIN3B and phosphorylated p53 .

The relationship between SIN3B and p53 in stress conditions follows this mechanism:

• Stress conditions (e.g., DNA damage) trigger p53 phosphorylation

• SIN3B expression increases in a p53-dependent manner

• SIN3B interacts preferentially with phosphorylated p53

• The SIN3B-p53 complex is recruited to p53 target gene promoters

• This recruitment leads to negative regulation of p53 target genes through histone deacetylation

Importantly, this represents a regulatory feedback loop where p53 initially induces SIN3B expression, and then SIN3B participates in fine-tuning p53 transcriptional activity. This mechanism allows for precise temporal control of the p53-mediated stress response, preventing excessive activation of p53 target genes that might lead to premature cell death .

What is the dual role of SIN3B in gene repression versus activation, and how can researchers investigate this context-dependent function?

The context-dependent switch between repression and activation appears to involve:

• Interaction with sulfatide, which induces conformational changes in SIN3B from compacted α-helices to a relaxed β-sheet in the PAH2 domain

• Reduced interaction with repressive partners like HDAC2 and MAD1

• Decreased recruitment of HDAC2 to target gene promoters, preventing histone deacetylation

To investigate this dual functionality, researchers should consider:

• Employing ChIP-seq to map SIN3B binding sites genome-wide under different cellular conditions

• Using proteomics approaches to identify SIN3B interacting partners in different cellular contexts

• Examining post-translational modifications of SIN3B that might regulate its repressive versus activating functions

• Utilizing structural approaches to understand how conformational changes in SIN3B affect its interaction with various partners

How specific is SIN3B in targeting different histone acetylation marks, and what methodological approaches can confirm this specificity?

SIN3B demonstrates remarkable specificity in targeting different histone acetylation marks. Studies have shown that the SIN3B complex specifically deacetylates p300-deposited histone marks on histone H3, particularly H3K27ac and H3K14ac, but not H3K9ac .

This specificity is achieved through:

• The structural arrangement of the SIN3B complex, where PHF12 presents the histone H3 tail to the catalytic tunnel of HDAC

• The insertion of the SIN3B "Gate loop" into the active site of HDAC, which stabilizes specific substrates

• Recognition of the histone H3 tail by the PHD1 domain of PHF12

To confirm this specificity in research settings, the following methodological approaches are recommended:

These approaches would provide complementary evidence for the specificity of SIN3B in targeting particular histone acetylation marks .

What technical considerations should researchers address when using SIN3B antibody for chromatin immunoprecipitation (ChIP) experiments?

When using SIN3B antibody for ChIP experiments, researchers should address several technical considerations:

• Antibody specificity validation: Before ChIP experiments, validate the specificity of the FITC-conjugated SIN3B antibody through Western blot and immunoprecipitation. For ChIP applications, confirm that the antibody recognizes SIN3B in its native conformation and when bound to chromatin.

• Crosslinking optimization: Since SIN3B is part of a multiprotein complex, optimize formaldehyde crosslinking conditions (typically 1% formaldehyde for 10-15 minutes) to efficiently capture protein-DNA interactions without over-crosslinking.

• Sonication parameters: Determine optimal sonication conditions to generate DNA fragments of 200-500 bp while preserving protein epitopes. Test different sonication times and amplitudes on your specific cell type.

• Antibody concentration: Titrate the antibody to determine the optimal concentration for ChIP. The FITC conjugation may require different antibody amounts compared to unconjugated antibodies.

• Negative controls: Include IgG control and, if possible, SIN3B-depleted samples as negative controls to assess background signal.

• Positive controls: Design primers for regions known to be bound by SIN3B, such as MYC-responsive gene promoters or p53 target genes under stress conditions .

• Sequential ChIP considerations: When investigating SIN3B complex components, consider sequential ChIP (re-ChIP) to confirm co-occupancy with partners like HDAC2, PHF12, or MORF4L1 .

• Fluorescence interference: The FITC conjugation may interfere with certain ChIP-qPCR detection methods. Ensure your detection system accounts for potential fluorescence interference.

How can researchers differentiate between direct and indirect effects of SIN3B on gene expression?

Differentiating between direct and indirect effects of SIN3B on gene expression requires a multi-faceted approach:

• Integrated genomic approaches: Combine SIN3B ChIP-seq with RNA-seq after SIN3B manipulation (overexpression, knockdown, or knockout). Direct targets will show both SIN3B binding and expression changes, while indirect targets will show expression changes without SIN3B binding.

• Time-course experiments: Use inducible systems (e.g., tetracycline-inducible SIN3B expression) to identify immediate early response genes (likely direct targets) versus delayed response genes (potential indirect targets).

• Mechanistic validation: For potential direct targets, perform reporter assays with wild-type and mutated promoters to confirm direct regulation. For instance, studies have shown that SIN3B directly activates the integrin αV promoter in the presence of sulfatide .

• Protein synthesis inhibition: Treat cells with protein synthesis inhibitors (e.g., cycloheximide) before SIN3B induction to distinguish direct effects (occurring despite protein synthesis inhibition) from indirect effects (requiring new protein synthesis).

• Complex component analysis: Since SIN3B functions within a multiprotein complex, manipulate other complex components (HDAC2, PHF12, MORF4L1) to determine whether the effects depend on the entire complex or specifically on SIN3B.

• Domain mutation studies: Create SIN3B constructs with mutations in functional domains (PAH domains, HID, Gate loop) to identify which domains are required for effects on specific genes.

This comprehensive approach allows researchers to confidently categorize genes as direct or indirect SIN3B targets and understand the underlying regulatory mechanisms.

What are the optimal fixation and permeabilization protocols when using FITC-conjugated SIN3B antibody for immunofluorescence?

When using FITC-conjugated SIN3B antibody for immunofluorescence, researchers should consider these optimal fixation and permeabilization protocols:

• Fixation options:

  • 4% paraformaldehyde (PFA) for 15 minutes at room temperature - Preserves cellular architecture while maintaining protein antigenicity

  • Methanol fixation (100% methanol at -20°C for 10 minutes) - May provide better nuclear antigen accessibility for transcription factors like SIN3B

  • Combined fixation: 4% PFA followed by methanol - Can provide benefits of both fixation methods

• Permeabilization options:

  • 0.1-0.5% Triton X-100 for 10 minutes (for PFA-fixed cells)

  • 0.1-0.5% Saponin (a milder detergent that may better preserve nuclear architecture)

  • Digitonin (0.01-0.1%) for selective plasma membrane permeabilization

• Blocking considerations:

  • Use 5-10% normal serum (from species unrelated to primary and secondary antibodies)

  • Include 0.1-0.3% Triton X-100 in blocking buffer for nuclear proteins

  • Consider adding 1% BSA to reduce non-specific binding

• Antibody incubation:

  • Since the antibody is directly FITC-conjugated, optimize dilution (starting with 1:100-1:200)

  • Incubate overnight at 4°C for optimal signal-to-noise ratio

  • Include 0.1% Triton X-100 in antibody dilution buffer

• Special considerations for SIN3B visualization:

  • Counter-stain nuclei with DAPI or Hoechst

  • Consider dual staining with markers of transcriptional repression complexes (HDAC1/2)

  • For stress-response studies, co-stain with phosphorylated p53 to visualize potential co-localization

• Photobleaching prevention:

  • Use anti-fade mounting media to prevent FITC photobleaching

  • Minimize exposure to light during all steps

  • Consider taking images promptly after staining or use appropriate storage in the dark at 4°C

Optimization of these protocols should be performed for each specific cell type being studied.

How can researchers investigate the relationship between SIN3B and histone acetylation patterns in different genomic contexts?

Investigating the relationship between SIN3B and histone acetylation patterns across different genomic contexts requires integrative approaches:

• Genome-wide mapping approaches:

  • Perform ChIP-seq for SIN3B alongside ChIP-seq for various histone acetylation marks (H3K27ac, H3K14ac, H3K9ac)

  • Use ATAC-seq to assess chromatin accessibility in relation to SIN3B binding

  • Consider CUT&RUN or CUT&Tag methods for improved signal-to-noise ratio

• Integrative bioinformatics analysis:

  • Generate heatmaps and metaplots to visualize the correlation between SIN3B binding and histone acetylation patterns

  • Classify genomic regions based on SIN3B binding and acetylation status (e.g., promoters, enhancers, gene bodies)

  • Perform motif analysis to identify transcription factors that might co-occur with SIN3B

• Experimental manipulation:

  • Use SIN3B knockdown/knockout approaches followed by ChIP-seq for histone acetylation marks

  • Employ HDAC inhibitors to assess how inhibiting the catalytic activity affects SIN3B genomic distribution

  • Create mutations in the SIN3B "Gate loop" to disrupt its insertion into the HDAC active site and observe effects on histone acetylation patterns

• Single-cell approaches:

  • Apply single-cell techniques to understand cell-to-cell variability in SIN3B-mediated regulation

  • Consider multiplexed immunofluorescence to visualize SIN3B and histone marks simultaneously

• Functional validation:

  • For key identified regions, perform focused ChIP-qPCR experiments under different conditions (e.g., stress, cell cycle phases)

  • Use CRISPR-based approaches to delete SIN3B binding sites and assess effects on local histone acetylation

  • Employ reporter assays to validate functional consequences of SIN3B binding at specific loci

These approaches collectively provide a comprehensive understanding of how SIN3B regulates histone acetylation in different genomic contexts and how this regulation affects gene expression.

What experimental design would best elucidate the dual functionality of SIN3B as both a repressor and an activator?

To elucidate the dual functionality of SIN3B as both a repressor and an activator, the following comprehensive experimental design is recommended:

• Global profiling experiments:

  • Perform RNA-seq and ChIP-seq for SIN3B across multiple cell types and conditions, including:

    • Normal versus stress conditions (e.g., DNA damage with bleomycin)

    • HCC versus normal liver cells

    • With and without sulfatide treatment

  • Classify SIN3B-bound genes as either activated or repressed based on expression changes after SIN3B manipulation

• Proteomics approach:

  • Conduct immunoprecipitation followed by mass spectrometry (IP-MS) to identify SIN3B interaction partners under different conditions

  • Perform proximity labeling (BioID or APEX) to identify context-specific protein interactions

  • Use sequential ChIP to identify co-occupancy of SIN3B with different partners at activated versus repressed genes

• Structural biology investigations:

  • Examine conformational changes in SIN3B under different conditions using:

    • Hydrogen-deuterium exchange mass spectrometry (HDX-MS)

    • Crosslinking mass spectrometry (XL-MS)

    • Cryo-EM of SIN3B complexes under activating versus repressive conditions

• Domain-specific manipulation:

  • Create a series of SIN3B constructs with mutations in:

    • PAH domains (particularly PAH2, which undergoes conformational changes upon sulfatide binding)

    • The Gate loop that inserts into the HDAC active site

    • The HDAC interaction domain (HID)

  • Test these mutants for their ability to either repress or activate target genes

• Chromatin environment assessment:

  • Perform ChIP-seq for histone modifications associated with active (H3K27ac, H3K4me3) and repressed (H3K27me3, H3K9me3) chromatin

  • Use CUT&RUN for chromatin accessibility in regions bound by SIN3B under different conditions

  • Employ Hi-C or similar approaches to assess 3D chromatin organization at SIN3B target loci

• Validation in model systems:

  • Create inducible SIN3B expression systems in relevant cell types

  • Generate CRISPR knock-in models with tagged endogenous SIN3B for live-cell tracking

  • Develop animal models with tissue-specific SIN3B manipulation to assess physiological consequences of its dual functionality

This comprehensive approach will provide mechanistic insights into how SIN3B switches between activating and repressive functions in different cellular contexts.

What controls should be included when using FITC-conjugated SIN3B antibody in flow cytometry experiments?

When using FITC-conjugated SIN3B antibody in flow cytometry experiments, a robust set of controls should be included:

• Unstained control: Cells without any antibody to establish autofluorescence baseline.

• Isotype control: FITC-conjugated rabbit IgG at the same concentration as the SIN3B antibody to assess non-specific binding.

• Fluorescence minus one (FMO) controls: When using multiple markers, include all antibodies except the SIN3B-FITC to accurately set gates.

• Expression controls:

  • Positive control: Cells known to express high levels of SIN3B (e.g., cells under stress conditions)

  • Negative control: Cells with SIN3B knockdown or knockout

  • Gradient control: Cells with varying SIN3B expression levels (e.g., time course after stress induction)

• Compensation controls: If using multiple fluorochromes, single-stained controls for each fluorochrome to correct for spectral overlap.

• Fixation/permeabilization controls:

  • Since SIN3B is predominantly nuclear, compare different fixation and permeabilization protocols

  • Include a known nuclear protein marker (e.g., histone H3) to validate nuclear permeabilization

• Specificity validation:

  • Pre-block with recombinant SIN3B protein to demonstrate specificity

  • Compare staining pattern with another SIN3B antibody (non-FITC conjugated) followed by a secondary antibody

• Technical controls:

  • Titration series of the antibody to determine optimal concentration

  • Time course of antibody incubation to establish optimal staining conditions

• Functional validation controls:

  • For stressed cells, include p53 staining as a functional correlate

  • For HCC studies, include integrin αV staining to assess correlation

These comprehensive controls ensure reliable and interpretable flow cytometry data when using the FITC-conjugated SIN3B antibody.

How can researchers resolve contradictory results between SIN3B expression and functional outcomes in different experimental systems?

Researchers encountering contradictory results between SIN3B expression and functional outcomes across different experimental systems should consider these systematic approaches:

• Context-dependent function analysis:

  • Map SIN3B binding partners in each experimental system using co-immunoprecipitation followed by mass spectrometry

  • Assess post-translational modifications of SIN3B that might differ between systems

  • Investigate the presence of sulfatide or similar molecules that could induce conformational changes in SIN3B

• Isoform-specific effects:

  • Verify which SIN3B isoform is predominantly expressed in each system (note that source observed similar effects across isoforms)

  • Use isoform-specific primers or antibodies to distinguish between isoforms

  • Perform rescue experiments with specific isoforms to determine functional differences

• Cell type-specific factors:

  • Compare the expression levels of known SIN3B interactors (HDAC1/2, PHF12, MORF4L1) across cell types

  • Investigate chromatin landscape differences that might affect SIN3B recruitment and function

  • Consider the p53 status, as SIN3B function can be p53-dependent

• Temporal considerations:

  • Conduct time-course experiments to capture dynamic changes in SIN3B function

  • Assess whether contradictory outcomes represent different phases of a single regulatory program

  • Consider cell cycle effects, as SIN3B can regulate cell cycle progression

• Technical validation:

  • Verify antibody specificity in each experimental system

  • Confirm SIN3B knockdown/overexpression efficiency at both mRNA and protein levels

  • Use multiple methodological approaches to validate key findings

• Mechanistic resolution strategies:

  • For genes with contradictory regulation, perform detailed promoter analysis to identify cis-regulatory elements

  • Use CRISPR-based approaches to mutate specific binding sites and resolve mechanism

  • Employ in vitro biochemical assays with purified components to dissect direct effects

This systematic approach will help researchers reconcile seemingly contradictory results and develop a more nuanced understanding of SIN3B's context-dependent functions.

What are the critical parameters to optimize when using SIN3B antibody for quantitative proteomics studies?

When using SIN3B antibody for quantitative proteomics studies, researchers should optimize these critical parameters:

• Sample preparation optimization:

  • Extract nuclear proteins efficiently, as SIN3B is predominantly nuclear

  • Use appropriate lysis buffers that preserve protein-protein interactions if studying the SIN3B complex

  • Consider crosslinking approaches to capture transient interactions

  • Optimize sonication/homogenization to release chromatin-bound SIN3B

• Immunoprecipitation conditions:

  • Determine optimal antibody-to-protein ratio through titration experiments

  • Test different coupling methods for the antibody (direct coupling vs. protein A/G beads)

  • Optimize wash stringency to remove non-specific binders without disrupting genuine interactions

  • Consider native versus denaturing conditions depending on study goals

• Controls and normalization:

  • Include IgG controls to identify non-specific interactions

  • Use SIN3B-depleted samples as negative controls

  • Incorporate isotope-labeled reference standards for accurate quantification

  • Consider spike-in controls for normalization across samples

• Digestion and peptide recovery:

  • Optimize protease digestion (trypsin, Lys-C, or combinations) for maximum sequence coverage

  • Test different digestion durations and temperatures

  • Evaluate on-bead versus in-solution digestion efficiency

  • Use specialized extraction methods to recover hydrophobic peptides

• Mass spectrometry parameters:

  • Optimize LC gradient conditions for complex samples

  • Adjust collision energies for SIN3B-specific peptides

  • Consider targeted approaches (PRM, MRM) for key SIN3B peptides and interactors

  • Use appropriate acquisition methods based on study goals (DDA, DIA)

• Data analysis considerations:

  • Select appropriate software and statistical approaches for quantitative analysis

  • Set stringent criteria for identifying genuine interactions

  • Use multiple biological replicates to ensure reproducibility

  • Apply appropriate normalization methods for accurate quantification

• Validation strategies:

  • Confirm key interactions by orthogonal methods (Western blot, proximity ligation assay)

  • Use sequential immunoprecipitation to validate complex composition

  • Consider structural validation of interactions through hydrogen-deuterium exchange or crosslinking mass spectrometry

By carefully optimizing these parameters, researchers can obtain reliable and reproducible results in quantitative proteomics studies using SIN3B antibody.

How can researchers dissect the specific contribution of SIN3B within multiprotein complexes that share overlapping functions?

Dissecting the specific contribution of SIN3B within multiprotein complexes that share overlapping functions requires sophisticated experimental approaches:

• Selective perturbation strategies:

  • Use degron-based approaches for rapid, inducible degradation of SIN3B

  • Employ CRISPR interference (CRISPRi) for targeted repression of SIN3B

  • Create domain-specific mutations that disrupt specific interactions while preserving others

  • Design peptide inhibitors that block specific protein-protein interactions

• Complex-specific biochemical separation:

  • Perform size-exclusion chromatography to separate different SIN3B-containing complexes

  • Use ion-exchange chromatography to exploit charge differences between complexes

  • Apply affinity purification using antibodies against different complex components

  • Implement blue native PAGE to separate intact protein complexes

• Proximity-based interaction mapping:

  • Use BioID or APEX2 proximity labeling with SIN3B as the bait protein

  • Apply FRET or BRET approaches to validate direct interactions in living cells

  • Implement PLA (proximity ligation assay) to visualize SIN3B interactions with specific partners

  • Consider split-protein complementation assays for binary interaction validation

• Functional genomics approaches:

  • Perform epistasis analysis by systematically depleting SIN3B and other complex components

  • Use synthetic genetic array (SGA) analysis to identify genetic interactions

  • Apply CRISPR screens with SIN3B overexpression or knockdown as background

  • Implement digital genomic footprinting to identify differential binding patterns

• Structural approaches:

  • Use cryo-EM to visualize the complete SIN3B complex architecture as demonstrated in source

  • Apply HDX-MS to map interaction surfaces and conformational changes

  • Implement integrative structural approaches combining multiple data types

  • Consider single-particle tracking to assess complex dynamics in living cells

• Domain-specific contribution analysis:

  • Create chimeric proteins swapping domains between SIN3B and related proteins

  • Use domain-specific antibodies to immunoprecipitate specific complexes

  • Design domain-focused CRISPR screens targeting interaction surfaces

  • Implement domain-specific proteomics to identify region-specific interactors

This multifaceted approach will allow researchers to disentangle the specific contributions of SIN3B from other overlapping functions within multiprotein complexes, as exemplified by the detailed structural and functional analysis in source .

What methodological considerations should be addressed when studying SIN3B in the context of cellular stress responses?

When studying SIN3B in the context of cellular stress responses, researchers should address these methodological considerations:

• Stress induction protocols:

  • Standardize stress conditions (e.g., bleomycin concentration, exposure time)

  • Consider multiple stress types (DNA damage, oxidative stress, hypoxia) to determine stress-type specificity

  • Use physiologically relevant stress levels to avoid artifactual responses

  • Implement time-course experiments to capture the dynamic nature of stress responses

• SIN3B expression analysis:

  • Monitor both protein and mRNA levels, as SIN3B increases at both levels during stress

  • Use quantitative methods (qPCR, Western blot with densitometry) for accurate measurement

  • Consider subcellular fractionation to assess potential translocation during stress

  • Implement pulse-chase experiments to determine protein stability changes

• p53 dependence evaluation:

  • Include p53-null or p53-mutant cells as controls

  • Monitor p53 phosphorylation status, as SIN3B interaction increases with phosphorylated p53

  • Use p53 inhibitors (e.g., pifithrin-α) to confirm p53 dependence

  • Consider p53 isoforms that might differentially interact with SIN3B

• Temporal resolution:

  • Implement early time points (minutes after stress) to capture immediate responses

  • Extend time courses to 24 hours or longer to observe sustained effects

  • Use synchronized cell populations to control for cell cycle effects

  • Consider microfluidic approaches for continuous monitoring with minimal perturbation

• Protein-protein interaction analysis:

  • Optimize immunoprecipitation protocols for stress conditions

  • Use crosslinking approaches to capture transient stress-induced interactions

  • Implement proximity labeling methods optimized for acute stress responses

  • Consider FRET-based biosensors for real-time interaction monitoring

• Chromatin binding dynamics:

  • Perform ChIP-seq at multiple time points after stress induction

  • Use spike-in controls for quantitative comparison across conditions

  • Implement CUT&RUN for improved sensitivity in detecting dynamic binding

  • Consider nascent transcription assays (e.g., PRO-seq) to correlate binding with immediate transcriptional effects

• Functional validation approaches:

  • Design rescue experiments with wild-type versus mutant SIN3B

  • Use inducible systems for precise temporal control of SIN3B expression

  • Implement CRISPR-based epigenome editing to mimic SIN3B effects at specific loci

  • Consider cell competition assays to assess fitness effects of SIN3B manipulation under stress

These methodological considerations will ensure robust and reproducible results when studying SIN3B's role in cellular stress responses.

How might combining SIN3B antibody with other epigenetic markers advance our understanding of dynamic chromatin regulation?

Combining SIN3B antibody with other epigenetic markers can significantly advance our understanding of dynamic chromatin regulation through these innovative approaches:

• Multiplexed ChIP-seq strategies:

  • Implement sequential ChIP (ChIP-reChIP) to identify genomic regions co-occupied by SIN3B and other factors

  • Use CUT&Tag with orthogonal tags for simultaneous profiling of SIN3B and histone modifications

  • Apply high-throughput ChIP-seq with combinatorial indexing to profile SIN3B binding across multiple conditions simultaneously

  • Implement cleavage under targets and release using nuclease (CUT&RUN) for improved signal-to-noise ratio

• Single-cell multi-omics approaches:

  • Combine single-cell ATAC-seq with SIN3B antibody staining to correlate chromatin accessibility with SIN3B levels

  • Implement scCUT&Tag approaches to profile SIN3B binding in heterogeneous cell populations

  • Apply cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) to correlate SIN3B protein levels with transcriptional states

  • Develop multi-modal approaches to simultaneously assess chromatin states and SIN3B binding in single cells

• Live-cell dynamics imaging:

  • Use CRISPR knock-in to tag endogenous SIN3B with fluorescent proteins compatible with FITC imaging

  • Implement FRAP (fluorescence recovery after photobleaching) to study SIN3B binding dynamics

  • Apply lattice light-sheet microscopy for high-resolution imaging of SIN3B and chromatin dynamics

  • Develop optogenetic tools to manipulate SIN3B function with spatiotemporal precision

• Integrative data analysis frameworks:

  • Develop computational approaches to integrate SIN3B binding data with histone modification maps

  • Implement machine learning algorithms to predict SIN3B binding patterns based on chromatin features

  • Apply network analysis to identify SIN3B-centered regulatory circuits

  • Develop predictive models of gene expression based on SIN3B binding and epigenetic modifications

• Domain-specific profiling:

  • Design approaches to map the binding sites of specific SIN3B domains (PAH domains, HID, Gate loop)

  • Implement crosslinking mass spectrometry to identify direct contacts between SIN3B domains and chromatin

  • Apply protein painting approaches to map domain-specific interactions in situ

  • Develop domain-specific CRISPR screens to identify functional interfaces