Crotonyl-HIST1H2AG (K119) Antibody

Which enzymes regulate H2AK119 crotonylation and decrotonylation?

The regulation of H2AK119 crotonylation involves specific enzymes for both writing and erasing this modification:

• Writers (Histone Crotonyltransferases, HCTs): P300/CBP are the major histone crotonyltransferases in mammalian cells. Other enzymes with HCT activity include members of the MYST family, such as human MOF and its yeast homolog Esa1 .

• Erasers (Decrotonylases): SIRT1, a member of the NAD⁺-dependent sirtuin family of histone deacetylases (HDACs), is the primary enzyme responsible for decrotonylation of H2AK119cr. Nicotinamide, which inhibits sirtuin family deacetylases, impairs the downregulation of H2AK119cr during replication stress, while trichostatin A, which inhibits class I and class II HDACs, does not affect H2AK119cr levels . Among nuclear sirtuins (SIRT1, SIRT6, and SIRT7), only SIRT1 overexpression significantly reduces H2AK119cr levels .

How can I validate the specificity of a Crotonyl-HIST1H2AG (K119) antibody?

To validate antibody specificity for H2AK119cr, implement multiple approaches:

• Peptide competition assay: Test the antibody against crotonylated and non-crotonylated peptides of H2A with specific modifications at K119. A specific antibody should recognize only the crotonylated K119 peptide.

• Metabolic manipulation: Treat cells with crotonate, which enhances histone crotonylation levels. A specific antibody should show increased signal after crotonate treatment .

• Genetic validation: Use SIRT1 knockout cells, which should show significantly increased H2AK119cr levels that can be detected by a specific antibody .

• Cross-reactivity testing: Evaluate reactivity against other histone modifications, particularly other acylations at K119, such as acetylation.

• Immunoblotting with positive and negative controls: Compare wild-type cells with cells exhibiting manipulated crotonylation levels through enzymatic or metabolic interventions.

What are the recommended techniques for detecting H2AK119cr in different experimental settings?

Several techniques can be employed to detect H2AK119cr, each with specific advantages:

How should I design experiments to study the dynamics between H2AK119 crotonylation and ubiquitination?

To study the dynamics between H2AK119cr and H2AK119ub:

• Time-course experiments: Induce replication stress using hydroxyurea and collect samples at different time points to monitor changes in both H2AK119cr and H2AK119ub levels using Western blotting.

• Modulation of crotonylation: Treat cells with crotonate to increase crotonylation levels and observe the consequent changes in ubiquitination .

• Enzyme manipulation:

  • Overexpress or knock out SIRT1 to modulate decrotonylation

  • Manipulate BMI1 expression to affect ubiquitination

  • Monitor both modifications simultaneously using specific antibodies

• Sequential ChIP (Re-ChIP): To determine if H2AK119cr and H2AK119ub occur on the same H2A molecules or on different molecules within the same genomic regions.

• Proximity ligation assays: To detect spatial relationships between crotonylated and ubiquitinated histones.

• In vitro conversion assays: Use purified histones, SIRT1, and BMI1 to recapitulate the sequential modification process in controlled conditions .

What controls should I include when performing ChIP experiments with a Crotonyl-HIST1H2AG (K119) antibody?

When performing ChIP with a Crotonyl-HIST1H2AG (K119) antibody, include these essential controls:

• Input control: Reserve 5-10% of chromatin before immunoprecipitation to normalize ChIP signals.

• Positive control loci: Include genomic regions known to be enriched for H2AK119cr, such as actively transcribed genes.

• Negative control loci: Include heterochromatic regions or silent genes expected to have low H2AK119cr levels.

• IgG control: Perform parallel immunoprecipitation with isotype-matched IgG to assess non-specific binding.

• Peptide competition: Pre-incubate the antibody with crotonylated H2AK119 peptides to demonstrate binding specificity.

• Biological manipulation controls:

  • Samples from cells with increased crotonylation (crotonate treatment)

  • Samples from SIRT1-KO cells (expected to have increased H2AK119cr)

  • Samples before and after replication stress induction

• Cross-antibody validation: Compare ChIP results using antibodies from different sources or that recognize different epitopes containing H2AK119cr.

How does H2AK119 crotonylation influence gene expression and chromatin structure?

H2AK119 crotonylation influences gene expression and chromatin structure through multiple mechanisms:

• Chromatin destabilization: Crotonylation of histones, including H2A, can destabilize nucleosome structure. For example, H3K122cr-containing tetrasomes show decreased thermal stability compared to unmodified H3-H4 tetrasomes .

• Transcriptional regulation: H2AK119cr and H2AK119ub exist in a dynamic equilibrium that regulates transcription. While H2AK119cr is generally associated with active chromatin, its conversion to H2AK119ub during replication stress leads to transcription repression near stalled replication forks .

• Recruitment of reader proteins: Crotonylation likely recruits specific reader proteins that recognize this modification and translate it into functional outcomes within the cell, similar to other histone modifications .

• Competition with other modifications: H2AK119cr competes with H2AK119ub for the same lysine residue, providing a mechanism for switching between different functional states of chromatin in response to cellular conditions .

• Metabolic sensing: The balance between crotonylation and other modifications may reflect cellular metabolic status, serving as an epigenetic mechanism that regulates diverse processes in response to metabolic changes .

What is the role of H2AK119 crotonylation in the DNA damage response and replication stress?

H2AK119 crotonylation plays a crucial role in the DNA damage response and replication stress:

• Dynamic regulation during replication stress: H2AK119cr and H2AK119ub are reversibly regulated in response to replication stress. Under normal conditions, H2AK119cr is present. During replication stress, SIRT1 mediates decrotonylation of H2AK119, which is a prerequisite for subsequent ubiquitination by BMI1 .

• Attenuation of transcription-replication conflicts (TRCs): The switch from H2AK119cr to H2AK119ub helps resolve conflicts between transcription and replication machinery. H2AK119ub accumulates at reversed replication forks, leading to:

  • Release of RNA Polymerase II

  • Transcription repression near stalled replication forks

  • Reduced R-loop formation

  • Decreased DNA double-strand breaks

• Cellular survival during replication stress: The proper regulation of the H2AK119cr-to-H2AK119ub switch by SIRT1 and BMI1 is important for cell survival under replication stress conditions .

• Interconnection with DNA repair mechanisms: Lysine crotonylation has been implicated in DNA repair processes, with CDYL-regulated crotonylation of RPA1 playing a role in homologous recombination (HR)-mediated DNA repair .

How does H2AK119 crotonylation interact with other histone modifications in the context of the histone code?

H2AK119 crotonylation interacts with other histone modifications in several ways:

• Competitive modification at the same residue: H2AK119cr directly competes with H2AK119ub for the same lysine residue, creating a switch mechanism where only one modification can exist at a time on a particular H2A molecule .

• Enzymatic cross-talk: The enzymes that regulate H2AK119cr also modify other histone residues. For example, SIRT1 acts on multiple histone marks, creating potential for coordinated regulation of different modifications .

• Functional associations with other active marks: As crotonylation is generally associated with active chromatin, H2AK119cr likely co-occurs with other active histone marks such as H3K4me3 and various histone acetylations, though this relationship needs further investigation in the specific context of H2AK119cr .

• Metabolic regulation: The availability of crotonyl-CoA, which competes with acetyl-CoA, creates a metabolic link between different acylation modifications. Changes in cellular metabolism can shift the balance between different acylations, including crotonylation and acetylation .

• Sequential modification patterns: The observed pattern where decrotonylation precedes ubiquitination suggests that histone modifications can occur in defined sequences as part of regulatory cascades, adding temporal dimension to the histone code .

Why might I observe inconsistent results when detecting H2AK119cr in different cell lines?

Inconsistent detection of H2AK119cr across cell lines can result from several factors:

• Cell type-specific expression of regulatory enzymes: Different cell lines may express varying levels of writers (p300/CBP), erasers (SIRT1), and other regulatory proteins that affect H2AK119cr levels .

• Metabolic variations: Cell lines have different metabolic profiles affecting the availability of crotonyl-CoA, the substrate for crotonylation. Differences in energy metabolism, redox state, and NAD⁺ levels (required for SIRT1 activity) can all influence crotonylation levels .

• Cell cycle distribution: H2AK119cr levels are regulated by replication stress, which is linked to cell cycle. Cell lines with different proliferation rates or cell cycle distributions may show varying baseline levels of H2AK119cr .

• Extraction method suitability: The standard acid extraction protocol (0.4 N H₂SO₄ followed by TCA precipitation) may need optimization for specific cell types to ensure complete and consistent histone extraction .

• Cross-reactivity issues: Some antibodies may exhibit differential cross-reactivity with other histone modifications depending on the specific pattern of modifications present in different cell lines.

To address these inconsistencies, standardize growth conditions, verify cell cycle status, optimize extraction protocols for each cell line, and validate results using multiple detection methods.

How can I distinguish between true H2AK119 crotonylation signal and antibody cross-reactivity with other acylations?

To distinguish true H2AK119cr signal from cross-reactivity:

• Validation with synthetic peptides: Test antibody reactivity against synthetic peptides containing various acylations (crotonylation, acetylation, butyrylation, etc.) at H2AK119 to establish specificity profiles.

• Metabolic labeling: Treat cells with crotonate to specifically enhance crotonylation without affecting other acylations. A true H2AK119cr antibody should show increased signal after crotonate treatment .

• Enzyme manipulation experiments:

  • SIRT1 knockout or inhibition should increase H2AK119cr specifically

  • Overexpression of p300/CBP may increase both crotonylation and acetylation

  • Compare results from these manipulations to identify modification-specific patterns

• Mass spectrometry validation: For critical experiments, confirm antibody-based results with mass spectrometry analysis of histones to precisely identify and quantify different acylations at H2AK119.

• Competition assays: Pre-incubate antibodies with crotonylated, acetylated, or other acylated peptides before immunodetection to determine if signal blocking is specific to crotonylated peptides.

• Sequential immunoprecipitation: Deplete samples of one modification using a highly specific antibody before testing for the presence of other modifications.

What are the most common pitfalls when optimizing ChIP protocols for H2AK119cr detection?

When optimizing ChIP for H2AK119cr, be aware of these common pitfalls:

• Insufficient crosslinking: Standard 1% formaldehyde for 10 minutes may not optimally preserve H2AK119cr. Test different crosslinking conditions or consider dual crosslinking approaches.

• Inadequate sonication: Over- or under-sonication affects chromatin fragmentation and epitope accessibility. Optimize sonication conditions (e.g., using Diagenode Bioruptor) for each cell type to achieve fragments of 200-500 bp .

• Antibody concentration: The optimal antibody amount needs calibration for each lot and application. Test different antibody concentrations using titration experiments.

• Buffer compatibility: Components in lysis or IP buffers may affect antibody binding to H2AK119cr. Test different buffer compositions, particularly regarding salt concentration and detergent types.

• Loss of modification during processing: Crotonylation may be unstable under certain conditions. Include HDAC inhibitors (like nicotinamide to inhibit SIRT1) in buffers to preserve the modification during lengthy procedures .

• Background issues: High background can mask specific signals. Optimize blocking conditions and include appropriate controls, such as IgG and input normalization.

• PCR inhibition: Components from the ChIP procedure may inhibit subsequent PCR steps. Include purification steps or dilute samples appropriately before PCR.

• Primer design for qPCR: Ensure primers target regions expected to contain H2AK119cr and design them to produce 80-150 bp amplicons for efficient quantification.

How can I investigate the interplay between H2AK119 crotonylation, replication stress, and R-loop formation?

To investigate the interplay between H2AK119cr, replication stress, and R-loops:

• Sequential ChIP-seq analysis: Perform ChIP-seq for H2AK119cr, H2AK119ub, and R-loop markers (e.g., S9.6 antibody that recognizes RNA:DNA hybrids) before and after inducing replication stress with hydroxyurea or other agents. Analyze overlapping and distinct genomic regions.

• Genetic manipulation experiments:

  • Create SIRT1 knockout cells to prevent the crotonylation-to-ubiquitination switch

  • Create BMI1 knockout cells to prevent H2AK119 ubiquitination

  • Analyze R-loop formation in these genetic backgrounds under replication stress using S9.6 immunofluorescence or DRIP-seq (DNA-RNA Immunoprecipitation)

• Live-cell imaging: Develop fluorescent reporters to monitor H2AK119cr, H2AK119ub, and R-loops simultaneously in living cells during replication stress.

• Genomic approaches: Identify genomic regions prone to both transcription-replication conflicts and R-loop formation. Assess how H2AK119cr-to-H2AK119ub switching correlates with these regions.

• Drug intervention studies: Test how SIRT1 inhibitors (e.g., nicotinamide) or enhancers affect R-loop formation during replication stress.

• RNA Polymerase II ChIP-seq: Analyze RNA Pol II occupancy in relation to H2AK119cr and H2AK119ub distribution to validate the model that H2AK119ub promotes RNA Pol II release from chromatin during replication stress .

What techniques can be used to study the dynamic exchange between H2AK119cr and H2AK119ub at specific genomic loci?

To study the dynamic exchange between H2AK119cr and H2AK119ub at specific loci:

• Time-resolved ChIP-seq: Perform ChIP-seq for both modifications at multiple time points after inducing replication stress to track the temporal dynamics of the switch at specific genomic locations.

• FRAP (Fluorescence Recovery After Photobleaching): Use fluorescently tagged reader proteins specific for H2AK119cr and H2AK119ub to monitor the real-time dynamics of these modifications at specific genomic loci.

• CUT&RUN or CUT&Tag methods: These methods offer higher resolution than traditional ChIP and can be performed with fewer cells, allowing more detailed analysis of modification dynamics at specific loci.

• Single-molecule imaging: Develop methods to visualize individual nucleosomes and their modification status in real-time using super-resolution microscopy.

• ChIP-bisulfite sequencing: Combine ChIP for H2AK119cr or H2AK119ub with bisulfite sequencing to correlate the presence of these modifications with DNA methylation patterns at specific loci.

• Nascent RNA sequencing: Correlate the presence of H2AK119cr/H2AK119ub with nascent transcription at specific genomic loci to understand the functional consequences of the modification switch.

• In vitro reconstitution systems: Establish defined systems with purified components to recapitulate the H2AK119cr-to-H2AK119ub switch on specific DNA templates, allowing detailed mechanistic studies .

How can I develop experimental models to investigate the role of H2AK119cr in diseases associated with replication stress?

To develop models investigating H2AK119cr in replication stress-associated diseases:

• Patient-derived cell lines: Establish cell lines from patients with conditions characterized by high replication stress (e.g., certain cancers, premature aging syndromes). Compare H2AK119cr/H2AK119ub dynamics with healthy controls.

• CRISPR-engineered disease models:

  • Create cell lines with mutations in SIRT1 or BMI1 that mimic disease-associated variants

  • Engineer mutations in H2A that prevent crotonylation or ubiquitination at K119

  • Assess consequences for replication stress response and genome stability

• Organoid systems: Develop 3D organoid cultures that better recapitulate tissue architecture and cellular heterogeneity to study H2AK119cr dynamics in a more physiologically relevant context.

• Mouse models: Generate conditional knockout models for SIRT1 or BMI1 in tissues prone to replication stress-associated pathologies. Analyze H2AK119cr levels and associated phenotypes.

• Drug screening platforms: Develop high-throughput assays to identify compounds that modulate the H2AK119cr-to-H2AK119ub switch, potentially leading to therapeutic approaches for diseases with dysregulated replication stress responses.

• Correlation studies: Analyze H2AK119cr levels in tissue samples from diseases associated with replication stress and correlate with clinical parameters and outcomes.

• Cell viability assays: Assess how disruption of H2AK119cr regulation affects cell survival under replication stress conditions that mimic disease states. For example, test sensitivity to hydroxyurea or doxorubicin in cells with altered SIRT1 or BMI1 expression .

How should I analyze ChIP-seq data for H2AK119cr to distinguish its unique distribution from other histone modifications?

For optimal H2AK119cr ChIP-seq data analysis:

• Differential binding analysis: Compare H2AK119cr distribution with other histone modifications (particularly H2AK119ub and various acetylations) to identify uniquely enriched regions using tools like DiffBind or MAnorm.

• Composite profile analysis: Generate metagene plots showing the distribution of H2AK119cr relative to transcription start sites, gene bodies, and transcription end sites. Compare these profiles with other modifications to identify unique patterns.

• Correlation heatmaps: Create correlation matrices comparing genome-wide distributions of H2AK119cr with other histone marks to quantify similarities and differences.

• Motif enrichment analysis: Identify DNA sequence motifs enriched in H2AK119cr-marked regions, which might indicate specific transcription factor associations.

• Integration with transcriptomic data: Correlate H2AK119cr peaks with RNA-seq data to determine associations with gene expression levels and transcriptional states.

• Chromatin state analysis: Use tools like ChromHMM or Segway to define chromatin states and determine which states are specifically associated with H2AK119cr.

• Peak shape analysis: Examine the breadth and intensity of H2AK119cr peaks compared to other modifications, as different histone marks can display characteristic peak morphologies.

• Replication timing correlation: Analyze the relationship between H2AK119cr distribution and replication timing domains to connect with its role in replication stress response.

What statistical approaches should I use when analyzing changes in H2AK119cr levels across different experimental conditions?

When analyzing changes in H2AK119cr levels:

• Normalization strategies:

  • For Western blot data: Normalize H2AK119cr signal to total H2A or loading controls like H3

  • For ChIP-seq data: Use spike-in normalization with exogenous chromatin (e.g., Drosophila) or normalization to unchanged regions

• Statistical tests for global level changes:

  • For Western blot quantification: Use paired t-tests or ANOVA with appropriate post-hoc tests for multiple conditions

  • For immunofluorescence quantification: Consider mixed-effects models to account for cell-to-cell variability

• Differential binding analysis for ChIP-seq:

  • Use specialized tools like DiffBind, MACS2 bdgdiff, or DESeq2

  • Apply appropriate multiple testing correction (e.g., Benjamini-Hochberg)

  • Consider log2 fold change thresholds in addition to p-values

• Time-course analysis:

  • Use regression models for temporal trends

  • Consider smoothing approaches for noisy time-series data

  • Apply repeated measures ANOVA for multiple time points

• Integration with other data types:

  • Use multivariate approaches when integrating with transcriptomic or other epigenomic data

  • Consider dimensionality reduction techniques like PCA or t-SNE

• Effect size estimation:

  • Calculate Cohen's d or similar metrics to quantify the magnitude of changes

  • Report confidence intervals for effect sizes

• Bayesian approaches:

  • Consider Bayesian statistics for small sample sizes or complex experimental designs

  • Use prior information from similar studies to improve estimation

How can I differentiate between direct and indirect effects when manipulating the H2AK119cr-to-H2AK119ub switch?

To differentiate direct from indirect effects when studying the H2AK119cr-to-H2AK119ub switch:

• Rapid induction systems:

  • Use degron-tagged SIRT1 or BMI1 for acute depletion

  • Employ chemical-genetic approaches for rapid enzyme activation/inhibition

  • Monitor early vs. late changes in H2AK119cr/H2AK119ub and downstream effects

• In vitro reconstitution:

  • Assemble defined systems with purified components

  • Verify that purified SIRT1 can directly decrotonylate H2AK119cr

  • Confirm that BMI1 can directly ubiquitinate K119 after decrotonylation

• Sequential ChIP experiments:

  • Perform time-resolved ChIP-seq after inducing replication stress

  • Identify genomic regions where H2AK119cr decreases before H2AK119ub increases

  • Map these changes to transcriptional effects and R-loop formation

• Rescue experiments:

  • Compare wildtype cells, enzyme knockout cells, and knockout cells complemented with catalytically inactive mutants

  • Design H2A mutants that can only be modified in one way (e.g., K119R or K119Q)

• Local manipulation approaches:

  • Use CRISPR-dCas9 fusions to recruit SIRT1 or BMI1 to specific genomic loci

  • Analyze local effects on H2AK119cr/H2AK119ub, transcription, and R-loop formation

• Correlation analysis with causality testing:

  • Apply Granger causality or similar statistical approaches to time-series data

  • Determine temporal precedence in modification changes and downstream effects

• Mathematical modeling:

  • Develop kinetic models of the H2AK119cr-to-H2AK119ub switch

  • Test different scenarios and compare with experimental data

  • Use modeling to identify which effects can be explained by direct mechanisms

How might single-cell technologies advance our understanding of H2AK119cr dynamics in heterogeneous cell populations?

Single-cell technologies offer transformative potential for understanding H2AK119cr dynamics:

• Single-cell ChIP-seq adaptations:

  • Modified CUT&Tag or CUT&RUN protocols compatible with single-cell workflows

  • Integration with cell sorting to analyze specific subpopulations

  • Correlation of H2AK119cr patterns with cell cycle phases or differentiation states

• Single-cell multi-omics approaches:

  • Simultaneous analysis of H2AK119cr distribution and transcriptome in the same cells

  • Integration with chromatin accessibility data (ATAC-seq)

  • Combined profiling of multiple histone modifications

• Mass cytometry applications:

  • Adaptation of CyTOF with H2AK119cr-specific antibodies

  • Simultaneous quantification of multiple protein modifications

  • High-throughput screening of cellular responses to perturbations

• Live-cell imaging at single-cell resolution:

  • Development of specific readers for H2AK119cr and H2AK119ub

  • Real-time monitoring of modification dynamics during replication stress

  • Correlation with cell fate decisions (proliferation, senescence, apoptosis)

• Single-cell computational approaches:

  • Trajectory inference to map dynamics of H2AK119cr during cellular processes

  • Network analysis to identify modification patterns associated with specific phenotypes

  • Machine learning to predict cellular responses based on epigenetic profiles

• Spatial transcriptomics integration:

  • Combining H2AK119cr analysis with spatial information

  • Investigating tissue microenvironment effects on crotonylation levels

• Lineage tracing experiments:

  • Tracking how H2AK119cr patterns are inherited through cell divisions

  • Determining if certain patterns predispose cells to specific fates or vulnerabilities

What are the potential implications of H2AK119cr dysregulation in cancer and other diseases?

Potential implications of H2AK119cr dysregulation in disease contexts:

• Cancer progression and therapy resistance:

  • Altered H2AK119cr-to-H2AK119ub switching may affect genome stability in cancer cells

  • Dysregulation of transcription-replication conflicts could drive genomic instability and mutation accumulation

  • Aberrant resolution of replication stress might contribute to therapy resistance

• Neurodegenerative diseases:

  • Neurons are particularly vulnerable to transcription-replication conflicts

  • H2AK119cr dysregulation might contribute to DNA damage in post-mitotic neurons

  • SIRT1 function is implicated in several neurodegenerative conditions

• Inflammatory and autoimmune disorders:

  • Histone crotonylation is linked to inflammatory responses

  • Dysregulation might affect immune cell function and inflammatory gene expression

  • R-loop accumulation due to improper H2AK119cr-to-H2AK119ub switching could trigger autoimmune responses

• Developmental disorders:

  • Proper epigenetic regulation is crucial during development

  • Defects in H2AK119cr dynamics might affect transcriptional programs during differentiation

  • BMI1 and SIRT1 have established roles in stem cell biology and development

• Metabolic diseases:

  • Crotonylation depends on metabolic factors like crotonyl-CoA availability

  • Metabolic disorders might disrupt the balance between different acylations

  • SIRT1 function is closely linked to metabolic regulation and energy homeostasis

• Aging-related pathologies:

  • Replication stress increases with age

  • SIRT1 function declines during aging

  • Impaired management of transcription-replication conflicts may contribute to age-related genome instability

• Biomarker potential:

  • H2AK119cr levels might serve as biomarkers for disease states or treatment responses

  • The H2AK119cr/H2AK119ub ratio could indicate cellular stress levels and disease progression

How can integrating multi-omics data enhance our understanding of H2AK119cr function in different cellular contexts?

Integrating multi-omics approaches offers comprehensive insights into H2AK119cr function:

• Combined epigenomic profiling:

  • Integrate H2AK119cr ChIP-seq with maps of other histone modifications

  • Correlate with DNA methylation patterns using WGBS or reduced representation methods

  • Incorporate chromatin accessibility data from ATAC-seq or DNase-seq

  • Include chromatin conformation data (Hi-C, Micro-C) to understand 3D context

• Transcriptome integration:

  • Correlate H2AK119cr distribution with RNA-seq to link to gene expression

  • Include nascent transcriptomics (PRO-seq, GRO-seq) to capture immediate transcriptional effects

  • Analyze RNA processing patterns (splicing, polyadenylation) in relation to H2AK119cr

• Proteomics approaches:

  • Identify proteins that interact with crotonylated H2A using techniques like RIME or BioID

  • Analyze global proteome changes following manipulation of H2AK119cr levels

  • Include post-translational modification profiling to understand broader signaling networks

• Metabolomics integration:

  • Measure levels of metabolic intermediates like crotonyl-CoA and acetyl-CoA

  • Correlate metabolic states with global H2AK119cr patterns

  • Understand how metabolic perturbations affect the H2AK119cr-to-H2AK119ub switch

• Computational integration frameworks:

  • Develop machine learning approaches to identify patterns across multi-omics datasets

  • Use network analysis to reveal functional modules connected to H2AK119cr

  • Implement causal inference methods to establish directional relationships

• Temporal multi-omics:

  • Collect multiple data types across time courses during replication stress

  • Establish temporal relationships between H2AK119cr changes and other molecular events

  • Build predictive models of cellular responses based on initial H2AK119cr states

• Spatial multi-omics:

  • Integrate imaging data with molecular profiles

  • Understand how nuclear organization affects H2AK119cr distribution

  • Correlate H2AK119cr patterns with replication factory locations and transcription hubs