Recombinant Rat E3 ubiquitin-protein ligase UHRF1 (Uhrf1)

What are the key functional domains of UHRF1 and their roles?

UHRF1 is a multi-domain epigenetic regulator comprising several distinct functional units that work in concert to perform its various cellular functions. The protein contains:

• Tandem Tudor Domain (TTD): Recognizes and binds to methylated lysine 9 on histone H3 (H3K9me2/3)

• Plant Homeodomain (PHD) finger: Specifically binds to unmodified arginine 2 on histone H3 (H3-R2)

• SET and RING-Associated (SRA) domain: Binds to hemi-methylated CpG sites on DNA

• RING domain: Possesses E3 ubiquitin ligase activity, responsible for histone H3 ubiquitylation

• Ubiquitin-like (UBL) domain: Essential for RING-mediated H3 ubiquitylation by stabilizing the E2-E3-chromatin complex

The linked TTD-PHD module allows UHRF1 to simultaneously recognize both unmodified H3-R2 and methylated H3-K9 on a single histone H3 tail, demonstrating its ability for combinatorial readout of histone modifications . This structural organization enables UHRF1 to connect specific histone modification patterns with DNA methylation maintenance.

How does UHRF1 recruit DNMT1 to maintain DNA methylation patterns?

UHRF1 plays a critical role in the inheritance of DNA methylation patterns through a well-orchestrated mechanism:

• During DNA replication, UHRF1 recognizes and binds to hemi-methylated CpG sites via its SRA domain

• The RING domain of UHRF1 catalyzes the ubiquitylation of histone H3 at specific lysine residues (K14, K18, and/or K23)

• These ubiquitin marks serve as docking sites for DNMT1, which recognizes them through a ubiquitin interaction motif in its replication foci targeting sequence (RFTS) domain

• Once recruited, DNMT1 methylates the newly synthesized DNA strand, thereby maintaining DNA methylation patterns across cell divisions

This process ensures the faithful inheritance of DNA methylation patterns during cell division, which is essential for maintaining cellular identity and genomic stability. The UBL domain of UHRF1 is particularly important in this process, as it helps stabilize the E2-E3-chromatin complex necessary for efficient H3 ubiquitylation .

What are the typical expression patterns of UHRF1 in different tissues and developmental stages?

UHRF1 shows distinctive temporal and spatial expression patterns across different tissues and developmental stages:

• In reproductive tissues:

  • UHRF1 is highly expressed in pre-mature Sertoli cells with gradually decreasing levels as they mature

  • In mice, UHRF1 levels in Sertoli cells decline gradually from postnatal day 1 (P1) to P14, after which it becomes barely detectable

  • UHRF1 is essential for germ cell development in both males and females

• In developmental contexts:

  • UHRF1 is required for early embryonic development

  • Global knockout of UHRF1 causes developmental arrest shortly after gastrulation in mice, highlighting its essential role in early development

  • UHRF1 expression is typically higher in proliferating cells and decreases upon differentiation, consistent with its role in cell cycle progression

• In adipose tissue:

  • UHRF1 serves as a key regulatory factor for adipogenesis

  • It regulates the expression of adipogenic transcription factors like PPAR-γ and C/EBP-α

This differential expression pattern suggests that UHRF1 functions are context-dependent and tightly regulated during development and tissue homeostasis.

How does the UBL domain of UHRF1 contribute to its E3 ligase activity at the molecular level?

The UBL domain plays a crucial and previously underappreciated role in UHRF1's E3 ligase function through several molecular mechanisms:

• Stabilization of the E2-E3-chromatin complex:

  • The UBL domain contains a hydrophobic patch that contacts a regulatory "backside" surface on the E2 ubiquitin conjugating enzyme UbcH5a/UBE2D1

  • This interaction significantly enhances the stability of the E2-E3-chromatin complex, which is essential for efficient ubiquitin transfer

  • Similar to ubiquitin itself, the UBL exerts its effects through this hydrophobic patch

• Structural rearrangements within UHRF1:

  • Crosslinking and mass spectrometry (XL-MS) studies have revealed that the UBL domain participates in distinct structural rearrangements within UHRF1

  • These rearrangements are triggered by UHRF1's engagement with chromatin and the E2 enzyme UbcH5a

  • The conformational changes likely position the RING domain optimally for ubiquitin transfer

• Experimental evidence of UBL domain importance:

  • Removal of the entire UBL domain or mutation of a single residue in its hydrophobic patch significantly interferes with the efficient recruitment of the E2 enzyme to chromatin

  • In mouse embryonic stem cells, mutation of the hydrophobic patch within UHRF1 results in reduced DNMT1 recruitment to newly replicated chromatin and loss of DNA methylation at repetitive elements

This detailed molecular understanding of the UBL domain's role has significant implications for understanding how UHRF1 coordinates epigenetic modifications and maintains DNA methylation patterns during cell division.

What methodologies are optimal for studying UHRF1-dependent histone ubiquitylation in vitro?

Studying UHRF1-dependent histone ubiquitylation requires specialized methodologies to recapitulate the complex chromatin environment. Based on recent research, the following approaches have proven effective:

• Chromatin substrate preparation:

  • 12 × 187 bp chromatin arrays (containing 12 nucleosomes regularly spaced by 601-nucleosome positioning sequences) yield the highest E3 activity compared to mono-nucleosomes or di- and tetra-nucleosomes

  • For enhanced activity, modified substrates containing H3K9me3 markedly stimulate both the rate and extent of H3 ubiquitylation in mono-nucleosomes and chromatin arrays

  • Fully CpG methylated DNA can increase UHRF1 auto-ubiquitylation rather than H3 ubiquitylation

• In vitro ubiquitylation assay components:

  • Recombinant UHRF1 (full-length or domain-specific variants)

  • E1 ubiquitin-activating enzyme

  • E2 ubiquitin-conjugating enzyme (preferably UbcH5a/UBE2D1)

  • Ubiquitin

  • ATP regeneration system

  • Appropriate chromatin substrate

• Analysis techniques:

  • Western blotting to detect mono-, di-, and tri-ubiquitylated H3 species

  • Mass spectrometry to identify specific ubiquitylation sites (predominant ubiquitylation at H3-K18 and H3-K23)

  • Crosslinking and mass spectrometry (XL-MS) to detect intra-molecular interactions and structural rearrangements within UHRF1

• Important experimental considerations:

  • The stimulation and targeting of UHRF1 E3 activity within the chromatin context appears to be a two-step process

  • Extent and position of pre-existing modifications on a nucleosome influence both the enzymatic rate and the correct transfer of ubiquitin to histone H3

  • Generating suitable long hemi-methylated chromatin substrates remains technically challenging

These methodological approaches provide a framework for investigating the molecular mechanisms of UHRF1-mediated histone ubiquitylation and its role in epigenetic regulation.

How can researchers effectively manipulate UHRF1 function in cellular systems to study its role in DNA methylation?

Researchers can employ several strategies to manipulate UHRF1 function in cellular systems for studying its role in DNA methylation:

• Genetic manipulation approaches:

  • CRISPR/Cas9-based knockout strategies to comprehensively analyze whole transcriptomic changes upon UHRF1 deletion

  • Domain-specific mutations, particularly in the hydrophobic patch of the UBL domain, which can disrupt UHRF1's E3 ubiquitin ligase activity without affecting its chromatin binding properties

  • N-terminal tagging considerations: N-terminally 3×FLAG-tagged UHRF1 has been shown to be unable to rescue DNA methylation in certain contexts, suggesting that tag position can significantly impact function

• Post-translational modification manipulation:

  • Phosphorylation of Ser-298 in the intermodule linker between the TTD and PHD finger has been shown to abrogate bivalent UHRF1:H3 interaction by altering the relative position of these two reader modules

  • This finding suggests that manipulating the phosphorylation state of the linker region can modulate UHRF1 function as a functional switch involved in multiple regulatory pathways

• Cell type considerations:

  • Mouse embryonic stem cells (mESCs) provide an excellent model system for studying UHRF1's role in maintaining DNA methylation patterns

  • For tissue-specific functions, cell types like pre-mature Sertoli cells, which naturally express high levels of UHRF1, can be valuable models

  • Adipocyte models can be used to study UHRF1's role in metabolism and adipogenesis

• Readout systems:

  • DNA methylation analysis at repetitive elements, which are particularly sensitive to loss of UHRF1 function

  • Immunofluorescence analysis of DNMT1 recruitment to newly replicated chromatin

  • RNA-seq to identify genes whose expression is altered upon UHRF1 manipulation

These approaches allow researchers to dissect the complex functions of UHRF1 in DNA methylation maintenance and its wider roles in epigenetic regulation.

What are the most effective strategies for producing high-quality recombinant rat UHRF1 for structural and functional studies?

Producing high-quality recombinant rat UHRF1 requires careful consideration of expression systems, purification strategies, and quality control measures:

• Expression systems:

  • Bacterial expression (E. coli): Suitable for individual domains (UBL, TTD, PHD, SRA, RING)

  • Insect cell expression (Sf9, Hi5): Preferred for full-length UHRF1 to ensure proper folding and post-translational modifications

  • Mammalian expression systems: Useful for studies requiring mammalian-specific modifications

• Expression constructs:

  • Full-length UHRF1 (for comprehensive functional studies)

  • Domain-specific constructs:

    • TTD-PHD module (aa 126-366): For histone binding studies

    • UBL domain (aa 1-77): For E3 ligase activity studies

    • SRA domain: For hemi-methylated DNA binding studies

• Purification strategies:

  • Affinity tags: 6×His, GST, or MBP tags facilitate initial purification

  • Chromatography sequence: Affinity chromatography followed by ion exchange and size exclusion chromatography

  • Tag removal: Include a protease cleavage site for tag removal if the tag might interfere with function

• Quality control measures:

  • SDS-PAGE and Western blotting to confirm protein identity and purity

  • Dynamic light scattering to assess homogeneity

  • Thermal shift assays to evaluate protein stability

  • Functional assays to confirm activity:

    • Histone binding assays for TTD-PHD module

    • DNA binding assays for SRA domain

    • In vitro ubiquitylation assays for full-length protein or RING domain

• Storage considerations:

  • Flash-freeze purified protein in small aliquots to avoid freeze-thaw cycles

  • Typical storage buffer: 20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT, 10% glycerol

Researchers should be aware that due to its conformational flexibility and size, full-length UHRF1 can be challenging to study by crystallography, electron microscopy, or nuclear magnetic resonance approaches . For structural studies, focus on individual domains or specific domain combinations may be more productive.

How can researchers effectively analyze the interaction between UHRF1 and chromatin substrates?

Analyzing UHRF1-chromatin interactions requires specialized techniques that can capture the complex nature of these interactions:

• Nucleosome binding assays:

  • Electrophoretic mobility shift assays (EMSAs) with recombinant UHRF1 and various nucleosomal substrates

  • Pull-down assays using immobilized nucleosomes or chromatin arrays

  • Surface plasmon resonance (SPR) for real-time binding kinetics

  • Microscale thermophoresis for quantitative binding analysis

• Chromatin substrate considerations:

  • Test different nucleosomal contexts: mono-nucleosomes, di-nucleosomes, tetra-nucleosomes, and 12 × 187 bp chromatin arrays

  • Compare unmodified nucleosomes with those containing specific modifications:

    • H3K9me3-modified octamers

    • Hemi-methylated or fully methylated CpG DNA

    • Combinations of histone and DNA modifications

• Structural analysis approaches:

  • Crosslinking and mass spectrometry (XL-MS) to identify intra-molecular interactions within UHRF1 when bound to chromatin

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify regions of UHRF1 that undergo conformational changes upon chromatin binding

  • Cryo-electron microscopy for visualizing UHRF1-nucleosome complexes

• Functional interaction analysis:

  • In vitro ubiquitylation assays to assess how different chromatin substrates affect UHRF1's E3 ligase activity

  • Assess both H3 ubiquitylation and UHRF1 auto-ubiquitylation rates

  • Mass spectrometric analysis to identify specific ubiquitylation sites on histone H3

• Cellular interaction analysis:

  • Chromatin immunoprecipitation (ChIP) to identify genomic regions bound by UHRF1

  • Proximity ligation assays to detect UHRF1-chromatin interactions in situ

  • FRAP (Fluorescence Recovery After Photobleaching) to analyze UHRF1 dynamics on chromatin

These methodologies provide complementary information about how UHRF1 interacts with chromatin, how these interactions are regulated, and how they contribute to UHRF1's function in epigenetic regulation.

What techniques are recommended for investigating UHRF1's role in tissue-specific contexts like spermatogenesis or adipogenesis?

Investigating UHRF1's role in tissue-specific contexts requires specialized techniques tailored to the biological system of interest:

• For spermatogenesis studies:

  • Temporal expression analysis:

    • Immunostaining assays to detect UHRF1 in testicular tissues at different developmental stages (e.g., E18.5, P1, P14 in mice)

    • Analysis of purified primary Sertoli cells from different stages (e.g., pre-mature P3 vs. mature P21)

  • Conditional knockout approaches:

    • Sertoli cell-specific Cre drivers (e.g., Amh-Cre) for UHRF1 deletion

    • Tamoxifen-inducible systems for temporal control of deletion

  • Functional assays:

    • Analysis of Sertoli-Germ cell adhesion and cytoskeletal organization

    • Assessment of ECM- and cell adhesion-related gene expression (e.g., Timp1, Trf, and Spp1)

    • Evaluation of CG methylation maintenance at specific target genes

• For adipogenesis studies:

  • Differentiation models:

    • Pre-adipocyte cell lines (e.g., 3T3-L1)

    • Primary stromal vascular fraction cells

    • CRISPR/Cas9-based UHRF1 knockout in these models

  • Transcriptomic analysis:

    • RNA-seq to identify genes affected by UHRF1 deletion

    • Bioinformatics analysis focused on adipogenesis regulators (e.g., PPAR-γ, C/EBP-α)

    • Analysis of TGF-β signaling pathway components

  • Secretome analysis:

    • Assessment of TGF-β1 and GPNMB secretion in UHRF1-depleted cells

    • Evaluation of fibrosis markers

• Common techniques applicable to both contexts:

  • DNA methylation analysis:

    • Bisulfite sequencing to assess maintenance of CG methylation

    • Targeted analysis of repetitive elements particularly sensitive to UHRF1 deletion

  • Chromatin immunoprecipitation (ChIP):

    • To identify UHRF1 binding sites in the genome

    • To assess changes in histone modifications upon UHRF1 deletion

  • Protein-protein interaction studies:

    • Co-immunoprecipitation to identify tissue-specific UHRF1 interactors

    • Proximity labeling approaches (BioID, APEX) for in vivo interaction mapping

These tissue-specific approaches allow researchers to dissect the context-dependent functions of UHRF1 in different biological systems, providing insights into its diverse roles in development and disease.

How should researchers interpret contradictory findings regarding UHRF1 function in different experimental systems?

Interpreting contradictory findings regarding UHRF1 function requires careful consideration of several factors that may contribute to these discrepancies:

• Domain-specific functions and interactions:

  • UHRF1 is a multi-domain protein with each domain contributing to distinct functions

  • Different experimental approaches may emphasize or disrupt specific domain functions

  • Consider whether contradictory findings might reflect domain-specific effects rather than global UHRF1 function

• Cell type and developmental stage considerations:

  • UHRF1 expression and function varies significantly across cell types and developmental stages

  • Pre-mature vs. mature Sertoli cells show dramatic differences in UHRF1 expression

  • Proliferating vs. differentiating cells may utilize UHRF1 for different functions

  • Consider whether contradictory findings reflect cell type-specific roles rather than universal functions

• Experimental system limitations:

  • In vitro reconstitution systems may lack essential cofactors or modifications

  • Different chromatin substrates (mono-nucleosomes vs. chromatin arrays) yield different results in UHRF1 activity assays

  • N-terminal tagging of UHRF1 can disrupt function in some contexts

  • Consider whether contradictory findings might result from specific experimental limitations

• Post-translational modifications:

  • Phosphorylation of the linker region between TTD and PHD finger can modulate UHRF1 function

  • Other modifications may similarly affect UHRF1 in context-specific ways

  • Consider whether contradictory findings might reflect different post-translational modification states

• Resolution approach for contradictory findings:

  • Perform comprehensive domain mapping to identify specific regions responsible for observed effects

  • Use multiple complementary techniques to validate findings

  • Test findings across different cell types or developmental stages

  • Consider how modifications or interacting partners might explain context-dependent functions

By systematically addressing these factors, researchers can better interpret seemingly contradictory findings and develop a more nuanced understanding of UHRF1's complex functions in different biological contexts.

What are the most common pitfalls in analyzing UHRF1-mediated histone modifications and how can they be avoided?

Analysis of UHRF1-mediated histone modifications presents several technical challenges that researchers should be aware of to avoid misinterpretation:

• Substrate complexity issues:

  • Pitfall: Using oversimplified chromatin substrates that fail to recapitulate physiological conditions

  • Solution: Use 12 × 187 bp chromatin arrays rather than mono-nucleosomes for more physiologically relevant results

  • Consideration: Different substrates can significantly affect both the rate and specificity of UHRF1-mediated ubiquitylation

• Modification cross-talk challenges:

  • Pitfall: Failing to account for pre-existing modifications that influence UHRF1 activity

  • Solution: Systematically test how H3K9me3 and DNA methylation status affect UHRF1's E3 ligase activity

  • Consideration: H3K9me3 modification has a stimulatory effect on the rate and extent of H3 ubiquitylation

• Target site specificity issues:

  • Pitfall: Not identifying the specific lysine residues that are ubiquitylated

  • Solution: Use mass spectrometric analysis to identify specific ubiquitylation sites (predominant ubiquitylation at H3-K18 and H3-K23)

  • Consideration: Different experimental conditions may alter the pattern of target lysine residues

• Auto-ubiquitylation complications:

  • Pitfall: Confusing UHRF1 auto-ubiquitylation with targeted H3 ubiquitylation

  • Solution: Use fully CpG methylated DNA with caution as it can increase UHRF1 auto-ubiquitylation rather than H3 ubiquitylation

  • Consideration: The pattern of auto-ubiquitylation vs. substrate ubiquitylation may provide insights into regulatory mechanisms

• Methodological standardization:

  • Pitfall: Lack of standardized assay conditions leading to inconsistent results

  • Solution: Establish clear protocols for in vitro ubiquitylation assays, including E2 enzyme choice (preferably UbcH5a/UBE2D1)

  • Consideration: The choice of E2 enzyme can significantly affect UHRF1's E3 ligase activity and specificity

• Technical limitations for physiological substrates:

  • Pitfall: Inability to generate suitable long hemi-methylated chromatin substrates

  • Solution: Acknowledge this limitation and interpret results accordingly

  • Consideration: The two-step process of stimulation and targeting of UHRF1 E3 activity within the chromatin context remains difficult to fully recapitulate in vitro

By addressing these common pitfalls, researchers can improve the reliability and physiological relevance of their analyses of UHRF1-mediated histone modifications.

How can researchers effectively distinguish between the direct and indirect effects of UHRF1 manipulation in cellular systems?

Distinguishing between direct and indirect effects of UHRF1 manipulation requires strategic experimental design and careful data interpretation:

• Acute vs. chronic depletion strategies:

  • Acute depletion (e.g., auxin-inducible degron systems) allows observation of immediate effects before compensatory mechanisms engage

  • Chronic depletion (e.g., stable knockout) may reveal more extensive but potentially indirect effects

  • Comparison between these approaches can help distinguish primary from secondary effects

• Rescue experiments with domain mutants:

  • Use domain-specific mutants that selectively disrupt specific UHRF1 functions:

    • UBL domain hydrophobic patch mutants specifically disrupt E3 ligase activity

    • SRA domain mutants disrupt hemi-methylated DNA binding

    • TTD-PHD module mutants disrupt histone binding

  • Compare phenotypes between these mutants to map specific functions to observed effects

• Direct target identification strategies:

  • ChIP-seq to identify genomic loci directly bound by UHRF1

  • Protein-protein interaction studies (IP-MS) to identify direct binding partners

  • Crosslinking approaches to capture transient interactions

  • Compare these direct targets with genes/proteins altered upon UHRF1 manipulation

• Temporal analysis of effects:

  • Time-course experiments following UHRF1 manipulation

  • Early changes are more likely to represent direct effects

  • Network analysis to model how early changes propagate to later effects

• Manipulation of downstream effectors:

  • Independent manipulation of putative downstream effectors (e.g., DNMT1)

  • If manipulating a downstream effector mimics UHRF1 depletion effects, this supports an indirect mechanism through that effector

  • Co-depletion experiments can help establish epistatic relationships

• Cell-free reconstitution systems:

  • Use in vitro systems with purified components to test direct biochemical activities

  • Validate that effects observed in cells can be recapitulated with purified components

  • This approach is particularly valuable for studying UHRF1's E3 ligase activity

By systematically applying these approaches, researchers can build a more accurate model of which cellular effects result directly from UHRF1 function versus those that arise as secondary consequences of disrupting UHRF1-dependent processes.

What are the most promising therapeutic applications of modulating UHRF1 function?

UHRF1's central role in epigenetic regulation makes it a promising therapeutic target with several potential applications:

• Cancer therapeutics:

  • UHRF1 is frequently overexpressed in various cancers, including breast cancer and malignant pleural mesothelioma

  • Targeting UHRF1's E3 ligase activity could disrupt DNA methylation maintenance in rapidly dividing cancer cells

  • Domain-specific inhibitors could selectively disrupt cancer-promoting functions while preserving essential functions

  • Potential therapeutic strategies include:

    • Small molecule inhibitors targeting the UBL hydrophobic patch to disrupt E2-E3 interactions

    • Compounds that interfere with the TTD-PHD module's recognition of histone marks

    • Approaches that disrupt UHRF1's interaction with DNMT1

• Metabolic disorders:

  • UHRF1's role in adipogenesis suggests potential applications in metabolic diseases

  • UHRF1 functions as a metabolic guardian restricting AMPK activity

  • Therapeutic modulation could help address aberrant adipocyte differentiation in obesity

  • Targeting UHRF1's role in TGF-β signaling could help address fibrosis in metabolic disorders

• Reproductive medicine:

  • UHRF1's essential role in germ cell development and Sertoli-Germ cell communication suggests applications in reproductive medicine

  • Modulating UHRF1 function might help address certain forms of infertility

  • Targeting tissue-specific functions could minimize side effects

• Considerations for therapeutic development:

  • Domain-specific approaches may offer greater specificity than targeting the entire protein

  • Tissue-specific delivery systems will be essential given UHRF1's widespread functions

  • Temporal control of UHRF1 modulation may be necessary to avoid developmental defects

  • Careful assessment of off-target effects will be critical given UHRF1's role in fundamental cellular processes

These promising therapeutic directions must be balanced with careful consideration of UHRF1's essential roles in normal cellular function, particularly in rapidly dividing cells and during development.

What novel technologies might advance our understanding of UHRF1's role in epigenetic regulation?

Several emerging technologies hold promise for deepening our understanding of UHRF1's complex roles in epigenetic regulation:

• Advanced structural biology approaches:

  • Cryo-electron microscopy (cryo-EM) for visualizing full-length UHRF1 in complex with nucleosomes

  • Integrative structural biology combining crystallography, NMR, SAXS, and computational modeling

  • Time-resolved structural techniques to capture dynamic conformational changes during UHRF1 function

• Single-molecule techniques:

  • Single-molecule FRET to observe real-time conformational changes in UHRF1 upon binding to chromatin and E2 enzymes

  • Optical tweezers or magnetic tweezers to study the mechanical aspects of UHRF1-chromatin interactions

  • Single-molecule tracking in living cells to observe UHRF1 dynamics during DNA replication

• Advanced genomic and epigenomic profiling:

  • CUT&Tag or CUT&RUN for high-resolution mapping of UHRF1 binding sites

  • Single-cell approaches to understand cell-to-cell variation in UHRF1 function

  • Long-read sequencing to examine UHRF1's role in maintaining methylation at repetitive elements

  • Spatial epigenomics to understand UHRF1's function in the context of nuclear organization

• Protein engineering approaches:

  • Optogenetic control of UHRF1 domains to manipulate function with spatial and temporal precision

  • Engineered allosteric switches to control UHRF1 conformational states

  • Split protein complementation systems to study domain interactions in living cells

• Advanced in vitro reconstitution systems:

  • Development of methods to generate physiologically relevant hemi-methylated chromatin arrays

  • Reconstitution of the complete DNA methylation maintenance machinery including replication factors

  • Microfluidic approaches for high-throughput analysis of UHRF1 function under various conditions

• Computational approaches:

  • Molecular dynamics simulations to understand the conformational dynamics of UHRF1

  • Machine learning approaches to integrate diverse datasets and predict context-specific functions

  • Systems biology modeling to understand how UHRF1 coordinates with other epigenetic regulators

These technological advances will help address current challenges in studying UHRF1, particularly its dynamic conformational changes, context-specific functions, and integration with other epigenetic regulatory mechanisms.

How might advanced genomic and proteomic techniques help uncover new functions of UHRF1 beyond its known roles?

Advanced genomic and proteomic techniques offer powerful approaches to discover novel UHRF1 functions beyond its established roles in DNA methylation maintenance:

• Unbiased interaction profiling approaches:

  • Proximity labeling methods (BioID, APEX) to identify context-specific UHRF1 interactors in different cell types and conditions

  • Crosslinking mass spectrometry to capture transient and weak interactions

  • Thermal proximity coaggregation (TPCA) to identify protein complexes containing UHRF1 in intact cells

  • These approaches might reveal unexpected binding partners suggesting novel functions

• Multi-omics integration strategies:

  • Integrated analysis of UHRF1 ChIP-seq, RNA-seq, and DNA methylation data from UHRF1-manipulated cells

  • Correlation with histone modification patterns and chromatin accessibility

  • Network analysis to identify regulatory circuits involving UHRF1

  • These approaches could reveal coordinated epigenetic programs controlled by UHRF1

• Systematic domain function analysis:

  • CRISPR-based domain-focused mutagenesis screens

  • Complementation assays with domain-swapped chimeric proteins

  • Targeted degradation of specific UHRF1 domains

  • These approaches could uncover domain-specific functions beyond current knowledge

• Spatial genomics and nuclear organization studies:

  • Hi-C analysis in UHRF1-depleted cells to examine effects on 3D genome organization

  • Imaging approaches (e.g., DNA-MERFISH) to visualize UHRF1's relationship to spatial genome organization

  • Studies of UHRF1's role in phase separation and biomolecular condensate formation

  • These approaches could reveal roles in higher-order chromatin organization

• Post-translational modification mapping:

  • Comprehensive PTM profiling of UHRF1 across different cellular contexts

  • Functional analysis of PTM-deficient mutants

  • Investigation of the phosphorylation of Ser-298 in the intermodule linker

  • These approaches could uncover regulatory mechanisms controlling UHRF1 function

• Tissue and cell type-specific profiling:

  • Single-cell multi-omics in tissues with differential UHRF1 expression

  • Comparison between pre-mature and mature Sertoli cells

  • Analysis of adipogenic differentiation stages

  • These approaches could reveal tissue-specific functions and regulatory networks

These advanced techniques, especially when applied in combination, have the potential to reveal unexpected UHRF1 functions in diverse biological processes, expanding our understanding of this multifaceted epigenetic regulator beyond its established roles in DNA methylation and histone modification.