Recombinant Human UHRF1-binding protein 1 (UHRF1BP1), partial

What is UHRF1BP1 and what is its relationship with UHRF1?

UHRF1BP1 (UHRF1-binding protein 1) is a protein encoded by a gene located on chromosome 6p21. It was first identified as an important component of the ICBP90 complex and functions as a putative binding protein of UHRF1 . The interaction between UHRF1 and UHRF1BP1 is significant because UHRF1 is a multidomain protein that acts as a key epigenetic regulator by bridging DNA methylation and chromatin modification . The binding of UHRF1BP1 to UHRF1 may lead to the relocation of UHRF1 within the cell, potentially modulating its function in epigenetic regulation . Current research suggests that UHRF1BP1 may function as a tumor suppressor, as overexpression appears to inhibit cell growth in certain cancer cell lines .

What are the known structural domains of UHRF1BP1 and how do they contribute to its function?

UHRF1BP1 contains several functional domains that contribute to its ability to interact with UHRF1 and other molecular partners. While the complete structural characterization remains ongoing, researchers have identified specific regions that mediate protein-protein interactions. For studying these domains, techniques such as X-ray crystallography, NMR spectroscopy, and cryo-electron microscopy are recommended for high-resolution structural analysis. Functional validation of these domains typically employs site-directed mutagenesis followed by co-immunoprecipitation assays to assess binding capabilities with partner proteins.

What cellular pathways is UHRF1BP1 involved in?

UHRF1BP1 participates in several cellular pathways, most notably those involving epigenetic regulation and tumor suppression. Experimental evidence indicates that UHRF1BP1 influences epithelial-mesenchymal transition (EMT), a crucial process in cancer progression . When UHRF1BP1 is down-regulated in bladder cancer cell lines, there is a significant increase in cell invasion and migration capabilities . At the molecular level, this process is accompanied by downregulation of epithelial markers (particularly E-cadherin, Desmoplakin, and EpCAM) and upregulation of mesenchymal markers like ZEB2 and N-cadherin . Additionally, UHRF1BP1 has been implicated in cell proliferation regulation, though the specific mechanisms may not involve direct cell cycle control .

How do genetic variations in UHRF1BP1 affect cancer susceptibility?

Genetic variations in UHRF1BP1 have been associated with increased cancer risk, particularly in bladder cancer. A comprehensive three-stage case-control study involving 3,399 bladder cancer patients and 4,647 controls identified a rare coding variant (rs35356162: G>T) in UHRF1BP1 that significantly increases bladder cancer risk in Han Chinese populations . This variant demonstrated an odds ratio of 4.332 (95% CI: 2.463–7.619, P = 3.62E-07), indicating a strong association with cancer susceptibility .

For researchers investigating genetic variations, a methodological approach should include:

• Exome or genome sequencing to identify novel variants

• Case-control association studies with appropriate population stratification controls

• Functional validation of identified variants using cell line models

• Gene-level analysis using methods such as SKAT-O, which showed significant association of UHRF1BP1 (P = 4.47E-03) with bladder cancer risk

What is the relationship between UHRF1BP1 and autoimmune diseases?

Beyond cancer, UHRF1BP1 has been linked to autoimmune conditions. Several non-synonymous variants of UHRF1BP1 have been associated with systemic lupus erythematosus (SLE) in both European descendants and Chinese populations . When studying these associations, researchers should consider:

• Genome-wide association studies (GWAS) designed specifically for autoimmune cohorts

• Analysis of protein expression in immune cell subsets from affected individuals

• Investigation of how UHRF1BP1 variants might affect immune system regulation

• Exploration of potential overlap between cancer-associated and autoimmune-associated variants

What experimental methods are optimal for studying UHRF1BP1 protein-protein interactions?

For investigating UHRF1BP1 protein interactions, particularly with UHRF1, researchers should consider:

• Co-immunoprecipitation followed by mass spectrometry to identify novel binding partners

• Proximity ligation assays to visualize protein interactions in situ

• FRET/BRET techniques to quantify protein interactions in living cells

• Yeast two-hybrid screening to identify specific interaction domains

• Protein fragment complementation assays to validate direct interactions

These approaches should be complemented by genetic manipulation techniques, such as CRISPR-Cas9 editing, to create specific mutations that disrupt binding interfaces.

How can UHRF1BP1 expression be effectively modulated in experimental settings?

Based on published research, modulating UHRF1BP1 expression can be achieved through several techniques:

• RNA interference: Short-hairpin RNA (shRNA) approaches have successfully down-regulated UHRF1BP1 in bladder cancer cell lines. Using two different shRNA sequences targeting UHRF1BP1 can reduce expression at both transcriptional and translational levels .

• CRISPR-Cas9 gene editing: For stable knockout models, CRISPR-Cas9 targeting of UHRF1BP1 provides longer-term expression modulation.

• Overexpression systems: For gain-of-function studies, expressing UHRF1BP1 using appropriate vectors in cell models that have low endogenous expression.

• Inducible expression systems: Doxycycline-inducible systems allow temporal control of UHRF1BP1 expression for studying acute versus chronic effects.

When assessing knockdown or overexpression efficiency, researchers should validate changes at both the mRNA level (using quantitative PCR) and protein level (using Western blotting) .

What is the role of UHRF1BP1 in epithelial-mesenchymal transition?

UHRF1BP1 appears to play a significant regulatory role in epithelial-mesenchymal transition (EMT), a critical process in cancer metastasis. Experimental evidence from functional validation studies demonstrates that down-regulation of UHRF1BP1 in bladder cancer cell lines significantly impacts EMT marker expression .

When UHRF1BP1 is knocked down:

• Epithelial markers are broadly down-regulated, with particularly significant decreases in E-cadherin, Desmoplakin, and EpCAM expression

• Mesenchymal markers, specifically ZEB2 and N-cadherin, are significantly up-regulated

• Cell invasion and migration capabilities dramatically increase

For researchers studying this phenomenon, recommended methodologies include:

• Quantitative real-time PCR analysis of a comprehensive panel of EMT markers

• Western blotting to confirm protein-level changes in key markers

• Transwell migration and invasion assays to assess functional consequences

• Wound healing assays to measure cell migration capabilities

• Immunofluorescence staining to visualize changes in cellular morphology and protein localization

What experimental designs best elucidate UHRF1BP1's tumor suppressor function?

Based on current research, UHRF1BP1 appears to function as a tumor suppressor in bladder cancer and potentially other cancer types . To investigate this function, researchers should consider:

• In vitro approaches:

  • Cell proliferation assays following UHRF1BP1 knockdown or overexpression

  • Colony formation assays to assess anchorage-dependent growth

  • Soft agar assays to evaluate anchorage-independent growth

  • Cell migration and invasion assays as described previously

  • Analysis of apoptotic markers and cell cycle distribution

• In vivo approaches:

  • Xenograft models using UHRF1BP1-modulated cancer cell lines

  • Patient-derived xenografts to assess clinical relevance

  • Orthotopic models to evaluate metastatic potential in appropriate tissue contexts

  • Genetic mouse models with tissue-specific UHRF1BP1 deletion or overexpression

• Clinical correlation studies:

  • Analysis of UHRF1BP1 expression in tumor versus normal tissues

  • Correlation of expression levels with patient survival and clinical outcomes

  • Association of UHRF1BP1 genetic variants with cancer risk and progression

How does the interaction between UHRF1 and UHRF1BP1 influence DNA methylation patterns?

The interaction between UHRF1 and UHRF1BP1 is particularly significant because UHRF1 plays a crucial role in epigenetic regulation. UHRF1 specifically recognizes and binds hemimethylated DNA at replication forks via its YDG domain and recruits DNMT1 methyltransferase to ensure faithful inheritance of DNA methylation patterns .

When studying how UHRF1BP1 influences this process, researchers should consider:

• Methylation-specific assays:

  • Bisulfite sequencing to analyze DNA methylation at specific loci

  • Methylated DNA immunoprecipitation (MeDIP) to identify regions affected by UHRF1BP1 modulation

  • Whole-genome bisulfite sequencing for comprehensive methylation analysis

• Chromatin association studies:

  • Chromatin immunoprecipitation (ChIP) to assess UHRF1 binding in the presence/absence of UHRF1BP1

  • ChIP-seq to identify genome-wide binding patterns

  • Re-ChIP experiments to determine co-localization of UHRF1 and UHRF1BP1

• Protein interaction analysis:

  • Co-immunoprecipitation to confirm direct interaction

  • Proximity ligation assays to visualize interactions in situ

  • Domain mapping to identify critical regions mediating interaction

What are the challenges in developing recombinant UHRF1BP1 for functional studies?

Production of recombinant UHRF1BP1 presents several technical challenges that researchers should consider when designing functional studies:

• Expression system selection:

  • Bacterial systems (E. coli) may not provide appropriate post-translational modifications

  • Insect cell systems (such as Baculovirus-infected Sf9 cells used for UHRF1 ) may offer better protein folding and modification

  • Mammalian expression systems provide the most physiologically relevant modifications but with lower yield

• Purification challenges:

  • Designing appropriate affinity tags that don't interfere with protein function

  • Maintaining protein solubility throughout purification

  • Preserving protein-protein interaction capabilities

• Functional validation:

  • Developing appropriate in vitro assays to confirm activity

  • Ensuring that recombinant protein retains native conformation

  • Validating interaction with known partners like UHRF1

• Partial versus full-length considerations:

  • Determining which domains are essential for specific functions

  • Optimizing expression of difficult domains

  • Ensuring proper folding of multi-domain constructs

What are the comparative effects of UHRF1BP1 across different cancer types?

UHRF1BP1 has shown tumor suppressive properties in bladder cancer cell lines , and previous research has also indicated growth inhibition effects in colon cancer cell lines . When conducting comparative studies across cancer types, researchers should:

• Employ consistent methodologies to enable direct comparisons, including:

  • Standardized expression modulation techniques

  • Common functional assays (proliferation, migration, invasion)

  • Uniform EMT marker panels

• Consider tissue-specific effects by:

  • Using cell lines derived from multiple tissue origins

  • Analyzing patient samples from different cancer types

  • Investigating tissue-specific binding partners

• Evaluate cancer-specific genetic contexts by:

  • Assessing how common oncogenic drivers in each cancer type interact with UHRF1BP1

  • Determining whether UHRF1BP1 variants have differential effects across cancer types

  • Investigating whether UHRF1BP1 regulation varies by cancer type

How might contradictory findings regarding UHRF1BP1 function be reconciled?

As with many proteins involved in complex cellular processes, contradictory findings regarding UHRF1BP1 function may emerge. To address such contradictions, researchers should:

• Consider context-dependent functions:

  • Cell type specificity (epithelial vs. mesenchymal backgrounds)

  • Cancer stage dependency (early vs. late stage effects)

  • Microenvironmental influences on UHRF1BP1 function

• Evaluate technical variables:

  • Differences in knockdown/overexpression efficiency

  • Variations in experimental timepoints

  • Discrepancies in assay sensitivity

• Analyze isoform-specific effects:

  • Determine whether specific UHRF1BP1 isoforms have distinct functions

  • Assess whether genetic variants differentially affect isoform expression or function

  • Ensure that experimental systems target the same isoforms when comparing results

• Apply integrative approaches:

  • Combine in vitro, in vivo, and clinical data to form comprehensive models

  • Use computational biology to predict context-dependent functions

  • Develop pathway models that incorporate UHRF1BP1 in different cellular contexts