HMG20B Antibody

What is HMG20B and what are its primary biological functions?

HMG20B (also known as BRAF35, HMGX2, HMGXB2) is a component of the CoREST repressor complex that regulates gene expression through chromatin modification. It has a calculated molecular weight of 36 kDa, though it often appears at approximately 40 kDa on western blots . HMG20B functions primarily in two major biological pathways:

• Transcriptional regulation: As part of the CoREST complex, HMG20B helps repress genes involved in erythroid differentiation by binding to target promoters via transcription factors like Gfi1b .

• Cell division regulation: HMG20B interacts with BRCA2 through its BRC repeats and plays a crucial role in the completion of cytokinesis. Depletion of HMG20B significantly delays and disrupts the completion of cell division .

The protein contains an HMG box DNA-binding domain and kinesin-like coiled coils, which contribute to its diverse cellular functions . HMG20B was first identified as a regulator of neuronal gene expression and has since been found to have essential roles in various cellular processes .

What cellular localization pattern does HMG20B exhibit?

HMG20B predominantly localizes to the nucleus, consistent with its function in chromatin remodeling and transcriptional regulation. Immunofluorescence studies in HeLa cells have confirmed this nuclear localization pattern . During mitosis, HMG20B has been shown to play a role in cytokinesis, suggesting a dynamic localization pattern throughout the cell cycle .

The protein's localization can be effectively visualized using immunofluorescence (IF) techniques with anti-HMG20B antibodies at dilutions of 1:50-1:500, depending on the specific antibody used . When performing IF studies, it's recommended to use appropriate positive controls such as HeLa cells, which have been validated to express detectable levels of endogenous HMG20B .

What types of HMG20B antibodies are available for research applications?

Several types of HMG20B antibodies are available for research purposes, including:

• Monoclonal antibodies:

  • Mouse monoclonal antibodies (e.g., clone 4.21, anti-BRAF35)

  • Mouse IgG1 monoclonal antibodies (e.g., 67354-1-Ig)

• Polyclonal antibodies:

  • Rabbit polyclonal antibodies (e.g., 14582-1-AP)

  • Rabbit polyclonal antibodies raised against specific regions (e.g., human HMG20B residues 138-245)

Each antibody has been validated for specific applications, with product-specific recommended dilutions. For example:

When selecting an HMG20B antibody, researchers should consider the specific application, species reactivity, and the region of HMG20B targeted by the antibody .

How should HMG20B antibodies be optimized for Western blot analysis?

For optimal Western blot analysis using HMG20B antibodies, follow these methodological guidelines:

• Sample preparation:

  • Use whole-cell extracts made in NP-40 lysis buffer (50 mM HEPES (pH 7.4), 100 mM NaCl, 0.5% NP-40, 10 mM EDTA, 20 mM β-glycerophosphate, 1 mM DTT, 1 mM sodium orthovanadate, 1 mM PMSF and complete protease inhibitor cocktail) .

  • Load approximately 3×10^5 cells for optimal detection .

• Gel separation:

  • Use 10-12% SDS polyacrylamide gels for standard HMG20B detection .

  • For studying interactions with larger proteins like BRCA2, 3-8% Tris-Acetate gels may be more appropriate .

• Antibody dilutions:

  • For monoclonal antibodies (e.g., 67354-1-Ig): Use at 1:5000-1:50000 dilution .

  • For polyclonal antibodies (e.g., 14582-1-AP): Use at 1:500-1:1000 dilution .

• Detection optimization:

  • Secondary staining can be performed using goat-anti-mouse/rabbit IR-Dye 680 or 800 antibodies in PBS with 5% (w/v) blotting grade non-fat dry milk powder and 0.05% (v/v) tween 20 .

  • Expected molecular weight is approximately 36-40 kDa, though the observed weight may vary slightly depending on the cell type and experimental conditions .

• Validated positive controls:

  • HeLa, LNCaP, HEK-293, Jurkat, K-562, HSC-T6, and NIH/3T3 cells have all been validated as positive controls for HMG20B Western blotting .

For troubleshooting, if background is high, increase the blocking time or adjust the antibody dilution. If the signal is weak, consider longer exposure times or loading more protein.

What are the critical considerations for immunohistochemistry with HMG20B antibodies?

When performing immunohistochemistry (IHC) using HMG20B antibodies, consider these critical methodological factors:

• Tissue preparation and antigen retrieval:

  • Primary suggestion: Use TE buffer pH 9.0 for antigen retrieval.

  • Alternative method: Citrate buffer pH 6.0 can also be effective .

  • Proper fixation is critical; overfixation may mask epitopes.

• Antibody selection and dilution:

  • For monoclonal antibodies like 67354-1-Ig: Use at 1:1000-1:4000 dilution .

  • For polyclonal antibodies like 14582-1-AP: Use at 1:20-1:200 dilution .

  • Always optimize dilutions for your specific tissue type.

• Validated tissue samples:

  • Human colon cancer tissue has been validated for positive staining with 14582-1-AP .

  • Human skin cancer tissue has been validated for positive staining with 67354-1-Ig .

• Controls:

  • Always include positive and negative controls in each IHC run.

  • For negative controls, omit primary antibody or use isotype-matched control antibodies.

  • For positive controls, use tissues known to express HMG20B.

• Signal detection and interpretation:

  • HMG20B primarily shows nuclear localization in IHC staining.

  • Evaluate staining intensity and pattern in comparison to controls.

  • Be aware that expression levels may vary across different tissue types and disease states.

The optimization of antigen retrieval methods is particularly critical for HMG20B detection, as improper retrieval can significantly affect staining quality and intensity.

How can HMG20B antibodies be effectively used in chromatin immunoprecipitation (ChIP)?

For effective chromatin immunoprecipitation (ChIP) using HMG20B antibodies, follow these methodological guidelines:

• Antibody selection:

  • Use antibodies specifically validated for ChIP applications, such as rabbit polyclonal 14582-1-AP, which has been cited in published ChIP studies .

  • Optimize antibody amount through titration experiments.

• Chromatin preparation:

  • For studying HMG20B binding to specific promoters (e.g., Gfi1b promoter), standard formaldehyde cross-linking protocols are appropriate .

  • Sonication conditions should be optimized to generate chromatin fragments of 200-500 bp.

• Immunoprecipitation procedure:

  • Pre-clear chromatin with protein A/G beads to reduce background.

  • For immunoprecipitation, couple antibodies (e.g., rabbit polyclonal anti-HMG20B) to protein A beads using DMP (dimethyl pimelimidate) .

  • Use 2 mg of cell extracts with 20 μg of protein A-coupled antibodies for optimal results .

• Controls and validation:

  • Include IgG control to assess non-specific binding.

  • As a positive control, examine enrichment at the Gfi1b promoter, which has been shown to bind HMG20B as part of the CoREST complex .

  • Lsd1 ChIP can be performed in parallel as a comparison for CoREST complex binding .

• Analysis considerations:

  • In proliferating proerythroblasts, HMG20B shows stronger enrichment on the Gfi1b promoter compared to differentiating cells, similar to the pattern observed for Lsd1 .

  • When analyzing ChIP data for HMG20B, consider its role as part of the CoREST complex, which is primarily associated with gene repression .

Research has shown that HMG20B enrichment patterns can change during cellular differentiation, making ChIP a valuable tool for studying its role in developmental processes .

How does HMG20B function in the regulation of erythroid differentiation?

HMG20B plays a critical role in erythroid differentiation as a repressor, with multiple layers of regulatory mechanisms:

• Role in the CoREST complex:

  • HMG20B functions as a subunit of the CoREST repressor complex, which is recruited to target promoters by the transcription factor Gfi1b in erythroblasts .

  • As part of this complex, HMG20B helps repress genes involved in erythroid differentiation, maintaining cells in a proliferative state .

• Experimental evidence from knockdown studies:

  • Knockdown of Hmg20b in mouse fetal liver proerythroblasts and I/11 cells (a differentiation-competent mouse fetal liver cell line) induces spontaneous differentiation .

  • Western blot and QRT-PCR analyses showed a modest decrease in Hmg20b expression during normal erythroid differentiation .

  • HMG20B-depleted cells display:

    • Reduced proliferation rate

    • G1 accumulation

    • Increased hemoglobinization

    • Enhanced enucleation (>50% compared to 10% in controls by day 7)

• Regulation of target genes:

  • Microarray analysis revealed that 85% (527 out of 620) of genes deregulated upon HMG20B knockdown are up-regulated, confirming its primarily repressive function .

  • Key targets include:

    • Embryonic β-like globins (up-regulated)

    • Hrasls3 (HRAS-like suppressor 3), a phospholipase that promotes differentiation (up-regulated)

    • Gfi1b, a repressor of erythroid differentiation (down-regulated)

• Mechanism of action:

  • ChIP analysis showed that HMG20B and Lsd1 (another CoREST component) bind to the Gfi1b promoter more strongly in proliferating than in differentiating cells .

  • This suggests a feedback loop where HMG20B activates Gfi1b, which in turn recruits the CoREST complex (including HMG20B) to repress genes involved in differentiation .

These findings establish HMG20B as a key inhibitor of erythroid differentiation that functions through the down-regulation of differentiation-promoting genes like Hrasls3 and the activation of differentiation repressors like Gfi1b .

What is the role of HMG20B in mitotic cell division and how does it interact with BRCA2?

HMG20B plays a crucial role in mitotic cell division through its interaction with the tumor suppressor BRCA2:

• Effects of HMG20B depletion on cell division:

  • Time-lapse microscopy of HeLa cells depleted of HMG20B shows significant delays in the time from anaphase onset to the completion of cell division .

  • Median time increases from 85 minutes (control) to 143 minutes (HMG20B siRNA) .

  • HMG20B-depleted cells show specific cytokinesis defects:

    • 49% of cells fail to complete cell division (vs. 15% in controls)

    • 37% form binucleate cells

    • 12% remain with advanced furrow ingression without abscission

• BRCA2-HMG20B interaction:

  • HMG20B binds directly to the BRC repeats of BRCA2, evolutionarily conserved motifs of ~35 residues .

  • The interaction exhibits specificity for particular BRC repeats:

    • Highest affinity for BRC5, which binds poorly to RAD51

    • Poor binding to BRC4, which binds strongly to RAD51

  • This suggests a separation of function, where different BRC repeats regulate either DNA recombination (via RAD51) or cytokinesis (via HMG20B).

• Molecular interaction details:

  • GST pull-down assays and streptavidin pull-down assays with biotinylated BRC peptides confirm direct interaction .

  • The C-terminal region of HMG20B mediates binding to the BRC5 motif in BRCA2 .

  • In vivo, BRC5 overexpression inhibits the BRCA2–HMG20B interaction and recapitulates defects in cell division similar to HMG20B depletion .

• Cancer-associated mutations:

  • Several mutations in the HMG20B gene have been detected in human cancer samples, including lung carcinomas .

  • The non-conservative substitution of HMG20b residue Ala247 with Pro disrupts HMG20b activities and impairs cytokinesis in a trans-dominant manner .

  • These heterozygous mutations affect cytokinesis regulation despite the presence of a normal allele .

This research establishes a novel function for HMG20B in cytokinesis that is regulated by its interaction with specific BRC repeats of BRCA2, separate from BRCA2's known function in DNA recombination .

How can researchers investigate cancer-associated mutations in HMG20B?

Researchers can employ several methodological approaches to investigate cancer-associated mutations in HMG20B:

• Mutation identification and analysis:

  • Consult databases like COSMIC (Catalogue of Somatic Mutations in Cancer) to identify known mutations in HMG20B across different cancer types .

  • Notable mutations include T189S, F192V, A247P, V303I, and V312G .

  • The A247P mutation in particular has been identified in human lung cancer and shown to have functional consequences .

• Functional impact assessment:

  • Generate expression constructs for wild-type and mutant HMG20B (e.g., FLAG-tagged) .

  • Test the ability of mutant proteins to bind to GST-tagged BRCA2 fragments containing the BRC5 motif (aa1613-1781) using pull-down assays .

  • Investigate effects on protein-protein interactions, particularly with BRCA2.

• Cellular phenotype evaluation:

  • Introduce mutations through site-directed mutagenesis and express mutant proteins in appropriate cell lines.

  • Use time-lapse microscopy to observe effects on cytokinesis, measuring:

    • Time from anaphase onset to completion of division

    • Frequency of binucleate cell formation

    • Furrow ingression abnormalities

  • Compare phenotypes to those observed with HMG20B depletion and wild-type overexpression.

• Structural biology approaches:

  • Employ X-ray crystallography or cryo-EM to determine how mutations affect the structure of HMG20B.

  • Focus particularly on the C-terminal region involved in BRCA2 binding .

• Cancer tissue analysis:

  • Utilize HMG20B antibodies for immunohistochemistry of cancer tissues .

  • Compare HMG20B expression and localization in cancer tissues with mutations versus normal tissues.

  • Human colon cancer and skin cancer tissues have been validated as suitable for HMG20B IHC studies .

• Trans-dominant effects:

  • Investigate how heterozygous mutations affect HMG20B function despite the presence of a wild-type allele .

  • Co-express wild-type and mutant HMG20B to assess dominant-negative effects.

By employing these methodologies, researchers can gain insights into how HMG20B mutations contribute to cancer pathogenesis through dysregulation of cytokinesis and potentially other cellular processes.

What are the methods for investigating HMG20B target genes in erythroid cells?

To investigate HMG20B target genes in erythroid cells, researchers can employ the following methodological approaches:

• Gene expression profiling after HMG20B knockdown:

  • Perform lentiviral-mediated knockdown of Hmg20b in appropriate cell models:

    • I/11 cells (differentiation-competent mouse fetal liver cell line)

    • Primary mouse fetal liver proerythroblasts (pMFL)

  • Verify knockdown efficiency by Western blot and QRT-PCR (target <20% of wild-type levels) .

  • Conduct microarray analysis on RNA extracted from biological triplicates of control and knockdown cells .

  • Use specific cutoffs (e.g., 1.5-fold change with P<0.01) to identify differentially expressed genes .

• Validation of target genes:

  • Perform QRT-PCR on selected targets from microarray data:

    • Up-regulated genes: Hrasls3, Trp53inp1, Cited2, Ccng2

    • Down-regulated genes: Rcor2, Kit, Gfi1b

  • For the most promising targets (e.g., Hrasls3), perform functional validation through:

    • Expression analysis during normal differentiation

    • Knockdown experiments to assess effects on differentiation

• ChIP analysis of direct targets:

  • Perform ChIP using anti-HMG20B antibodies to identify direct binding sites .

  • Compare HMG20B enrichment patterns with other CoREST complex components (e.g., Lsd1) .

  • Focus on promoters of key regulated genes (e.g., Gfi1b) .

  • Compare enrichment patterns between proliferating and differentiating cells .

• Functional classification of target genes:

  • Categorize regulated genes based on function:

    • 85% of deregulated genes (527 out of 620) are up-regulated upon HMG20B knockdown, consistent with its repressive role .

    • 15% (93 out of 620) are down-regulated, suggesting HMG20B may also function as an activator for some genes .

  • Pay particular attention to genes involved in:

    • Globin expression (e.g., embryonic β-like globins)

    • Cell cycle regulation

    • Terminal differentiation

• Pathway analysis:

  • Identify key pathways affected by HMG20B regulation.

  • Investigate feedback loops, such as the reciprocal regulation between HMG20B and Gfi1b .

Using these approaches, researchers have identified HMG20B as a repressor of erythroid differentiation that functions by downregulating differentiation-promoting genes (e.g., Hrasls3) and activating differentiation repressors (e.g., Gfi1b) .

How should researchers validate the specificity of HMG20B antibodies?

Proper validation of HMG20B antibodies is critical for ensuring reliable experimental results. Researchers should follow these methodological steps:

• Western blot validation:

  • Test antibodies on multiple cell lines known to express HMG20B (e.g., LNCaP, HeLa, HEK-293, Jurkat, K-562, HSC-T6, NIH/3T3) .

  • Verify molecular weight (expected ~36-40 kDa) .

  • Include positive controls and, if available, HMG20B-knockdown or knockout samples as negative controls.

  • Different antibodies may show slightly different band patterns; for example, monoclonal antibody 67354-1-Ig detects a band at approximately 40 kDa, while polyclonal antibody 14582-1-AP detects a band at 36 kDa .

• RNA interference controls:

  • Perform siRNA or shRNA knockdown of HMG20B and verify reduction in signal intensity.

  • Examples from research include lentiviral-mediated knockdown with two independent shRNA constructs that reduced Hmg20b expression to less than 20% of wild-type levels .

  • Western blot signal should decrease proportionally to the knockdown efficiency.

• Immunoprecipitation validation:

  • Perform IP with anti-HMG20B antibodies followed by Western blot detection with a different HMG20B antibody.

  • Example protocol: Use 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate .

  • For antibody coupling, protein A beads can be used with DMP (dimethyl pimelimidate) .

• Immunofluorescence specificity:

  • Compare staining patterns in cells with and without HMG20B knockdown.

  • Verify nuclear localization pattern consistent with HMG20B function .

  • Include appropriate blocking controls and secondary-only controls.

• Cross-reactivity assessment:

  • Test antibody on samples from different species to confirm specified cross-reactivity.

  • Many HMG20B antibodies show reactivity with human, mouse, and rat samples .

  • For human studies, antibodies have been validated on multiple cell lines and tissue samples .

• Epitope mapping:

  • Consider the antibody's target region when interpreting results.

  • Different antibodies target different regions of HMG20B:

    • Some target the N-terminus (e.g., A41835)

    • Others target specific internal regions (e.g., HPA053157 targets the sequence REKQQYMKELRAYQQSEAYKMCTEKIQEKKIKKEDSSSGLMNTLLNGHKGGDCDGFSTFDVP)

Through comprehensive validation using these methods, researchers can ensure the specificity and reliability of HMG20B antibodies for their experimental applications.

What are common challenges when using HMG20B antibodies and how can they be addressed?

Researchers working with HMG20B antibodies may encounter several technical challenges. Here are methodological solutions to address these issues:

• Variable detection sensitivity across applications:

  • Challenge: Some antibodies perform well in Western blot but poorly in IHC or IF.

  • Solution: Choose application-specific validated antibodies. For example:

    • 67354-1-Ig: Validated for WB (1:5000-1:50000) and IHC (1:1000-1:4000)

    • 14582-1-AP: Validated for WB (1:500-1:1000), IHC (1:20-1:200), IF/ICC (1:50-1:500), and IP (0.5-4.0 μg)

  • Always perform preliminary titration experiments to determine optimal conditions for your specific sample type.

• Cross-reactivity with related proteins:

  • Challenge: HMG20B belongs to the high-mobility group protein family, which shares structural similarities.

  • Solution:

    • Use monoclonal antibodies for higher specificity when cross-reactivity is a concern.

    • Include appropriate knockdown controls to confirm specificity.

    • For critical experiments, validate results with multiple antibodies targeting different epitopes.

• Inefficient immunoprecipitation:

  • Challenge: Poor IP efficiency can limit co-immunoprecipitation and ChIP studies.

  • Solution:

    • Optimize extraction conditions: For BRCA2-HMG20B complexes, extract cells with buffer A (50 mM HEPES pH 7.4, 420 mM NaCl, 0.2% NP-40, 1 mM EDTA, 25% glycerol, 1 mM DTT, and protease inhibitors) and dilute with buffer B (50 mM HEPES pH 7.4, 0.2% NP-40, and 1 mM EDTA) .

    • Couple antibodies to beads: Use protein A beads with DMP for stable coupling .

    • Use 2 mg of diluted extracts with 20 μg of protein A-coupled antibodies for optimal results .

• Cell cycle-dependent expression and interactions:

  • Challenge: HMG20B interactions (e.g., with BRCA2) may vary throughout the cell cycle.

  • Solution:

    • For mitotic cell studies, treat cells with nocodazole (40 ng/ml) overnight to enrich cells in prometaphase .

    • For cytokinesis studies, treat nocodazole-arrested cells with Purvalanol A (22.5 μM) for 40 minutes .

    • Synchronize cells appropriately for the specific cell cycle phase of interest.

• Detection in tissue samples:

  • Challenge: Variable staining quality in tissue sections.

  • Solution:

    • Optimize antigen retrieval: Use TE buffer pH 9.0 as primary suggestion or citrate buffer pH 6.0 as an alternative .

    • Test different fixation protocols to preserve epitope integrity.

    • For human tissues, colon cancer and skin cancer samples have been validated as positive controls .

• Functional studies interpretation:

  • Challenge: Distinguishing direct from indirect effects of HMG20B.

  • Solution:

    • Combine antibody-based detection methods with functional studies (e.g., knockdown experiments).

    • Use ChIP to identify direct binding targets .

    • Validate results with rescue experiments (e.g., re-expressing wild-type or mutant HMG20B in knockdown cells).

By implementing these methodological approaches, researchers can overcome common challenges and optimize their experiments with HMG20B antibodies.

What are the best practices for storing and handling HMG20B antibodies?

Proper storage and handling of HMG20B antibodies is critical for maintaining their specificity and activity. Here are the best practices based on manufacturer recommendations and scientific protocols:

Following these best practices will help ensure consistent and reliable results when working with HMG20B antibodies across various experimental applications.

What are the emerging research areas involving HMG20B in cancer biology?

Recent advances have highlighted several promising research areas involving HMG20B in cancer biology:

• Cancer-associated mutations and their functional consequences:

  • The identification of heterozygous HMG20B mutations in various human epithelial cancers, particularly lung carcinomas, has opened new avenues for research .

  • The A247P mutation in particular has been shown to disrupt HMG20B function in a trans-dominant manner, impairing cytokinesis despite the presence of a wild-type allele .

  • Future research should focus on:

    • Comprehensive screening of additional cancer types for HMG20B mutations

    • Detailed functional characterization of identified mutations

    • Investigation of mutation-specific effects on different HMG20B functions

• Connection to BRCA2-dependent tumor suppression:

  • The interaction between HMG20B and the tumor suppressor BRCA2 suggests a role in BRCA2-dependent cancer pathways .

  • HMG20B binds specifically to the BRC5 repeat of BRCA2, which binds poorly to RAD51, suggesting a separation of BRCA2's functions in DNA recombination and cytokinesis .

  • This indicates that divergent tumor-suppressive pathways regulating chromosome segregation and structure may be governed by different BRC motifs in BRCA2 .

  • Future studies should explore:

    • How HMG20B mutations affect BRCA2-dependent tumor suppression

    • The potential synthetic lethality between HMG20B and BRCA2 deficiencies

    • Therapeutic opportunities targeting this interaction

• Role in genomic instability:

  • HMG20B's function in cytokinesis suggests its deficiency could contribute to genomic instability, a hallmark of cancer .

  • The formation of binucleate cells following HMG20B depletion or mutation could lead to aneuploidy and chromosomal instability .

  • Future research directions include:

    • Long-term consequences of HMG20B deficiency on genomic stability

    • Correlation between HMG20B status and aneuploidy in cancer samples

    • Potential synergistic effects with other genomic instability factors

• Therapeutic targeting opportunities:

  • Understanding HMG20B's role in cancer may reveal new therapeutic targets.

  • As part of the CoREST complex, HMG20B is involved in chromatin remodeling and gene repression, processes that can be targeted pharmacologically .

  • Future studies could explore:

    • Small molecule inhibitors of HMG20B or its interactions

    • Synthetic lethal interactions that could be exploited in cancer therapy

    • Biomarker potential of HMG20B expression or mutation status

These emerging research areas highlight the potential significance of HMG20B in cancer biology and offer promising directions for future investigation that may lead to new diagnostic and therapeutic approaches.

How can researchers apply new technologies to better understand HMG20B function?

Researchers can leverage several cutting-edge technologies to advance our understanding of HMG20B function:

• CRISPR-Cas9 genome editing:

  • Generate precise knockout or knockin cell lines to study HMG20B function.

  • Create cellular models expressing cancer-associated mutations (e.g., A247P) .

  • Develop HMG20B domain deletion mutants to map functional regions.

  • Engineer tagged versions of endogenous HMG20B for live-cell imaging.

  • Applications include:

    • Studying effects on cytokinesis in real-time

    • Assessing changes in gene expression patterns

    • Investigating interaction with BRCA2 and other partners

• Proteomics approaches:

  • Employ BioID or APEX proximity labeling to identify novel HMG20B interaction partners.

  • Use principles from published biotinylation techniques for HMG20B:

    • Clone Bio-HA-HMG20B into lentiviral vectors

    • Express in BirA-expressing cells for biotinylation

    • Purify complexes using streptavidin and analyze by mass spectrometry

  • Apply phosphoproteomics to identify post-translational modifications regulating HMG20B function.

  • Investigate how the interactome changes across the cell cycle or during differentiation.

• Advanced microscopy techniques:

  • Implement super-resolution microscopy to visualize HMG20B localization with nanometer precision.

  • Use live-cell imaging to track HMG20B dynamics during cell division.

  • Apply FRET or BRET approaches to study HMG20B-BRCA2 interactions in real-time.

  • Correlative light and electron microscopy could reveal ultrastructural details of HMG20B function during cytokinesis.

• Next-generation sequencing applications:

  • ChIP-seq to comprehensively map HMG20B binding sites genome-wide .

  • Cut&Run or CUT&Tag as alternatives to traditional ChIP for higher resolution and lower background.

  • RNA-seq to profile transcriptional changes upon HMG20B manipulation with greater depth than microarrays .

  • ATAC-seq to investigate changes in chromatin accessibility regulated by HMG20B.

  • HiChIP to study long-range chromatin interactions mediated by HMG20B.

• Structural biology approaches:

  • Cryo-EM to determine the structure of HMG20B alone or in complex with BRCA2 fragments.

  • X-ray crystallography of the C-terminal region that mediates binding to BRCA2 .

  • NMR studies of specific domains and their interactions.

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces.

• Single-cell technologies:

  • scRNA-seq to resolve heterogeneous responses to HMG20B manipulation.

  • CyTOF to simultaneously measure multiple parameters in individual cells.

  • Spatial transcriptomics to map HMG20B-dependent gene expression changes in tissue context.