HIST1H2BC Antibody Pair

What is HIST1H2BC and why is it significant in epigenetic research?

HIST1H2BC (Histone cluster 1 H2B family member c) is one of several homomorphic variants of the canonical histone H2B, located at the HIST1 locus on chromosome 6p21-22. It belongs to a group of five variants (HIST1H2BC, HIST1H2BE, HIST1H2BF, HIST1H2BG, and HIST1H2BI) that share identical amino acid sequences despite being encoded by distinct genes . As a core histone protein, HIST1H2BC plays a crucial role in the organization of nucleosomes, which are the fundamental units of chromatin . The significance of HIST1H2BC in epigenetic research stems from emerging evidence suggesting that specific histone variants contribute to chromatin dynamics and gene regulation beyond the role of canonical histones. Recent studies have linked altered expression of histone variants, including HIST1H2BC and related H2B variants, to various pathological conditions including cancer and developmental disorders .

How does HIST1H2BC differ from other H2B variants in terms of function and expression patterns?

While HIST1H2BC shares identical amino acid sequences with four other H2B variants (HIST1H2BE, HIST1H2BF, HIST1H2BG, and HIST1H2BI), research suggests these variants may have distinct functions based on their differential expression patterns across tissues and in pathological conditions . For instance, HIST1H2BE shows tissue-specific expression with notably high levels in testes and thymus . The functional diversity likely stems from differences in genomic regulation rather than protein structure, as these variants differ at the nucleotide level despite encoding identical proteins . Studies have shown that H2B variants can undergo post-translational modifications, particularly ubiquitination, which affects nucleosomal dynamics and influences gene expression through cross-talk with other histone modifications . Though research on the specific functions of HIST1H2BC is still emerging, evidence suggests that precise control of H2B variant expression is critical for normal cellular function, as both overexpression and downregulation of variants like HIST1H2BE have been shown to decrease proliferation in breast cancer cell lines .

What is the relationship between HIST1H2BC expression and disease pathology?

Emerging research has revealed significant connections between HIST1H2BC expression levels and various pathological conditions, particularly in cancer. Studies have identified altered methylation patterns and expression of histone variants, including HIST1H2BC and related variants like HIST1H2BE, in endocrine-resistant breast cancer models . Analysis of HIST1H2BE mRNA expression in estrogen receptor-positive (ER+) aromatase inhibitor (AI)-treated breast tumors demonstrated significantly higher expression in resistant tumors compared to sensitive ones (p = 0.01) . More broadly, nanostring analysis has identified significant overexpression of 22 variant histone genes in tumors resistant to AI therapy . The Cancer Genome Atlas (TCGA) data analysis has shown frequent amplification of the HIST1 locus, which contains the HIST1H2BC gene . This evidence suggests that dysregulation of histone variants, including HIST1H2BC, may contribute to treatment resistance mechanisms in cancer, highlighting their potential as therapeutic targets or biomarkers.

What are the critical parameters to consider when selecting a HIST1H2BC antibody for research applications?

When selecting a HIST1H2BC antibody, researchers should evaluate several critical parameters to ensure experimental success. First, consider the specificity challenge: since HIST1H2BC shares 100% amino acid sequence identity with four other H2B variants (HIST1H2BE/F/G/I), it's essential to determine whether absolute specificity is required or if cross-reactivity with related variants is acceptable for your research question . Next, evaluate the validated applications for each antibody candidate; for instance, the monoclonal AMAb91337 antibody is validated for immunohistochemistry (IHC) and Western blot (WB), while the polyclonal PACO60504 antibody is validated for ELISA, WB, IHC, and immunofluorescence (IF) . Consider the host species (mouse or rabbit) and ensure compatibility with your experimental system and secondary detection methods . Examine species reactivity; AMAb91337 reacts with human, mouse, and rat proteins, while PACO60504 reacts with human and rat proteins . Additionally, assess the targeted epitope - for example, PACO60504 targets a peptide sequence around Lysine 16, which may be significant if studying post-translational modifications near this residue . Finally, review published validation data, including Western blot band sizes (typically 14 kDa for HIST1H2BC) and staining patterns in relevant tissues to ensure the antibody performs as expected in systems similar to yours .

How can I validate a HIST1H2BC antibody for specificity given the high sequence homology with other H2B variants?

Validating HIST1H2BC antibody specificity presents unique challenges due to the identical amino acid sequences of five H2B variants (HIST1H2BC/E/F/G/I). A comprehensive validation approach should include multiple complementary strategies. Begin with knockout/knockdown controls: use CRISPR-Cas9 or siRNA to specifically reduce HIST1H2BC expression, targeting the unique nucleotide sequences of the HIST1H2BC gene despite protein sequence homology . Combine this with overexpression studies using tagged HIST1H2BC constructs to create positive controls . For distinguishing between highly similar variants, perform parallel quantitative RT-PCR using variant-specific primers to correlate protein detection with mRNA expression levels across experimental conditions . Implement peptide competition assays using peptides derived from regions where nucleotide differences occur, even if amino acid sequences are identical. Conduct immunoprecipitation followed by mass spectrometry to identify which specific H2B variants are being captured by the antibody . Cross-validate results using multiple antibodies targeting different epitopes of HIST1H2BC, particularly those recognizing post-translationally modified regions that might differ between variants . Finally, include tissue-specific expression controls based on known differential expression patterns of H2B variants; for example, using tissues where HIST1H2BE shows characteristically high expression (testes, thymus) versus tissues with lower expression .

What are the optimal protocols for detecting HIST1H2BC ubiquitination in chromatin studies?

Detection of HIST1H2BC ubiquitination requires carefully optimized protocols to preserve this labile post-translational modification. Begin with proper sample preparation: harvest cells directly into lysis buffer containing deubiquitinase inhibitors (N-ethylmaleimide, PR-619) and proteasome inhibitors (MG132) to prevent rapid deubiquitination that occurs during standard extraction . For immunoprecipitation-based detection, use a dual-antibody approach with anti-HIST1H2BC antibody for initial pull-down, followed by anti-ubiquitin detection, or specialized antibodies that directly recognize H2B ubiquitination at lysine 120 (H2BubiK120) . When performing Western blot analysis, the ubiquitinated form will appear as a band approximately 8-10 kDa larger than unmodified HIST1H2BC (which typically runs at 14 kDa) . For chromatin immunoprecipitation (ChIP) applications targeting ubiquitinated HIST1H2BC, formaldehyde crosslinking must be carefully optimized, as excessive crosslinking can mask ubiquitination sites . Recent studies have shown success using tandem mass spectrometry (MS/MS) approaches, which allow precise identification and quantification of ubiquitination sites on histones, distinguishing between different H2B variants despite sequence similarities . To validate the specificity of ubiquitination detection, include controls with ubiquitination site mutations (K120R) or treatment with deubiquitinating enzymes as negative controls . For visualization of ubiquitinated HIST1H2BC in nuclear contexts, proximity ligation assays (PLA) combining H2B and ubiquitin antibodies provide superior spatial resolution compared to conventional immunofluorescence.

How can I optimize immunohistochemistry protocols for HIST1H2BC detection in FFPE tissue samples?

Optimizing immunohistochemistry (IHC) for HIST1H2BC detection in formalin-fixed, paraffin-embedded (FFPE) tissues requires addressing several histone-specific challenges. Begin with rigorous antigen retrieval: histones are tightly bound to DNA and heavily crosslinked during fixation, necessitating extended heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) . Testing both buffer conditions is recommended as epitope exposure can vary significantly between them. For antibody concentration, start with the manufacturer's recommended dilutions - 1:10 to 1:100 for PACO60504 and adjust based on signal strength and background . Implement a blocking step with 5-10% normal serum from the species of the secondary antibody plus 1% BSA to minimize non-specific binding, which is particularly important given the nuclear abundance of histones . Include phosphatase inhibitors in wash buffers to prevent loss of potential phosphorylation modifications on HIST1H2BC that may affect antibody recognition. For detection systems, amplification methods like tyramide signal amplification (TSA) can enhance sensitivity when detecting potentially low-abundance specific variants . Counterstain optimization is crucial - use lighter hematoxylin counterstaining to avoid masking the DAB signal in the nucleus where HIST1H2BC resides. Always run parallel positive controls using tissues known to express HIST1H2BC (such as testes or thymus) and negative controls using isotype-matched non-specific antibodies . For multiplex detection of HIST1H2BC alongside other markers, sequential immunostaining with appropriate antibody stripping between rounds is recommended to prevent cross-reactivity.

What is the recommended protocol for quantifying HIST1H2BC expression levels using qRT-PCR?

Accurate quantification of HIST1H2BC expression by qRT-PCR requires careful consideration of the high sequence homology among H2B variants. Begin with RNA extraction using TRIzol or column-based methods with additional DNase treatment to eliminate genomic DNA contamination, which is particularly crucial when working with intronless histone genes like HIST1H2BC . For cDNA synthesis, use oligo(dT) primers combined with random hexamers to capture both polyadenylated and non-polyadenylated histone transcripts . The critical step is primer design: create primers that target the unique nucleotide sequences that differentiate HIST1H2BC from other H2B variants (HIST1H2BE/F/G/I), focusing on regions with multiple distinguishing nucleotides . Validated primer sequences should span at least 18-20 nucleotides with 2-3 variant-specific nucleotides positioned centrally to ensure specificity. For the PCR reaction, use a touchdown protocol starting at 68°C and decreasing to 62°C to maximize specific amplification . Include melt curve analysis to confirm single-product amplification, and verify amplicon size by gel electrophoresis. For absolute quantification, generate a standard curve using plasmids containing the HIST1H2BC sequence . For relative quantification, carefully select reference genes, avoiding cell cycle-regulated genes; GAPDH, ACTB, and RPL13A in combination provide reliable normalization . Validate the specificity of your assay by testing expression in tissues with known differential expression of H2B variants, such as testes and thymus where HIST1H2BE shows characteristically high expression . For comparison across multiple H2B variants, ensure all primer sets have similar amplification efficiencies (95-105%) to allow accurate relative comparison.

How can I resolve weak or non-specific signals when using HIST1H2BC antibodies in Western blot applications?

Resolving weak or non-specific signals when detecting HIST1H2BC in Western blot applications requires a systematic approach addressing histone-specific challenges. For weak signals, first optimize protein extraction using histone-specific extraction protocols with 0.2N HCl or specialized histone extraction kits to ensure efficient release of histone proteins from chromatin . Increase loading amounts incrementally, as histones may require higher protein concentrations (20-30 µg) compared to standard proteins . Enhance transfer efficiency by using PVDF membranes instead of nitrocellulose and adding 0.1% SDS to transfer buffer to facilitate movement of basic histone proteins . Adjust blocking conditions; excessive blocking can mask histone epitopes, so consider reducing blocking time to 30 minutes or using 2-3% BSA instead of 5% milk . For antibody incubation, extend primary antibody incubation to overnight at 4°C, and adjust concentrations - HIST1H2BC antibodies often work optimally at 1:500 to 1:1000 dilutions rather than standard 1:1000 to 1:5000 . For non-specific signals, improve gel resolution using higher percentage (15-18%) gels or specialized Triton-Acid-Urea (TAU) gels that better separate histone variants . Implement more stringent washing with PBST containing 0.1-0.2% Tween-20 and adding 100-150mM NaCl to reduce non-specific binding . If background persists, pre-adsorb antibodies with non-specific proteins or use more specific detection systems like enhanced chemiluminescence plus (ECL+) . When optimized, Western blots should show a clean band for HIST1H2BC at approximately 14 kDa across various cell lines including HeLa, 293, A549, and HepG2 .

What strategies can mitigate cross-reactivity issues when studying HIST1H2BC in systems that express multiple H2B variants?

Mitigating cross-reactivity between HIST1H2BC and other H2B variants requires multi-faceted approaches combining antibody selection, experimental design, and validation strategies. First, use complementary detection methods: pair antibody-based detection with nucleic acid-based techniques that can distinguish variants at the mRNA level using variant-specific primers . For antibody selection, prioritize monoclonal antibodies like AMAb91337 that may offer greater specificity compared to polyclonal options , and consider antibodies raised against synthetic peptides representing regions with subtle sequence differences or unique post-translational modification patterns between variants . Implement sequential immunodepletion: first immunoprecipitate with antibodies specific to other H2B variants before probing for HIST1H2BC to reduce cross-reactivity . Use recombinant expression systems to create competitive binding controls - express tagged versions of each H2B variant and use these as competitors in immunoassays to determine relative binding affinities . For tissue studies, leverage known differential expression patterns; compare antibody signals in tissues with established expression profiles for different H2B variants (e.g., high HIST1H2BE expression in testes and thymus) . Employ post-separation identification by excising protein bands from gels after Western blotting and performing mass spectrometry to definitively identify which H2B variants are present . Consider new technologies like Cleavable ICAT (isotope-coded affinity tags) labeling combined with mass spectrometry to distinguish and quantify closely related histone variants in complex samples . When cross-reactivity cannot be eliminated, acknowledge the limitation and report results as "H2B variant detection" rather than claiming absolute specificity to HIST1H2BC.

How should I address epitope masking issues when detecting HIST1H2BC in different chromatin states?

Epitope masking presents a significant challenge when detecting HIST1H2BC across different chromatin states, as the antibody binding site may be occluded in certain conformations or by specific protein-protein interactions. To address this, implement a multi-pronged approach beginning with epitope mapping: determine which region of HIST1H2BC your antibody targets (e.g., PACO60504 recognizes the region around Lysine 16) and consider how this epitope might be affected by chromatin compaction or protein interactions . Employ parallel detection with multiple antibodies recognizing different HIST1H2BC epitopes to ensure at least one remains accessible across various chromatin states . Adjust fixation protocols based on the target chromatin state; for detecting HIST1H2BC in heterochromatin, reduce formaldehyde concentration to 0.5-1% and extend fixation time, while for euchromatin, standard 4% formaldehyde for 10 minutes may be sufficient . Implement differential extraction methods: use sequential extraction with increasing salt concentrations (0.3M, 0.6M, and 1.2M NaCl) to selectively release histones from euchromatin through heterochromatin, analyzing each fraction separately . For immunofluorescence applications, combine antigen retrieval methods (heat and enzymatic) to maximize epitope exposure, and consider pre-treating samples with histone deacetylase inhibitors like sodium butyrate to partially relax chromatin structure . When studying HIST1H2BC in the context of ubiquitination, note that H2Bub has been shown to enhance linker histone H1 binding to nucleosomes, potentially further masking epitopes in ubiquitinated regions . For chromatin immunoprecipitation (ChIP) applications, sonication conditions should be optimized separately for open and closed chromatin regions, with more intensive sonication needed for compacted regions to ensure adequate epitope exposure.

How can HIST1H2BC antibodies be utilized in studying epigenetic mechanisms in cancer progression?

HIST1H2BC antibodies offer powerful tools for investigating epigenetic mechanisms in cancer progression through multiple advanced applications. For mapping genome-wide distribution patterns, chromatin immunoprecipitation followed by sequencing (ChIP-seq) using validated HIST1H2BC antibodies can reveal how this variant's distribution changes during cancer progression, particularly in models of treatment resistance . Studies have shown that H2B variants, including HIST1H2BC, undergo differential methylation and expression changes in endocrine-resistant breast cancer models, suggesting potential roles in resistance mechanisms . For studying HIST1H2BC in the context of chromatin modifications, sequential ChIP (re-ChIP) can be employed to identify genomic regions where HIST1H2BC co-occurs with specific histone modifications like H3K4me3 or H3K27me3, as H2B ubiquitination has been shown to stimulate H3K4 and K79 tri-methylation through histone cross-talk mechanisms . To investigate HIST1H2BC ubiquitination, which affects nucleosomal dynamics and gene expression, researchers can use specialized antibodies that recognize ubiquitinated H2B in combination with gene expression analysis . For mechanistic studies, researchers can combine HIST1H2BC ChIP with transcription factor binding analysis to determine how this variant influences transcription factor recruitment at specific genomic loci . In patient-derived xenograft models, immunohistochemistry with HIST1H2BC antibodies can track changes in expression and localization patterns during tumor evolution and in response to therapy . Finally, for high-resolution analysis of HIST1H2BC in nuclear architecture, super-resolution microscopy combined with HIST1H2BC antibodies can visualize how this variant's distribution in the nucleus changes during cancer progression, potentially revealing altered chromatin organizational patterns associated with malignant transformation .

What are the most effective approaches for studying HIST1H2BC post-translational modifications beyond ubiquitination?

Studying post-translational modifications (PTMs) of HIST1H2BC beyond ubiquitination requires specialized approaches that can detect and quantify these modifications with high specificity. Begin with enrichment strategies: use phospho-specific antibodies for immunoprecipitation, titanium dioxide for phosphorylation enrichment, or hydrophilic interaction liquid chromatography (HILIC) to enrich modified histones prior to analysis . For comprehensive PTM mapping, employ bottom-up proteomics with chemical derivatization (propionylation or acetylation) of lysine residues prior to tryptic digestion to improve detection of histone peptides by mass spectrometry . Middle-down proteomics approaches using electron transfer dissociation (ETD) or electron capture dissociation (ECD) fragmentation can preserve labile modifications and analyze larger histone fragments to identify co-occurring modifications on the same molecule . For site-specific analysis, implement targeted proteomics using parallel reaction monitoring (PRM) or multiple reaction monitoring (MRM) to quantify specific modified peptides across experimental conditions . To study the dynamics of HIST1H2BC modifications, combine pulse-chase experiments using heavy isotope labeled amino acids (SILAC) with mass spectrometry to track modification turnover rates . For visualizing modification patterns in cellular contexts, proximity ligation assays (PLA) can detect specific modified forms of HIST1H2BC at the single-cell level . When investigating cross-talk between modifications, sequential ChIP experiments can reveal genomic regions where modified HIST1H2BC co-occurs with other histone modifications . Finally, for functional studies, CRISPR-Cas9 mediated mutation of specific modification sites (e.g., changing lysine to arginine to prevent acetylation) combined with phenotypic assays can reveal the biological significance of specific HIST1H2BC modifications .

How can I design experiments to investigate the role of HIST1H2BC in treatment resistance mechanisms?

Designing experiments to investigate HIST1H2BC's role in treatment resistance requires multilayered approaches that connect molecular changes to functional outcomes. Begin with clinical correlation studies: analyze HIST1H2BC expression and modification patterns in matched pre-treatment and post-resistance patient samples using immunohistochemistry with validated antibodies . Evidence already suggests significantly higher expression of HIST1H2BE in aromatase inhibitor-resistant breast tumors compared to sensitive tumors (p = 0.01) . For mechanistic studies, implement CRISPR-Cas9 mediated knockout or overexpression of HIST1H2BC in sensitive cell lines, followed by treatment to determine if altered expression affects the development of resistance . Use inducible expression systems to modulate HIST1H2BC levels at different treatment stages, as studies have shown that both overexpression and downregulation of related H2B variants affect cellular proliferation . To identify HIST1H2BC-associated gene networks, perform RNA-seq after HIST1H2BC manipulation, focusing on pathways previously implicated in resistance mechanisms . Combine this with ChIP-seq to map genome-wide distribution changes of HIST1H2BC before and after resistance development . For epigenetic regulation studies, analyze DNA methylation patterns at the HIST1H2BC locus in sensitive versus resistant cells, as hypomethylation of related H2B variants has been observed in resistance models . Implement proteomics approaches to identify HIST1H2BC-interacting proteins in resistant versus sensitive cells, potentially revealing novel cofactors in resistance mechanisms . For translational relevance, test whether pharmacological targeting of HIST1H2BC-related pathways (such as inhibitors of histone-modifying enzymes) can resensitize resistant cells to treatment . Finally, develop xenograft models with manipulated HIST1H2BC levels to validate in vitro findings in vivo, measuring both tumor growth and treatment response .

How should I interpret changes in HIST1H2BC nuclear localization patterns in immunofluorescence experiments?

Interpreting changes in HIST1H2BC nuclear localization patterns requires careful analysis that connects spatial distribution to functional significance. Begin with pattern recognition: classify observed patterns into distinct categories such as diffuse nuclear, punctate foci, peripheral/lamina-associated, or nucleolar-associated distribution . Quantify pattern frequencies across cell populations and experimental conditions using automated image analysis software with consistent thresholding parameters . For co-localization studies, implement dual or triple immunofluorescence to correlate HIST1H2BC distribution with known heterochromatin markers (H3K9me3, HP1) or euchromatin markers (H3K4me3, H3K27ac) to determine the chromatin context of observed patterns . Calculate Pearson's correlation coefficients or Manders' overlap coefficients to quantify co-localization precisely . When analyzing treatment-induced changes, perform time-course experiments to distinguish between direct effects on HIST1H2BC and secondary consequences of altered cell cycle distribution, as histone variant localization can change throughout the cell cycle . For mechanistic insights, correlate localization patterns with chromatin accessibility measured by ATAC-seq or DNase-seq from parallel samples . In disease models, compare HIST1H2BC localization patterns between normal and pathological states, such as sensitive versus resistant cancer cells, as evidence suggests altered histone variant expression in treatment-resistant tumors . For high-resolution analysis, implement super-resolution microscopy techniques (STORM, PALM, or STED) that can resolve HIST1H2BC distribution at the nanoscale level, potentially revealing associations with specific nuclear substructures not visible by conventional microscopy . Finally, correlate localization changes with functional outcomes such as changes in gene expression, DNA repair efficiency, or cell cycle progression to establish the biological significance of observed pattern alterations .

What statistical approaches are most appropriate for analyzing HIST1H2BC expression data across different tissue samples?

Analyzing HIST1H2BC expression data across tissue samples requires statistical approaches that address the unique characteristics of histone variant data. For comparison between different tissue types or disease states, begin with data normalization: use global normalization methods for microarray data or specialized methods like DESeq2 or edgeR for RNA-seq data, with particular attention to accounting for total histone expression differences between samples . When comparing HIST1H2BC expression relative to other H2B variants, implement compositional data analysis approaches using centered log-ratio transformations to accurately represent the proportional nature of variant expression . For hierarchical clustering of samples based on histone variant expression profiles, use Pearson correlation with average linkage to identify tissue-specific patterns, as studies have shown tissue-specific expression of variants like HIST1H2BE in testes and thymus . When analyzing clinical samples, implement multivariate logistic regression to identify associations between HIST1H2BC expression and clinical outcomes while controlling for confounding variables . Consider using bootstrap resampling methods to generate confidence intervals for expression differences, particularly with smaller sample sizes . For time-course experiments monitoring HIST1H2BC expression changes during disease progression or treatment response, apply mixed-effects models to account for within-subject correlation while identifying significant temporal trends . When correlating HIST1H2BC expression with other molecular features (DNA methylation, gene expression), use regularized methods like elastic net regression to handle high-dimensional data and identify the most robust associations . For meta-analysis across multiple datasets, implement random-effects models to account for between-study heterogeneity . In all analyses, correct for multiple testing using Benjamini-Hochberg procedure to control false discovery rate, particularly when analyzing HIST1H2BC alongside multiple histone variants simultaneously .

How can I integrate HIST1H2BC ChIP-seq data with other omics datasets to gain comprehensive biological insights?

Integrating HIST1H2BC ChIP-seq data with other omics datasets requires sophisticated computational approaches that reveal functional relationships across multiple molecular layers. Begin with peak calling optimization: use specialized algorithms like MACS2 with parameters optimized for histone ChIP-seq to accurately identify HIST1H2BC enrichment regions . For integration with transcriptomic data, implement correlation analyses between HIST1H2BC occupancy at promoters/enhancers and gene expression levels measured by RNA-seq, potentially revealing regulatory roles in transcription as suggested by studies on H2B ubiquitination and transcriptional elongation . When analyzing DNA methylation data alongside HIST1H2BC ChIP-seq, use segmentation algorithms to identify regions with coordinated changes in both datasets, as studies have shown relationships between histone variant distribution and DNA methylation patterns in resistance models . For multi-omics integration, implement dimension reduction techniques like multi-omics factor analysis (MOFA) or joint non-negative matrix factorization (jNMF) to identify latent factors that explain coordinated variation across datasets . To identify potential transcription factor interactions, perform motif enrichment analysis within HIST1H2BC binding sites and integrate with transcription factor ChIP-seq data to identify co-binding patterns . For pathway-level insights, use gene set enrichment analysis (GSEA) on genes associated with HIST1H2BC binding sites to identify biological processes potentially regulated by this histone variant . When comparing HIST1H2BC distribution between conditions (e.g., treatment-sensitive vs. resistant cells), implement differential binding analysis using DiffBind or MAnorm, followed by functional annotation of differentially bound regions . For integrating chromatin accessibility data (ATAC-seq), calculate the overlap between HIST1H2BC binding sites and accessible chromatin regions, potentially revealing roles in chromatin organization . Finally, visualize multi-omics integration using genome browsers with multiple tracks or circular plots to identify genomic hotspots with coordinated changes across multiple molecular features .