Biology subjects

Dalal, Y.

Publications and source records attributed to Dalal, Y..

15 recordsLinked to original sources

bioRxiv2025

MetaChrome: An Open-Source, User-Friendly Tool for Automated Metaphase Chromosome Analysis

DNA Fluorescence In Situ Hybridization (FISH) is an essential technique to study chromosome biology and genetics, enabling precise visualization of specific genomic loci to study structural abnormalities, gene mapping, and chromosomal rearrangements. High-Throughput Imaging (HTI) can automate the analysis of DNA-FISH chromosome images, but the accurate and automated segmentation of mitotic chromosomes and simultaneous colocalization of FISH signals remains a challenge. While several commercial automated karyotyping tools partially solve these issues, open-source software that effectively combines robust chromosome segmentation with comprehensive colocalization analysis capabilities remains necessary. To address this unmet need, we developed MetaChrome, an open-source software platform built around a graphical user interface and explicitly designed for automated metaphase chromosome analysis. MetaChrome leverages fine-tuned deep learning models to automate metaphase chromosome segmentation, together with colocalization analysis of chromosome-specific FISH probes and immunofluorescent-labeled proteins. Importantly, MetaChrome achieves enhanced segmentation accuracy compared to traditional image processing methods by adopting a Cellpose segmentation model fine-tuned with manually annotated metaphase chromosome datasets. The fine-tuned model ensures precise assignment of DNA-FISH spots to individual chromosomes in an automated manner. This facilitates rapid identification of chromosomal abnormalities, reduces human error, and advances high-throughput chromosome analysis workflows, addressing a key bottleneck in chromosome biology research.

genetics↗

bioRxiv2025

Linker Histone H1.5 Contributes to Centromere Integrity in Human Cells

Mammalian H1 linker histones comprise a group of 11 non-allelic variants which have key roles in modulating chromatin. H1 variant specific genomic distribution contributes to fine tuning regulation of gene expression and chromatin architecture. Contradictory reports on the presence and role of H1 histones at centromeres led us to further investigate whether H1s impact centromeric chromatin. In this study, we focused on H1.5 and by in vitro assays we showed that H1.5 directly interacts with centromeric-protein A (CENP-A) mononucleosomes. Notably, our in vitro findings revealed that H1 variants H1.0 and H1.2 can also bind CENP-A nucleosomes, although with differing affinities and signatures, asserting centromeric localization may not be unique to H1.5. In human cells, H1.5 localized to the centromere and chromatin immuno-precipitation revealed an interaction between H1.5 with CENP-A nucleosomes. Knocking down of H1.5 resulted in the loss of centromeric -satellite transcription, reduction in loading of new CENP-A, and the accumulation of mitotic defects. These data point to an unreported role for histone H1 in the regulation of mitotic integrity in human cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/657682v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@26a35borg.highwire.dtl.DTLVardef@aecc44org.highwire.dtl.DTLVardef@1e49939org.highwire.dtl.DTLVardef@107a485_HPS_FORMAT_FIGEXP M_FIG Graphical AbstractProposed model of H1.5 function at the centromere. When H1.5 is bound to the CENP-A nucleosomes at the centromere, the centromeric chromatin allows RNAPII based transcription to generate -satellite transcripts, which in turn allows for de novo CENP-A loading. The absence of H1.5 blocks this transcription, leading to an accumulation of mitotic defects. C_FIG

biochemistry↗

bioRxiv2025

Oncogenic lncRNA transgene transcription modulates epigenetic memory at a naive chromosomal locus.

Maintaining genome integrity is crucial for the proper functioning and development of organisms. One intriguing aspect of genome integrity is the formation and function of neocentromeres at non-centromeric sites. CENP-A, a centromere-specific protein, is essential for centromere identification and function. However, in many cancers, CENP-A is often found to be ectopically misplaced when overexpressed. Moreover, CENP-A deposition at the centromere depends on the transcription of centromeric non-coding RNAs. Consequently, ectopic CENP-A is found at transcriptionally active and frequent breakpoint regions. To further explore ectopic CENP-A localization, we previously engineered a stable ectopic CENP-A site on a naive chromosome by overexpressing a non-centromeric oncogenic lncRNA, PCAT2, which was capable of recruiting CENP-A to its transcription site. In this work, we tracked cells carrying this stable transgene to understand the longevity of the induced ectopic CENP-A site at the chromosome that harbors it. Our findings revealed that the induced epigenetic memory was eventually lost due to the suppression of the transgene through competing epigenetic silencing mechanisms. This epigenetic restoration naturally reversed the ectopic CENP-A level to its previous levels at the engineered site. These data suggest that cells may have evolved failsafe mechanisms to prevent neocentromere formation at ectopic sites by suppressing transcription, unless otherwise favored by selection involving multiple components.

genetics↗

bioRxiv2024

Incorporating histone H2B variants into chromatin modifies chromatin accessibility to induce epithelial to mesenchymal transition in breast cancer

Histones scaffold genomic DNA and regulate access to the transcriptional machinery. However, naturally occurring histone variants can alter histone-DNA interactions, DNA and histone modifications, and the chromatin interactome. Hence, alterations in histone variant deposition can disrupt chromatin, and are increasingly recognized as a way to trigger various disease, including cancer. While significant attention has been placed on the biochemical and functional roles of H2A, H3, and H4 histone variants, the variants of H2B remain largely understudied. Here, we show that H2B variants are dysregulated in breast cancer and that certain variants are associated with specific breast cancer subtypes. HIST1H2BO overexpression (in particular) is more common in Asian, African American/Black, and young female populations and is associated with a worse prognosis. In vitro studies show that H2B1O compacts nucleosome structure. Incorporating H2B1O into chromatin activates pro-inflammatory and oncogenic pathways, induces the epithelial-to-mesenchymal transition (EMT), and generates resistance to first-line chemotherapeutic agents. Thus, H2B1O acts much like an onco-histone, with H2B variant expression being a prognostic biomarker for breast cancer and a potential new target for drug therapies to enhance treatment efficacy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/627414v1_ufig1.gif" ALT="Figure 1"> View larger version (106K): org.highwire.dtl.DTLVardef@12c21dcorg.highwire.dtl.DTLVardef@631f3aorg.highwire.dtl.DTLVardef@1972244org.highwire.dtl.DTLVardef@14ab3b1_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Aberrant expression of histone H2B variants reshape chromatin and alter oncogenic gene expression programs

Chromatin architecture governs DNA accessibility and gene expression. Thus, any perturbations to chromatin can significantly alter gene expression programs and promote disease. Prior studies demonstrate that every amino acid in a histone is functionally significant, and that even a single amino acid substitution can drive specific cancers. We previously observed that naturally occurring H2B variants are dysregulated during the epithelial to mesenchymal transition (EMT) in bronchial epithelial cells. Naturally occurring H2B variants differ from canonical H2B by only a few amino acids, yet single amino acid changes in other histone variants (e.g., H3.3) can drive cancer. We therefore hypothesized that H2B variants might function like oncohistones, and investigated how they modify chromatin architecture, dynamics, and function. We find that H2B variants are frequently dysregulated in many cancers, and correlate with patient prognosis. Despite high sequence similarity, mutations in each H2B variant tend to occur at specific "hotspots" in cancer. Some H2B variants cause tighter DNA wrapping around nucleosomes, leading to more compact chromatin structures and reduced transcription factor accessibility to nucleosomal DNA. They also altered genome-wide accessibility to oncogenic regulatory elements and genes, with concomitant changes in oncogenic gene expression programs. Although we did not observe changes in cell proliferation or migration in vitro, our Gene Ontology (GO) analyses of ATAC-seq peaks and RNA-seq data indicated significant changes in oncogenic pathways. These findings suggest that H2B variants may influence early-stage, cancer-associated regulatory mechanisms, potentially setting the stage for oncogenesis later on. Thus, H2B variant expression could serve as an early cancer biomarker, and H2B variants might be novel therapeutic targets.

cancer biology↗

bioRxiv2023

Unraveling the mechanism of centromere inactivation in human aging

Aging involves a range of genetic, epigenetic, and physiological alterations. A key characteristic of aged cells is the loss of global heterochromatin, accompanied by a reduction in canonical histone levels. In this study, we track the fate of centromeres during aging in human cells. Our findings reveal that the centromeric histone H3 variant CENP-A is downregulated in aged cells, in a p53-dependent manner. We observe repression of centromeric noncoding transcription through an epigenetic mechanism via recruitment of a lysine-specific demethylase 1 (LSD1/KDM1A) to centromeres. This suppression results in defective de novo CENP-A loading at aging centromeres. By dual inhibition of p53 and LSD1/KDM1A in aged cells, we mitigate the reduction in centromeric proteins and centromeric transcripts, leading to mitotic rejuvenation of these cells. These results offer insights into a novel mechanism for centromeric inactivation during aging and provide potential strategies to reactivate centromeres.

molecular biology↗

bioRxiv2023

A hyper-quiescent chromatin state formed during aging is reversed by regeneration

Epigenetic alterations are a key hallmark of aging but have been limitedly explored in tissues. Here, using naturally aged murine liver as a model and extending to other quiescent tissues, we find that aging is driven by temporal chromatin alterations that promote a refractory cellular state and compromise cellular identity. Using an integrated multi-omics approach, and the first direct visualization of aged chromatin we find that globally, old cells show H3K27me3-driven broad heterochromatinization and transcription suppression. At the local level, site-specific loss of H3K27me3 over promoters of genes encoding developmental transcription factors leads to expression of otherwise non-hepatocyte markers. Interestingly, liver regeneration reverses H3K27me3 patterns and rejuvenates multiple molecular and physiological aspects of the aged liver.

genomics↗

bioRxiv2022

The Molecular and Evolutionary Principles of Histone Folding in Eukarya and Archaea

Histones are the dominant proteins to compact and store DNA in both Eukarya and Archaea. For a long time, histones are observed to exist in the unit of dimers but diverge into different formats such as heterodimers in Eukarya or homodimers in Archaea. Here, by studying 11 types of histone proteins, both monomers and their dimeric complexes, using multiscale molecular dynamics (MD) simulations combined with NMR and circular dichroism experiments, we confirm the widely applied "folding upon binding" mechanism of histone structures. A histone dimer appears to form the longest 2 helices followed by other shorter helices and inter-molecular tertiary structures. We report an alternative conformation, namely, the inverted non-native dimer, which has a minimum free energy state. Protein sequence analysis indicates that the inverted conformation can be attributed to a hidden head-tail sequence symmetry underlying all histone proteins. This finding strongly support previously proposed histone evolution hypotheses. Finally, we separately used the MD-based AWSEM and AI-based AlphaFold-Multimer model to predict eukaryotic histone homodimer structures and performed extensive allatom MD simulations to examine their structural stabilities. Our results suggest that eukaryotic histones can also form stable homodimers, whereas their disordered tails-- the structurally asymmetrical region--may tip the balance towards the formation of heterotypic dimers.

biophysics↗

bioRxiv2022

Mobility of nucleosomes is regulated by their binding partners

Chromatin accessibility is modulated in a variety of ways to create open and closed chromatin states, both of which are critical for eukaryotic gene regulation. At the single molecule level, how accessibility is regulated in the chromatin fiber composed of canonical or variant nucleosomes is a fundamental question in the field. Here, we developed a single-molecule tracking method where we could analyze thousands of canonical H3 and centromeric variant nucleosomes imaged by high-speed atomic force microscopy. This approach allowed us to investigate how changes in nucleosome dynamics in vitro inform us about chromatin accessibility in vivo. By high-speed atomic force microscopy, we tracked chromatin dynamics in real time and determined the MSD and diffusion constant for the variant centromeric CENP-A nucleosome. Furthermore, an essential kinetochore protein CENP-C reduces the diffusion constant and mobility of centromeric nucleosomes along the chromatin fiber. We subsequently interrogated how CENP-C modulates CENP-A chromatin dynamics in vivo. Overexpressing CENP-C resulted in reduced centromeric transcription and impaired loading of new CENP-A molecules. Thus, changes which alter chromatin accessibility in vitro, also correspondingly alter transcription in vivo. These data suggest a model in which variant nucleosomes encode their own diffusion kinetics and mobility, and where binding partners can suppress or enhance mobility.

biophysics↗

bioRxiv2020

Binding Dynamics of Disordered Linker Histone H1 with a Nucleosomal Particle

Linker histone H1 is an essential regulatory protein for many critical biological processes, such as eukaryotic chromatin packaging and gene expression. Mis-regulation of H1s is commonly observed in tumor cells, where the balance between different H1 subtypes has been shown to alter the cancer phenotype. Consisting of a rigid globular domain and two highly charged terminal domains, H1 can bind to multiple sites on a nucleosomal particle to alter chromatin hierarchical condensation levels. In particular, the disordered H1 amino- and carboxyl-terminal domains (NTD/CTD) are believed to enhance this binding affinity, but their detailed dynamics and functions remain unclear. In this work, we used a coarse-grained computational model AWSEM-DNA to simulate the H1.0b-nucleosome complex, namely chromatosome. Our results demonstrate that H1 disordered domains restrict the dynamics of both globular H1 and linker DNA arms, resulting in a more compact and rigid chromatosome particle. Furthermore, we identified regions of H1 disordered domains that are tightly tethered to DNA near the entry-exit site. Overall, our study elucidates at near atomic resolution the way the disordered linker histone H1 modulates nucleosomes structural preferences and conformational dynamics.

biophysics↗

bioRxiv2019

CENP-C stabilizes the conformation of CENP-A nucleosomes within the inner kinetochore at human centromere

The centromere is the chromosomal locus that seeds the kinetochore, allowing for a physical connection between the chromosome and the mitotic spindle. At the heart of the centromere is the centromere-specific histone H3 variant CENP-A/CENH3. Throughout the cell cycle the constitutive centromere associated network is bound to CENP-A chromatin, but how this protein network modifies CENP-A nucleosome dynamics in vivo is unknown. Here, we purify kinetochore associated native centromeric chromatin and analyze its biochemical features using a combinatorial approach. We report that kinetochore bound chromatin has strongly reduced DNA accessibility and a distinct stabilized nucleosomal configuration. Disrupting the balance between CENP-A and CENP-C result in reduced centromeric occupancy of RNA polymerase 2 and impaired de novo CENP-A loading on the centromeric chromatin fiber, correlating with significant mitotic defects. CENP-A mutants that restore the ratio rescue the mitotic defects. These data support a model in which CENP-C bound centromeric nucleosomes behave as a barrier to the transcriptional machinery and suggest that maintaining the correct ratio between CENP-A and CENP-C levels is critical for centromere homeostasis.

biochemistry↗

bioRxiv2018

The Oligomerization Landscape of Histones

In eukaryotes, DNA is packaged through nucleosomes. Each nucleosome is typically centered around an octameric histone protein core: one central tetramer plus two separate dimers. Studying the assembly mechanisms of histones is essential for understanding the dynamics of entire nucleosomes and higher-order DNA packaging. Here we investigate the canonical histone assembly and that of the centromere-specific histone variant CENP-A using molecular dynamics simulations. We quantitatively characterize their thermodynamical and dynamical features, showing that the canonical H3 tetramer exhibits large instability around the central interface manifested via a swiveling motion of two halves, supporting the recently observed DNA handedness flipping of the tetrasome. In contrast, the variant CENP-A encodes a distinctive stability to its tetramer with a rigid but twisted interface compared to the crystal structure, implying the diverse structural possibilities of the histone variant. Interestingly, the observed tetramer dynamics alter significantly and appear to reach a new dynamics balance when H2A/H2B dimers are present. In all, these data reveal key mechanistic insights and structural details for the assembly of canonical and variant CENP-A histone tetramers and octamers, providing theoretical quantifications and physical interpretations for longstanding and recent experimental observations.

biophysics↗

bioRxiv2018

Elastic and Rigidified CENP-A Nucleosomes govern Centromeric Chromatin Plasticity

Histone variants fine-tune transcription, replication, DNA damage repair, and faithful chromosome segregation. Whether and how nucleosome variants encode unique mechanical properties to their cognate chromatin structures remains elusive. Here, using novel in silico and in vitro nanoindentation methods, extending to in vivo dissections, we report that histone variant nucleosomes are intrinsically more elastic than their canonical counterparts. Furthermore, binding proteins which discriminate between histone variant nucleosomes suppress this innate elasticity and also compact chromatin. Interestingly, when we overexpress the binding proteins in vivo, we also observe increased compaction of chromatin enriched for histone variant nucleosomes, correlating with diminished access. Together, these data suggest a plausible link between innate mechanical properties possessed by histone variant nucleosomes, the adaptability of chromatin states in vivo, and the epigenetic plasticity of the underlying locus.\n\nSignificanceNucleosomes are the base unit which organize eukaryotic genomes. Besides the canonical histone, histone variants create unique local chromatin domains that fine-tune transcription, replication, DNA damage repair, and faithful chromosome segregation. We developed computational and single-molecule nanoindentation tools to determine mechanical properties of histone variant nucleosomes. We found that the CENP-A nucleosome variant is more elastic than the canonical H3 nucleosome but becomes stiffer when bound to its partner CENP-C. In addition, CENP-C induces cross-array clustering, creating a chromatin state that less accessible. These data suggest that innate material properties of nucleosomes can influence the ultimate chromatin state, thereby influence biological outcomes.

biochemistry↗

bioRxiv2017

Internal modifications in the CENP-A nucleosome modulate centromeric dynamics

Post-translational modifications (PTMs) of core histones have studied for over 2 decades, and are correlated with changes in transcriptional status, chromatin fiber folding, and nucleosome dynamics. However, within the centromere-specific histone H3 variant CENP-A, few modifications have been reported, and their functions remain largely unexplored. In this multidisciplinary report, we utilize in silico computational and in vivo approaches to dissect lysine 124 of human CENP-A, which was previously reported to be acetylated in advance of replication. Computational modeling demonstrates that acetylation of K124 causes tightening of the histone core, and hinders accessibility to its C-terminus, which in turn diminishes CENP-C binding. Additionally, CENP-A K124ac/H4 K79ac containing nucleosomes are prone to DNA sliding. In vivo experiments using an acetyl or unacetylatable mimic (CENP-A K124Q and K124A respectively) reveal alterations in CENP-C levels, and a modest increase in mitotic errors. Furthermore, mutation of K124 results in alterations in centromeric replication timing, with the permanently acetylated form replicating centromeres early, and the unacetylable form replicating centromeres late. Purification of native CENP-A proteins followed by mass spectrometry analysis reveal that while CENP-A K124 is acetylated at G1/S, it switches to monomethylation during early and mid-S phase. Finally, we provide evidence that the HAT p300 is involved in this cycle. Taken together, our data suggest that cyclical modifications within the CENP-A nucleosome can influence the binding of key kinetochore proteins, the integrity of mitosis and centromeric replication. These data support the emerging paradigm that core modifications in histone variant nucleosomes transduce defined changes to key biological processes.

biochemistry↗

bioRxiv2017

CENP-A associated lncRNAs influence chromosome segregation in human cells

Transcription occurs ubiquitously throughout non-coding parts of the genome, including at repetitive -satellite DNA elements which comprise the majority of human centromeres. The function of temporally regulated centromeric transcription, and transcripts, is consequently a topic of intense investigation. In this study, we use high throughput approaches to identify and describe lncRNAs associated with the centromere specific histone variant CENP-A that arise from the transcription of specific centromeres at early G1, which we then show are physically associated with centromeres, and which are functionally necessary for accurate chromosome segregation. Targeted depletion of one such centromeric RNA, which originates from a single centromere, is sufficient to increase the frequency of chromosome segregation defects. These data support the emerging paradigm of the necessity of centromere-specific lncRNAs in the integrity of faithful chromosome segregation.

cell biology↗