Biology subjects

Meshorer, E.

Publications and source records attributed to Meshorer, E..

12 recordsLinked to original sources

bioRxiv2026

BLeaching In-cell Single-molecule burstS (BLISS) reveals a small dynamic fraction of HP1α clusters in undifferentiated embryonic stem cells

Confocal fluorescence microscopy measurements of dense cellular regions of interest (ROIs) in cells using fluorescence lifetime imaging microscopy (FLIM) provide detailed pixelated images. Yet, those pixels report ensemble- and time-averaged biomolecular data, due to the diffraction limit and acquisition times that are slower than typical biomolecular dynamics. The ability to acquire data on one biomolecule at a time within a given ROI can help recover some of the underlying biomolecular subpopulations that are otherwise masked out. Here, we present a simple approach to achieving single-biomolecule photon bursts in live cells, dubbed BLeaching In-cell Single-molecule burstS (BLISS), which does not necessarily require super-resolution modalities. Using BLISS, we show that mCherry-tagged heterochromatin protein 1 (mCherry-HP1) in chromocenters of undifferentiated mouse embryonic stem cells (ESCs), exhibit photon bursts with millisecond durations arising from dynamic clusters. Fluorescence lifetimes of these bursts are substantially lower than the pixel-wise averaged values observed in FLIM, attributed to higher density in HP1 clusters. These higher density clusters are observed primarily in undifferentiated ESCs, while two days post retinoic acid (RA)-induced differentiation (2d-RA), these bursts are rarely observed. Using BLISS, we also detect dynamic clusters of the chromocenter-associated protein, CENP-V, and the nucleolar protein, nucleolin; however, in both cases the fluorescence lifetimes of their clusters closely resemble the pixel-wise averaged values recovered from FLIM. In conclusion, we introduce BLISS as a method for revealing rare and dynamic subpopulation in dense ROIs that are otherwise hidden by averaging in diffraction-limited imaging techniques. Importantly, using BLISS we demonstrate that denser-than-average HP1 clusters are hyperdynamic.

biophysics↗

bioRxiv2024

Distinct Roles for SETα and SETβ in Early Cell Fate Decisions

SET, the nuclear proto-oncogene, is primarily expressed as SET in embryonic stem cells. Upon pluripotency exit, a transcriptional switch driven by alternative promoters causes SET{beta} to largely replace SET expression. Functional distinctions between the two isoforms have been difficult to ascertain, partly due to the redundancy between SET and SET{beta} in their protein structure and activity. In this study, we use embryonic stem cells (ESCs) with inducible SET isoform-specific expression to investigate the differences between both SET isoforms. Time-course RNA-seq analyses in SET-KO backgrounds as well as isoform-specific ChIP-seq experiments reveal regulatory functions for SET and SET{beta}. Despite sharing many binding sites and binding partners, SET has unique regulatory functions on its target genes, while SET{beta} downregulates FGF4. As KLF5 specifically regulates SET, this implicates SET isoform switching at the KLF5/FGF signalling axis during primitive endoderm specification. Together, we propose a model of how distinct roles of SET and SET{beta} may regulate cell identity in the early blastocyst.

developmental biology↗

bioRxiv2024

RoAM: computational reconstruction of ancient methylomes and identification of differentially methylated regions

Identifying evolutionary changes in DNA methylation bears a huge potential for unraveling adaptations that have occurred in modern humans. Over the past decade, computational methods to reconstruct DNA methylation patterns from ancient DNA sequences have been developed, allowing for the exploration of DNA methylation changes during the past hundreds of thousands of years of human evolution. Here, we introduce a new version of RoAM (Reconstruction of Ancient Methylation), a flexible tool that allows for the reconstruction of ancient methylomes, as well as the identification of differentially methylated regions between ancient populations. RoAM incorporates a series of filtering and quality control steps, resulting in highly reliable DNA methylation maps that exhibit similar characteristics to modern maps. To showcase RoAMs capabilities, we used it to compare ancient methylation patterns between pre- and post-Neolithic revolution samples from the Balkans. Differentially methylated regions separating these populations are shown to be associated with genes related to regulation of sugar metabolism. Notably, we provide evidence for overexpression of the gene PTPRN2 in post-Neolithic revolution samples. PTPRN2 is a key regulator of insulin secretion, and our finding is compatible with hypoinsulinism in pre-Neolithic revolution hunter-gatherers. Additionally, we observe methylation changes in the genes EIF2AK4 and SLC2A5, which provide further evidence to metabolic adaptations to a changing diet during the Neolithic transition. RoAM offers powerful algorithms that position it as a key asset for researchers seeking to identify evolutionary regulatory changes through the lens of paleoepigenetics.

evolutionary biology↗

bioRxiv2024

H3.3 deposition counteracts the replication-dependent enrichment of H3.1 at chromocenters in embryonic stem cells

Chromocenters in mouse cells are membrane-less nuclear compartments that represent typical heterochromatin stably maintained during the cell cycle. Here, we explore how histone H3 variants, replicative H3.1/H3.2 or replacement H3.3, mark these domains during the cell cycle. In mouse embryonic stem cells (ESCs), neuronal precursor cells (NPCs) as well as immortalized 3T3 cells, we find a strong and distinct H3.1 enrichment at chromocenters, with some variation in ESCs. Mechanistically, this H3.1 selective enrichment depends on the DNA Synthesis Coupled (DSC) deposition pathway operating in S phase. Yet, this selective enrichment is challenged when we target H3.3 deposition through the DNA Synthesis Independent (DSI) deposition pathway mediated by HIRA. Altering the H3.1/H3.3 equilibrium at chromocenters in ESCs affects its heterochromatin properties leading to mitotic defects. We thus reveal opposing mechanisms for H3.1 and H3.3 deposition with different enforcement according to cell cycle and potency which determine their ratio at chromocenters and are critical for genome stability and cell survival.

cell biology↗

bioRxiv2023

FRET-sensitized acceptor emission localization (FRETsael) - nanometer localization of biomolecular interactions using fluorescence lifetime imaging

Super-resolution light microscopy techniques facilitate the observation of nanometer-size biomolecules, which are 1-2 orders of magnitude smaller than the diffraction limit of light. Using super-resolution microscopy techniques, it is possible to observe fluorescence from two biomolecules in close proximity, however not necessarily in direct interaction. Using FRET-sensitized acceptor emission localization (FRETsael), we localize biomolecular interactions exhibiting FRET with nanometer accuracy, from two-color fluorescence lifetime imaging data. The concepts of FRETsael were tested first against simulations, in which the recovered localization accuracy is 20-30 nm for true-positive detections of FRET pairs. Further analyses of the simulation results report the conditions in which true-positive rates are maximal. We then show the capabilities of FRETsael on simulated samples of Actin-Vinculin and ER-ribosomes interactions, as well as on experimental samples of actin-myosin two-color confocal imaging. Conclusively, the FRETsael approach paves the way towards studying biomolecular interactions with improved spatial resolution from laser scanning confocal two-color fluorescence lifetime imaging. SignificanceFRET is used in fluorescence microscopy to report whether dye-labeled biomolecules of choice are close within distances of 10 nm or less, hence typical interaction distances. However, in many cases, using FRET imaging for the study of biomolecular interactions is difficult due to the high density of dye-labeled biomolecules and due to the existence of unbound dye-labeled biomolecules. In addition, the resolution of localizing molecules using light microscopy is diffraction limited. This work presents FRETsael, a new approach for localizing interacting biomolecules undergoing FRET, with improved resolution of 20-30 nm for confocal microscopy using search algorithms for local extrema in contribution to FRET using two-channel fluorescence intensity and lifetime data.

biophysics↗

bioRxiv2023

Improved detection of methylation in ancient DNA

Reconstructing premortem DNA methylation levels in ancient DNA (aDNA) has led to breakthrough studies such as the prediction of anatomical features of the Denisovan, as well as the castration status of ancient horses. These studies relied on computationally inferring methylation levels from damage signals in naturally deaminated cytosines. Because of statistical constraints, this inference requires high-coverage sequencing, and is thus not only expensive but also restricted to samples with exceptional DNA preservation. Instead, a method to directly measure methylation levels in aDNA, as exists in modern DNA samples, would open the door to a more thorough and cost effective ability to study ancient DNA methylation. We have tested two methods for direct methylation measurement developed for modern DNA based on either bisulfite or enzymatic methylation treatments. We find that both methods preserve sufficient DNA yields to allow for methylation measurement. Bisulfite treatment, combined with a single stranded library preparation, shows the least reduction in DNA yields compared to no methylation treatment, as well as the least biases during methylation conversion. In addition, we show that applying bisulfite treatment to [~]0.4-fold coverage sample provides a methylation signal that is comparable to, or even better, than the computationally inferred one. We thus present a method to directly measure methylation in ancient DNA that is cost effective and can be used on a wide variety of ancient samples.

genomics↗

bioRxiv2023

SMARCAD1 and TOPBP1 contribute to heterochromatin maintenance at the transition from the 2C-like to the pluripotent state

Chromocenters are established after the 2-cell (2C) stage during mouse embryonic development, but the factors that mediate chromocenter formation remain largely unknown. To identify regulators of 2C heterochromatin establishment, we generated an inducible system to convert embryonic stem cells (ESCs) to 2C-like cells. This conversion is marked by a global reorganization and dispersion of H3K9me3-heterochromatin foci, which are then reversibly formed upon re-entry into pluripotency. By profiling the chromatin-bound proteome (chromatome) through genome capture of ESCs transitioning to 2C-like cells, we uncover chromatin regulators involved in de novo heterochromatin formation. We identified TOPBP1 and investigated its binding partner SMARCAD1. SMARCAD1 and TOPBP1 associate with H3K9me3-heterochromatin in ESCs. Interestingly, the nuclear localization of SMARCAD1 is lost in 2C-like cells. SMARCAD1 or TOPBP1 depletion in mouse embryos leads to developmental arrest, reduction of H3K9me3, and remodeling of heterochromatin foci. Collectively, our findings contribute to comprehending the maintenance of chromocenters during early development.

developmental biology↗

bioRxiv2023

Fluorescent protein lifetimes report increased local densities and phases of nuclear condensates during embryonic stem cell differentiation

Fluorescent proteins (FP) are frequently used for studying proteins inside cells. In advanced fluorescence microscopy, FPs can report on additional intracellular variables. One variable is the local density near FPs, which can be useful in studying densities within cellular bio-condensates. Here, we show that a reduction in fluorescence lifetimes of common monomeric FPs reports increased levels of local densities. We demonstrate the use of this fluorescence-based variable to report the distribution of local densities within heterochromatin protein 1 (HP1) in mouse embryonic stem cells (ESCs), before and after early differentiation. We find that local densities within HP1 condensates in pluripotent ESCs are heterogeneous and cannot be explained by a single liquid phase. Early differentiation, however, induces a change towards a more homogeneous distribution of local densities, which can be explained as a liquid-like phase. In conclusion, we provide a fluorescence-based method to report increased local densities and apply it to distinguish between homogeneous and heterogeneous local densities within bio-condensates.

biophysics↗

bioRxiv2021

Transposable elements as sensors of SARS-CoV-2 infection

Transposable element (TE) transcription is induced in response to viral infections. TE induction triggers a robust and durable interferon (IFN) response, providing a host defense mechanism. Still, the connection between SARS-CoV-2 IFN response and TEs remains largely unknown. Here, we analyzed TE expression changes in response to SARS-CoV-2 infection in different human cellular models. We find that compared to other viruses, which cause global upregulation of TEs, SARS-CoV-2 infection results in a significantly milder TE response in both primary lung epithelial cells and in iPSC-derived lung alveolar type 2 cells. TE activation precedes, and correlates with, the induction of IFN-related genes, suggesting that the limited activation of TEs following SARS-CoV-2 infection may be the reason for the weak IFN response. Diminished TE activation was not observed in lung cancer cell lines with very high viral load. Moreover, we identify two variables which explain most of the observed diverseness in immune responses: basal expression levels of TEs in the pre-infected cells, and the viral load. Finally, analyzing the SARS-CoV-2 interactome, as well as the epigenetic landscape around the TEs that are activated following infection, we identify SARS-CoV-2 interacting proteins, which may regulate chromatin structure and TE transcription in response to a high viral load. This work provides a functional explanation for SARS-CoV-2s success in its fight against the host immune system, and suggests that TEs could be used as sensors and serve as potential drug targets for COVID-19. Key points O_LIUnlike other viruses, SARS-CoV-2 invokes a weak and inefficient transposable element (TE) response C_LIO_LITE induction precedes and predicts IFN response C_LIO_LIBasal TE expression and viral load explain immune responses C_LIO_LIDistinct chromatin and enhancer binding factors occupancy on TEs induced by SARS-CoV-2 C_LI

bioinformatics↗

bioRxiv2020

Pluripotent stem cell derived models of neurological diseases reveal early transcriptional heterogeneity

BackgroundMany neurodegenerative diseases (NDs) develop only later in life, when cells in the nervous system lose their structure or function. In genetic forms of NDs, this late onset phenomenon remains largely unexplained. ResultsAnalyzing single cell RNA sequencing (scRNA-seq) from Alzheimers disease (AD) patients, we find increased transcriptional heterogeneity in AD excitatory neurons. We hypothesized that transcriptional heterogeneity precedes ND pathologies. To test this idea experimentally, we used juvenile forms (72Q; 180Q) of Huntingtons disease (HD) iPSCs, differentiated them into committed neuronal progenitors, and obtained single cell expression profiles. We show a global increase in gene expression variability in HD. Autophagy genes become more stable, while energy and actin-related genes become more variable in the mutant cells. Knocking-down several differentially-variable genes resulted in increased aggregate formation, a pathology associated with HD. We further validated the increased transcriptional heterogeneity in CHD8+/- cells, a model for autism spectrum disorder. ConclusionsOverall, our results suggest that although NDs develop over time, transcriptional regulation imbalance is present already at very early developmental stages. Therefore, an intervention aimed at this early phenotype may be of high diagnostic value.

molecular biology↗

bioRxiv2020

CloneSeq: A Highly Sensitive Single-cell Analysis Platform for Comprehensive Characterization of Cells from 3D Culture

Single-cell assays have revealed the scope and importance of heterogeneity in many biological systems. However, in many cases, single cell limited sensitivity is a major hurdle for uncovering the full range of cellular variation. To overcome this limitation, we developed a complementary single cell technology, CloneSeq that combines clonal expansion under controlled culture conditions inside three-dimensional (3D) hydrogel spheres and droplet-based RNA sequencing (RNA-seq). We show that unlike single cell transcriptomes, clonal cells maintain cell states and share similar transcriptional profiles. CloneSeq analysis of Non-small-cell lung carcinoma (NSCLC) cells revealed the presence of novel cancer-specific subpopulations, including cancer stem-like cells (CSLCs). Standard single cell RNA-seq assays as well as cell-to-clone tracing by genetic barcoding failed to identify these rare CSLCs. In addition to CSLCs, clonal expansion within 3D soft microenvironments supported cellular stemness of embryonic stem cells (ESCs) that retained their pluripotent state in the absence of pluripotent media and improved epigenetic reprogramming efficiency of mouse embryonic fibroblasts. Our results demonstrate the capacity of CloneSeq, which can be effectively adapted to different biological systems, to discover rare and previously hidden subpopulations of cells, including CSLCs, by leveraging the broader expression space within clones.

molecular biology↗

bioRxiv2017

Recent Regulatory Changes Shaped the Human Facial and Vocal Anatomy

SummaryRegulatory changes are broadly accepted as key drivers of phenotypic divergence. However, identifying regulatory changes that underlie human-specific traits has proven very challenging. Here, we use 63 DNA methylation maps of ancient and present-day humans, as well as of six chimpanzees, to detect differentially methylated regions that emerged in modern humans after the split from Neanderthals and Denisovans. We show that genes affecting the face and vocal tract went through particularly extensive methylation changes. Specifically, we identify widespread hypermethylation in a network of face- and voice-affecting genes (SOX9, ACAN, COL2A1, NFIX and XYLT1). We propose that these repression patterns appeared after the split from Neanderthals and Denisovans, and that they might have played a key role in shaping the modern human face and vocal tract.View Full Text

evolutionary biology↗