Biology subjects

Biran, A.

Publications and source records attributed to Biran, A..

9 recordsLinked to original sources

bioRxiv2026

CREPAS: a reproducible nascent chromatin sequencing analysis pipeline for epigenome replication studies

Chromatin-based genomics data are essential for understanding genome regulation and the mechanisms underlying epigenetic memory. Recent methods such as ChOR-seq and SCAR-seq assess histone modifications and chromatin-associated proteins during and after replication, capturing chromatin states that contribute to memory across cell divisions. Current tools for chromatin data analysis lack scalability and reproducibility across computing infrastructures, offer limited parameters, and are applicable only to a few sequencing techniques, ignoring the information from nascent chromatin assays. To address these challenges, we developed CREPAS, a Nextflow pipeline for analyzing nascent and parental chromatin sequencing data, including ChIP-seq, ChOR-seq, SCAR-seq, OK-seq, ATAC-seq, CUT&RUN, and CUT&Tag, and derivative protocols. CREPAS provides an end-to-end solution, from quality control to advanced analyses, including downsampling, peak calling, annotation, and visualization. By harnessing quantitative assays such as qChIP-seq and qChOR-seq, the normalization methods in CREPAS allow to compare the restoration kinetics of individual marks or proteins across replication timepoints. Moreover, the pipeline includes calculations such as fork directionality and partitioning using OK-seq and SCAR-seq data, linking replication dynamics to epigenetic inheritance. CREPAS is a valuable resource that enhances the efficiency and reproducibility of nascent chromatin sequencing data analyses, enabling the study of chromatin replication and propagation of epigenetic states. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/732899v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@11634b8org.highwire.dtl.DTLVardef@df2e07org.highwire.dtl.DTLVardef@46d117org.highwire.dtl.DTLVardef@14950a2_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2025

Receptor dimerization enables ligand discrimination through tunable response heterogeneity

Signaling pathways enable cells to coordinate collective behaviors by exchanging specific information. Many pathways utilize multiple ligand variants to activate the same intracellular signaling cascade, raising the question of how cells discriminate between these seemingly redundant signals. It has been shown that individual cells can discriminate between signals based on their induced level of activity, temporal dynamics or combinatorial effect. Here, we demonstrate that ligand discrimination could also emerge at the population level. Using mathematical models of ligand-receptor interactions, we examine how response heterogeneity at the population level can encode ligand identity. We introduce a local scaling metric to quantify how variation in pathway components affects the cellular response. Our results reveal that for pathways with dimeric receptors, and more significantly for heterodimeric receptors, biochemical parameters of the ligands control the resulting heterogeneity in the response of a population of cells. Furthermore, we show that the population-level heterogeneity encodes the enzymatic activity of the resulting receptor complex. This suggests a functional advantage for utilizing heterodimeric receptor complexes in pathways acting across a population of cells, such as the type I interferon pathway, which shows several of the characteristics of our model. This contrasts to juxtacrine pathways, such as Notch, that do not act at the population level and use a single component receptor. Overall, our findings highlight a novel mechanism by which receptor architecture enables cells to encode ligand-specific information through population-level heterogeneity, offering insights into immune regulation, tissue development, and synthetic biology. Significance StatementCells often communicate using distinct molecular signals (ligands) that activate the same pathway. Yet, how cells distinguish between these signals remains unclear. This study reveals that ligand discrimination can emerge at the level of entire cell populations, not just individual cells. By analyzing mathematical models, we show that certain receptor architectures, especially those involving heterodimeric receptors, allow signals to control the heterogeneity of the response level across a population. This mechanism enables cell populations to decode specific information about their environment, impacting processes like immune responses and tissue development. Our findings provide new insights into how signals coordinate collective behaviors and suggest strategies for designing synthetic systems to precisely control biological responses.

systems biology↗

bioRxiv2024

Investigating the p21 Ubiquitin-Independent Degron Reveals a Dual Degron Module Regulating p21 Degradation and Function.

A group of intrinsically disordered proteins (IDPs) are subjected to 20S proteasomal degradation in a ubiquitin-independent manner. Recently, we have reported that many IDP/IDR are targeted to the 20S proteasome via interaction with the C-terminus of PSMA3 subunit, termed the PSMA3 Trapper. In this study, we investigated the biological significance of the IDP-Trapper interaction using the IDP p21. Using split luciferase reporter assay and conducting detailed p21 mutagenesis, we first identified the p21 RRLIF box, localized at the C-terminus, as mediating the Trapper interaction in cells. To demonstrate the role of this box in p21 degradation, we edited the genome of HEK293 and HeLa cell lines using a CRISPR strategy. We found that the p21 half-life increased in cells with either a deleted or mutated p21 RRLIF box. The edited cell lines displayed an aberrant cell cycle pattern under normal conditions and in response to DNA damage. Remarkably, these cells highly expressed senescence hallmark genes in response to DNA damage, highlighting that the increased p21 half-life, not its actual level, regulates senescence. Our findings suggest that the p21 RRLIF box, which mediates interaction with the PSMA3 Trapper, acts as a ubiquitin-independent degron. This degron is positioned adjacent to the previously identified ubiquitin-dependent degron, forming a dual degron module that functionally regulates p21 degradation and its physiological outcomes.

molecular biology↗

bioRxiv2024

Acute multi-level response to defective de novo chromatin assembly in S-phase

Long-term perturbation of de novo chromatin assembly during DNA replication has profound effects on epigenome maintenance and cell fate. The early mechanistic origin of these defects is unknown. Here, we combine acute degradation of Chromatin Assembly Factor 1 (CAF-1), a key player in de novo chromatin assembly, with single-cell genomics, quantitative proteomics, and live-microscopy to uncover these initiating mechanisms in human cells. CAF-1 loss immediately slows down DNA replication speed and renders nascent DNA hyper-accessible. A rapid cellular response, distinct from canonical DNA damage signaling, is triggered and lowers histone mRNAs. As a result, histone variants usage and their modifications are altered, limiting transcriptional fidelity and delaying chromatin maturation within a single S-phase. This multi-level response induces a cell-cycle arrest after mitosis. Our work reveals the immediate consequences of defective de novo chromatin assembly during DNA replication, explaining how at later times the epigenome and cell fate can be altered. HighlightsO_LIThe histone chaperone CAF-1 sustains DNA replication speed in single cells. C_LIO_LICAF-1 loss alters histone repertoire and delays chromatin maturation. C_LIO_LIH3K9me3 and H3K27me3 regions respond differently to acute CAF-1 depletion. C_LIO_LIImpaired S-phase chromatin assembly triggers an immediate response and a G0 arrest. C_LI

cell biology↗

bioRxiv2023

Repair of DNA double-strand breaks leaves heritable impairment to genome function

Upon DNA breakage, a genomic locus undergoes alterations in 3-D chromatin architecture to facilitate signaling and repair. While cells possess mechanisms to repair damaged DNA, it is unknown whether the surrounding chromatin is restored to its naive state. We show that a single DNA double-strand break (DSB) within a topologically-associated domain (TAD) harboring conformation-sensitive genes causes lasting chromatin alterations, which persist after completion of DNA repair and feature structural changes, chromatin compaction and loss of local RNA species. Unexpectedly, these newly-acquired features of post-repair chromatin are transmitted to daughter cells and manifest as heritable impairments of gene expression. These findings uncover a hitherto concealed dimension of DNA breakage, which we term post-repair chromatin fatigue, and which confers heritable impairment of gene function beyond DNA repair.

cell biology↗

bioRxiv2022

CAF-1 deposits newly synthesized histones during DNA replication using distinct mechanisms on the leading and lagging strands

During every cell cycle, both the genome and the associated chromatin must be accurately replicated. Chromatin Assembly Factor-1 (CAF-1) is a key regulator of chromatin replication, but how CAF-1 cooperates with the DNA replication machinery is unknown. Here, we reveal that this crosstalk differs between the leading and lagging strand at replication forks. Using biochemical reconstitution, we show that DNA and histones promote CAF-1 recruitment to its binding partner PCNA and reveal that two CAF-1 complexes are required for efficient nucleosome assembly under these conditions. Remarkably, in the context of the replisome, CAF-1 competes with the leading strand DNA polymerase epsilon (Pol{varepsilon}) for PCNA binding, but not with the lagging strand DNA polymerase Delta (Pol{delta}). Yet, in cells, CAF-1 deposits newly synthesized histones equally on both daughter strands. Thus, on the leading strand, chromatin assembly by CAF-1 cannot occur simultaneously to DNA synthesis, while on the lagging strand both processes are coupled. We propose that these differences may facilitate distinct parental histone recycling mechanisms and accommodate the inherent asymmetry of DNA replication.

biochemistry↗

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

Oncogenic addiction to high 26S proteasome levels

Proteasomes are large intracellular complexes responsible for the degradation of cellular proteins. The altered protein homeostasis of cancer cells results in increased dependency on proteasome function. There are several different proteasome complexes that may be assembled in cells, with the 20S catalytic core common to them all. 20S proteasomes can function in isolation, or as part of larger complexes (26S) with regulatory particles (RP) such as the 19S that is needed for the targeting and processing of ubiquitinated substrates. Proteasome inhibitors target the catalytic barrel (20S) and thus this inhibition does not allow the deconvolution of the distinct roles of 20S vs. 26S proteasomes in cancer progression. We examined the degree of dependency of cancer cells specifically to the level of the 26S proteasome complex. We found that oncogenic transformation of human and mouse immortalized cells with mutant Ras induced a strong increase in the translation of the 26S proteasome subunits, giving rise to high 26S complex levels. We show that depletion of a single subunit of the 19S RP was sufficient to significantly reduce the 26S proteasome level and lower the cellular 26S/20S ratio. We further demonstrate that the accumulated 26S proteasome was essential for the viability of the transformed cells. Moreover, the viability of 20 different cancer cell lines, but not normal human fibroblasts, was severely compromised upon specific 26S proteasome suppression regardless of their p53 status. Suppression of 26S activated the UPR and Caspase-3, which at least partially explains the cell-killing effect. Morphologically, suppression of the 26S proteasome resulted in cytoplasm shrinkage and nuclear deformation. Thus, the tumor cell-specific addiction to high 26S proteasome levels sets the stage for future strategies in cancer therapy.

cancer biology↗

bioRxiv2017

A 20S proteasome receptor for degradation of intrinsically disordered proteins

Degradation of intrinsically disordered proteins (IDPs) by the 20S proteasome, unlike ubiquitin-dependent 26S proteasomal degradation, does not require proteasomal targeting by polyubiquitin. However, how these proteins are recognized by the proteasome was unknown. We report here on a mechanism of 20S proteasome targeting. Analysis of protein interactome datasets revealed that the proteasome subunit PSMA3 interacts with many IDPs. By employing in vivo and cell-free experiments we demonstrated that the PSMA3 C-terminus binds p21, c-Fos and p53, all IDPs and 20S proteasome substrates. A 69 amino-acids long fragment is autonomously functional in interacting with IDP substrates. Remarkably, this fragment in isolation blocks the degradation of a large number of IDPs in vitro and increases the half-life of proteins in vivo. We propose a model whereby the PSMA3 C-terminal region plays a role of substrate receptor in the process of proteasomal degradation of many IDPs.

molecular biology↗