Biology subjects

Fleifel, D.

Publications and source records attributed to Fleifel, D..

6 recordsLinked to original sources

bioRxiv2026

Molecular control of replication licensing speed by c-MYC

A critical prerequisite for DNA replication is genome-wide origin licensing, a process tightly restricted to the G1 cell cycle phase. This restriction means that short G1 cells must license quickly or risk incomplete replication and genome instability, but mechanisms to increase licensing speed are largely unknown. Using quantitative single cell approaches, we identify here a non-canonical role for c-MYC as a potent origin licensing accelerator. Importantly, a point mutation separates c-MYC-induced rapid licensing from the well-known c-MYC gene expression function. c-MYC accelerates licensing through the TRRAP lysine acetyltransferase adapter and GCN5 acetyltransferase. In addition c-MYC has the unexpected ability to induce widespread linker histone H1 eviction. Moreover, directly reducing chromatin-bound histone H1 is sufficient to accelerate licensing. In addition, c-MYC stimulates licensing in heterochromatin, a typically slow-licensing genome compartment. We suggest that global chromatin opening promotes rapid licensing when G1 phase is short such as during early development, rapid progenitor cell proliferation, and adaptation to oncogene activation.

molecular biology↗

bioRxiv2026

ELDR-Glo, a biosensor for cell age and quiescence depth

Fluorescent reporters are powerful tools for revealing intercellular heterogeneity among proliferating cells. However, there are few tools to analyze differences among quiescent (G0) cells, though such differences are relevant for development, tissue maintenance, and cancer cell behavior. Quiescence heterogeneity, also known as quiescence depth, typically correlates with time after cell cycle arrest, yet directly measuring cell age is not feasible for all cell types or most tissues. Here, we describe ELDR-Glo, a genetically-encoded fluorescent biosensor that estimates relative cell age, or time since the last cell cycle. The biosensor integrates replication-coupled degradation in S phase with a slow-maturing mCherry and a normalization module. We demonstrate that ELDR-Glo signal correlates with true cell age by both live-cell imaging and in fixed cells. ELDR-Glo distinguishes early and late G0 cells and functions as a relative quiescence depth reporter in situ. The biosensor is compatible with multiplexed immunofluorescence and flow cytometry. ELDR-Glo provides a unique and scalable tool to investigate cell proliferation control. TeaserA fluorescent biosensor to distinguish cells of different ages and thus, likelihood of returning to proliferation from quiescence.

molecular biology↗

bioRxiv2025

Extrachromosomal DNA Gives Cancer a New Evolutionary Pathway

During tumor progression, it has been assumed that individual cells that have acquired advantageous mutations overtake the population. Cancers driven by extrachromosomal DNA (ecDNA) do not follow this paradigm. Instead, these tumors have a spectrum of oncogene copy numbers across cells, and graded ecDNA variation may function as a form of bet-hedging that equips tumors with a broad range of phenotypes. Using imaging, single-cell multiomics, and multiplexed proteomics, we systematically characterized ecDNA levels across thousands of single cells. Higher ecDNA dosage produces proportional changes in transcript abundance, chromatin accessibility, protein levels, cell-cycle progression, and proliferation. Genes amplified on ecDNA exhibit distinct transcriptional scaling regimes that shift when the same genes are reintegrated into chromosomal homogeneous staining regions. When we experimentally disrupted the continuum of ecDNA dosage by sorting cells into low- and high-copy number states, the population rapidly recovered its original, continuous distribution. Our time-course data, live-cell imaging, and stochastic models collectively show that restoring this spectrum is an active, deterministic process rather than the passive outcome of random segregation. Together, these findings position ecDNA-mediated expression as a distinct evolutionary mechanism that endows tumors with rapid, population-level adaptability. These findings offer insight into why ecDNA-driven cancers are among the most aggressive and treatment-resistant.

genomics↗

bioRxiv2024

USP37 prevents unscheduled replisome unloading through MCM complex deubiquitination

The CMG helicase (CDC45-MCM2-7-GINS) unwinds DNA as a component of eukaryotic replisomes. Replisome (dis)assembly is tightly coordinated with cell cycle progression to ensure genome stability. However, factors that prevent premature CMG unloading and replisome disassembly are poorly described. Since disassembly is catalyzed by ubiquitination, deubiquitinases (DUBs) represent attractive candidates for safeguarding against untimely and deleterious CMG unloading. We combined a targeted loss-of-function screen with quantitative, single-cell analysis to identify human USP37 as a key DUB preventing replisome disassembly. We demonstrate that USP37 maintains active replisomes on S-phase chromatin and promotes normal cell cycle progression. Proteomics and enzyme assays revealed USP37 interacts with the CMG complex to deubiquitinate MCM7, thus antagonizing replisome disassembly. Significantly, USP37 protects normal epithelial cells from oncoprotein-induced replication stress. Our findings reveal USP37 to be critical to the maintenance of replisomes in S-phase and suggest USP37-targeting as a potential strategy for treating malignancies with defective DNA replication control.

cell biology↗

bioRxiv2023

APC/C prevents non-canonical order of cyclin/CDK activity to maintain CDK4/6 inhibitor-induced arrest

Regulated cell cycle progression ensures homeostasis and prevents cancer. In proliferating cells, premature S phase entry is avoided by the E3 ubiquitin ligase APC/C (anaphase promoting complex/cyclosome), although the APC/C substrates whose degradation restrains G1-S progression are not fully known. The APC/C is also active in arrested cells that exited the cell cycle, but it is not clear if APC/C maintains all types of arrest. Here by expressing the APC/C inhibitor, EMI1, we show that APC/C activity is essential to prevent S phase entry in cells arrested by pharmacological CDK4/6 inhibition (Palbociclib). Thus, active protein degradation is required for arrest alongside repressed cell cycle gene expression. The mechanism of rapid and robust arrest bypass from inhibiting APC/C involves cyclin-dependent kinases acting in an atypical order to inactivate RB-mediated E2F repression. Inactivating APC/C first causes mitotic cyclin B accumulation which then promotes cyclin A expression. We propose that cyclin A is the key substrate for maintaining arrest because APC/C-resistant cyclin A, but not cyclin B, is sufficient to induce S phase entry. Cells bypassing arrest from CDK4/6 inhibition initiate DNA replication with severely reduced origin licensing. The simultaneous accumulation of S phase licensing inhibitors, such as cyclin A and geminin, with G1 licensing activators disrupts the normal order of G1-S progression. As a result, DNA synthesis and cell proliferation are profoundly impaired. Our findings predict that cancers with elevated EMI1 expression will tend to escape CDK4/6 inhibition into a premature, underlicensed S phase and suffer enhanced genome instability. SignificanceAppropriate stable cell cycle arrest is critical to prevent cancer. However, it is not well-understood how cells maintain arrest. It is known that arrest requires repressing proliferation-stimulating genes, but the role of targeted protein degradation is unclear. This work demonstrates that continuous degradation of cyclin A through the action of the anaphase promoting complex/cyclosome (APC/C) is required to maintain arrest induced by a cancer drug that blocks cell cycle kinase enzymes. APC/C activity is required to prevent cell cycle re-entry. Impaired APC/C activity causes arrest bypass, inefficient DNA replication, and ultimately long-term proliferation defects. These results suggest that the activity level of the APC/C in tumors may profoundly influence the response to drugs that target cell cycle kinases.

cell biology↗

bioRxiv2021

Quantitative Profiling of Adaptation to Cyclin E Overproduction

Cyclin E/CDK2 drives cell cycle progression from G1 to S phase. Cyclin E overproduction is toxic to mammalian cells, although the gene encoding cyclin E (CCNE1) is overexpressed in some cancers. To gain insight into how cancer cells tolerate high cyclin E, we extensively characterized non-transformed epithelial cells throughout a time course of chronic cyclin E overproduction. Cells overproducing human cyclin E, but not cyclin D or cyclin A, briefly experienced truncated G1 phases, then endured a transient period of DNA replication origin underlicensing, replication stress, and severely impaired proliferation. Individual cells displayed substantial intercellular heterogeneity in cell cycle dynamics and CDK activity. Each phenotype improved rapidly despite maintaining high cyclin E-associated activity. Transcriptome analysis revealed that adapted cells downregulated a cohort of G1-regulated genes. Withdrawing cyclin E induction only partially reversed the intermediate licensing phenotype of adapted cells indicating that adaptation is at least partly independent of mutations. This study provides evidence that mammalian cyclin E/CDK inhibits origin licensing by an indirect mechanism through premature S phase onset and provides further insight into the relationship between CDK activity and licensing in mammals. It serves as an example of specific oncogene adaptation that may recapitulate molecular changes during tumorigenesis.

cell biology↗