Biology subjects

Swaffer, M. P.

Publications and source records attributed to Swaffer, M. P..

11 recordsLinked to original sources

bioRxiv2026

Retrograde signalling mediates cellular adaptation to mitochondrial DNA copy number alterations

Eukaryotic cells maintain multiple copies of the mitochondrial genome, which is essential for cellular metabolism. Accordingly, alterations in the mitochondrial DNA (mtDNA) copy number are associated with severe human diseases and ageing. However, the mechanisms through which cells regulate mtDNA copy number and the cellular consequences of altered copy number remain poorly understood. Here, using budding yeast as a model, we show that mtDNA copy number is determined by the amount of three limiting factors, Mip1, Abf2, and Rim1. By synthetically tuning the concentrations of only these three proteins, we can modulate mtDNA dosage inside the cell. This revealed that cells are surprisingly robust to mtDNA copy number alterations, with increased copy numbers even accelerating cell growth. Our findings suggest that this robustness is due to protein dosage compensation and independent regulation of mitochondrial morphology. Mechanistically, we identified a critical role of the retrograde signalling pathway for this adaptation. We show that signalling from mitochondria to the nucleus is upregulated in cells with higher mtDNA copy number, and disruption of this regulation diminishes their faster growth. Taken together, our work reveals regulatory principles that enable cells to adapt to mtDNA copy number alterations.

cell biology↗

bioRxiv2026

Cell Size Modulates SBF and Whi5 Chromatin Binding to Regulate the Start of the Budding Yeast Cell Cycle

Cell growth and division are tightly coordinated to cell size. In budding yeast, increasing cell size promotes the G1/S transition, called Start, by activating the transcription factor SBF, which drives a large fraction of cell-cycle-dependent gene expression. Part of this regulation arises because the concentration of the SBF inhibitor Whi5 decreases as cells grow. However, cells lacking Whi5 can still maintain a relatively accurate size when the SBF activator Cln3 is also removed, indicating that there are additional size control mechanisms. To understand how cell size is mechanistically translated into the activity of SBF-regulated promoters, we quantified the binding kinetics of Whi5 and SBF in live cells using single-molecule fluorescence microscopy. We found that increasing cell size is associated with both a decreased chromatin affinity of Whi5 and an increased chromatin affinity of SBF, accompanied by a higher SBF:Whi5 cell copy-number ratio. Chromatin-binding trends under basal and Whi5 overexpression conditions indicate that Whi5 restricts SBF association with chromatin. The transition point at which SBF binding overtakes Whi5 binding coincides with the onset of the expression of the G1 cyclins CLN1 and CLN2, two SBF targets that are important for committing cells to division. Reduced Whi5 binding reflects changes in its chromatin-association rate, as Whi5 and SBF dwell times on chromatin remain [~]10 s and are largely independent of cell size. Together, these results show how changes in SBF and Whi5 abundance and chromatin association transmit cell size information to the genome to regulate the size-dependent Start transition in budding yeast.

cell biology↗

bioRxiv2025

Cell size-dependent mRNA transcription drives proteome remodeling

Increasing cell size drives proteomic changes that impact cell physiology. However, the molecular basis of size-dependent proteome remodeling has remained unclear. Here, we develop an inducible Cyclin D1 expression system in human cells to generate populations of proliferating cells spanning over a two-fold size range. We use this genetic system to make comprehensive genome-wide measurements of mRNA and protein concentrations and stability. We find that protein and mRNA turnover rates are weakly related to cell size, but that mRNA concentrations are strongly size-dependent. This establishes that transcriptional regulation is the basis of proteome remodeling. Live-cell imaging of endogenous mRNAs using MS2 fluorescent protein binding motifs is used to measure how transcriptional dynamics change with cell size. Larger cells prolong transcriptional bursts and shorten inactive periods between bursts but maintain similar burst amplitudes to achieve transcriptional scaling. Taken together, our results show how transcription is modulated by cell size to remodel the proteome and alter cell physiology.

cell biology↗

bioRxiv2024

Somatic polyploidy supports biosynthesis and tissue function by increasing transcriptional output

Cell size and biosynthetic capacity generally increase with increased DNA content. Polyploidy has therefore been proposed to be an adaptive strategy to increase cell size in specialized tissues with high biosynthetic demands. However, if and how DNA concentration limits cellular biosynthesis in vivo is not well understood, and the impacts of polyploidy in non-disease states is not well studied. Here, we show that polyploidy in the C. elegans intestine is critical for cell growth and yolk biosynthesis, a central role of this organ. Artificially lowering the DNA/cytoplasm ratio by reducing polyploidization in the intestine gave rise to smaller cells with more dilute mRNA. Highly-expressed transcripts were more sensitive to this mRNA dilution, whereas lowly-expressed genes were partially compensated - in part by loading more RNA Polymerase II on the remaining genomes. DNA-dilute cells had normal total protein concentration, which we propose is achieved by increasing production of translational machinery at the expense of specialized, cell-type specific proteins.

cell biology↗

bioRxiv2023

Genome dilution by cell growth drives starvation-like proteome remodeling in mammalian and yeast cells

Cell size is tightly controlled in healthy tissues and single-celled organisms, but it remains unclear how size influences cell physiology. Increasing cell size was recently shown to remodel the proteomes of cultured human cells, demonstrating that large and small cells of the same type can be biochemically different. Here, we corroborate these results in mouse hepatocytes and extend our analysis using yeast. We find that size-dependent proteome changes are highly conserved and mostly independent of metabolic state. As eukaryotic cells grow larger, the dilution of the genome elicits a starvation-like proteome phenotype, suggesting that growth in large cells is limited by the genome in a manner analogous to a limiting nutrient. We also find that this phenomenon explains many proteomic changes ascribed to yeast aging. Overall, our data suggest that genome concentration is a universal determinant of proteome content in growing cells.

cell biology↗

bioRxiv2023

Genome-wide distribution of 5-hydroxymethyluracil and chromatin accessibility in the Breviolum minutum genome

In dinoflagellates, a unique and extremely divergent genomic and nuclear organization has evolved. The highly unusual features of dinoflagellate nuclei and genomes include permanently condensed liquid crystalline chromosomes, primarily packaged by proteins other than histones, genes organized in very long unidirectional gene arrays, a general absence of transcriptional regulation, high abundance of the otherwise very rare DNA modification 5-hydroxymethyluracil (5-hmU), and many others. While most of these fascinating properties were originally identified in the 1970s and 1980s, they have not yet been investigated using modern genomic tools. In this work, we address some of the outstanding questions regarding dinoflagellate genome organization by mapping the genome-wide distribution of 5-hmU (using both immunoprecipitation-based and basepair-resolution chemical mapping approaches) and of chromatin accessibility in the genome of the Symbiodiniaceae dinoflagellate Breviolum minutum. We find that the 5-hmU modification is preferentially enriched over certain classes of repetitive elements, often coincides with the boundaries between gene arrays, and is generally correlated with decreased chromatin accessibility, the latter otherwise being largely uniform along the genome. We discuss the potential roles of 5-hmU in the functional organization of dinoflagellate genomes and its relationship to the transcriptional landscape of gene arrays.

genomics↗

bioRxiv2021

RNA polymerase II dynamics and mRNA stability feedback determine mRNA scaling with cell size

A fundamental feature of cellular growth is that protein and RNA amounts scale with cell size so that concentrations remain constant. A key component to this is that global transcription rates increase in larger cells, but the underlying mechanism has remained unknown. Here, we identify RNAPII as the major limiting factor increasing transcription with cell size in budding yeast as transcription is highly sensitive to the dosage of RNAPII but not to other components of the general transcriptional machinery. Our experiments support a dynamic equilibrium model where global transcription at a given size is set by the mass-action recruitment kinetics of unengaged nucleoplasmic RNAPII, and DNA content. This drives a sub-linear increase in transcription with size, which is precisely compensated for by a decrease in mRNA decay rates as cells enlarge. Thus, limiting RNAPII and feedback on mRNA stability work in concert to ensure mRNA concentration homeostasis in growing cells.

molecular biology↗

bioRxiv2021

Increasing cell size remodels the proteome and promotes senescence

Cell size is tightly controlled in healthy tissues, but it is poorly understood how variations in cell size affect cell physiology. To address this, we employed a high-accuracy mass spectrometry-based approach to measure how the proteome changes with cell size. Protein concentration changes are widespread, measurable in both asynchronous and G1-arrested cell populations, and predicted by subcellular localization, size-dependent changes in mRNA concentrations, and protein turnover. As proliferating cells grow larger, protein concentration changes typically associated with cellular senescence are increasingly pronounced. This suggests that large size is a cause rather than just a consequence of cell senescence. Consistent with this hypothesis, larger cells are prone to replicative, DNA damage-, and CDK4/6i-induced senescence. Size-dependent changes to the proteome, including those associated with senescence, are not observed when an increase in cell size is accompanied by a similar increase in ploidy. This shows that proteome composition is determined by the DNA-to-ploidy ratio rather than cell size per se and that polyploidization is an elegant method to generate large non-senescent cells as is commonly found in nature. Together, our findings show how cell size could impact many aspects of cell physiology through remodeling the proteome, thereby providing a rationale for cell size control and polyploidization.

cell biology↗

bioRxiv2021

Localized phosphorylation of RNA Polymerase II by G1 cyclin-Cdk promotes cell cycle entry

The cell cycle is thought to be initiated by cyclin-dependent kinases (Cdk) inactivating transcriptional inhibitors of cell cycle gene-expression(1, 2). In budding yeast, the G1 cyclin Cln3-Cdk1 complex is thought to directly phosphorylate Whi5, thereby releasing the transcription factor SBF and committing cells to division(3-7). Here, we report that Cln3-Cdk1 does not phosphorylate Whi5, but instead phosphorylates the RNA Polymerase II subunit Rpb1s C-terminal domain (CTD) on S5 of its heptapeptide repeats. Cln3-Cdk1 binds SBF-regulated promoters(8) and Cln3s function can be performed by the canonical S5 kinase(9) Ccl1-Kin28 when synthetically recruited to SBF. Thus, Cln3-Cdk1 triggers cell division by phosphorylating Rpb1 at SBF-regulated promoters to activate transcription. Our findings blur the distinction between cell cycle and transcriptional Cdks to highlight the ancient relationship between these processes.

cell biology↗

bioRxiv2020

Size-independent mRNA synthesis and chromatin-based partitioning mechanisms generate and maintain constant amounts of protein per cell

Biosynthesis scales with cell size such that protein concentrations generally remain constant as cells grow. As an exception, synthesis of the cell-cycle inhibitor Whi5 sub-scales with cell size so that its concentration is lower in larger cells to promote cell-cycle entry. Here, we find that a transcriptional control uncouples Whi5 synthesis from cell size and, screening for similar genes, identify histones as the major class of sub-scaling transcripts besides WHI5. Histone synthesis is thereby matched to genome content rather than cell size. Such sub-scaling proteins are challenged by asymmetric cell division because proteins are typically partitioned in proportion to new-born cell volume. To avoid this fate, Whi5 uses chromatin-binding to partition similar protein amounts to each new-born cell regardless of cell size. Finally, disrupting both Whi5 synthesis and chromatin-based partitioning compromises G1 size control. Thus, specific transcriptional and partitioning mechanisms determine protein sub-scaling to control cell size.

cell biology↗

bioRxiv2018

Long-range single-molecule mapping of chromatin accessibility in eukaryotes

Active regulatory elements in eukaryotes are typically characterized by an open, nucleosome-depleted chromatin structure; mapping areas of open chromatin has accordingly emerged as a widely used tool in the arsenal of modern functional genomics. However, existing approaches for profiling chromatin accessibility are limited by their reliance on DNA fragmentation and short read sequencing, which leaves them unable to provide information about the state of chromatin on larger scales or reveal coordination between the chromatin state of individual distal regulatory elements. To address these limitations, we have developed a method for profiling accessibility of individual chromatin fibers at multi-kilobase length scale (SMAC-seq, or Single-Molecule long-read Accessible Chromatin mapping sequencing assay), enabling the simultaneous, high-resolution, single-molecule assessment of the chromatin state of distal genomic elements. Our strategy is based on combining the preferential methylation of open chromatin regions by DNA methyltransferases (CpG and GpC 5-methylcytosine (5mC) and N6-methyladenosine (m6A) enzymes) and the ability of long-read single-molecule nanopore sequencing to directly read out the methylation state of individual DNA bases. Applying SMAC-seq to the budding yeast Saccharomyces cerevisiae, we demonstrate that aggregate SMAC-seq signals match bulk-level accessibility measurements, observe single-molecule protection footprints of nucleosomes and transcription factors, and quantify the correlation between the chromatin states of distal genomic elements.

genomics↗