Biology subjects

Papalopulu, N.

Publications and source records attributed to Papalopulu, N..

16 recordsLinked to original sources

bioRxiv2025

Engineering protein expression dynamics with Tet-ON and dTAG degron systems: from precise control to oscillations

Precise temporal control of protein expression is essential for dissecting protein function and dynamic cellular processes. We present a framework for engineering tunable oscillatory protein expression (repeated pulses in expression) using widely adopted molecular tools, applying them to modulate NGN3 expression. Single-cell time-lapse microscopy reveals that the Tet-On system unexpectedly generates asynchronous oscillations in protein expression under continuous doxycycline administration. These oscillations are dependent on protein instability and are not tunable by doxycycline concentration. In contrast, the dTAG degron system enables precise, reversible, concentration-dependent control of protein degradation and reaccumulation. Coupled with a constitutive promoter, we achieve synchronous oscillatory protein expression (COD: Constituitive promoter driving Oscillations via Degradation). Mathematical modelling identifies optimal dTAG drug addition and removal timings using the COD system to flexibly tune NGN3 oscillation periods while maintaining other oscillatory parameters (mean level and peak-to-trough fold-change). Using microfluidics (COD+CHIPS) we reproduce model-predicted 5 and 10 hours periodicities while maintaining similar mean levels and peak-to-trough fold-changes. This work introduces a generalisable, programmable approach for generating and modulating protein oscillations, allowing investigation into how dynamic protein expression governs cellular function.

developmental biology↗

bioRxiv2025

HES1 oscillations are required for cell cycle progression and re-entry in oestrogen receptor positive breast cancer cells

Long-term recurrence in breast cancer is driven by reactivation of dormant disseminated tumour cells (DTCs) and remains a major clinical challenge, particularly in oestrogen receptor positive (ER) tumours. This process is underpinned by regulation of the cell cycle machinery that controls quiescence maintenance and exit. HES1, a Notch pathway transcription factor, regulates key cell cycle genes and has been shown to demonstrate protein expression oscillations. Here, we sought to establish whether HES1 oscillations may regulate ER+ cancer cell quiescence and reactivation. To investigate this, we developed a fundamental in vitro model of cell cycle arrest and re-entry based on reversible CDK4/6 inhibition (CDK4/6i) with palbociclib, compatible with quantitative single-cell live-imaging of a knock-in endogenous HES1 reporter. Consistent with earlier findings, HES1 exhibited [~]24-hour protein oscillations in cycling cells demonstrating a reproducible dip in protein expression prior to S-Phase. During CDK4/6i-mediated arrest, the [~]24h HES1 oscillation was lost, HES1 levels were maintained at a moderately higher level and HES1 exhibited smaller dips. Similar changes were observed in unperturbed, spontaneously quiescent cells. Following release from CDK4/6i and cell cycle re- entry, these alterations were reversed and the characteristic G1/S HES1 dip was observed. Preventing this dip at the point of release, by inducibly sustaining HES1 with a Tet-On system, upregulated the cell cycle inhibitor p21, impeded cell cycle re-entry and induced cell death. These findings suggest that manipulating HES1 dynamics could represent a promising therapeutic approach to prevent reactivation of dormant tumour cells. Significance StatementBreast cancer can recur years after initial treatment due to reactivation of dormant tumour cells. Understanding how these cells exit dormancy is crucial for preventing relapse. We investigated HES1, a transcription factor with rhythmic protein oscillations, and its role in regulating quiescence in oestrogen receptor-positive (ER) breast cancer cells. Using live-cell imaging and a reversible cell cycle arrest model, we show that HES1 dynamics change during dormancy and reactivation, and that disrupting these oscillations prevents cell cycle re-entry and induces cell death. These findings reveal HES1 protein dynamics as a potential therapeutic vulnerability and highlight a novel strategy to target dormant cancer cells to prevent their reactivation.

cancer biology↗

bioRxiv2025

Oscillatory Co-expression of HES1 and HES5 Enables a Hybrid State in a Bistable Transcription Factor Regulatory Motif

Many cell fate decisions in the developing neural tube are directed by cross-repressive transcription factor (TF) motifs that generate bistability, enforcing expression of one dominant TF. However, evidence of hybrid states, where cells co-express opposing fate determinants, challenges this model. We hypothesised that oscillatory expression enables co-existence of cross-repressive TFs within single cells, allowing hybrid states in bistable motifs. To test this, we focused on HES1 and HES5, oscillatory, cross-repressive TFs that regulate neural progenitor maintenance and are expressed in adjacent dorsoventral domains in the developing spinal cord. Using live-cell imaging of fluorescent reporters and computational modelling, we show that HES1 and HES5 co-express and oscillate in-phase within single cells. Differences in protein stability result in distinct free-running periodicity, but co-expression results in entrainment and phase-locking. Modulating cross-repression strength and/or abundance shifts the system towards bistability and dominance of a single TF oscillator. Consistent with this, we observe progressive separation of the HES expression domains in vivo, through a decrease in oscillatory co-expression. Our findings provide a mechanism for hybrid states to emerge in a developmental bistable motif.

developmental biology↗

bioRxiv2024

Cellular signalling protrusions enable dynamic distant contacts in spinal cord neurogenesis

In the developing mouse ventral spinal cord, HES5, a transcription factor downstream of Notch signalling, is expressed as evenly spaced clusters of high HES5-expressing neural progenitor cells along the dorsoventral axis. While Notch signalling requires direct membrane contact for its activation, we have previously shown mathematically that contact needs to extend beyond neighbouring cells for the HES5 pattern to emerge. However, the presence of cellular structures that could enable such long-distance signalling was unclear. Here, we report that cellular protrusions are present all along the apicobasal axis of individual neural progenitor cells. Through live imaging, we show that these protrusions dynamically extend and retract reaching lengths of up to [~]20m, enough to extend membrane contact beyond adjacent cells. The Notch ligand DLL1 was found to colocalise with protrusions, further supporting the idea that Notch signalling can be transduced at a distance. The effect of protrusions on the HES5 pattern was tested by reducing the density of protrusions using the CDC42 inhibitor ML141, leading to a tendency to decrease the distance between high HES5 cell clusters. However, this tendency was not significant and leaves an open question about their role in the fine-grained organisation of neurogenesis.

developmental biology↗

bioRxiv2024

NGN3 oscillatory expression controls the timing of human pancreatic endocrine differentiation

Understanding protein expression dynamics is crucial for the mechanistic understanding of cell differentiation. We investigate the dynamics of NGN3, a transcription factor critical for pancreatic endocrine development, including their function and decoding mechanisms. A knock-in endogenous reporter shows that the expression of NGN3 protein oscillates with a 13-hour periodicity in human iPS-derived endocrine progenitors and is switched off as cells differentiate to {beta}-like and -like cells. Increasing the stability of NGN3 protein results in one broad peak of expression instead of oscillations, with a larger peak to trough fold-change. This leads to precocious endocrine differentiation to both {beta}-like and -like cells and precocious expression of key NGN3 target genes. Single-cell analysis of dynamics, mathematical modelling and bioinformatics suggest that decoding of NGN3 oscillations occurs by fold-change detection via an incoherent feedforward motif that explains both normal and precocious differentiation. Our findings suggest that oscillatory NGN3 dynamics control the timing of differentiation, but not fate specification.

developmental biology↗

bioRxiv2023

Identification of genes with oscillatory expression in glioblastoma: The paradigm of SOX2

Quiescence, a reversible state of cell-cycle arrest, is an important state during both normal development and cancer progression. For example, in glioblastoma (GBM) quiescent glioblastoma stem cells (GSCs) play an important role in re-establishing the tumour, leading to relapse. While most studies have focused on identifying differentially expressed genes between proliferative and quiescent cells as potential drivers of this transition, recent studies have shown the importance of protein oscillations in controlling the exit from quiescence of neural stem cells. Here, we have undertaken a genome-wide bioinformatic inference approach to identify genes whose expression oscillates and which may be good candidates for controlling the transition to and from the quiescent cell state in GBM. Our analysis identified, among others, a list of important transcription regulators as potential oscillators, including the stemness gene SOX2, which we verified to oscillate in quiescent GSCs. These findings expand on the way we think about gene regulation and introduce new candidate genes as key regulators of quiescence.

cancer biology↗

bioRxiv2022

Cell coupling compensates for changes in single-cell Her6 dynamics and provides phenotypic robustness

her6 is a zebrafish ortholog of Hes1, known for its role in maintaining neural progenitors during neural development. Here, we characterise the population-level effect of altering Her6 protein expression dynamics at the single-cell level in the embryonic zebrafish telencephalon. Using an endogenous Her6:Venus reporter and 4D single-cell tracking, we show that Her6 oscillates in neural telencephalic progenitors and that fusion of a protein destabilisation domain (PEST) to Her6:Venus alters its expression dynamics causing most cells to downregulate Her6 prematurely. However, in PEST mutants, a higher proportion of cells exhibit Her6 oscillations and while expression is reduced in most cells, some cells express Her6 at wild-type levels resulting in increased heterogeneity of Her6 expression in the population. Despite the profound differences in the single-cell Her6 dynamics, differentiation markers do not exhibit major differences early on, while an increase in differentiation is observed at later developmental stages (vglut2a, gad1 and gad2). At the same time, at late stage the overall size of the telencephalon remains the same. Computational modelling that simulates changes in Her6 protein stability reveals that the increase in population Her6 expression heterogeneity is an emergent property of finely tuned Notch signalling coupling between single cells. Our study suggests that such cell coupling provides a compensation strategy whereby a normal phenotype is maintained while single-cell dynamics are abnormal, although the limit of this compensation is reached at late developmental stages. We conclude that in the neural progenitor population, cell coupling controls Her6 expression heterogeneity and in doing so, it provides phenotypic robustness when individual cells lose Her6 expression prematurely.

developmental biology↗

bioRxiv2021

Inferring kinetic parameters of oscillatory gene regulation from single cell time series data

Gene expression dynamics, such as stochastic oscillations and aperiodic fluctuations, have been associated with cell fate changes in multiple contexts, including development and cancer. Single cell live imaging of protein expression with endogenous reporters is widely used to observe such gene expression dynamics. However, the experimental investigation of regulatory mechanisms underlying the observed dynamics is challenging, since these mechanisms include complex interactions of multiple processes, including transcription, translation, and protein degradation. Here, we present a Bayesian method to infer kinetic parameters of oscillatory gene expression regulation using an auto-negative feedback motif with delay. Specifically, we use a delay-adapted nonlinear Kalman filter within a Metropolis-adjusted Langevin algorithm to identify posterior probability distributions. Our method can be applied to time series data on gene expression from single cells and is able to infer multiple parameters simultaneously. We apply it to published data on murine neural progenitor cells and show that it outperforms alternative methods. We further analyse how parameter uncertainty depends on the duration and time resolution of an imaging experiment, to make experimental design recommendations. This work demonstrates the utility of parameter inference on time course data from single cells and enables new studies on cell fate changes and population heterogeneity.

developmental biology↗

bioRxiv2021

HES1 protein oscillations are necessary for neural stem cells to exit from quiescence

Quiescence is a dynamic process of reversible cell-cycle arrest. High-level sustained expression of the HES1 transcriptional repressor, which oscillates with an ultradian periodicity in proliferative neural stem cells (NSCs), is thought to mediate quiescence. However, it is not known whether this is due to a change in levels or in dynamics. Here, we induce quiescence in NSCs with BMP4, which does not increase HES1 level, and we find that HES1 continues to oscillate. To assess the role of HES1 dynamics, we express sustained HES1 under a moderate-strength promoter, which overrides the endogenous oscillations while maintaining the total HES1 level within physiological range. We find that sustained HES1 does not affect proliferation or entry into quiescence, however, exit from quiescence is impeded. Thus, oscillatory expression of HES1 is specifically required for NSCs to exit quiescence, a finding of potential importance for controlling reactivation of stem cells in tissue regeneration and cancer.

cell biology↗

bioRxiv2021

Phase register of Hes1 oscillations with mitoses underlies cell-cycle heterogeneity in ER+ breast cancer cells.

Here, we study the dynamical expression of endogenously labelled Hes1, a transcriptional repressor implicated in controlling cell proliferation, to understand how cell-cycle length heterogeneity is generated in ER+ breast cancer cells. We find that Hes1 shows oscillatory expression with approximately 25h periodicity and during each cell-cycle has a variable peak in G1, a trough around G1-S transition and a less variable second peak in G2/M. Compared to other subpopulations, the cell-cycle in CD44HighCD24Low cancer stem cells is longest and most variable. Most cells divide around the peak of the Hes1 expression wave but preceding mitoses in slow dividing CD44HighCD24Low cells appear phase-shifted, resulting in a late-onset Hes1 peak in G1. The position, duration and shape of this peak, rather than the Hes1 expression levels, are good predictors of cell-cycle length. Diminishing Hes1 oscillations by enforcing sustained expression slows down the cell-cycle, impairs proliferation, abolishes the dynamic expression of p21, and increases the percentage of CD44HighCD24Low cells. Reciprocally, blocking the cell-cycle causes an elongation of Hes1 periodicity, suggesting a bidirectional interaction of the Hes1 oscillator and the cell-cycle. We propose that Hes1 oscillations are functionally important for the efficient progression of the cell-cycle and that the position of mitosis in relation to the Hes1 wave underlies cell-cycle length heterogeneity in cancer cell subpopulations. Significance statementTumours exhibit heterogeneities that are not due to mutations, including Cancer Stem Cells with different potencies. We show that the cancer stem cell state predisposed to dormancy in vivo has a highly variable and long cell-cycle. Using single-cell live-imaging for the transcriptional repressor Hes1 (a key molecule in cancer), we show a new type of circadian-like oscillatory expression of Hes1 in all cells in the population. The most potent cancer stem cells tend to divide around the trough of the Hes1 oscillatory wave, a feature predictive of a long cell-cycle. A novel concept proposed here is that the position of cell division with respect to the Hes1 wave is predictive of its prospective cell-cycle length and cancer cellular sub-state.

cell biology↗

bioRxiv2020

Sequential onset and concurrent expression of miR-9 genomic loci in single cells contributes to the temporal increase of mature miR-9 in zebrafish neurogenesis

MicroRNAs (miRs) have important quantitative roles in tuning dynamical gene expression. Hes/Her transcription factor dynamics are sensitive to the increasing amount of miR-9 in the cell, transitioning from noisy high-level expression to oscillatory expression and then to downregulation. However, the mechanism by which miR-9 is quantitatively controlled is not known. In vertebrates, several distinct genomic loci produce the same mature miR-9, but the functional significance of multiple primary transcripts remains unknown. Here, we show that the amount of mature miR-9 increases during zebrafish neurogenesis in a sharp stepwise manner. We characterize the spatiotemporal profile of 7 distinct pri-mir-9s and show that they are sequentially expressed during hindbrain neurogenesis. Quantitative analysis of expression at the single-cell level, shows that expression of late-onset pri-mir-9-1 is added on, rather than replacing the expression of early onset pri-mir-9-4 and 9-5. Mutating the late-onset pri-mir-9-1 with CRISPR/Cas9 prevents the developmental increase of mature miR-9 and reduces late neuronal differentiation. Finally, we use mathematical modelling to explore possible benefits of a stepwise increase of miR-9 over a linear increase. We find that an adaptive network containing Her6 can be insensitive to a linear increase in miR-9 and show that such adaptation can be overcome by step-wise increases of miR-9. In conclusion, our work suggests that a sharp stepwise increase of mature miR-9 is contributed by sequential temporal activation of distinct loci. This may be a strategy to overcome adaptation and facilitate a transition to a new state of Her6 dynamics or level.

developmental biology↗

bioRxiv2020

A dynamic and spatially periodic micro-pattern of HES5 expression underlies the probability of neuronal differentiation in the mouse spinal cord

Ultradian oscillations of HES Transcription Factors (TFs) at the single cell level, enable cell state transitions. However, the tissue level organisation of HES5 dynamics in neurogenesis is unknown. Here, we analyse the expression of HES5 ex-vivo in the developing mouse ventral spinal cord and identify microclusters of 4-6 cells with positively correlated HES5 level and ultradian dynamics. These microclusters are spatially periodic along the dorsoventral axis and temporally dynamic, alternating between high and low expression with a supra-ultradian persistence time. We show that Notch signaling is required for temporal dynamics but not the spatial periodicity of HES5. Few Neurogenin-2 cells are observed per cluster, irrespective of high or low state, suggesting that the microcluster organization of HES5 enables the stable selection of differentiating cells. Computational modelling predicts that different cell coupling strengths underlie the HES5 spatial patterns and rate of differentiation, which is consistent with comparison between the motoneuron and interneuron progenitor domains. Our work shows a previously unrecognised spatiotemporal organisation of neurogenesis, emergent at the tissue level from the synthesis of single cell dynamics. SynopsisLive imaging of HES5 expression in the ventral mouse spinal cord together with computational modelling is used to identify and analyse spatially periodic HES5 micro-patterns that emerge from the synthesis of single cell dynamics. O_LIHES5 is expressed in spatially periodic microclusters along the dorsal-ventral axis in spinal cord that are dynamically maintained by Notch signalling. C_LIO_LIMicroclusters can arise, in part, from single cell oscillators that are synchronous and weakly coupled via Notch. C_LIO_LISpatial patterns are different between motorneuron and interneuron progenitor domains and the probability for progenitor differentiation is regulated by the coupling strength between cells. C_LIO_LINGN2 is also spatially periodic along the dorso-ventral axis and microclusters of HES5 may act to pick a single NGN2 high cell for differentiation. C_LI

developmental biology↗

bioRxiv2020

Feedback motifs tuned by microRNAs in human transcriptional networks

Concepts from dynamical systems theory, including multi-stability, oscillations, robustness and stochasticity, are increasingly implicated in the control of gene expression during cell fate decisions, inflammation and stem cell heterogeneity. However, the prevalence of the underlying structures within gene networks which drive these dynamical behaviours, such as direct autoregulation or feedback by microRNAs, is unknown. We integrate transcription factor binding site (TFBS) and microRNA target data to generate a gene interaction network across 28 human tissues. This network was interrogated to identify network motifs capable of driving dynamical gene expression, in particular oscillations. Autoregulatory motifs were identified in 56% of transcription factors (TFs) investigated, 89% of which were also found in dual feedback motifs with a microRNA. Both the autoregulatory and dual feedback motifs were enriched in the network. TFs that autoregulate were found to be highly conserved between tissues. Dual feedback motifs with microRNAs were also conserved, but less so. Such dual feedback motifs were conserved between tissues, although TFs regulate different combinations of microRNAs in a tissue-dependent manner. TFs which autoregulate are prevalent among human TFs and have more interactions with microRNAs than non-autoregulatory genes. The enrichment of such motifs within the human transcriptional network indicates that more genes may have interesting expression dynamics than previously thought. These data provide a resource for the identification of TFs which regulate the dynamical properties of human gene expression. These findings support the development of dynamical conceptual frameworks for the study of fundamental biological processes.

bioinformatics↗

bioRxiv2019

miR-9 mediated noise optimization of the her6 oscillator is needed for cell state progression in the Zebrafish hindbrain

Ultradian oscillations of key transcription factors, such as members of the Hes family, are thought to be important in Neural Progenitor Cell (NPC) maintenance and miR-9 acts as a tuner of these oscillations in vitro. However, the existence and the role of such dynamic oscillatory expression in vivo is poorly understood. Here, we have generated a Zebrafish CRISPR knock-in Her6::venus fusion (Hes1 orthologue) to study endogenous dynamic gene expression in the embryonic hindbrain. We show that Her6 undergoes a transition from irregular, noisy, fluctuations to periodic oscillations as neurogenesis proceeds. In the absence of miR-9 input, noise in the Her6 oscillator increases and NPCs are unable to transit away from an intermediary state where they co-express progenitor and early differentiation markers. Thus, Her6 oscillations are facilitated by noise optimization mediated by miR-9 and this noise-tuning step is functionally important for cells to transition to differentiation.

developmental biology↗

bioRxiv2019

OscoNet: Inferring oscillatory gene networks

MotivationOscillatory genes, with periodic expression at the mRNA and/or protein level, have been shown to play a pivotal role in many biological contexts. However, with the exception of the circadian clock and cell cycle, only a few such genes are known. Detecting oscillatory genes from snapshot single-cell experiments is a challenging task due to the lack of time information. Oscope is a recently proposed method to identify co-oscillatory gene pairs using single-cell RNA-seq data. Although promising, the current implementation of Oscope does not provide a principled statistical criterion for selecting oscillatory genes.\n\nResultsWe improve the optimisation scheme underlying Oscope and provide a well-calibrated non-parametric hypothesis test to select oscillatory genes at a given FDR threshold. We evaluate performance on synthetic data and three real datasets and show that our approach is more sensitive than the original Oscope formulation, discovering larger sets of known oscillators while-avoiding the need for less interpretable thresholds. We also describe how our proposed pseudo-time estimation method is more accurate in recovering the true cell order for each gene cluster while-requiring substantially less computation time than the extended nearest insertion approach.\n\nConclusionOscoNet is a robust and versatile approach to detect oscillatory gene networks from snapshot single-cell data addressing many of the limitations of the original Oscope method.

bioinformatics↗

bioRxiv2018

Quantitative, real-time, single cell analysis in tissue reveals expression dynamics of neurogenesis

During embryogenesis cells make fate decisions within complex tissue environments. The levels and dynamics of transcription factor expression regulate these decisions. Here we use single cell live imaging of an endogenous HES5 reporter and absolute protein quantification to gain a dynamic view of neurogenesis in the embryonic mammalian spinal cord. We report that dividing neural progenitors show both aperiodic and periodic HES5 protein fluctuations. Mathematical modelling suggests that in progenitor cells the HES5 oscillator operates close to its bifurcation boundary where stochastic conversions between dynamics are possible. HES5 expression becomes more frequently periodic as cells transition to differentiation which, coupled with an overall decline in HES5 expression, creates a transient period of oscillations with higher fold expression change. This increases the decoding capacity of HES5 oscillations and correlates with interneuron versus motor neuron cell fate. Thus, HES5 undergoes complex changes in gene expression dynamics as cells differentiate.

developmental biology↗