Biology subjects

Rowntree, A.

Publications and source records attributed to Rowntree, A..

5 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↗

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↗

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↗