A Hybrid Model of Mammalian Cell Cycle Regulation

The timing of DNA synthesis, mitosis and cell division is regulated by a complex network of biochemical reactions that control the activities of a family of cyclin-dependent kinases. The temporal dynamics of this reaction network is typically modeled by nonlinear differential equations describing the rates of the component reactions. This approach provides exquisite details about molecular regulatory processes but is hampered by the need to estimate realistic values for the many kinetic constants that determine the reaction rates. It is difficult to estimate these kinetic constants from available experimental data. To avoid this problem, modelers often resort to ‘qualitative’ modeling strategies, such as Boolean switching networks, but these models describe only the coarsest features of cell cycle regulation. In this paper we describe a hybrid approach that combines the best features of continuous differential equations and discrete Boolean networks. Cyclin abundances are tracked by piecewise linear differential equations for cyclin synthesis and degradation. Cyclin synthesis is regulated by transcription factors whose activities are represented by discrete variables (0 or 1) and likewise for the activities of the ubiquitin-ligating enzyme complexes that govern cyclin degradation. The discrete variables change according to a predetermined sequence, with the times between transitions determined in part by cyclin accumulation and degradation and as well by exponentially distributed random variables. The model is evaluated in terms of flow cytometry measurements of cyclin proteins in asynchronous populations of human cell lines. The few kinetic constants in the model are easily estimated from the experimental data. Using this hybrid approach, modelers can quickly create quantitatively accurate, computational models of protein regulatory networks in cells

Telomeres in ICF syndrome cells are vulnerable to DNA damage due to elevated DNA:RNA hybrids.

DNA:RNA hybrids, nucleic acid structures with diverse physiological functions, can disrupt genome integrity when dysregulated. Human telomeres were shown to form hybrids with the lncRNA TERRA, yet the formation and distribution of these hybrids among telomeres, their regulation and their cellular effects remain elusive. Here we predict and confirm in several human cell types that DNA:RNA hybrids form at many subtelomeric and telomeric regions. We demonstrate that ICF syndrome cells, which exhibit short telomeres and elevated TERRA levels, are enriched for hybrids at telomeric regions throughout the cell cycle. Telomeric hybrids are associated with high levels of DNA damage at chromosome ends in ICF cells, which are significantly reduced with overexpression of RNase H1. Our findings suggest that abnormally high TERRA levels in ICF syndrome lead to accumulation of telomeric hybrids that, in turn, can result in telomeric dysfunction

Characterization of Sirt2 using conditional RNAi in mice

Within the past eight years, RNA interference (RNAi) has emerged as a powerful experimental tool for gene function analysis in mice. Reversible control of shRNA mediated RNAi has been achieved by using a tetracycline (tet)-inducible promoter. In the presence of the inductor doxycycline (dox), shRNA mediated gene silencing is initiated, whereas RNAi mechanism is blocked in the absence of dox. To achieve spatially and temporally regulated RNAi, the tet inducible system was combined with a Cre/loxP based strategy for tissue specific activation of shRNA constructs. To this end, a loxP-flanked "promoter inhibitory element" (PIE) was placed between the proximal (PSE) and distal sequence element (DSE) of a dox inducible promoter such that promoter function is completely blocked. Re-activation can be achieved through Cre mediated excision of PIE. To allow for gene silencing in a selected tissue, Cre expression can be regulated by a tissue-specific promoter. In mouse ES cells, the system mediated tight regulation of shRNA expression upon Cre mediated activation and dox administration, reaching knockdown efficiencies of >80%. Unexpectedly, the system showed a limited activity in transgenic mice when applied for conditional silencing of two different targets, LacZ and Sirt2. Sirt2 is a member of the sirtuin family which has considerably gained attention in vitro for its possible role in many physiological processes, including adipogenesis and neurodegenerative diseases. To investigate the function of Sirt2 in vivo, the unmodified dox-responsive and tet-inducible promoter was further used for conditional RNAi in transgenic mice. Inducible shRNA expression resulted in efficient silencing of Sirt2 (>90%) in all tissues which have been analyzed. Suppression of Sirt2 during embryogenesis resulted in offspring consisting of equal ratios of wild type and transgenic pups, indicating that Sirt2 is not indispensable for development. In adult animals, glucose metabolism, insulin sensitivity and energy balance appeared to be unaffected by Sirt2 deficiency. Likewise, expression of PPARγ, a downstream target of Sirt2, was not found to be altered upon Sirt2 inhibition. Finally, Sirt2 silencing was induced in an experimental model of Parkinson disease (PD). Data from Rotarod performances to study motor behaviour did not provide any evidence for a role of Sirt2 in PD pathogenesis as suggested by previous in vitro studies. Taken together, conditional Sirt2 silencing in vivo does not support speculation concerning a central role of Sirt2 in physiological processes, embryogenesis and in a mouse model of Parkinson disease

Multiscale modelling of cancer progression and treatment control : the role of intracellular heterogeneities in chemotherapy treatment

Cancer is a complex, multiscale process involving interactions at intracellular, intercellular and tissue scales that are in turn susceptible to microenvironmental changes. Each individual cancer cell within a cancer cell mass is unique, with its own internal cellular pathways and biochemical interactions. These interactions contribute to the functional changes at the cellular and tissue scale, creating a heterogenous cancer cell population. Anticancer drugs are effective in controlling cancer growth by inflicting damage to various target molecules and thereby triggering multiple cellular and intracellular pathways, leading to cell death or cell-cycle arrest. One of the major impediments in the chemotherapy treatment of cancer is drug resistance driven by multiple mechanisms, including multi-drug and cell-cycle mediated resistance to chemotherapy drugs. In this article, we discuss two hybrid multiscale modelling approaches, incorporating multiple interactions involved in the sub-cellular, cellular and microenvironmental levels to study the effects of cell-cycle, phase-specific chemotherapy on the growth and progression of cancer cells.PostprintPeer reviewe

Chloride channels regulate differentiation and barrier functions of the mammalian airway.

The conducting airway forms a protective mucosal barrier and is the primary target of airway disorders. The molecular events required for the formation and function of the airway mucosal barrier, as well as the mechanisms by which barrier dysfunction leads to early onset airway diseases, remain unclear. In this study, we systematically characterized the developmental landscape of the mouse airway using single-cell RNA sequencing and identified remarkably conserved cellular programs operating during human fetal development. We demonstrated that in mouse, genetic inactivation of chloride channel Ano1/Tmem16a compromises airway barrier function, results in early signs of inflammation, and alters the airway cellular landscape by depleting epithelial progenitors. Mouse Ano1-/-mutants exhibited mucus obstruction and abnormal mucociliary clearance that resemble the airway defects associated with cystic fibrosis. The data reveal critical and non-redundant roles for Ano1 in organogenesis, and show that chloride channels are essential for mammalian airway formation and function

Rif1 controls DNA replication by directing Protein Phosphatase 1 to reverse Cdc7-mediated phosphorylation of the MCM complex

Peer reviewedPublisher PD

TRIP13PCH-2 promotes Mad2 localization to unattached kinetochores in the spindle checkpoint response.

The spindle checkpoint acts during cell division to prevent aneuploidy, a hallmark of cancer. During checkpoint activation, Mad1 recruits Mad2 to kinetochores to generate a signal that delays anaphase onset. Yet, whether additional factors contribute to Mad2's kinetochore localization remains unclear. Here, we report that the conserved AAA+ ATPase TRIP13(PCH-2) localizes to unattached kinetochores and is required for spindle checkpoint activation in Caenorhabditis elegans. pch-2 mutants effectively localized Mad1 to unattached kinetochores, but Mad2 recruitment was significantly reduced. Furthermore, we show that the C. elegans orthologue of the Mad2 inhibitor p31(comet)(CMT-1) interacts with TRIP13(PCH-2) and is required for its localization to unattached kinetochores. These factors also genetically interact, as loss of p31(comet)(CMT-1) partially suppressed the requirement for TRIP13(PCH-2) in Mad2 localization and spindle checkpoint signaling. These data support a model in which the ability of TRIP13(PCH-2) to disassemble a p31(comet)/Mad2 complex, which has been well characterized in the context of checkpoint silencing, is also critical for spindle checkpoint activation