Governing Cell Fate Decisions Research Articles

Reverse hierarchical DED assembly in the cFLIP-procaspase-8 and cFLIP-procaspase-8-FADD complexes

cFLIP, a master anti-apoptotic regulator, targets the FADD-induced DED complexes of procaspase-8 in death receptor and ripoptosome signaling pathways. Several tumor cells maintain relatively high levels of cFLIP in achieving their immortality. However, understanding the three-dimensional regulatory mechanism initiated or mediated by elevated levels of cFLIP has been limited by the absence of the atomic coordinates for cFLIP-induced DED complexes. Here we report the crystal plus cryo-EM structures to uncover an unconventional mechanism where cFLIP and procaspase-8 autonomously form a binary tandem DED complex, independent of FADD. This complex gains the ability to recruit FADD, thereby allosterically modulating cFLIP assembly and partially activating caspase-8 for RIPK1 cleavage. Our structure-guided mutagenesis experiments provide critical insights into these regulatory mechanisms, elucidating the resistance to apoptosis and necroptosis in achieving immortality. Finally, this research offers a unified model for the intricate bidirectional hierarchy-based processes using multiprotein helical assembly to govern cell fate decisions.

Redox heterogeneity in mouse embryonic stem cells individualizes cell fate decisions

Pluripotent embryonic stem cells (ESCs) can develop into any cell type in the body. Yet, the regulatory mechanisms that govern cell fate decisions during embryogenesis remain largely unknown. We now demonstrate that mouse ESCs (mESCs) display large natural variations in mitochondrial reactive oxygen species (mitoROS) levels that individualize their nuclear redox state, H3K4me3 landscape, and cell fate. While mESCs with high mitoROS levels (mitoROSHIGH) differentiate toward mesendoderm and form the primitive streak during gastrulation, mESCs, which generate less ROS, choose the alternative neuroectodermal fate. Temporal studies demonstrated that mesendodermal (ME) specification of mitoROSHIGH mESCs is mediated by a Nrf2-controlled switch in the nuclear redox state, triggered by the accumulation of redox-sensitive H3K4me3 marks, and executed by a hitherto unknown ROS-dependent activation process of the Wnt signaling pathway. In summary, our study explains how ESC heterogeneity is generated and used by individual cells to decide between distinct cellular fates.

ScMaSigPro: Differential Expression Analysis along Single-Cell Trajectories.

Understanding the dynamics of gene expression across different cellular states is crucial for discerning the mechanisms underneath cellular differentiation. Genes that exhibit variation in mean expression as a function of Pseudotime and between branching trajectories are expected to govern cell fate decisions. We introduce scMaSigPro, a method for the identification of differential gene expression patterns along Pseudotime and branching paths simultaneously. We assessed the performance of scMaSigPro using synthetic and public datasets. Our evaluation shows that scMaSigPro outperforms existing methods in controlling the False Positive Rate and is computationally efficient. scMaSigPro is available as a free R package (version 4.0 or higher) under the GPL(≥2) license on GitHub at 'github.com/BioBam/scMaSigPro' and archived with version 0.03 on Zenodo at 'zenodo.org/records/12568922'.

Divergent Roles of MLL3 and MLL4 in Hematopoietic Stem Cell Fate Decision

Complex gene regulatory mechanisms, orchestrated by transcription factors (TFs) and epigenetic regulators, govern cell fate decisions in normal and malignant hematopoiesis. While prior works have extensively characterized the TF-networks in blood development, we currently have limited understanding of epigenetic regulation, in particular molecular rules underlying cofactor usage, in hematopoietic cell fate decisions. Kmt2c and Kmt2d are homologous genes that encode transcriptional coactivators, MLL3 and MLL4, respectively. They have been shown to coordinate embryonic stem cell fate transitions and are recurrently mutated in leukemias, offering an ideal paradigm to study epigenetic regulation of hematopoiesis. While MLL3 and MLL4 are often believed to play redundant roles in stem cell fate decisions, few has characterized their molecular distinctions. MLL3 and MLL4 each nucleate a chromatin-bound complex called the Complex of Proteins Associated with Set1 (COMPASS) and both deposit H3K4me1 at enhancers. Despite similar biochemical functions, Kmt2c and Kmt2d loss-of-function mutations have been reported with contrasting phenotypes in hematopoiesis. Kmt2d loss impairs HSC self-renewal and enhances myeloid differentiation at the expense of lymphopoiesis, whereas Kmt2c deletions enhance HSC self-renewal and impair differentiation. We hypothesize that MLL3 and MLL4 are recruited by different transcription factors and bind chromatin at both overlapping and distinct enhancers to convey specific hematopoietic fates. To test whether MLL3 and MLL4 are redundant in licensing myeloid differentiation, we transplanted wildtype, Kmt2c/d single knockout, and Kmt2c/d double knockout (DKO) donor cells into lethally irradiated recipients. Surprisingly, DKO recipients showed significant expansion of phenotypic HSCs, MPPs, and pre-granulocyte-monocyte progenitors (pGMs), typically associated with a myeloid differentiation block. DKO mice progressively developed bone marrow failure 9-months after transplantation, indicating that MLL3/4-COMPASS is necessary to sustain hematopoiesis. To dissect how Kmt2c/d deletions shape lineage specification and whether DKO imposes a differentiation block, we performed Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) on donor Lin -Kit +Sca1 + (LSK) cells from the transplantation described above. While Kmt2c deletion does not markedly reprogram HSC/MPP identity, Kmt2d deletion results in precocious myeloid differentiation. Most notably, we observed that compound Kmt2c/d deletions fundamentally reprogramed HSCs/MPPs toward a B-lymphoid-like state, instead of imposing a myeloid differentiation block. The data establish a model wherein MLL3 and MLL4 have redundant roles in licensing HSC identity and myeloid potential. However, within the HSC to myeloid differentiation trajectory, MLL3 promotes differentiation while MLL4 promotes self-renewal. To understand how MLL3- and MLL4-COMPASS differentially regulate enhancer accessibility, we performed single-cell transposase-accessible chromatin sequencing (scATAC-seq) on the same donor LSK cells as in the CITE-seq. Compound Kmt2c/d deletions impose a unique epigenomic state on HSCs/MPPs (Figure 1A). Motif enrichment analyses revealed that DKO cells (i) were enriched for PAX5 and EBF1 motifs, consistent with B-lymphoid priming. HSCs (ii) were enriched for AP-1 and NFE2L2 motifs, suggesting that MLL4 may preferentially bind AP-1 and antioxidant response elements associated with HSC identity. Clusters of myeloid progenitors (iii and iv) were populated primarily by Kmt2d-deficient cells and enriched for CEBP and SPI1 motifs, suggesting that MLL3 is preferentially recruited to enhancers bound by myeloid TFs. This study provides several novel insights into epigenetic regulation of hematopoiesis. We have shown that HSC identity and myeloid fate specification are licensed by redundant MLL3/4-COMPASS-dependent TF-cofactor interactions, but once HSCs are established, MLL3- and MLL4-COMPASS engage distinct, antagonistic programs. Furthermore, early B-lymphoid priming is mediated via MLL3/4-COMPASS-independent mechanisms (Figure 1B). Ongoing work will link precise transcriptional codes to divergent cofactor utilization patterns and provide critical insights into how epigenetic regulators are deployed to effect distinct cell fates.

Quadra-Stable Dynamics of p53 and PTEN in the DNA Damage Response

Cell fate determination is a complex process that is frequently described as cells traveling on rugged pathways, beginning with DNA damage response (DDR). Tumor protein p53 (p53) and phosphatase and tensin homolog (PTEN) are two critical players in this process. Although both of these proteins are known to be key cell fate regulators, the exact mechanism by which they collaborate in the DDR remains unknown. Thus, we propose a dynamic Boolean network. Our model incorporates experimental data obtained from NSCLC cells and is the first of its kind. Our network's wild-type system shows that DDR activates the G2/M checkpoint, and this triggers a cascade of events, involving p53 and PTEN, that ultimately lead to the four potential phenotypes: cell cycle arrest, senescence, autophagy, and apoptosis (quadra-stable dynamics). The network predictions correspond with the gain-and-loss of function investigations in the additional two cell lines (HeLa and MCF-7). Our findings imply that p53 and PTEN act as molecular switches that activate or deactivate specific pathways to govern cell fate decisions. Thus, our network facilitates the direct investigation of quadruplicate cell fate decisions in DDR. Therefore, we concluded that concurrently controlling PTEN and p53 dynamics may be a viable strategy for enhancing clinical outcomes.

2001 – THE ENDOSOMAL ADAPTOR PROTEIN MYCT1 CONTROLS ENVIRONMENTAL SENSING IN HUMAN HEMATOPOIETIC STEM CELLS

Hematopoietic stem cells (HSC) integrate diverse environmental cues to balance between quiescence, self-renewal and differentiation. However, the molecular programs governing HSC stemness become dysregulated during culture, compromising HSC self-renewal and engraftment ability. Despite recent advances, our ability to expand functional human HSCs in culture for therapeutic use is still limited. We uncovered MYCT1 as a novel regulator of human HSCs that becomes drastically downregulated during culture, concomitantly with loss of engraftment potential. We discovered that MYCT1 is essential for human HSPC ex vivo expansion and engraftment upon transplantation. Immunoprecipitation-mass spectrometry revealed that MYCT1 is a membrane-associated endosomal protein that interacts with signaling receptors with critical functions in HSC biology. MYCT1 knockdown (KD) in human ECs and HSPCs led to hyperactivation of endocytosis, a crucial regulatory step determining the responsiveness to extracellular cues and governing cell fate decisions. Phospho-proteomics, single-cell RNAseq, and functional assays revealed that MYCT1 KD causes widespread dysregulation of signaling pathways, including hypersensitivity to the cytokines in the media, loss of stemness programs, and defective proliferation. Importantly, overexpression of MYCT1 in human HSPCs rescued endocytosis, signaling, and transcriptomic programs that were dysregulated in cultured human HSPCs and further disrupted by MYCT1 KD. These data suggest that MYCT1 governs human HSC stemness by moderating environmental sensing through the control of endocytosis. As MYCT1 expression is downregulated in cultured HSPCs, the inability to properly sense microenvironmental signals may be a key mechanism contributing to culture-associated HSC dysfunction that will need to be overcome to restore transplantability of ex vivo expanded human HSCs. Hematopoietic stem cells (HSC) integrate diverse environmental cues to balance between quiescence, self-renewal and differentiation. However, the molecular programs governing HSC stemness become dysregulated during culture, compromising HSC self-renewal and engraftment ability. Despite recent advances, our ability to expand functional human HSCs in culture for therapeutic use is still limited. We uncovered MYCT1 as a novel regulator of human HSCs that becomes drastically downregulated during culture, concomitantly with loss of engraftment potential. We discovered that MYCT1 is essential for human HSPC ex vivo expansion and engraftment upon transplantation. Immunoprecipitation-mass spectrometry revealed that MYCT1 is a membrane-associated endosomal protein that interacts with signaling receptors with critical functions in HSC biology. MYCT1 knockdown (KD) in human ECs and HSPCs led to hyperactivation of endocytosis, a crucial regulatory step determining the responsiveness to extracellular cues and governing cell fate decisions. Phospho-proteomics, single-cell RNAseq, and functional assays revealed that MYCT1 KD causes widespread dysregulation of signaling pathways, including hypersensitivity to the cytokines in the media, loss of stemness programs, and defective proliferation. Importantly, overexpression of MYCT1 in human HSPCs rescued endocytosis, signaling, and transcriptomic programs that were dysregulated in cultured human HSPCs and further disrupted by MYCT1 KD. These data suggest that MYCT1 governs human HSC stemness by moderating environmental sensing through the control of endocytosis. As MYCT1 expression is downregulated in cultured HSPCs, the inability to properly sense microenvironmental signals may be a key mechanism contributing to culture-associated HSC dysfunction that will need to be overcome to restore transplantability of ex vivo expanded human HSCs.

Single-cell transcriptome analysis defines mesenchymal stromal cells in the mouse incisor dental pulp

The dental pulp is known to be highly heterogenous, comprising distinct cell types including mesenchymal stromal cells (MSCs), which represent neural-crest-derived cells with the ability to differentiate into multiple cell lineages. However, the cellular heterogeneity and the transcriptome signature of different cell clusters within the dental pulp remain to be established. To better understand discrete cell types, we applied a single-cell RNA sequencing strategy to establish the RNA expression profiles of individual dental pulp cells from 5- to 6-day-old mouse incisors. Our study revealed distinct subclasses of cells representing osteoblast, odontoblast, endothelial, pancreatic, neuronal, immune, pericyte and ameloblast lineages. Collectively, our research demonstrates the complexity and diversity of cell subclasses within the incisor dental pulp, thus providing a foundation for uncovering the molecular processes that govern cell fate decisions and lineage commitment in dental pulp-derived MSCs.

TET-Mediated Epigenetic Regulation in Immune Cell Development and Disease.

TET proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidation products in DNA. The oxidized methylcytosines (oxi-mCs) facilitate DNA demethylation and are also novel epigenetic marks. TET loss-of-function is strongly associated with cancer; TET2 loss-of-function mutations are frequently observed in hematological malignancies that are resistant to conventional therapies. Importantly, TET proteins govern cell fate decisions during development of various cell types by activating a cell-specific gene expression program. In this review, we seek to provide a conceptual framework of the mechanisms that fine tune TET activity. Then, we specifically focus on the multifaceted roles of TET proteins in regulating gene expression in immune cell development, function, and disease.

Deciphering the multifaceted roles of TET proteins in T‐cell lineage specification and malignant transformation

TET proteins are DNA demethylases that can oxidize 5-methylcytosine (5mC) to generate 5-hydroxymethylcytosine (5hmC) and other oxidized mC bases (oxi-mCs). Importantly, TET proteins govern cell fate decisions during development of various cell types by activating a cell-specific gene expression program. In this review, we focus on the role of TET proteins in T-cell lineage specification. We explore the multifaceted roles of TET proteins in regulating gene expression in the contexts of T-cell development, lineage specification, function, and disease. Finally, we discuss the future directions and experimental strategies required to decipher the precise mechanisms employed by TET proteins to fine-tune gene expression and safeguard cell identity.

RIP1-dependent linear and nonlinear recruitments of caspase-8 and RIP3 respectively to necrosome specify distinct cell death outcomes

There remains a significant gap in our quantitative understanding of crosstalk between apoptosis and necroptosis pathways. By employing the SWATH-MS technique, we quantified absolute amounts of up to thousands of proteins in dynamic assembling/de-assembling of TNF signaling complexes. Combining SWATH-MS-based network modeling and experimental validation, we found that when RIP1 level is below ~1000 molecules/cell (mpc), the cell solely undergoes TRADD-dependent apoptosis. When RIP1 is above ~1000 mpc, pro-caspase-8 and RIP3 are recruited to necrosome respectively with linear and nonlinear dependence on RIP1 amount, which well explains the co-occurrence of apoptosis and necroptosis and the paradoxical observations that RIP1 is required for necroptosis but its increase down-regulates necroptosis. Higher amount of RIP1 (>~46,000 mpc) suppresses apoptosis, leading to necroptosis alone. The relation between RIP1 level and occurrence of necroptosis or total cell death is biphasic. Our study provides a resource for encoding the complexity of TNF signaling and a quantitative picture how distinct dynamic interplay among proteins function as basis sets in signaling complexes, enabling RIP1 to play diverse roles in governing cell fate decisions.

The Impact of Notch Signaling for Carcinogenesis and Progression of Nonmelanoma Skin Cancer: Lessons Learned from Cancer Stem Cells, Tumor Angiogenesis, and Beyond.

Since many decades, nonmelanoma skin cancer (NMSCs) is the most common malignancy worldwide. Basal cell carcinomas (BCC) and squamous cell carcinomas (SCC) are the major types of NMSCs, representing approximately 70% and 25% of these neoplasias, respectively. Because of their continuously rising incidence rates, NMSCs represent a constantly increasing global challenge for healthcare, although they are in most cases nonlethal and curable (e.g., by surgery). While at present, carcinogenesis of NMSC is still not fully understood, the relevance of genetic and molecular alterations in several pathways, including evolutionary highly conserved Notch signaling, has now been shown convincingly. The Notch pathway, which was first developed during evolution in metazoans and that was first discovered in fruit flies (Drosophila melanogaster), governs cell fate decisions and many other fundamental processes that are of high relevance not only for embryonic development, but also for initiation, promotion, and progression of cancer. Choosing NMSC as a model, we give in this review a brief overview on the interaction of Notch signaling with important oncogenic and tumor suppressor pathways and on its role for several hallmarks of carcinogenesis and cancer progression, including the regulation of cancer stem cells, tumor angiogenesis, and senescence.

Tracking leukemic T-cell transcriptional dynamics in vivo with a blood-based reporter assay.

Transcriptional dynamics of cancer cells govern cell fate decisions and are therapeutically actionable drug targets. In this study, we engineered a circulating cancer cell line that secretes a luciferase reporter to capture constitutive and circadian clock-driven transcription dynamics over the course of a day. Engineered human leukemic T cells (Jurkat) were observed to rhythmically secrete luciferase in a continuous flow cell culture system. When transplanted invivo, engineered leukemic cells caused circadian plasma luciferase activity and had expected pathological signs of leukemic disease. This technique is rapid and noninvasive, requiring only a few microliters of media or blood, and can aid in investigating relationships between invivo cancer cell signaling and behavior, such as diet or sleep.

Desmoglein-3 acts as a pro-survival protein by suppressing reactive oxygen species and doming whilst augmenting the tight junctions in MDCK cells

Kidney disease prevalence increases with age, with a common feature of the disease being defects in the epithelial tight junctions. Emerging evidence suggests that the desmosomal adhesion protein Desmoglein-3 (Dsg3) functions beyond the desmosomal adhesion and plays a role in regulating the fundamental pathways that govern cell fate decisions in response to environmental chemical and mechanical stresses. In this study, we explored the role of Dsg3 on dome formation, reactive oxygen species (ROS) production and transepithelial electrical resistance (TER) in MDCK cells, a kidney epithelial cell model widely used to study cell differentiation and tight junction formation and integrity. We show that overexpression of Dsg3 constrained nuclear ROS production and cellular doming in confluent cell cultures and these features coincided with augmented TER and enhanced tight junction integrity. Conversely, cells expressing dominant-negative Dsg3ΔC mutants exhibited heightened ROS production and accelerated doming, accompanied by increased apoptosis, as well as cell proliferation, with massive disruption in F-actin organization and accumulation, and alterations in tight junctions. Inhibition of actin polymerization and protein synthesis was able to sufficiently block dome formation in mutant populations. Taken together, these findings underscore that Dsg3 has a role in controlling cellular viability and differentiation as well as the functional integrity of tight junctions in MDCK cells.

Quantifying the interplay between genetic and epigenetic regulations in stem cell development

Waddington epigenetic landscape, as a classic metaphor, has been used to explain cellular development and differentiation. However, it remains challenging to quantify the epigenetic landscape. Especially, a key issue arises as what are the underlying mechanisms for the interplay between genetic and epigenetic regulations to govern cell fate decisions in development. Based on a developmental epigenetic model combining histone modifications and gene regulations, we studied state switching mechanisms of histone modifications for stem cell development, and uncovered corresponding epigenetic landscape. The topography of landscape provides a quantitative measure for the relative stability of different attractors or phenotypes. We showed that histone regulations facilitate the occurrence of intermediate states or multistability. From the epigenetic landscape of stem cell differentiation, we identified key cellular states characterized by attractors, including pluripotent stem cell state, differentiated state and intermediate states. We also quantified representative kinetic transition paths for differentiation, reprogramming and transdifferentiation, which agree well with previous experimental observations. Specifically, previous experiments indicate that transdifferentiation can go through a mixed, unspecific intermediate or progenitor-like state. By calculating the kinetic transition paths, our developmental epigenetic models are able to replicate all these three experimental results, and therefore provide theoretical explanations for these experimental observations. We propose that epigenetic regulations play critical roles on the kinetic transitions for differentiation, reprogramming and transdifferentiation, which also provide a source for the heterogeneity of gene expressions observed in developmental process. Our work provides new insights into the roles of epigenetic modifications on controlling gene expression and stem cell differentiation, and facilitates our mechanistic understanding for the cell fate determinations regarding the interplay between genetic and epigenetic regulations.

Local Auxin Biosynthesis Is a Key Regulator of Plant Development

Auxin is a major phytohormone that controls numerous aspects of plant development and coordinates plant responses to the environment. Morphogenic gradients of auxin govern cell fate decisions and underlie plant phenotypic plasticity. Polar auxin transport plays a central role in auxin maxima generation. The discovery of the exquisite spatiotemporal expression patterns of auxin biosynthesis genes of the WEI8/TAR and YUC families suggested that local auxin production may contribute to the formation of auxin maxima. Herein, we systematically addressed the role of local auxin biosynthesis in plant development and responses to the stress phytohormone ethylene by manipulating spatiotemporal patterns of WEI8. Our study revealed that local auxin biosynthesis and transport act synergistically and are individually dispensable for root meristem maintenance. In contrast, flower fertility and root responses to ethylene require local auxin production that cannot be fully compensated for by transport in the generation of morphogenic auxin maxima.

Design of Large-Scale Reporter Construct Arrays for Dynamic, Live Cell Systems Biology.

Dynamic systems biology aims to identify the molecular mechanisms governing cell fate decisions through the analysis of living cells. Large scale molecular information from living cells can be obtained from reporter constructs that provide activities for either individual transcription factors or multiple factors binding to the full promoter following CRISPR/Cas9 directed insertion of luciferase. In this report, we investigated the design criteria to obtain reporters that are specific and responsive to transcription factor (TF) binding and the integration of TF binding activity with genetic reporter activity. The design of TF reporters was investigated for the impact of consensus binding site spacing sequence and off-target binding on the reporter sensitivity using a library of 25 SMAD3 activity reporters with spacers of random composition and length. A spacer was necessary to quantify activity changes after TGFβ stimulation. TF binding site prediction algorithms (BEEML, FIMO and DeepBind) were used to predict off-target binding, and nonresponsiveness to a SMAD3 reporter was correlated with a predicted competitive binding of constitutively active p53. The network of activity of the SMAD3 reporter was inferred from measurements of TF reporter library, and connected with large-scale genetic reporter activity measurements. The integration of TF and genetic reporters identified the major hubs directing responses to TGFβ, and this method provided a systems-level algorithm to investigate cell signaling.

Abstract A177: Role of promoter methylation in regulation of Notch4 and JAG2 gene expression in human glioblastoma

Abstract In cancer, DNA methylation causes alteration in gene expression affecting important signal transduction pathways. Since methylation occurs at a very early stage and is reversible, hypermethylated promoters hold great promise as biomarkers for early detection and effective drug targets for gene reactivation. The Notch signaling pathway is one such important developmental pathway governing cell fate decisions. Dysregulated Notch signaling is found to have a prominent role in the development of various cancers. Glioblastoma (GBM) is the most common primary brain tumor, with a very poor prognosis despite advanced treatment facilities due to poor molecular stratification. It is therefore important to study genetic and epigenetic events leading to gliomagenesis and to guide new treatment strategies. The aim of this study was to detect Notch pathway genes potentially regulated by promoter methylation in human GBM. We used real-time PCR and quantitative methylation-specific PCR to study methylation status of underexpressed Notch pathway genes from human GBM formalin-fixed, paraffin-embedded sections. 50% of samples showed methylation at Notch4 gene promoter at levels higher than 25 % (P < 0.05). However, JAG2 gene promoter was estimated to be methylated at a negligible level (<2 %) (P < 0.05). Our study showed poor gene expression and high-level methylation of Notch4 gene, suggesting a possibility of epigenetic silencing. However, low JAG2 gene expression despite not being highly methylated suggests lack of correlation between gene expression and promoter methylation and the presence of alternative regulatory mechanisms. This study for the first time provides gene expression and DNA methylation profiles of Notch pathway genes from GBM patient samples. We have identified genes whose expression may be regulated by epigenetic mechanisms and thus can be used as potential biomarkers. This study will eventually give clues to improve diagnosis, prognosis, and treatment of GBM. Citation Format: Madhuri G S Aithal, Rajeswari Narayanappa. Role of promoter methylation in regulation of Notch4 and JAG2 gene expression in human glioblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A177.

Cell reprogramming modelled as transitions in a hierarchy of cell cycles

We construct a model of cell reprogramming (the conversion of fully differentiated cells to a state of pluripotency, known as induced pluripotent stem cells, or iPSCs) which builds on key elements of cell biology viz. cell cycles and cell lineages. Although reprogramming has been demonstrated experimentally, much of the underlying processes governing cell fate decisions remain unknown. This work aims to bridge this gap by modelling cell types as a set of hierarchically related dynamical attractors representing cell cycles. Stages of the cell cycle are characterised by the configuration of gene expression levels, and reprogramming corresponds to triggering transitions between such configurations. Two mechanisms were found for reprogramming in a two level hierarchy: cycle specific perturbations and a noise induced switching. The former corresponds to a directed perturbation that induces a transition into a cycle-state of a different cell type in the potency hierarchy (mainly a stem cell) whilst the latter is a priori undirected and could be induced, e.g. by a (stochastic) change in the cellular environment. These reprogramming protocols were found to be effective in large regimes of the parameter space and make specific predictions concerning reprogramming dynamics which are broadly in line with experimental findings.

MTOR Controls Mitochondrial Dynamics and Cell Survival via MTFP1

The mechanisms that link environmental and intracellular stimuli to mitochondrial functions, including fission/fusion, ATP production, metabolite biogenesis, and apoptosis, are not well understood. Here, we demonstrate that the nutrient-sensing mechanistic/mammalian target of rapamycin complex 1 (mTORC1) stimulates translation of mitochondrial fission process 1 (MTFP1) to control mitochondrial fission and apoptosis. Expression of MTFP1 is coupled to pro-fission phosphorylation and mitochondrial recruitment of the fission GTPase dynamin-related protein 1 (DRP1). Potent active-site mTOR inhibitors engender mitochondrial hyperfusion due to the diminished translation of MTFP1, which is mediated by translation initiation factor 4E (eIF4E)-binding proteins (4E-BPs). Uncoupling MTFP1 levels from the mTORC1/4E-BP pathway upon mTOR inhibition blocks the hyperfusion response and leads to apoptosis by converting mTOR inhibitor action from cytostatic to cytotoxic. These data provide direct evidence for cell survival upon mTOR inhibition through mitochondrial hyperfusion employing MTFP1 as a critical effector of mTORC1 to govern cell fate decisions.

Reversed graph embedding resolves complex single-cell trajectories.

Single-cell trajectories can unveil how gene regulation governs cell fate decisions. However, learning the structure of complex trajectories with two or more branches remains a challenging computational problem. We present Monocle 2, which uses reversed graph embedding to describe multiple fate decisions in a fully unsupervised manner. Applied to two studies of blood development, Monocle 2 revealed that mutations in key lineage transcription factors diverts cells to alternative fates.