Cell Cycle Exit Research Articles

The synaptosome-associated protein 23 (SNAP23) is necessary for proper myogenesis.

Vesicle-mediated transport is necessary for maintaining cellular homeostasis and proper signaling. The synaptosome-associated protein23 (SNAP23) is a member of the SNARE protein family and mediates the vesicle docking and membrane fusion steps of secretion during exocytosis. Skeletal muscle has been established as a secretory organ; however, the role of SNAP23 in the context of skeletal muscle development is still unknown. Here, we show that depletion of SNAP23 in C2C12 mouse myoblasts reduces their ability to differentiate into myotubes as a result of premature cell cycle exit and early activation of the myogenic transcriptional program. This effect is rescued when cells are seeded at a high density or when cultured in conditioned medium from wild type cells. Proteomic analysis of collected medium indicates that SNAP23 depletion leads to a misregulation of exocytosis, including decreased secretion of the insulin-like growth factor 1 (IGF1), a critical protein for muscle growth, development, and function. We further demonstrate that treatment of SNAP23-depleted cells with exogenous IGF1 rescues their myogenic capacity. We propose that SNAP23 mediates the secretion of specific proteins, such as IGF1, that are important for achieving proper differentiation of skeletal muscle cells during myogenesis. This work highlights the underappreciated role of skeletal muscle as a secretory organ and contributes to the understanding of factors necessary for myogenesis.

P97/VCP ATPase inhibitors can rescue p97 mutation-linked motor neuron degeneration.

Mutations in p97/VCP cause two motor neuron diseases: inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia and familial amyotrophic lateral sclerosis. How p97 mutations lead to motor neuron degeneration is, however, unknown. Here we used patient-derived induced pluripotent stem cells to generate p97 mutant motor neurons. We reduced the genetic background variation by comparing mutant motor neurons to its isogenic wild type lines. Proteomic analysis reveals that p97R155H/+ motor neurons upregulate several cell cycle proteins at Day 14, but this effect diminishes by Day 20. Molecular changes linked to delayed cell cycle exit are observed in p97 mutant motor neurons. We also find that two p97 inhibitors, CB-5083 and NMS-873, restore some dysregulated protein levels. In addition, two p97 inhibitors and a food and drug administration-approved cyclin-dependent kinase 4/6 inhibitor, Abemaciclib, can rescue motor neuron death. Overall, we successfully used iPSC-derived motor neurons, identified dysregulated proteome and transcriptome and showed that p97 inhibitors rescue phenotypes in this disease model.

Dissecting mechanisms of chamber-specific cardiac differentiation and its perturbation following retinoic acid exposure.

The specification of distinct cardiac lineages occurs before chamber formation and acquisition of bona fide atrial or ventricular identity. However, the mechanisms underlying these early specification events remain poorly understood. Here, we performed single cell analysis at the murine cardiac crescent, primitive heart tube and heart tube stages to uncover the transcriptional mechanisms underlying formation of atrial and ventricular cells. We find that progression towards differentiated cardiomyocytes occurs primarily based on heart field progenitor identity, and that progenitors contribute to ventricular or atrial identity through distinct differentiation mechanisms. We identify new candidate markers that define such differentiation processes and examine their expression dynamics using computational lineage trajectory methods. We further show that exposure to exogenous retinoic acid causes defects in ventricular chamber size, dysregulation in FGF signaling and a shunt in differentiation towards orthogonal lineages. Retinoic acid also causes defects in cell-cycle exit resulting in formation of hypomorphic ventricles. Collectively, our data identify, at a single cell level, distinct lineage trajectories during cardiac specification and differentiation, and the precise effects of manipulating cardiac progenitor patterning via retinoic acid signaling.

Transcription factor-driven coordination of cell cycle exit and lineage-specification in vivo during granulocytic differentiation

Differentiation of multipotent stem cells into mature cells is fundamental for development and homeostasis of mammalian tissues, and requires the coordinated induction of lineage-specific transcriptional programs and cell cycle withdrawal. To understand the underlying regulatory mechanisms of this fundamental process, we investigated how the tissue-specific transcription factors, CEBPA and CEBPE, coordinate cell cycle exit and lineage-specification in vivo during granulocytic differentiation. We demonstrate that CEBPA promotes lineage-specification by launching an enhancer-primed differentiation program and direct activation of CEBPE expression. Subsequently, CEBPE confers promoter-driven cell cycle exit by sequential repression of MYC target gene expression at the G1/S transition and E2F-meditated G2/M gene expression, as well as by the up-regulation of Cdk1/2/4 inhibitors. Following cell cycle exit, CEBPE unleashes the CEBPA-primed differentiation program to generate mature granulocytes. These findings highlight how tissue-specific transcription factors coordinate cell cycle exit with differentiation through the use of distinct gene regulatory elements.

Proteolytic control in ciliogenesis: Temporal restriction or early initiation?

Cellular processes are highly dependent on a dynamic proteome that undergoes structural and functional rearrangements to allow swift conversion between different cellular states. By inducing proteasomal degradation of inhibitory or stimulating factors, ubiquitylation is particularly well suited to trigger such transitions. One prominent example is the remodelling of the centrosome upon cell cycle exit, which is required for the formation of primary cilia - antenna-like structures on the surface of most cells that act as integrative hubs for various extracellular signals. Over the last decade, many reports on ubiquitin-related events involved in the regulation of ciliogenesis have emerged. Very often, these processes are considered to be initiated ad hoc, that is, directly before its effect on cilia biogenesis becomes evident. While such a temporal restriction may hold true for the majority of events, there is evidence that some of them are initiated earlier during the cell cycle. Here, we provide an overview of ubiquitin-dependent processes in ciliogenesis and discuss available data that indicate such an early onset of proteolytic regulation within preceding cell cycle stages.

Glucocorticoid receptor antagonization propels endogenous cardiomyocyte proliferation and cardiac regeneration.

In mammals, the physiological activation of the glucocorticoid receptor (GR) by glucocorticoids (GCs) promotes the maturation of cardiomyocytes during late gestation, but the effect on postnatal cardiac growth and regenerative plasticity is unclear. Here we demonstrate that the GC-GR axis restrains cardiomyocyte proliferation during postnatal development. Cardiomyocyte-specific GR ablation in conditional knockout (cKO) mice delayed the postnatal cardiomyocyte cell cycle exit, hypertrophic growth and cytoarchitectural maturation. GR-cKO hearts showed increased expression of genes involved in glucose catabolism and reduced expression of genes promoting fatty acid oxidation and mitochondrial respiration. Accordingly, oxygen consumption in GR-cKO cardiomyocytes was less dependent on fatty acid oxidation, and glycolysis inhibition reverted GR-cKO effects on cardiomyocyte proliferation. GR ablation or transient pharmacological inhibition after myocardial infarction in juvenile and/or adult mice facilitated cardiomyocyte survival, cell cycle re-entry and division, leading to cardiac muscle regeneration along with reduced scar formation. Thus, GR restrains heart regeneration and may represent a therapeutic target.

CEND1 deficiency induces mitochondrial dysfunction and cognitive impairment in Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of neurodegenerative disease featured with memory loss and cognitive function impairments. Chronic mitochondrial stress is a vital pathogenic factor for AD and finally leads to massive neuronal death. However, the underlying mechanism is unclear. By proteomic analysis, we identified a new mitochondrial protein, cell-cycle exit and neuronal differentiation 1 (CEND1), which was decreased significantly in the brain of 5xFAD mice. CEND1 is a neuronal specific protein and locates in the presynaptic mitochondria. Depletion of CEND1 leads to increased mitochondrial fission mediated by upregulation of dynamin related protein 1 (Drp1), resulting in abnormal mitochondrial functions. CEND1 deficiency leads to cognitive impairments in mice. Overexpression of CEND1 in the hippocampus of 5xFAD mice rescued cognitive deficits. Moreover, we identified that CDK5/p25 interacted with and phosphorylated CEND1 which promoted its degradation. Our study provides new mechanistic insights in mitochondrial function regulations by CEND1 in Alzheimer's disease.

Abstract 905: Targeting muscarinic acetylcholine receptors in glioma stem like cells

Abstract Primary gliomas arising within the brain remain the deadliest form of brain cancer and account for 78% of all malignant brain tumors. Glioblastoma patients have a 5 percent five-year survival rate and drug-resistant tumors often recur following surgical resection and treatment with radiation or chemotherapy. The cancer stem cell hypothesis suggests the presence of a subset of undifferentiated cells, namely glioma stem cells (GSCs), in the heterogenous tumor mass that are likely responsible for tumor initiation and recurrence of tumors post resection and give rise to drug resistant tumors. Determining a way to suppress these malignant characteristics and/or depleting the GSC population could improve current cancer treatments and the survival of glioma patients. Some GSCs are similar to oligodendrocyte precursor cells (OPCs), found during neural development and also residing in the adult brain. OPCs are prone to malignant transformation and believed to be a cellular origin for glioma. Recent findings indicate that the well-characterized neurotransmitter acetylcholine (ACh) maintains the primitive state of normal OPCs via muscarinic ACh receptors (mAChRs) preventing maturation and cell cycle exit. We investigated the functional characterization of ACh and mAChRs in modulating malignancy in OPC-like cells. We studied cultures of primary mouse OPC-like GSCs. We used the primary cells to culture three-dimensional tumor organoids in vitro to better represent tumor heterogeneity. We also used patient derived xenografts (PDX) of glioma to grow flank xenografts in NSG mice for an in vivo model. Publicly available data and studies in our lab show high levels of expression of CHRM3 (M3mAChR) in glioma patients and in primary OPC like GSCs. A drug screen conducted in the context of multiple sclerosis determined that benztropine (BZT), an anti muscarinic drug targeting M3mAChR, causes normal OPCs to exit the cell cycle, lose stem cell like characteristics, and differentiate. In our lab, electrophysiological studies demonstrated that activation of mAChRs in OPC-like cells from mouse and patient tumors generates rapid (< 1 second) increases in cytosolic calcium. Pharmacologic studies indicated that treatment with FDA approved anti muscarinic benztropine suppresses proliferation in cultured glioma cells. We also observed that serially passaging flank tumors treated with BZT into new host mice slowed down recurrence of new tumors. In order to determine key receptors mediating cholinergic responses in OPC-like cells we performed protein quantification, which displayed lowered phosphorylation of ERK. Electrophysiological studies are being conducted to dissect the mechanisms by which ACh evokes calcium release. These studies improve understanding of how cholinergic microenvironment influences stem-like glioma cells, providing a platform for repositioning available small molecule modifiers for treatment of glioma. Citation Format: Sumyuktha V. Anand, Alexander G. Skorput, Allan T. Gulledge, Isabella B. Fox, Damian A. Bonnin, Alison L. Young, Matthew C. Havrda. Targeting muscarinic acetylcholine receptors in glioma stem like cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 905.

HGprt deficiency disrupts dopaminergic circuit development in a genetic mouse model of Lesch–Nyhan disease

In Lesch–Nyhan disease (LND), deficiency of the purine salvage enzyme hypoxanthine guanine phosphoribosyl transferase (HGprt) leads to a characteristic neurobehavioral phenotype dominated by dystonia, cognitive deficits and incapacitating self-injurious behavior. It has been known for decades that LND is associated with dysfunction of midbrain dopamine neurons, without overt structural brain abnormalities. Emerging post mortem and in vitro evidence supports the hypothesis that the dopaminergic dysfunction in LND is of developmental origin, but specific pathogenic mechanisms have not been revealed. In the current study, HGprt deficiency causes specific neurodevelopmental abnormalities in mice during embryogenesis, particularly affecting proliferation and migration of developing midbrain dopamine (mDA) neurons. In mutant embryos at E14.5, proliferation was increased, accompanied by a decrease in cell cycle exit and the distribution and orientation of dividing cells suggested a premature deviation from their migratory route. An abnormally structured radial glia-like scaffold supporting this mDA neuronal migration might lie at the basis of these abnormalities. Consequently, these abnormalities were associated with an increase in area occupied by TH+ cells and an abnormal mDA subpopulation organization at E18.5. Finally, dopaminergic innervation was disorganized in prefrontal and decreased in HGprt deficient primary motor and somatosensory cortices. These data provide direct in vivo evidence for a neurodevelopmental nature of the brain disorder in LND. Future studies should not only focus the specific molecular mechanisms underlying the reported neurodevelopmental abnormalities, but also on optimal timing of therapeutic interventions to rescue the DA neuron defects, which may also be relevant for other neurodevelopmental disorders.

AP-1 activation mediates post-natal cardiomyocyte maturation.

Post-natal maturation of mammalian cardiomyocytes proceeds rapidly after birth, with most of the myocytes exiting cell cycle, becoming binucleated, and adopting oxidative phosphorylation as the primary metabolic route. The triggers and transcriptional programmes regulating cardiomyocyte maturation have not been fully understood yet. We performed single-cell RNA-Seq in post-natal rat hearts in order to identify the important factors for this process. Single-cell RNA-Seq profiling was performed of post-natal Day 1 and Day 7 rat hearts, and we found that members of the activating protein 1 (AP-1) transcription factors showed a transient up-regulation in the maturing cardiomyocytes, suggesting their functional involvement in the process. Activating members of the AP-1 family by palmitate or adrenergic stimulation inhibited cardiomyocyte cytokinesis and promoted cardiomyocyte maturation. In contrast, knocking down AP-1 members Atf3 and Jun promoted cardiomyocyte cytokinesis, reduced polyploidy, and inhibited maturation. Mechanistically, RNA-Seq results and rescue experiments indicated that AP-1 members activate the expression of fatty acid metabolic genes to promote cardiomyocyte maturation. Finally, intraperitoneal injection of AP-1 inhibitor T-5224 in neonatal mice inhibits cardiomyocyte maturation in vivo. Our results are the first evidence implicating AP-1 transcription factors in post-natal cardiomyocyte maturation both in vitro and in vivo, which expand our understanding of the molecular mechanism of cardiomyocyte maturation, and may lead to novel therapies to treat congenital heart diseases.

Induction of enucleation in primary and immortalized erythroid cells.

Enucleation is a crucial event during the erythropoiesis, implicating drastic morphologic and transcriptomic/proteomic changes. While many genes deletion lead to failed or impaired enucleation have been identified, directly triggering the erythroid maturation, particularly enucleation, is still challenging. Inducing enucleation at the desired timing is necessary to develop efficient methods to generate mature, fully functional red blood cells in vitro for future transfusion therapies. However, there are considerable differences between primary erythroid cells and cultured cell sources, particularly pluripotent stem cell-derived erythroid cells and immortalized erythroid cell lines. For instance, the difference in the proliferative status between those cell types could be a critical factor, as cell cycle exit is closely connected to the terminal maturation of primary. In this review, we will discuss previous findings on the enucleation machinery and current challengings to trigger the enucleation of infinite erythroid cell sources.

Retinoblastoma-E2F Transcription Factor Interplay Is Essential for Testicular Development and Male Fertility.

The retinoblastoma (RB) protein family members (pRB, p107 and p130) are key regulators of cell cycle progression, but also play crucial roles in apoptosis, and stem cell self-renewal and differentiation. RB proteins exert their effects through binding to E2F transcription factors, which are essential developmental and physiological regulators of tissue and organ homeostasis. According to the canonical view, phosphorylation of RB results in release of E2Fs and induction of genes needed for progress of the cell cycle. However, there are eight members in the E2F transcription factor family with both activator (E2F1-3a) and repressor (E2F3b–E2F8) roles, highlighting the functional diversity of RB-E2F pathway. In this review article we summarize the data showing that RB-E2F interaction is a key cell-autonomous mechanism responsible for establishment and maintenance of lifelong male fertility. We also review the expression pattern of RB proteins and E2F transcription factors in the testis and male germ cells. The available evidence supports that RB and E2F family members are widely and dynamically expressed in the testis, and they are known to have versatile roles during spermatogenesis. Knowledge of the function and significance of RB-E2F interplay for testicular development and spermatogenesis comes primarily from gene knock-out (KO) studies. Several studies conducted in Sertoli cell-specific pRB-KO mice have demonstrated that pRB-mediated inhibition of E2F3 is essential for Sertoli cell functional maturation and cell cycle exit, highlighting that RB-E2F interaction in Sertoli cells is paramount to male fertility. Similarly, ablation of either pRB or E2F1 in the germline results in progressive testicular atrophy due to germline stem cell (GSC) depletion, emphasizing the importance of proper RB-E2F interplay for germline maintenance and lifelong sperm production. In summary, while balanced RB-E2F interplay is essential for cell-autonomous maintenance of GSCs and, the pRB-E2F3 system in Sertoli cells is critical for providing GSC niche thus laying the basis for spermatogenesis.

PU.1 and MYC transcriptional network defines synergistic drug responses to KIT and LSD1 inhibition in acute myeloid leukemia.

Responses to kinase-inhibitor therapy in AML are frequently short-lived due to the rapid development of resistance, limiting the clinical efficacy. Combination therapy may improve initial therapeutic responses by targeting pathways used by leukemia cells to escape monotherapy. Here we report that combined inhibition of KIT and lysine-specific demethylase 1 (LSD1) produces synergistic cell death in KIT-mutant AML cell lines and primary patient samples. This drug combination evicts both MYC and PU.1 from chromatin driving cell cycle exit. Using a live cell biosensor for AKT activity, we identify early adaptive changes in kinase signaling following KIT inhibition that are reversed with the addition of LSD1 inhibitor via modulation of the GSK3a/b axis. Multi-omic analyses, including scRNA-seq, ATAC-seq and CUT&Tag, confirm these mechanisms in primary KIT-mutant AML. Collectively, this work provides rational for a clinical trial to assess the efficacy of KIT and LSD1 inhibition in patients with KIT-mutant AML.

Cell-cycle exit and stem cell differentiation are coupled through regulation of mitochondrial activity in the Drosophila testis

SummaryWhereas stem and progenitor cells proliferate to maintain tissue homeostasis, fully differentiated cells exit the cell cycle. How cell identity and cell-cycle state are coordinated during differentiation is still poorly understood. The Drosophila testis niche supports germline stem cells and somatic cyst stem cells (CySCs). CySCs give rise to post-mitotic cyst cells, providing a tractable model to study the links between stem cell identity and proliferation. We show that, while cell-cycle progression is required for CySC self-renewal, the E2f1/Dp transcription factor is dispensable for self-renewal but instead must be silenced by the Drosophila retinoblastoma homolog, Rbf, to permit differentiation. Continued E2f1/Dp activity inhibits the expression of genes important for mitochondrial activity. Furthermore, promoting mitochondrial biogenesis rescues the differentiation of CySCs with ectopic E2f1/Dp activity but not their cell-cycle exit. In sum, E2f1/Dp coordinates cell-cycle progression with stem cell identity by regulating the metabolic state of CySCs.

Elucidating the Role of the Electron Transport Chain in Retinal Progenitor Cells and Neuro‐ regeneration

In the retinas of non‐mammalian vertebrates, such as zebrafish, neural regeneration is mediated by the molecular reprogramming of MGs into progenitor‐like cells. While mammalian MGs do not exhibit the regenerative potential of the zebrafish, it has been shown that MGs of mammals display limited‐ and transient‐entry into the cell cycle in response to damage. Therefore, an intrinsic proliferative and/or regenerative block likely prevents mammalian MGs from undergoing sustained cell cycle re‐entry and acquiring a multipotential retinal progenitor‐like state. Using a gain‐of‐function strategy we bypassed the Hippo signaling pathway and induced expression of a mutant version of YAP (YAP5SA) in MGs. Lineage tracing and histological analyses of these YAP5SA‐mutant retinas showed that MGs that expressed YAP5SA entered the cell cycle and formed radial clusters of proliferative cells strikingly similar to clonally expanding MG‐derived progenitors of the regenerating zebrafish retina. This finding indicated that YAP5SA is capable of spontaneously driving MGs into the cell cycle. Whether this YAP‐mediated reprogramming of MGs is sufficient to induce differentiation of MGs into fully functional neurons is undetermined. Subsequent single cell RNA sequencing analysis of the YAP5SA‐mutant retinas revealed that the proliferative event of YAP5SA‐positive cells occurred coincident with a dramatic downregulation of genes required for mitochondrial metabolic processes indicating that a metabolic shift is part of the MG reprogramming event. In a variety of cellular contexts, mitochondrial function and the electron transport chain (ETC) activity have emerged as integral regulators of the cell cycle and cell fate. The factors regulating a mitochondrial metabolic reprogramming and cell cycle are coupled but unknown. We generated a mouse line to conditionally knock out (CKO) the mitochondrial transcription factor A (Tfam) to target the ETC. In the CKO developing retinas, loss of Tfam results in loss of ETC activity. Preliminary histological analysis of CKO developing retinas show that loss of Tfam delays progenitor cell cycle exit and reduces neurogenesis resulting in a hypoplastic adult retina. Our findings so far indicate that unless retinal progenitor cells have a functional ETC‐driven oxidative activity, they remain within the cell cycle compromising the normal neural developmental program. The overall goal of this studies will be to determine how the RPC cell cycle machinery interfaces with ETC to drive cell cycle exit and neural differentiation. A better understanding of these requirements will have broad implications for the entire field of neural regeneration.

Loss of Adhesion G‐Protein‐Coupled Receptor L2 Expression Impacts Colonic Epithelial Proliferation

BackgroundThe role of adhesion G‐protein‐coupled receptor L2 (ADGRL2, a.k.a. Latrophilin‐2) in human health has been understudied despite discoveries that the receptor is required for both heart development and neural synaptogenesis. This project tests the physiological function of ADGRL2 in the colon where our prior findings that ADGRL2 is lost in human colorectal cancer (CRC), downregulated during hyperproliferative recovery from murine DSS colitis, enriched in stem and progenitor cells, and is inversely correlated with growth arrest. This raises the intriguing possibility that ADGRL2 restricts stem cell expansion and therefore downregulation of ADGRL2 is required for stem‐cell driven regeneration and repair.MethodsThe effect of Adgrl2 on cell proliferation was assessed by histological staining for Ki‐67 antigen or for the thymidine analogue EdU (5‐ethynyl‐2’‐deoxyuridine, administered by intraperitoneal injection 2hr prior to harvest) in intestinal‐epithelial specific Adgrl2 knockout mice (Adgrl2 IE KO). Percent positive cells were quantified per crypt and per crypt region (crypt base, mid crypt, upper crypt) in Image J.Results/ConclusionsAdgrl2 IE KO mice have overall reduced numbers of Ki‐67+ cells (8.2% difference, p = 0.0016) in the colonic epithelium. This seems paradoxical given that we hypothesize a tumor suppressor role for ADGRL2. However, this effect is driven by a pronounced decrease in Ki‐67+ cells in the post‐mitotic upper crypt of Adgrl2 IE KO mice (15.2% difference, p = 0.0121) while percentages of Ki‐67+ cells in the more proliferative mid crypt and crypt base were determined to be roughly equal between groups, suggesting that the observed effect is likely not driven by a reduction in proliferating stem or progenitor cells but is perhaps due to increased cell cycle exit or terminal differentiation with Adgrl2 knockout. Given that Ki‐67 antigen is expressed in all active cell cycle phases, we used EdU labeling of S‐phase proliferating cells to further assess the impact of Adgrl2 loss on colonocyte proliferation. Proliferation is not impaired with loss of Adgrl2 and instead showed a trend towards increased EdU positive cells in the crypt base (6.7% increase, p= 0.0907). As expected, overall number of S‐phase cells in the upper crypt compartment was low for both Adgrl2 KO and WT animals, however a significant decrease in EdU positive cells was still detected in Adgrl2 KO mice (2.98% change, p=0.0500). Based on these data, the loss of Adgrl2 does not decrease proliferative capacity of the stem cell or progenitor niche, but does effect the distribution of active cycling or differentiating cells along the crypt axis, likely by promoting cell cycle exit or terminal differentiation. Further studies will investigate this possibility and the role of Adgrl2 loss on cell renewal in the context of disease. These data support our hypothesis that ADGRL2 modulates epithelial regeneration in the colon, potentially as a stem‐cell specific negative regulator. Mechanistic studies on dysregulated growth and repair in the colon may also illuminate targets to prevent colitis‐associated cancer in IBD patients.

SIN-Like Pathway Kinases Regulate the End of Mitosis in the Methylotrophic Yeast Ogataea polymorpha.

The mitotic exit network (MEN) is a conserved signalling pathway essential for the termination of mitosis in the budding yeast Saccharomyces cerevisiae. All MEN components are highly conserved in the methylotrophic budding yeast Ogataea polymorpha, except for Cdc15 kinase. Instead, we identified two essential kinases OpHcd1 and OpHcd2 (homologue candidate of ScCdc15) that are homologous to SpSid1 and SpCdc7, respectively, components of the septation initiation network (SIN) of the fission yeast Schizosaccharomyces pombe. Conditional mutants for OpHCD1 and OpHCD2 exhibited significant delay in late anaphase and defective cell separation, suggesting that both genes have roles in mitotic exit and cytokinesis. Unlike Cdc15 in S. cerevisiae, the association of OpHcd1 and OpHcd2 with the yeast centrosomes (named spindle pole bodies, SPBs) is restricted to the SPB in the mother cell body. SPB localisation of OpHcd2 is regulated by the status of OpTem1 GTPase, while OpHcd1 requires the polo-like kinase OpCdc5 as well as active Tem1 to ensure the coordination of mitotic exit (ME) signalling and cell cycle progression. Our study suggests that the divergence of molecular mechanisms to control the ME-signalling pathway as well as the loss of Sid1/Hcd1 kinase in the MEN occurred relatively recently during the evolution of budding yeast.

The Histone Deacetylase Inhibitor I13 Induces Differentiation of M2, M3 and M5 Subtypes of Acute Myeloid Leukemia Cells and Leukemic Stem-Like Cells

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by reduced differentiation of myeloid cells and uncontrolled cell proliferation. AML is prone to drug resistance and has a high recurrence rate during treatment with cytarabine-based chemotherapy. Our study aims to explore the cell differentiation effect of a potent histone deacetylase inhibitor (HDACi), I13, and its possible mechanism on AML cell lines (Kasumi-1, KG-1, MOLM-13 and NB4). It has been shown that I13 can significantly inhibit proliferation and colony formation of these AML cells by inducing cell differentiation coupled with cell-cycle exit at G0/G1. Mechanically, I13 presented the property of HDAC inhibition, as assessed by the acetylation of histone H3, which led to the differentiation of Kasumi-1 cells. In addition, the HDAC inhibition of I13 likely dictated the activation of the antigen processing and presentation pathway, which maybe has the potential to promote immune cells to recognize leukemic cells and respond directly against leukemic cells. These results indicated that I13 could induce differentiation of M3 and M5 subtypes of AML cells, M2 subtype AML cells with t(8;21) translocation and leukemic stem-like cells. Therefore, I13 could be an alternative compound which is able to overcome differentiation blocks in AML.

Myoblast deactivation within engineered human skeletal muscle creates a transcriptionally heterogeneous population of quiescent satellite-like cells

Satellite cells (SCs), the adult Pax7-expressing stem cells of skeletal muscle, are essential for muscle repair. However, in vitro investigations of SC function are challenging due to isolation-induced SC activation, loss of native quiescent state, and differentiation to myoblasts. In the present study, we optimized methods to deactivate in vitro expanded human myoblasts within a 3D culture environment of engineered human skeletal muscle tissues (“myobundles”). Immunostaining and gene expression analyses revealed that a fraction of myoblasts within myobundles adopted a quiescent phenotype (3D-SCs) characterized by increased Pax7 expression, cell cycle exit, and activation of Notch signaling. Similar to native SCs, 3D-SC quiescence is regulated by Notch and Wnt signaling while loss of quiescence and reactivation of 3D-SCs can be induced by growth factors including bFGF. Myobundle injury with a bee toxin, melittin, induces robust myofiber fragmentation, functional decline, and 3D-SC proliferation. By applying single cell RNA-sequencing (scRNA-seq), we discover the existence of two 3D-SC subpopulations (quiescent and activated), identify deactivation-associated gene signature using trajectory inference between 2D myoblasts and 3D-SCs, and characterize the transcriptomic changes within reactivated 3D-SCs in response to melittin-induced injury. These results demonstrate the ability of an in vitro engineered 3D human skeletal muscle environment to support the formation of a quiescent and heterogeneous SC population recapitulating several aspects of the native SC phenotype, and provide a platform for future studies of human muscle regeneration and disease-associated SC dysfunction.

Elevated ASCL1 activity creates de novo regulatory elements associated with neuronal differentiation

BackgroundThe pro-neural transcription factor ASCL1 is a master regulator of neurogenesis and a key factor necessary for the reprogramming of permissive cell types to neurons. Endogenously, ASCL1 expression is often associated with neuroblast stem-ness. Moreover, ASCL1-mediated reprogramming of fibroblasts to differentiated neurons is commonly achieved using artificially high levels of ASCL1 protein, where ASCL1 acts as an “on-target” pioneer factor. However, the genome-wide effects of enhancing ASCL1 activity in a permissive neurogenic environment has not been thoroughly investigated. Here, we overexpressed ASCL1 in the neuronally-permissive context of neuroblastoma (NB) cells where modest endogenous ASCL1 supports the neuroblast programme.ResultsIncreasing ASCL1 in neuroblastoma cells both enhances binding at existing ASCL1 sites and also leads to creation of numerous additional, lower affinity binding sites. These extensive genome-wide changes in ASCL1 binding result in significant reprogramming of the NB transcriptome, redirecting it from a proliferative neuroblastic state towards one favouring neuronal differentiation. Mechanistically, ASCL1-mediated cell cycle exit and differentiation can be increased further by preventing its multi-site phosphorylation, which is associated with additional changes in genome-wide binding and gene activation profiles.ConclusionsOur findings show that enhancing ASCL1 activity in a neurogenic environment both increases binding at endogenous ASCL1 sites and also results in additional binding to new low affinity sites that favours neuronal differentiation over the proliferating neuroblast programme supported by the endogenous protein. These findings have important implications for controlling processes of neurogenesis in cancer and cellular reprogramming.