28. Delaney, Sean P.; Julian, Lisa M.; Pietrobon, Adam; Yockell-Lelièvre, Julien; Doré, Carole; Wang, Ting T.; Doyon, Valerie C.; Raymond, Angela; Patten, David A.; Kristof, Arnold S.; Harper, Mary-Ellen; Sun, Hongyu; Stanford, William L. (2020) Human pluripotent stem cell modeling of tuberous sclerosis complex reveals lineage-specific therapeutic vulnerabilities.bioRxivHuman pluripotent stem cell modeling of tuberous sclerosis complex reveals lineage-specific therapeutic vulnerabilities
mTORC1 hyperactivation resulting from inactivating TSC2 mutations underlie the multi-system tumor disorder tuberous sclerosis complex (TSC) and the rare pulmonary neoplasm lymphangioleiomyomatosis (LAM). Mutation-bearing neural precursor cells (NPCs) lead to the formation of TSC brain tumors during development, while the cell of origin of TSC mesenchymal tumors such as LAM is unknown. We report the first model of multi-system TSC cell types, characterized by NPCs and neural crest cells (NCCs) differentiated in parallel from multiple engineered TSC2−/− human pluripotent stem cell (hPSC) lines. These cells successfully model defining phenotypes of neural and mesenchymal TSC, with transcriptomic signatures reflecting those observed in patient tumors, thus establishing TSC2−/− NCCs as a powerful model of LAM. Employing this rich cellular and transcriptomic resource, we identified lineage-specific catabolic signaling mechanisms that drive divergent cell behavior and therapeutic sensitivities that, in turn, demonstrate the power of employing lineage-specific stem cell models to dissect multi-system diseases.Website DOI
27.Julian, LM; Stanford, WL. (2020) Organelle Cooperation in Stem Cell Fate: Lysosomes as Emerging Regulators of Cell Identity.Front. Cell. Dev. Biol. 8 Organelle Cooperation in Stem Cell Fate: Lysosomes as Emerging Regulators of Cell Identity
lysosomes; stem cell identity and fate; metabolism; neural stem cell (NSC); pluripotent stem cell (PSC); neural crest (NC); cancer stem cell (CSC)
Regulation of stem cell fate is best understood at the level of gene and protein regulatory networks, though it is now clear that multiple cellular organelles also have critical impacts. A growing appreciation for the functional interconnectedness of organelles suggests that an orchestration of integrated biological networks functions to drive stem cell fate decisions and regulate metabolism. Metabolic signaling itself has emerged as an integral regulator of cell fate including the determination of identity, activation state, survival, and differentiation potential of many developmental, adult, disease, and cancer-associated stem cell populations and their progeny. As the primary adenosine triphosphate-generating organelles, mitochondria are well-known regulators of stem cell fate decisions, yet it is now becoming apparent that additional organelles such as the lysosome are important players in mediating these dynamic decisions. In this review, we will focus on the emerging role of organelles, in particular lysosomes, in the reprogramming of both metabolic networks and stem cell fate decisions, especially those that impact the determination of cell identity. We will discuss the inter-organelle interactions, cell signaling pathways, and transcriptional regulatory mechanisms with which lysosomes engage and how these activities impact metabolic signaling. We will further review recent data that position lysosomes as critical regulators of cell identity determination programs and discuss the known or putative biological mechanisms. Finally, we will briefly highlight the potential impact of elucidating mechanisms by which lysosomes regulate stem cell identity on our understanding of disease pathogenesis, as well as the development of refined regenerative medicine, biomarker, and therapeutic strategies. DOI PubMed
24. Huang, TW; Gonzalez, YR; Qu, DB; Huang, E; Safarpour, F; Wang, E; Joselin, A; Im, DS; Callaghan, SM; Boonying, W; Julian, L; Dunwoodie, SL; Slack, RS; Park, DS. (2019) The pro-death role of Cited2 in stroke is regulated by E2F1/4 transcription factors.J. Biol. Chem. 294: 8617-8629 The pro-death role of Cited2 in stroke is regulated by E2F1/4 transcription factors
stroke; ischemia; neuron; hypoxia; E2F transcription factor; brain injury; Cbp; P300-interacting transactivator with Glu; Asp-rich carboxyl-terminal domain 2; cell death; gene promoter; gene regulation
We previously reported that the cell cycle-related cyclin-dependent kinase 4-retinoblastoma (RB) transcriptional corepressor pathway is essential for stroke-induced cell death both in vitro and in vivo. However, how this signaling pathway induces cell death is unclear. Previously, we found that the cyclin-dependent kinase 4 pathway activates the pro-apoptotic transcriptional co-regulator Cited2 in vitro after DNA damage. In the present study, we report that Cited2 protein expression is also dramatically increased following stroke/ischemic insult. Critically, utilizing conditional knockout mice, we show that Cited2 is required for neuronal cell death, both in culture and in mice after ischemic insult. Importantly, determining the mechanism by which Cited2 levels are regulated, we found that E2F transcription factor (E2F) family members participate in Cited2 regulation. First, E2F1 expression induced Cited2 transcription, and E2F1 deficiency reduced Cited2 expression. Moreover, determining the potential E2F-binding regions on the Cited2 gene regulatory sequence by ChIP analysis, we provide evidence that E2F1/4 proteins bind to this DNA region. A luciferase reporter assay to probe the functional outcomes of this interaction revealed that E2F1 activates and E2F4 inhibits Cited2 transcription. Moreover, we identified the functional binding motif for E2F1 in the Cited2 gene promoter by demonstrating that mutation of this site dramatically reduces E2F1-mediated Cited2 transcription. Finally, E2F1 and E2F4 regulated Cited2 expression in neurons after stroke-related insults. Taken together, these results indicate that the E2F-Cited2 regulatory pathway is critically involved in stroke injury. DOI PubMed
23. Tam, RY; Yockell-Lelievre, J; Smith, LJ; Julian, LM; Baker, AEG; Choey, C; Hasim, MS; Dimitroulakos, J; Stanford, WL; Shoichet, MS. (2019) Rationally Designed 3D Hydrogels Model Invasive Lung Diseases Enabling High-Content Drug Screening.Adv. Mater. 31 Rationally Designed 3D Hydrogels Model Invasive Lung Diseases Enabling High-Content Drug Screening
3D hydrogels; cancer; cell invasion; disease modeling; drug screening
Cell behavior is highly dependent upon microenvironment. Thus, to identify drugs targeting metastatic cancer, screens need to be performed in tissue mimetic substrates that allow cell invasion and matrix remodeling. A novel biomimetic 3D hydrogel platform that enables quantitative analysis of cell invasion and viability at the individual cell level is developed using automated data acquisition methods with an invasive lung disease (lymphangioleiomyomatosis, LAM) characterized by hyperactive mammalian target of rapamycin complex 1 (mTORC1) signaling as a model. To test the lung-mimetic hydrogel platform, a kinase inhibitor screen is performed using tuberous sclerosis complex 2 (TSC2) hypomorphic cells, identifying Cdk2 inhibition as a putative LAM therapeutic. The 3D hydrogels mimic the native niche, enable multiple modes of invasion, and delineate phenotypic differences between healthy and diseased cells, all of which are critical to effective drug screens of highly invasive diseases including lung cancer. DOI PubMed
21. Ho, MS; Ho, M; Julian, LM; Stanford, W; Stewart, DJ. (2018) The X-factor involved in apoptosis and proliferation-reciprocal exosome cross-talk between lymphangioleiomyomatosis-derived smooth muscle cells (LAM-SMC) and endothelial cells (ECs) mediates ec network disruption/apoptosis and promotes LAM-SMC growth.Cytotherapy 20: S16 (abstract only) The X-factor involved in apoptosis and proliferation-reciprocal exosome cross-talk between lymphangioleiomyomatosis-derived smooth muscle cells (LAM-SMC) and endothelial cells (ECs) mediates ec network disruption/apoptosis and promotes LAM-SMC growth.
Lymphangioleiomyomatosis (LAM) is a progressive and incurable disease caused by mutations in the TSC2 gene, which is involved in mTOR pathway regulation. Fragile endothelial cell (EC) neovessels are stabilized by mural smooth muscle cells (SMCs). However, SMC-like LAM cells invade and destroy the lung through unknown mechanism(s). We postulate that LAM-SMCs will disrupt EC capillary-like networks and promote EC apoptosis.
DOI
20. Ho, MS; Ho, M; Julian, LM; Stanford, WL; Stewart, DJ. (2018) Exosome-Mediated Endothelial Cell Apoptosis and Network Disruption by Lymphangioleiomyomatosis-Smooth Muscle Cells (LAM-SMCs) Derived from Human Induced Pluripotent Stem Cell (iPSC).Circulation 138:A16591 (Suppl_1) Exosome-Mediated Endothelial Cell Apoptosis and Network Disruption by Lymphangioleiomyomatosis-Smooth Muscle Cells (LAM-SMCs) Derived from Human Induced Pluripotent Stem Cell (iPSC)
Abstract
Introduction: In LAM, SMC-like cells invade the lung thus compromising respiratory function which is dependent on normal vascular and alveoli structure. We posit that while healthy mural SMCs stabilize fragile neovessels and maintain vascular homeostasis, LAM-SMCs promote disruption of lung microvasculature by inducing endothelial cell (EC) apoptosis.
Methods/Results: iPSCs derived from LAM patients or healthy subjects were differentiated into SMCs using a teratoma protocol. Co-culture of healthy iSMCs with HUVECs on Matrigel improved EC network persistence for ≥72Hrs, while LAM-SMC co-culture led to rapid network collapse in <15Hrs. Culture of ECs with LAM-SMC conditioned media (24-72Hrs) increased EC apoptosis as assessed by Annexin V/PI flow cytometry, confirmed by increases in cleaved Caspase-3 protein expression. To further elucidate mechanism and kinetics of EC apoptosis, Annexin V/PI flow cytometry and Western Blot with Caspases 8,9,12 were performed in parallel at 3hr intervals from 0-12 Hrs. Increase in EC apoptosis at 9hrs, corresponded with a 6-fold increase in Cas-12 protein expression, followed by a 3-fold increase in Cas-9 at 12hrs with little change in Cas-8. This is in line with our RNAseq study showing dysregulation in endoplasmic reticulum and mitochondria related genes in LAM-SMC. Culture of HUVECs with LAM-SMC exosomes similarly induced EC apoptosis, with a profound decrease in EC gene expression (eNOS,TIE2,TAL1; 5-7 fold; p<0.05) but increase expression of vessel-destabilizing, ANGPT2 (6-fold; p<0.05). Scratch-wound assay also showed ~60% reduction (p<0.05) in EC migratory capacity. Intriguingly, apoptotic ECs released exosomes containing translationally controlled tumor protein, TCTP, which were taken up by LAM-SMCs, as evidenced by immunofluorescence studies, associated with increases in the levels of proliferation-associated phospho-mTOR-and-S6k protein.
Conclusions: LAM-SMCs derived exosomes resulted in EC apoptosis and network destabilization. Conversely, apoptotic ECs enhanced LAM-SMC growth, possibly by Caspase-dependent exosome release of TCTP, a positive regulator of mTOR signaling. These novel mechanisms may promote both microvasculature disruption and uncontrolled growth of LAM-SMCs.Website
19. Lavictoire, SJ; Gont, A; Julian, LM; Stanford, WL; Vlasschaert, C; Gray, DA; Jomaa, D; Lorimer, IAJ. (2018) Engineering PTEN-L for Cell-Mediated Delivery.Mol.Ther.-Methods Clin. Dev. 9: 12-22 Engineering PTEN-L for Cell-Mediated Delivery
The tumor suppressor PTEN is frequently inactivated in glioblastoma. PTEN-L is a long form of PTEN produced by translation from an alternate upstream start codon. Unlike PTEN, PTEN-L has a signal sequence and a tract of six arginine residues that allow PTEN-L to be secreted from cells and be taken up by neighboring cells. This suggests that PTEN-L could be used as a therapeutic to restore PTEN activity. However, effective delivery of therapeutic proteins to treat CNS cancers such as glioblastoma is challenging. One method under evaluation is cell-mediated therapy, where cells with tumor-homing abilities such as neural stem cells are genetically modified to express a therapeutic protein. Here, we have developed a version of PTEN-L that is engineered for enhanced cell-mediated delivery. This was accomplished by replacement of the native leader sequence of PTEN-L with a leader sequence from human light-chain immunoglobulin G (IgG). This version of PTEN-L showed increased secretion and an increased ability to transfer to neighboring cells. Neural stem cells derived from human fibroblasts could be modified to express this version of PTEN-L and were able to deliver catalytically active light-chain leader PTEN-L (lclPTEN-L) to neighboring glioblastoma cells. DOI PubMed
18.Julian, LM; Delaney, SP; Wang, Y; Goldberg, AA; Dore, C; Yockell-Lelievre, J; Tam, RY; Giannikou, K; McMurray, F; Shoichet, MS; Harper, ME; Henske, EP; Kwiatkowski, DJ; Darling, TN; Moss, J; Kristof, AS; Stanford, WL. (2017) Human Pluripotent Stem Cell-Derived TSC2-Haploinsufficient Smooth Muscle Cells Recapitulate Features of Lymphangioleiomyomatosis.Cancer Res. 77: 5491-5502 Human Pluripotent Stem Cell-Derived TSC2-Haploinsufficient Smooth Muscle Cells Recapitulate Features of Lymphangioleiomyomatosis
Lymphangioleiomyomatosis (LAM) is a progressive destructive neoplasm of the lung associated with inactivating mutations in the TSC1 or TSC2 tumor suppressor genes. Cell or animal models that accurately reflect the pathology of LAM have been challenging to develop. Here, we generated a robust human cell model of LAM by reprogramming TSC2 mutation-bearing fibroblasts from a patient with both tuberous sclerosis complex (TSC) and LAM (TSC-LAM) into induced pluripotent stem cells (iPSC), followed by selection of cells that resemble those found in LAM tumors by unbiased in vivo differentiation. We established expandable cell lines under smooth muscle cell (SMC) growth conditions that retained a patient-specific genomic TSC2(-/-) mutation and recapitulated the molecular and functional characteristics of pulmo-nary LAM cells. These include multiple indicators of hyperactive mTORC1 signaling, presence of specific neural crest and SMC markers, expression of VEGF-D and female sex hormone receptors, reduced autophagy, and metabolic reprogramming. Intriguingly, the LAM-like features of these cells suggest that haploin-sufficiency at the TSC2 locus contributes to LAM pathology, and demonstrated that iPSC reprogramming and SMC lineage differentiation of somatic patient cells with germline mutations was a viable approach to generate LAM-like cells. The patient-derived SMC lines we have developed thus represent a novel cellular model of LAM that can advance our understanding of disease pathogenesis and develop therapeutic strategies against LAM. (C) 2017 AACR. DOI PubMed
17.Julian, LM; McDonald, ACH; Stanford, WL. (2017) Direct reprogramming with SOX factors: masters of cell fate.Curr. Opin. Genet. Dev. 46: 24-36 Direct reprogramming with SOX factors: masters of cell fate
Over the last decade significant advances have been made toward reprogramming the fate of somatic cells, typically by overexpression of cell lineage-determinant transcription factors. As key regulators of cell fate, the SOX family of transcription factors has emerged as potent drivers of direct somatic cell reprogramming into multiple lineages, in some cases as the sole overexpressed factor. The vast capacity of SOX factors, especially those of the SOXB1, E and F subclasses, to reprogram cell fate is enlightening our understanding of organismal development, cancer and disease, and offers tremendous potential for regenerative medicine and cell-based therapies. Understanding the molecular mechanisms through which SOX factors reprogram cell fate is essential to optimize the development of novel somatic cell transdifferentiation strategies. DOI PubMed
14.Julian, LM; Carpenedo, RL; Rothberg, JLM; Stanford, WL. (2016) Formula G1: Cell cycle in the driver's seat of stem cell fate determination.Bioessays 38: 325-332 Formula G1: Cell cycle in the driver's seat of stem cell fate determination
bivalent promoter; cell cycle; cell fate; differentiation; G1 phase; pluripotency
Cell cycle dynamics has emerged as a key regulator of stem cell fate decisions. In particular, differentiation decisions are associated with the G1 phase, and recent evidence suggests that self-renewal is actively regulated outside of G1. The mechanisms underlying these phenomena are largely unknown, but direct control of gene regulatory programs by the cell cycle machinery is heavily implicated. A recent study sheds important mechanistic insight by demonstrating that in human embryonic stem cells (hESCs) the Cyclin-dependent kinase CDK2 controls a wide-spread epigenetic program that drives transcription at differentiation-related gene promoters specifically in G1. Here, we discuss this finding and explore whether similar mechanisms are likely to function in multipotent stem cells. The implications of this discovery toward our understanding of stem cell-related disease are discussed, and we postulate novel mechanisms that position the cell cycle as a regulator of cell fate gene networks at epigenetic, transcriptional and post-transcriptional levels. DOI PubMed
13.Julian, LM; Liu, Y; Pakenham, CA; Dugal-Tessier, D; Ruzhynsky, V; Bae, S; Tsai, SY; Leone, G; Slack, RS; Blais, A. (2016) Tissue-specific targeting of cell fate regulatory genes by E2f factors.Cell Death Differ. 23: 565-575 Tissue-specific targeting of cell fate regulatory genes by E2f factors
Cell cycle proteins are important regulators of diverse cell fate decisions, and in this capacity have pivotal roles in neurogenesis and brain development. The mechanisms by which cell cycle regulation is integrated with cell fate control in the brain and other tissues are poorly understood, and an outstanding question is whether the cell cycle machinery regulates fate decisions directly or instead as a secondary consequence of proliferative control. Identification of the genes targeted by E2 promoter binding factor (E2f) transcription factors, effectors of the pRb/E2f cell cycle pathway, will provide essential insights into these mechanisms. We identified the promoter regions bound by three neurogenic E2f factors in neural precursor cells in a genome-wide manner. Through bioinformatic analyses and integration of published genomic data sets we uncovered hundreds of transcriptionally active E2f-bound promoters corresponding to genes that control cell fate processes, including key transcriptional regulators and members of the Notch, fibroblast growth factor, Wnt and Tgf-beta signaling pathways. We also demonstrate a striking enrichment of the CCCTC binding factor transcription factor (Ctcf) at E2f3-bound nervous system-related genes, suggesting a potential regulatory co-factor for E2f3 in controlling differentiation. Finally, we provide the first demonstration of extensive tissue specificity among E2f target genes in mammalian cells, whereby E2f3 promoter binding is well conserved between neural and muscle precursors at genes associated with cell cycle processes, but is tissue-specific at differentiation-associated genes. Our findings implicate the cell cycle pathway as a widespread regulator of cell fate genes, and suggest that E2f3 proteins control cell type-specific differentiation programs by regulating unique sets of target genes. This work significantly enhances our understanding of how the cell cycle machinery impacts cell fate and differentiation, and will importantly drive further discovery regarding the mechanisms of cell fate control and transcriptional regulation in the brain, as well as in other tissues. DOI PubMed
12.Julian, LM; Blais, A. (2015) Transcriptional control of stem cell fate by E2Fs and pocket proteins.Front. Genet. 6 Transcriptional control of stem cell fate by E2Fs and pocket proteins
E2F transcription factors and their regulatory partners, the pocket proteins (PPs), have emerged as essential regulators of stem cell fate control in a number of lineages. In mammals, this role extends from both pluripotent stem cells to those encompassing all embryonic germ layers, as well as extra-embryonic lineages. E2F/PP-mediated regulation of stem cell decisions is highly evolutionarily conserved, and is likely a pivotal biological mechanism underlying stem cell homeostasis. This has immense implications for organismal development, tissue maintenance, and regeneration. In this article, we discuss the roles of E2F factors and PPs in stem cell populations, focusing on mammalian systems. We discuss emerging findings that position the E2F and PP families as widespread and dynamic epigenetic regulators of cell fate decisions. Additionally, we focus on the ever expanding landscape of E2F/PP target genes, and explore the possibility that E2Fs are not simply regulators of general 'multi-purpose' cell fate genes but can execute tissue- and cell type-specific gene regulatory programs. DOI PubMed
11.Julian, LM; Vandenbosch, R; Pakenham, CA; Andrusiak, MG; Nguyen, AP; McClellan, KA; Svoboda, DS; Lagace, DC; Park, DS; Leone, G; Blais, A; Slack, RS. (2013) Opposing Regulation of Sox2 by Cell-Cycle Effectors E2f3a and E2f3b in Neural Stem Cells.Cell Stem Cell 12: 440-452 Opposing Regulation of Sox2 by Cell-Cycle Effectors E2f3a and E2f3b in Neural Stem Cells
The mechanisms through which cell-cycle control and cell-fate decisions are coordinated in proliferating stem cell populations are largely unknown. Here, we show that E2f3 isoforms, which control cell-cycle progression in cooperation with the retinoblastoma protein (pRb), have critical effects during developmental and adult neurogenesis. Loss of either E2f3 isoform disrupts Sox2 gene regulation and the balance between precursor maintenance and differentiation in the developing cortex. Both isoforms target the Sox2 locus to maintain baseline levels of Sox2 expression but antagonistically regulate Sox2 levels to instruct fate choices. E2f3-mediated regulation of Sox2 and precursor cell fate extends to the adult brain, where E2f3a loss results in defects in hippocampal neurogenesis and memory formation. Our results demonstrate a mechanism by which E2f3a and E2f3b differentially regulate Sox2 dosage in neural precursors, a finding that may have broad implications for the regulation of diverse stem cell populations. DOI PubMed
9. Ghanem, N; Andrusiak, MG; Svoboda, D; Al Lafi, SM; Julian, LM; McClellan, KA; De Repentigny, Y; Kothary, R; Ekker, M; Blais, A; Park, DS; Slack, RS. (2012) The Rb/E2F Pathway Modulates Neurogenesis through Direct Regulation of the Dlx1/Dlx2 Bigene Cluster.J. Neurosci. 32: 8219-8230 The Rb/E2F Pathway Modulates Neurogenesis through Direct Regulation of the Dlx1/Dlx2 Bigene Cluster
During brain morphogenesis, the mechanisms through which the cell cycle machinery integrates with differentiation signals remain elusive. Here we show that the Rb/E2F pathway regulates key aspects of differentiation and migration through direct control of the Dlx1 and Dlx2 homeodomain proteins, required for interneuron specification. Rb deficiency results in a dramatic reduction of Dlx1 and Dlx2 gene expression manifested by loss of interneuron subtypes and severe migration defects in the mouse brain. The Rb/E2F pathway modulates Dlx1/Dlx2 regulation through direct interaction with a Dlx forebrain-specific enhancer, I12b, and the Dlx1/Dlx2 proximal promoter regions, through repressor E2F sites both in vitro and in vivo. In the absence of Rb, we demonstrate that repressor E2Fs inhibit Dlx transcription at the Dlx1/Dlx2 promoters and Dlx1/2-I12b enhancer to suppress differentiation. Our findings support a model whereby the cell cycle machinery not only controls cell division but also modulates neuronal differentiation and migration through direct regulation of the Dlx1/Dlx2 bigene cluster during embryonic development. DOI PubMed
8. Andrusiak, MG; McClellan, KA; Dugal-Tessier, D; Julian, LM; Rodrigues, SP; Park, DS; Kennedy, TE; Slack, RS. (2011) Rb/E2F Regulates Expression of Neogenin during Neuronal Migration.Mol. Cell. Biol. 31: 238-247 Rb/E2F Regulates Expression of Neogenin during Neuronal Migration
The Rb/E2F pathway has long been appreciated for its role in regulating cell cycle progression. Emerging evidence indicates that it also influences physiological events beyond regulation of the cell cycle. We have previously described a requirement for Rb/E2F mediating neuronal migration; however, the molecular mechanisms remain unknown, making this an ideal system to identify Rb/E2F-mediated atypical gene regulation in vivo. Here, we report that Rb regulates the expression of neogenin, a gene encoding a receptor involved in cell migration and axon guidance. Rb is capable of repressing E2F-mediated neogenin expression while E2F3 occupies a region containing E2F consensus sites on the neogenin promoter in native chromatin. Absence of Rb results in aberrant neuronal migration and adhesion in response to netrin-1, a known ligand for neogenin. Increased expression of neogenin through ex vivo electroporation results in impaired neuronal migration similar to that detected in forebrain-specific Rb deficiency. These findings show direct regulation of neogenin by the Rb/E2F pathway and demonstrate that regulation of neogenin expression is required for neural precursor migration. These studies identify a novel mechanism through which Rb regulates transcription of a gene beyond the classical E2F targets to regulate events distinct from cell cycle progression. DOI PubMed
7. Dugal-Tessier, D; Andrusiak, MG; Ruzhynsky, VA; Julian, LM; Park, DS; Slack, RS. (2010) E2F4 is essential for development of the ventral telencephalon.Int. J. Dev. Neurosci. 28: 672-672 E2F4 is essential for development of the ventral telencephalon
Cell cycle; Neural precursors; Sonic hedgehog; Telencephalon development
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5. Pakenham, CA; Julian, LM; Park, DS; Leone, G; Slack, RS. (2010) Maintenance of neural stem cell self-renewal by E2f3.Int. J. Dev. Neurosci. 28: 679-679 Maintenance of neural stem cell self-renewal by E2f3
E2f3; Polycomb Group proteins; Stem cell; Cell cycle
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4. McClellan, KA; Vanderluit, JL; Julian, LM; Andrusiak, MG; Dugal-Tessier, D; Park, DS; Slack, RS. (2009) The p107/E2F Pathway Regulates Fibroblast Growth Factor 2 Responsiveness in Neural Precursor Cells.Mol. Cell. Biol. 29: 4701-4713 The p107/E2F Pathway Regulates Fibroblast Growth Factor 2 Responsiveness in Neural Precursor Cells
We have previously shown that p107, a member of the retinoblastoma (Rb) cell cycle regulatory family, has a unique function in regulating the pool of neural precursor cells. As the pool of progenitors is regulated by a limiting supply of trophic factors, we asked if the Rb/E2F pathway may control the size of the progenitor population by regulating the levels of growth factors or their receptors. Here, we demonstrate that fibroblast growth factor 2 (FGF2) is aberrantly upregulated in the brains of animals lacking Rb family proteins and that the gene encoding the FGF2 ligand is directly regulated by p107 and E2F3. Chromatin immunoprecipitation assays demonstrated that E2F3 and p107 occupy E2F consensus sites on the FGF2 promoter in the context of native chromatin. To evaluate the physiological consequence of FGF2 deregulation in both p107 and E2F3 mutants, we measured neural progenitor responsiveness to growth factors. Our results demonstrate that E2F3 and p107 are each mediators of FGF2 growth factor responsiveness in neural progenitor cells. These results support a model whereby p107 regulates the pool of FGF-responsive progenitors by directly regulating FGF2 gene expression in vivo. By identifying novel roles for p107/E2F in regulating genes outside of the classical cell cycle machinery targets, we uncover a new mechanism whereby Rb/E2F mediates proliferation through regulating growth factor responsiveness. DOI PubMed
3.Julian, LM; Palander, O; Seifried, LA; Foster, JEG; Dick, FA. (2008) Characterization of an E2F1-specific binding domain in pRB and its implications for apoptotic regulation.Oncogene 27: 1572-1579 Characterization of an E2F1-specific binding domain in pRB and its implications for apoptotic regulation
cell cycle; retinoblastoma; E2F; apoptosis; transcription
The retinoblastoma protein (pRB) has the dual capability to negatively regulate both E2F-induced cell cycle entry and E2F1-induced apoptosis. In this report, we characterize a unique pRB-E2F1 interaction. Using mutagenesis to disrupt E2F1 binding, we find that the ability of pRB to regulate E2F1-induced apoptosis is diminished when this interaction is lost. Strikingly, this mutant form of pRB retains the ability to control E2F responsive cell cycle genes and blocks cell proliferation. These functional properties are the reciprocal of a previously described E2F binding mutant of pRB that interacts with E2F1, but lacks the ability to interact with other E2Fs. Our work shows that these distinct interactions allow pRB to separately regulate E2F-induced cell proliferation and apoptosis. This suggests a novel form of regulation whereby separate types of binding contacts between the same types of molecules can confer distinct functional outcomes. DOI PubMed
2. Seifried, LA; Talluri, S; Cecchini, M; Julian, LM; Myrnryk, JS; Dick, FA. (2008) PRB-E2F1 complexes are resistant to adenovirus E1A-mediated disruption.J. Virol. 82: 4511-4520 PRB-E2F1 complexes are resistant to adenovirus E1A-mediated disruption
Disruption of pRB-E2F interactions by E1A is a key event in the adenoviral life cycle that drives expression of early viral transcription and induces cell cycle progression. This function of E1A is complicated by E2F1, an E2F family member that controls multiple processes besides proliferation, including apoptosis and DNA repair. Recently, a second interaction site in pRB that only contacts E2F1 has been discovered, allowing pRB to control proliferation separately from other E2F1-dependent activities. Based on this new insight into pRB-E2F1 regulation, we investigated how EIA affects control of E2F1 by pRB. Our data reveal that pRB-E2F1 interactions are resistant to E1A-mediated disruption. Using mutant forms of pRB that selectively force E2F1 to bind through only one of the two binding sites on pRB, we determined that EIA is unable to disrupt E2F1's unique interaction with pRB. Furthermore, analysis of pRB-E2F complexes during adenoviral infection reveals the selective maintenance of pRB-E2F1 interactions despite the presence of E1A. Our experiments also demonstrate that E2F1 functions to maintain cell viability in response to E1A expression. This suggests that adenovirus E1A's seemingly complex mechanism of disrupting pRB-E2F interactions provides selectivity in promoting viral transcription and cell cycle advancement, while maintaining cell viability. DOI PubMed
1. Isaac, CE; Francis, SM; Martens, AL; Julian, LM; Seifried, LA; Erdmann, N; Binne, UK; Harrington, L; Sicinski, P; Berube, NG; Dyson, NJ; Dick, FA. (2006) The retinoblastoma protein regulates pericentric heterochromatin.Mol. Cell. Biol. 26: 3659-3671 The retinoblastoma protein regulates pericentric heterochromatin
The retinoblastoma protein (pRb) has been proposed to regulate cell cycle progression in part through its ability to interact with enzymes that modify histone tails and create a repressed chromatin structure. We created a mutation in the murine Rb1 gene that disrupted pRb's ability to interact with these enzymes to determine if it affected cell cycle control. Here, we show that loss of this interaction slows progression through mitosis and causes aneuploidy. Our experiments reveal that while the LXCXE binding site mutation does not disrupt pRb's interaction with the Suv4-20h histone methyltransferases, it dramatically reduces H4-K20 trimethylation in pericentric heterochromatin. Disruption of heterochromatin structure in this chromosomal region leads to centromere fusions, chromosome missegregation, and genomic instability. These results demonstrate the surprising finding that pRb uses the LXCXE binding cleft to control chromatin structure for the regulation of events beyond the G(1)-to-S-phase transition. DOI PubMed
