Nontelomeric TRF2-REST Interaction Modulates Neuronal Gene Silencing and Fate of Tumor and Stem Cells

SummaryRemoval of TRF2, a telomere shelterin protein, recapitulates key aspects of telomere attrition including the DNA-damage response and cell-cycle arrest [1]. Distinct from the response of proliferating cells to loss of TRF2 [2, 3], in rodent noncycling cells, TRF2 inhibition promotes differentiation and growth [4, 5]. However, the mechanism that couples telomere gene-silencing features [6–8] to differentiation programs has yet to be elucidated. Here we describe an extratelomeric function of TRF2 in the regulation of neuronal genes mediated by the interaction of TRF2 with repressor element 1-silencing transcription factor (REST), a master repressor of gene networks devoted to neuronal functions [9–12]. TRF2-REST complexes are readily detected by coimmunoprecipitation assays and are localized to aggregated PML-nuclear bodies in undifferentiated pluripotent human NTera2 stem cells. Inhibition of TRF2, either by a dominant-negative mutant or by RNA interference, dissociates TRF2-REST complexes resulting in ubiquitin-proteasomal degradation of REST. Consequentially, REST-targeted neural genes (L1CAM, β3-tubulin, synaptophysin, and others) are derepressed, resulting in acquisition of neuronal phenotypes. Notably, selective damage to telomeres without affecting TRF2 levels causes neither REST degradation nor cell differentiation. Thus, in addition to protecting telomeres, TRF2 possesses a novel role in stabilization of REST thereby controlling neural tumor and stem cell fate

Embryonic Stem Cell-Derived Cardiomyocyte Heterogeneity and the Isolation of Immature and Committed Cells for Cardiac Remodeling and Regeneration

Pluripotent stem cells represent one promising source for cell replacement therapy in heart, but differentiating embryonic stem cell-derived cardiomyocytes (ESC-CMs) are highly heterogeneous and show a variety of maturation states. In this study, we employed an ESC clonal line that contains a cardiac-restricted ncx1 promoter-driven puromycin resistance cassette together with a mass culture system to isolate ESC-CMs that display traits characteristic of very immature CMs. The cells display properties of proliferation, CM-restricted markers, reduced mitochondrial mass, and hypoxia-resistance. Following transplantation into rodent hearts, bioluminescence imaging revealed that immature cells, but not more mature CMs, survived for at least one month following injection. These data and comparisons with more mature cells lead us to conclude that immature hypoxia resistant ESC-CMs can be isolated in mass in vitro and, following injection into heart, form grafts that may mediate long-term recovery of global and regional myocardial contractile function following infarction

RECQL5 cooperates with Topoisomerase II alpha in DNA decatenation and cell cycle progression

DNA decatenation mediated by Topoisomerase II is required to separate the interlinked sister chromatids post-replication. SGS1, a yeast homolog of the human RecQ family of helicases interacts with Topoisomerase II and plays a role in chromosome segregation, but this functional interaction has yet to be identified in higher organisms. Here, we report a physical and functional interaction of Topoisomerase IIα with RECQL5, one of five mammalian RecQ helicases, during DNA replication. Direct interaction of RECQL5 with Topoisomerase IIα stimulates the decatenation activity of Topoisomerase IIα. Consistent with these observations, RECQL5 co-localizes with Topoisomerase IIα during S-phase of the cell cycle. Moreover, cells with stable depletions of RECQL5 display a slow proliferation rate, a G2/M cell cycle arrest and late S-phase cycling defects. Metaphase spreads generated from RECQL5-depleted cells exhibit undercondensed and entangled chromosomes. Further, RECQL5-depleted cells activate a G2/M checkpoint and undergo apoptosis. These phenotypes are similar to those observed when Topoisomerase II catalytic activity is inhibited. These results reveal an important role for RECQL5 in the maintenance of genomic stability and a new insight into the decatenation process

Zscan4 regulates telomere elongation and genomic stability in ES cells

Exceptional genomic stability is one of the hallmarks of mouse embryonic stem (ES) cells. However, the genes contributing to this stability remain obscure. We previously identified Zscan4 as a specific marker for two-cell embryo and ES cells. Here we show that Zscan4 is involved in telomere maintenance and long-term genomic stability in ES cells. Only 5% of ES cells express Zscan4 at a given time, but nearly all ES cells activate Zscan4 at least once during nine passages. The transient Zscan4-positive state is associated with rapid telomere extension by telomere recombination and upregulation of meiosis-specific homologous recombination genes, which encode proteins that are colocalized with ZSCAN4 on telomeres. Furthermore, Zscan4 knockdown shortens telomeres, increases karyotype abnormalities and spontaneous sister chromatid exchange, and slows down cell proliferation until reaching crisis by passage eight. Together, our data show a unique mode of genome maintenance in ES cells. © 2010 Macmillan Publishers Limited. All rights reserved

B-MYB Is Essential for Normal Cell Cycle Progression and Chromosomal Stability of Embryonic Stem Cells

Background: The transcription factor B-Myb is present in all proliferating cells, and in mice engineered to remove this gene, embryos die in utero just after implantation due to inner cell mass defects. This lethal phenotype has generally been attributed to a proliferation defect in the cell cycle phase of G1. Methodology/Principal Findings: In the present study, we show that the major cell cycle defect in murine embryonic stem (mES) cells occurs in G2/M. Specifically, knockdown of B-Myb by short-hairpin RNAs results in delayed transit through G2/M, severe mitotic spindle and centrosome defects, and in polyploidy. Moreover, many euploid mES cells that are transiently deficient in B-Myb become aneuploid and can no longer be considered viable. Knockdown of B-Myb in mES cells also decreases Oct4 RNA and protein abundance, while over-expression of B-MYB modestly up-regulates pou5f1 gene expression. The coordinated changes in B-Myb and Oct4 expression are due, at least partly, to the ability of B-Myb to directly modulate pou5f1 gene promoter activity in vitro. Ultimately, the loss of B-Myb and associated loss of Oct4 lead to an increase in early markers of differentiation prior to the activation of caspase-mediated programmed cell death. Conclusions/Significance: Appropriate B-Myb expression is critical to the maintenance of chromosomally stable and pluripotent ES cells, but its absence promotes chromosomal instability that results in either aneuploidy or differentiation-associated cell death

Enhanced Proliferation of Monolayer Cultures of Embryonic Stem (ES) Cell-Derived Cardiomyocytes Following Acute Loss of Retinoblastoma

Background: Cardiomyocyte (CM) cell cycle analysis has been impeded because of a reliance on primary neonatal cultures of poorly proliferating cells or chronic transgenic animal models with innate compensatory mechanisms. Methodology/Principal Findings: We describe an in vitro model consisting of monolayer cultures of highly proliferative embryonic stem (ES) cell-derived CM. Following induction with ascorbate and selection with puromycin, early CM cultures are.98 % pure, and at least 85 % of the cells actively proliferate. During the proliferative stage, cells express high levels of E2F3a, B-Myb and phosphorylated forms of retinoblastoma (Rb), but with continued cultivation, cells stop dividing and mature functionally. This developmental transition is characterized by a switch from slow skeletal to cardiac TnI, an increase in binucleation, cardiac calsequestrin and hypophosphorylated Rb, a decrease in E2F3, B-Myb and atrial natriuretic factor, and the establishment of a more negative resting membrane potential. Although previous publications suggested that Rb was not necessary for cell cycle control in heart, we find following acute knockdown of Rb that this factor actively regulates progression through the G1 checkpoint and that its loss promotes proliferation at the expense of CM maturation. Conclusions/Significance: We have established a unique model system for studying cardiac cell cycle progression, and show in contrast to previous reports that Rb actively regulates both cell cycle progression through the G1 checkpoint an

IDENTIFICATION AND CHARACTERIZATION OF HUMAN METABOLITES OF CAl [5-AMINO-I -1 (4'-CHLOROBENZOYL-3,5-DICHLOROBENZYL)-1 ,2,3-TRIAZOLE- 4-CARBOXAMIDE)

ABSTRACT: The calcium Influx inhibitor and cytostatic agent, 5-amlno-1-1(4'

Stepwise Maturation of Lytic Granules during Differentiation and Activation of Human CD8+ T Lymphocytes

During differentiation, cytotoxic T lymphocytes (CTL) acquire their killing potential through the biogenesis and maturation of lytic granules that are secreted upon target cell recognition. How lytic granule load in lytic molecules evolves during CTL differentiation and which subsets of lytic granules are secreted following activation remains to be investigated. We set up a flow cytometry approach to analyze single lytic granules isolated from primary human CTL according to their size and molecular content. During CTL in vitro differentiation, a relatively homogeneous population of lytic granules appeared through the progressive loading of Granzyme B, Perforin and Granzyme A within LAMP1+ lysosomes. PMA/ionomycin-induced lytic granule exocytosis was preceded by a rapid association of the docking molecule Rab27a to approximately half of the lytic granules. Activated CTL were found to limit exocytosis by sparing lytic granules including some associated to Rab27a. Our study provides a quantification of key steps of lytic granule biogenesis and highlights the potential of flow cytometry to study organelle composition and dynamics

Spinal Cord Injury Reveals Multilineage Differentiation of Ependymal Cells

Spinal cord injury often results in permanent functional impairment. Neural stem cells present in the adult spinal cord can be expanded in vitro and improve recovery when transplanted to the injured spinal cord, demonstrating the presence of cells that can promote regeneration but that normally fail to do so efficiently. Using genetic fate mapping, we show that close to all in vitro neural stem cell potential in the adult spinal cord resides within the population of ependymal cells lining the central canal. These cells are recruited by spinal cord injury and produce not only scar-forming glial cells, but also, to a lesser degree, oligodendrocytes. Modulating the fate of ependymal progeny after spinal cord injury may offer an alternative to cell transplantation for cell replacement therapies in spinal cord injury