Academic literature on the topic '2C-like cell'

In mouse embryonic stem cell (ESC), a small cell population displays totipotent features by expressing a set of genes that are transiently active in 2-cell–stage embryos. These 2-cell–like (2C-like) cells spontaneously transit back into the pluripotent state. We previously dissected the transcriptional dynamics of the transition from pluripotency to the totipotent 2C-like state and identified factors that modulate the process. However, how 2C-like cells transit back into the pluripotent state remains largely unknown. In this study, we analyzed the transcriptional dynamics from the 2C-like state to pluripotent ESCs and identified an intermediate state. The intermediate state characterized by two-wave step up-regulation of pluripotent genes is different from the one observed during the 2C-like entry transition. Nonsense-mediated Dux mRNA decay plays an important role in the 2C-like state exit. Thus, our study not only provides a transcriptional roadmap for 2C-like–to–pluripotent state transition but also reveals a key molecular event driving the transition.

Upon fertilization, the mammalian embryo must switch from dependence on maternal transcripts to transcribing its own genome, and in mice this involves the transient up-regulation of MERVL transposons and MERVL-driven genes at the two-cell stage. The mechanisms and requirement for MERVL and two-cell (2C) gene up-regulation are poorly understood. Moreover, this MERVL-driven transcriptional program must be rapidly shut off to allow two-cell exit and developmental progression. Here, we report that robust ribosomal RNA (rRNA) synthesis and nucleolar maturation are essential for exit from the 2C state. 2C-like cells and two-cell embryos show similar immature nucleoli with altered structure and reduced rRNA output. We reveal that nucleolar disruption via blocking RNA polymerase I activity or preventing nucleolar phase separation enhances conversion to a 2C-like state in embryonic stem cells (ESCs) by detachment of the MERVL activator Dux from the nucleolar surface. In embryos, nucleolar disruption prevents proper nucleolar maturation and Dux silencing and leads to two- to four-cell arrest. Our findings reveal an intriguing link between rRNA synthesis, nucleolar maturation, and gene repression during early development.

Zinc finger protein Zscan4 is selectively expressed in mouse two-cell (2C) embryos undergoing zygotic genome activation (ZGA) and in a rare subpopulation of embryonic stem cells with 2C-like features. Here, we show that Zscan4 specifically recognizes a subset of (CA)n microsatellites, repeat sequences prone to genomic instability. Zscan4-associated microsatellite regions are characterized by low nuclease sensitivity and high histone occupancy. In vitro, Zscan4 binds nucleosomes and protects them from disassembly upon torsional strain. Furthermore, Zscan4 depletion leads to elevated DNA damage in 2C mouse embryos in a transcription-dependent manner. Together, our results identify Zscan4 as a DNA sequence–dependent microsatellite binding factor and suggest a developmentally regulated mechanism, which protects fragile genomic regions from DNA damage at a time of embryogenesis associated with high transcriptional burden and genomic stress.

BackgroundOvarian cancer (OC) responds poorly to immunotherapies. Regulatory T cells (Treg) engage IL-2 by high-affinity CD25 for differentiation and function,1 and anti-tumor effector T cells (Teff) use intermediate affinity CD122. We studied IL-2 complexes (IL-2c) that selectively activate CD122 (Teff) over CD25 (Tregs).MethodsOrthotopic ID8agg-luc mouse OC burden was measured by in vivo imaging. Tumor, ascites and draining lymph nodes (TDLN) were analyzed by flow and tSNE. IL-2c was complexed using 1.5 µg/mouse IL-2 and 7.5 µg/mouse aIL-2 (clone JES6-5H4) before i.p. injection every other day x 4 starting at day 7. antiPD-L1 was given at 100ug/mouse every 3 days x 4 starting from Day 11. FIR mice2 were used to sort live Tregs.ResultsIL-2c but not antiPD-L1 potently inhibits ID8agg (figure 1). IL-2c decreased ascites Treg functional markers (e.g., CD25, granzymeB) while upregulating the same markers on Teffs (figure 2). IL-2c inhibited Treg suppression in ascites while TDLN Tregs were unaffected (figure 3). tSNE showed great similarity of TDLN Tregs treated with isotype and IL-2c while ascites Tregs after IL-2c showed a fragile phenotype (e.g., increased PD-1, T-bet, and IFNgamma with maintained FoxP3 expression [figure 4]) which is known to contribute to better response to cancer immunotherapy.3 4 We observed a complete reduction of tumor bioluminescence with IL-2c and antiPD-L1 combo treatment in nearly all subjects significantly exceeding effect of IL-2c alone (figure 5). A CD8+CXCR5+TCF-1+ T cell stem cell (TCSC) population reportedly improves immune checkpoint blockade efficacy.5 6 Since CD122 is regulated by TCF-1,7 we explored the effect of IL-2c on these TCSC. IL-2c significantly induced a CD8+TCF-1+ TCSC population in ID8agg tumors (figure 6), possibly through a positive feedback loop by further enhancing CD122 expression on TCF-1+, but not TCF-1- cells (figure 7). tSNE analysis of detailed immune phenotype of IL-2c induced TCSC revealed that these TCSC differed from those induced by antiPD-L1. In ID8agg, antiPD-L1-induced TCSC are mostly CXCR5+ and PD1+, consistent with previous reports in other cancers3 4 while IL-2c-induced TCSC were PD1- (figure 8), expressed CCR2 and CXCR3, and produced TNFalpha (figure 9).Abstract 690 Figure 1IL-2c but not aPD-L1 treats ID8aggLuciferase signal of ID8agg-luc tumors treated with isotype, ?PD-L1, or IL-2c measured by in vivo imaging. Arrows indicate treatments.Abstract 690 Figure 2IL-2c inhibits functional markers on tregs and promotes teffExpression of CD25 and granzymeB were measured by flow cytometry in indicated population from isotype or IL-2c treated ascites 3 weeks after final IL-2c dose.Abstract 690 Figure 3IL-2c reduces ascites Treg suppressive functionTregs sorted from ascites or TDLN of isotype or IL-2c treated FIR mice co-cultured with Teff cells. Percent suppression of Teff cells proliferation shown.Abstract 690 Figure 4IL-2c induces Treg fragility in ascites but not TDLN tSNE analysis on CD45+CD3+CD4+FoxP3+ cells from ascites and TDLN of isotype and IL-2c treated ID8agg-luc challenged mice. Right, representative bar graphs of flow data.Abstract 690 Figure 5IL-2c and aPD-L1 combo treatment effectively cures ID8aggLuciferase signal of ID8agg-luc tumors treated with isotype, ?PD-L1, IL-2c or combo measured by in vivo imaging. Arrows indicate treatments.Abstract 690 Figure 6IL-2c induces a CD8+TCF-1+ population in ID8aggID8agg-luc tumors analyzed by flow cytometry gated on CD45+CD3+CD8+ cells.Abstract 690 Figure 7IL-2c induces CD122 on CD8+TCF-1+ but not TCF-1- cellsCD122 expression was measured by flow cytometry in CD8+TCF-1+ and CD8+ TCF-1- cells from tumors treated with isotype or IL-2c.Abstract 690 Figure 8CD8+TCF1+ cells from ID8agg analyzed by flow cytometry plus tSNE. Cells expressing CXCR5 (blue) and PD1 (red) indicated in overlayAbstract 690 Figure 9CD8+TCF1+ cells from ID8agg analyzed by flow cytometryConclusionsWe define two novel IL-2c effects: inducing Treg fragility therefore reducing immunosuppression while promoting TCSC that could enhance effective anti-tumor immunity. Current work tests if effects are related and help efficacy, and mechanisms for IL-2c Treg effects. We also show that elicited TCSC differ by treatment and tumor, requiring additional investigations.AcknowledgementsThis work is supported by CPRIT Research Training Award (RP 170345), Ovarian Cancer Research Alliance Ann and Sol Schreiber Mentored Investigator Award to YD and R01 CA205965to TC.Ethics ApprovalAll mice studies were approved by UT Health San Antonio Institutional Animal Care and Use Committee (IACUC). Approval number 20150093AR, 20140001AR, 20170035AR, 20140039AR, 20140027AR, 20090128AR, 20120071AR, 20180021AR.ReferencesMalek TR: The biology of interleukin-2. Annu Rev Immunol 2008, 26:453–479.Fantini MC, Dominitzki S, Rizzo A, Neurath MF, Becker C: In vitro generation of CD4+ CD25+ regulatory cells from murine naive T cells. Nat Protoc 2007, 2(7):1789–1794.Overacre-Delgoffe AE, Chikina M, Dadey RE, Yano H, Brunazzi EA, Shayan G, Horne W, Moskovitz JM, Kolls JK, Sander C, et al: Interferon-gamma Drives Treg Fragility to Promote Anti-tumor Immunity. Cell 2017, 169(6):1130–1141e1111.Overacre-Delgoffe AE, Vignali DAA: Treg Fragility: A Prerequisite for Effective Antitumor Immunity? Cancer Immunol Res 2018, 6(8):882–887.Brummelman J, Mazza EMC, Alvisi G, Colombo FS, Grilli A, Mikulak J, Mavilio D, Alloisio M, Ferrari F, Lopci E, et al: High-dimensional single cell analysis identifies stem-like cytotoxic CD8(+) T cells infiltrating human tumors. J Exp Med 2018.Sade-Feldman M, Yizhak K, Bjorgaard SL, Ray JP, de Boer CG, Jenkins RW, Lieb DJ, Chen JH, Frederick DT, Barzily-Rokni M, et al: Defining T Cell States Associated with Response to Checkpoint Immunotherapy in Melanoma. Cell 2018, 175(4):998–1013 e1020.Jeevan-Raj B, Gehrig J, Charmoy M, Chennupati V, Grandclement C, Angelino P, Delorenzi M, Held W: The Transcription Factor Tcf1 Contributes to Normal NK Cell Development and Function by Limiting the Expression of Granzymes. Cell Rep 2017, 20(3):613–626.

The chalcogenide compound CuNbGaSe3, belonging to the system I-II-III-VI3, has been investigated by means of X-ray powder diffraction and its crystal structure has been refined by the Rietveld method.This is a material of the semiconductor type, which improves the properties of a simple semiconductor like CuGaSe2 because it ads spintronic applications due to its magnetic behavior. The powder pattern was composed by 94.2% of the principal phase CuNbGaSe3 and 5.8% of the secondary phase Cu0.667NbSe2. This material crystallizes with a CuFeInSe3-type structure in the tetragonal space group P4 2c (Nº 112), unit cell parameters a = 5.6199(4) Å, c = 11.0275(2) Å, V = 348.28(4) Å3, with a normal adamantane-structure where occurs a degradation of symmetry from the chalcopyrite structure I4 2d to a related structure P4 2c.

Minor splicing plays an important role in vertebrate development. Zrsr1 and Zrsr2 paralog genes have essential roles in alternative splicing, mainly participating in the recognition of minor (U12) introns. To further explore their roles during early embryo development, we produced Zrsr1mu and Zrsr2mu mutant mice, containing truncating mutations within the second zinc finger domain. Both homozygous mutant mice were viable with a normal lifespan. When we crossed a homozygous Zrsr2mu/mu female with Zrsr1mu/mu male, the double heterozygotes were non-viable, giving rise to embryos that stopped developing mainly between the 2- and 4-cell stages, just after zygotic gene activation. RNA-seq analysis of Zrsr1/2mu 2-cell embryos showed altered gene and isoform expression of thousands of genes enriched in gene ontology terms and biological pathways related to ribosome, RNA transport, spliceosome, and essential zygotic gene activation steps. Alternative splicing was analyzed, showing a significant increase in intron retention in both U2 and U12 intron-containing genes related to cell cycle and mitotic nuclear division. Remarkably, both Zrsr1 and Zrsr2 were required for the conversion of mouse-induced pluripotent stem cells into 2C-like cells. According to our results, Zrsr1 or Zrsr2 are necessary for ZGA and both are indispensable for the conversion of induced pluripotent stem cells into 2C-like cells.

Embryonic stem cells (ESCs) present a characteristic pluripotency heterogeneity correspondent to specific metastates. We recently demonstrated that retinoic acid (RA) induces an increase in a specific 2C-like metastate marked by target genes specific to the two-cell embryo stage in preimplantation. Prame (Preferentially expressed antigen in melanoma) is one of the principal actors of the pluripotency stage with a specific role in RA responsiveness. Additionally, PRAME is overexpressed in a variety of cancers, but its molecular functions are poorly understood. To further investigate Prame’s downstream targets, we used a chromatin immunoprecipitation sequencing (ChIP-seq) assay in RA-enriched 2C-like metastates and identified two specific target genes, Cdk8 and Cdkn2d, bound by Prame. These two targets, involved in cancer dedifferentiation and pluripotency, have been further validated in RA-resistant ESCs. Here, we observed for the first time that Prame controls the Cdk8 and Cdkn2d genes in ESCs after RA treatment, shedding light on the regulatory network behind the establishment of naïve pluripotency.

Embryonic stem cells (ESCs) are characterized by a rapid cell cycle, which leads to high replication stress (RS) in otherwise unperturbed conditions. The mechanisms that ESCs adopt to cope with their endogenous RS, however, remain to this day elusive. In our recent work we demonstrated that the activation of the checkpoint kinase ATR in response to RS leads to a broad activation of 2-cells stage specific genes in mouse ESCs. This response relies on the up-regulation of Dux, a transcription factor encoded in a macrosatellite sequence repeated in tandem. Dux is repressed by variant Polycomb repressive complex 1 (vPRC1) in unperturbed ESCs, independently from PRC2 presence. Here we demonstrate that RS causes a major rearrangement of both PRC1 and PRC2 in ESCs nuclei, resulting in a major loss of both repressive marks in correspondence to target promoters. Surprisingly, Dux undergoes an increase in vPRC1 occupancy upon RS in an ATR-dependent manner, possibly due to PRC1 involvement in the replication of highly repeated DNA sequences. More interestingly, Dux activation upon RS requires the presence of PRC2. This result is possibly due to PRC2 proved role in the processing of stalled replication forks, which are the main structure signaling RS. In agreement to this data, also the fork remodeling translocases HLTF and ZRANB3 displayed an effect in Dux activation following RS. Taken together, our results show that the up-regulation of 2-cells genes following RS not only requires ATR activation, but also downstream remodeling processes.

Chromocenters are established after the 2-cell (2C) stage during mouse embryonic development, but the factors that mediate chromocenter formation remain largely unknown. To identify regulators of 2C heterochromatin establishment, we generated an inducible system to convert embryonic stem cells (ESCs) to 2C-like cells, thus modeling early embryogenesis in vitro. This conversion is marked by a global reorganization of H3K9me3-heterochromatin foci, which are then reversibly formed upon re-entry into pluripotency. Profiling the chromatin-bound proteome of ESCs transitioning to 2C-like cells, we uncover chromatin regulators involved in de novo heterochromatin formation and a relationship between cell cycle regulation and the establishment of the 2C-like state. We identified SMARCAD1, which associates with H3K9me3-heterochromatin in ESCs, but its nuclear localization is lost in 2C-like cells. SMARCAD1 depletion in mouse embryos leads to developmental arrest and loss of H3K9me3. Collectively, our findings contribute to comprehending the establishment of chromocenters during early development, a key step to instruct the embryonic totipotent program toward pluripotency.
Los cromocentros se forman después del estadio de 2 células (2C) durante el desarrollo embrionario de ratón, pero los factores que median su formación siguen siendo en gran medida desconocidos. Para identificar los reguladores del establecimiento de la heterocromatina 2C, generamos un sistema inducible capaz de convertir células madre embrionarias (ESCs) en células similares a embriones 2C (células 2C-like), modelando así el desarrollo temprano in vitro. Esta conversión está caracterizada por una reorganización global de las regiones heterocromáticas marcadas por H3K9me3, las cuales se forman de nuevo al volver a la pluripotencia. Describimos el proteoma unido a la cromatina de ESCs reprogramándose a células 2C-like donde descubrimos reguladores de la cromatina implicados en la formación de novo de heterocromatina así como una relación entre la regulación del ciclo celular y el establecimiento del estado 2C-like. Identificamos el factor SMARCAD1, el cual se asocia con la heterocromatina marcada por H3K9me3 en ESCs, pero su localización nuclear se pierde en las células 2C-like. Finalmente, eliminamos SMARCAD1 en embriones de ratón observando una detención en su desarrollo y una pérdida de H3K9me3. Estos hallazgos contribuyen a comprender el establecimiento de los cromocentros durante el desarrollo temprano, un paso crucial para instruir el programa totipotente embrionario hacia la pluripotencia.

This study extends a micromechanics approach based upon the computational cell methodology to model ductile crack extension of longitudinal crack-like defects in a high strength pipeline steel. Laboratory testing of an API 5L X60 steel at room temperature using standard, deep crack C(T) specimens provide the data needed to measure the crack growth resistance curve for the material. In the computational cell model, ductile crack extension occurs through void growth and coalescence (by cell extinction) within a thin layer of material ahead of crack tip. A simple scheme to calibrate material-specific parameters for the cells is also described. A central focus of the paper is the application of the cell methodology to predict experimentally measured burst pressures for pre-cracked pipe specimens with different crack sizes. The experimental program includes longitudinally precracked 20” (508 mm) O.D. pipe specimens with 15.8 mm thickness containing an internal crack with notch depth (a) and notch length (2c) 7 × 140 mm. Large-scale, full 3-D computations are conducted on detailed finite element models for the pipe specimens to describe crack extension with increased pressure. The numerical simulations demonstrate the effectiveness of the cell approach to describe crack growth response and to predict the burst pressure for the tested pipes.