Marion Dejosez

Caspase activity mediates the differentiation of embryonic stem cells

Embryonic stem cells (ESCs) are capable of indefinite self-renewal while retaining the ability to differentiate to any of the three germ layers that give rise to all somatic cell types. An emerging view is that a core set of transcription factors, including Oct4, Sox2, and Nanog, form a robust autoregulatory circuit that maintains ESCs in a self-renewing state. To accommodate the capacity of such cells to undergo germ layer-specific differentiation, we predicted a posttranslational mechanism that could negatively regulate these core self-renewal factors. Here we report caspase-induced cleavage of Nanog in differentiating ESCs. Stem cells lacking the Casp3 gene showed marked defects in differentiation, while forced expression of a caspase cleavage-resistant Nanog mutant in ESCs strongly promoted self-renewal. These results link a major component of the programmed cell-death pathway to the regulation of ESC development.

Ronin/Hcf-1 binds to a hyperconserved enhancer element and regulates genes involved in the growth of embryonic stem cells

Self-renewing embryonic stem (ES) cells have an exceptional need for timely biomass production, yet the transcriptional control mechanisms responsible for meeting this requirement are largely unknown. We report here that Ronin (Thap11), which is essential for the self-renewal of ES cells, binds with its transcriptional coregulator, Hcf-1, to a highly conserved enhancer element that previously lacked a recognized binding factor. The subset of genes bound by Ronin/Hcf-1 function primarily in transcription initiation, mRNA splicing, and cell metabolism; genes involved in cell signaling and cell development are conspicuously underrepresented in this target gene repertoire. Although Ronin/Hcf-1 represses the expression of some target genes, its activity at promoter sites more often leads to the up-regulation of genes essential to protein biosynthesis and energy production. We propose that Ronin/Hcf-1 controls a genetic program that contributes to the unimpeded growth of ES cells.

Safeguards for Cell Cooperation in Mouse Embryogenesis Shown by Genome-Wide Cheater Screen

Keeping Cells Cooperating Multicellular organisms have certain advantages over those that are single-celled. To evolve, however, they must surmount a persistent challenge: ensuring that their constituent cells cooperate with one another rather than “cheat” or compete with each other for resources. Dejosez et al. (p. 1511, published online 12 September) performed a genome-wide screen in induced pluripotent stem cells to search for genes that promote cell cooperation. A number of genes were identified of which knockdown allowed competitive behavior to dominate. These genes formed a network centered on p53, topoisomerase 1, and olfactory receptors. Thus, a genetic mechanism may promote cooperation in the developing embryo. During embryogenesis, a network of genes centered on p53, topoisomerase 1, and olfactory receptors helps to ensure cell cooperation. Ensuring cooperation among formerly autonomous cells has been a central challenge in the evolution of multicellular organisms. One solution is monoclonality, but this option still leaves room for exploitative behavior, as it does not eliminate genetic and epigenetic variability. We therefore hypothesized that embryonic development must be protected by robust regulatory mechanisms that prevent aberrant clones from superseding wild-type cells. Using a genome-wide screen in murine induced pluripotent stem cells, we identified a network of genes (centered on p53, topoisomerase 1, and olfactory receptors) whose down-regulation caused the cells to replace wild-type cells in vitro and in the mouse embryo—without perturbing normal development. These genes thus appear to fulfill an unexpected role in fostering cell cooperation.

The terminal complement complex inhibits apoptosis in vascular smooth muscle cells by activating an autocrine IGF-1 loop

Two counteracting processes determine accumulation of human vascular smooth muscle cells (SMCs) in atherosclerotic lesions: cell proliferation and apoptosis. SMCs synthesize insulin‐like growth factor‐1 (IGF‐1), which potently inhibits apoptosis. The terminal complement complex C5b‐9 interacts with SMCs in early human atherogenesis. In this study, we investigated whether C5b‐9 may activate the IGF‐1 system in SMCs, resulting in the inhibition of SMC apoptosis. C5b‐9 generation on SMCs in vitro markedly reduced CD95‐mediated apoptosis as assessed by flowcytometric analysis of annexin V binding and in caspase 3 assays. C5b‐9 induced both significant IGF‐1 release and up‐regulation of IGF‐1 binding sites in SMCs. Immunoneutralization of IGF‐1 with a monoclonal IGF‐1 antibody abolished the antiapoptotic effects of C5b‐9. We conclude that C5b‐9 inhibits apoptosis in SMCs by inducing an autocrine IGF‐1 loop. This mechanism may contribute to the accumulation of SMCs in early human atherosclerotic lesions.—Taylor, N. A., Van De Ven, W. J. M., Creemers, J. W. M. Curbing activation: proprotein convertases in homeostasis and pathology. FASEB J. 17, 1215–1227 (2003)

H1 and H9 Human Embryonic Stem Cell Lines Are Heterozygous for the ABO Locus

The establishment of human Embryonic Stem (ES) cell lines holds great promise for regenerative medicine (1). This is because ES cells can be propagated indefi nitely in vitro and they have the potential to differentiate into a variety of cell lineages (2). However, to use human ES cells and their derivatives for therapeutic cell transplantation, as it is in all other transplantation treatments, the fi rst obstacle encountered is the histocompatibility problem. One possible way to overcome this histocompatibility barrier is to genetically modify the histocompatibility loci within the human ES cell genome (3, 4). ABO is the most important histocompatibility locus that determines the ABO histo-blood type. The hyperacute rejection in an ABO-incompatible transfusion or solid organ transplantation may lead to a lethal consequence. Therefore it is important to avoid ABO incompatibility when transplanting human ES cell-derived tissues. Here we describe the characterization of ABO alleles in H1 and H9, two of the most widely distributed and researched human ES cell lines among the 78 human ES cell lines on the NIH Human Embryonic Stem Cell Registry (http://stemcells.nih. gov/research/registry/). Our fi ndings suggest that H1 is heterozygous for the ABO locus and bears two different O type alleles, O101 and O201. H9 carries ABO alleles of 2 different types, one is A1 type (A101) and the other is O type (O101). The ABO blood group system was fi rst described by Karl Landsteiner more than a century ago. It is a phenomenon observed only in Catarhinni (human, apes, and old word monkeys)(5). The presence of A and/or B antigens on the surface of the red blood cell, endothelium and almost all the epithelia is determined by different galactosyltransferase alleles carried in a highly polymorphic locus, the ABO locus (6–9). So far there are at least 180 ABO alleles in the Blood Group Antigen Gene Mutation Database (http://www.ncbi. nlm.nih.gov/projects/mhc/xslcgi.fcgi). Three types ABO alleles, A type, B type, and O type, are commonly found in the human population. The A type alleles encodes A transferase (α1-3 N-acetylgalactosaminyltransferase), which puts N-acetylgalactosamnine (UDP-GalNAc) to the H antigens (Fuctose-α1-2-Galactose-β1-R). The most common A allele found in the human population is A101, which is used as the reference sequence for the ABO gene. A101 also represents the A1 subtype alleles (responsible for 80% A phenotypes). There are A2 subtype alleles, observed at a lower frequency (about 20% of A phenotype), which create a phenotype with only about 1/5 A antigens on the surface of red blood cells compared to that of the A1 subtype. The O alleles are loss-offunction alleles. A frameshift mutation, 261ΔG, which results in the mistranslation of the glactosyltransferase, is found in the majority of the O alleles. The B alleles encode B transferase (α1-3 galactosyltransferase), which possesses different substrate specifi city. Instead of building an A antigen, B transferases use galactose (UDP-Gal) as a substrate to build B antigens. There are seven B allele-specifi c single nucleotide polymorphisms (SNPs), four of them (C526G, G703A, C796A, and G803C) fall within the enzyme catalytic domain and result in 4 amino acid changes in the substrate binding pocket, Arg176Gly, Gly235Ser, Leu266Met, and Gly268Ala, thus changing the substrate specifi city (10). In rare cases, cisAB (AAAB), which possesses both A transferase and B transferase activity, and B(A)(BABB), which possesses a strong B weak A transferase activity, are also observed. To examine the ABO genotypes of H1 and H9 human ES cells, genomic sequences of exon 6, 7, and intron 2 were amplifi ed by polymerase chain reactions (PCR) and sequenced. Related primer sequences used in this study were listed in Table 1. PCR were performed using a high fi delity DNA polymerase kit (Expand Long Template PCR System, Roche Applied Science, Indianapolis, IN) on genomic templates of H1 cells (passage 29) and H9 cells (passage 27) (WiCell,

Ronin influences the DNA damage response in pluripotent stem cells

Early mammalian embryonic cells must maintain a particularly robust DNA repair system, as mutations at this developmental point have detrimental consequences for the organism. How the repair system can be tuned to fulfill such elevated requirements is largely unknown, but it may involve transcriptional regulation. Ronin (Thap11) is a transcriptional regulator responsible for vital programs in pluripotent cells. Here, we report that this protein also modulates the DNA damage response of such cells. We show that conditional Ronin knockout sensitizes embryonic stem cells (ESCs) to UV-C-induced DNA damage in association with Atr pathway activation and G2/M arrest. Ronin binds to and regulates the genes encoding several DNA repair factors, including Gtf2h4 and Rad18, providing a potential mechanism for this phenotype. Our results suggest that the unique DNA repair requirements of the early embryo are not met by a static system, but rather via highly regulated processes.