Publications by authors named "Ido Sagi"

16 Publications

  • Page 1 of 1

RNA-Mediated Feedback Control of Transcriptional Condensates.

Cell 2021 01 16;184(1):207-225.e24. Epub 2020 Dec 16.

Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Electronic address:

Regulation of biological processes typically incorporates mechanisms that initiate and terminate the process and, where understood, these mechanisms often involve feedback control. Regulation of transcription is a fundamental cellular process where the mechanisms involved in initiation have been studied extensively, but those involved in arresting the process are poorly understood. Modeling of the potential roles of RNA in transcriptional control suggested a non-equilibrium feedback control mechanism where low levels of RNA promote condensates formed by electrostatic interactions whereas relatively high levels promote dissolution of these condensates. Evidence from in vitro and in vivo experiments support a model where RNAs produced during early steps in transcription initiation stimulate condensate formation, whereas the burst of RNAs produced during elongation stimulate condensate dissolution. We propose that transcriptional regulation incorporates a feedback mechanism whereby transcribed RNAs initially stimulate but then ultimately arrest the process.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2020.11.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8128340PMC
January 2021

Partitioning of cancer therapeutics in nuclear condensates.

Science 2020 06;368(6497):1386-1392

Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany.

The nucleus contains diverse phase-separated condensates that compartmentalize and concentrate biomolecules with distinct physicochemical properties. Here, we investigated whether condensates concentrate small-molecule cancer therapeutics such that their pharmacodynamic properties are altered. We found that antineoplastic drugs become concentrated in specific protein condensates in vitro and that this occurs through physicochemical properties independent of the drug target. This behavior was also observed in tumor cells, where drug partitioning influenced drug activity. Altering the properties of the condensate was found to affect the concentration and activity of drugs. These results suggest that selective partitioning and concentration of small molecules within condensates contributes to drug pharmacodynamics and that further understanding of this phenomenon may facilitate advances in disease therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.aaz4427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7735713PMC
June 2020

Distinct Imprinting Signatures and Biased Differentiation of Human Androgenetic and Parthenogenetic Embryonic Stem Cells.

Cell Stem Cell 2019 09;25(3):419-432.e9

Department of Obstetrics and Gynecology and Columbia University Fertility Center, Columbia University, College of Physicians & Surgeons, New York, NY 10032, USA; Department of Pediatrics, Naomi Berrie Diabetes Center, Columbia Stem Cell Initiative, Columbia University, New York, NY 10032, USA. Electronic address:

Genomic imprinting is an epigenetic mechanism that results in parent-of-origin monoallelic expression of specific genes, which precludes uniparental development and underlies various diseases. Here, we explored molecular and developmental aspects of imprinting in humans by generating exclusively paternal human androgenetic embryonic stem cells (aESCs) and comparing them with exclusively maternal parthenogenetic ESCs (pESCs) and bi-parental ESCs, establishing a pluripotent cell system of distinct parental backgrounds. Analyzing the transcriptomes and methylomes of human aESCs, pESCs, and bi-parental ESCs enabled the characterization of regulatory relations at known imprinted regions and uncovered imprinted gene candidates within and outside known imprinted regions. Investigating the consequences of uniparental differentiation, we showed the known paternal-genome preference for placental contribution, revealed a similar bias toward liver differentiation, and implicated the involvement of the imprinted gene IGF2 in this process. Our results demonstrate the utility of parent-specific human ESCs for dissecting the role of imprinting in human development and disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stem.2019.06.013DOI Listing
September 2019

Genome-wide Screen for Culture Adaptation and Tumorigenicity-Related Genes in Human Pluripotent Stem Cells.

iScience 2019 Jan 3;11:398-408. Epub 2019 Jan 3.

The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem, Israel. Electronic address:

Human pluripotent stem cells (hPSCs) acquire genetic changes during their propagation in culture that can affect their use in research and future therapies. To identify the key genes involved in selective advantage during culture adaptation and tumorigenicity of hPSCs, we generated a genome-wide screening system for genes and pathways that provide a growth advantage either in vitro or in vivo. We found that hyperactivation of the RAS pathway confers resistance to selection with the hPSC-specific drug PluriSIn-1. We also identified that inactivation of the RHO-ROCK pathway gives growth advantage during culture adaptation. Last, we demonstrated the importance of the PI3K-AKT and HIPPO pathways for the teratoma formation process. Our screen revealed key genes and pathways relevant to the tumorigenicity and survival of hPSCs and should thus assist in understanding and confronting their tumorigenic potential.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.isci.2018.12.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6348297PMC
January 2019

Mice from Same-Sex Parents: CRISPRing Out the Barriers for Unisexual Reproduction.

Cell Stem Cell 2018 11;23(5):625-627

The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem 91904, Israel. Electronic address:

Genomic imprinting results in the molecular and functional inequality of maternal and paternal alleles, precluding mammalian unisexual development. In this issue of Cell Stem Cell, Li et al. (2018) employ sophisticated manipulations of gametes and engineered haploid embryonic stem cells to successfully generate both all-maternal and all-paternal mice, effectively overcoming the roadblocks of imprinting.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stem.2018.10.012DOI Listing
November 2018

Defining essential genes for human pluripotent stem cells by CRISPR-Cas9 screening in haploid cells.

Nat Cell Biol 2018 05 16;20(5):610-619. Epub 2018 Apr 16.

The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem, Israel.

The maintenance of pluripotency requires coordinated expression of a set of essential genes. Using our recently established haploid human pluripotent stem cells (hPSCs), we generated a genome-wide loss-of-function library targeting 18,166 protein-coding genes to define the essential genes in hPSCs. With this we could allude to an intrinsic bias of essentiality across cellular compartments, uncover two opposing roles for tumour suppressor genes and link autosomal-recessive disorders with growth-retardation phenotypes to early embryogenesis. hPSC-enriched essential genes mainly encode transcription factors and proteins related to cell-cycle and DNA-repair, revealing that a quarter of the nuclear factors are essential for normal growth. Our screen also led to the identification of growth-restricting genes whose loss of function provides a growth advantage to hPSCs, highlighting the role of the P53-mTOR pathway in this context. Overall, we have constructed an atlas of essential and growth-restricting genes in hPSCs, revealing key aspects of cellular essentiality and providing a reference for future studies on human pluripotency.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41556-018-0088-1DOI Listing
May 2018

Haploidy in Humans: An Evolutionary and Developmental Perspective.

Dev Cell 2017 06;41(6):581-589

The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem 91904, Israel. Electronic address:

Although haploidy has not been observed in vertebrates, its natural occurrence in various eukaryotic species that had diverged from diploid ancestors suggests that there is an innate capacity for an organism to regain haploidy and that haploidy may confer evolutionary benefits. Haploid embryonic stem cells have been experimentally generated from mouse, rat, monkey, and humans. Haploidy results in major differences in cell size and gene expression levels while also affecting parental imprinting, X chromosome inactivation, and mitochondrial metabolism genes. We discuss here haploidy in evolution and the barriers to haploidy, in particular in the human context.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.devcel.2017.04.019DOI Listing
June 2017

Stem cells: Aspiring to naivety.

Nature 2016 12 30;540(7632):211-212. Epub 2016 Nov 30.

The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature20484DOI Listing
December 2016

Haploid Human Embryonic Stem Cells: Half the Genome, Double the Value.

Cell Stem Cell 2016 11;19(5):569-572

The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem 91904, Israel. Electronic address:

Recent advances in the generation of haploid embryonic stem cells (ESCs), capable of self-renewal and differentiation, have laid the groundwork for numerous biomedical applications in developmental biology and reproductive medicine. When combined with the power of genetic screening, haploid human ESCs could advance cancer research, regenerative medicine, and disease modeling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stem.2016.10.009DOI Listing
November 2016

Identification and propagation of haploid human pluripotent stem cells.

Nat Protoc 2016 Nov 20;11(11):2274-2286. Epub 2016 Oct 20.

The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem, Israel.

Haploid human pluripotent stem cells (PSCs) integrate haploidy and pluripotency, providing a novel system for functional genomics and developmental research in humans. We have recently derived haploid human embryonic stem cells (ESCs) by parthenogenesis and demonstrated their wide differentiation potential and applicability for genetic screening. Because haploid cells can spontaneously become diploid, their enrichment at an early passage is key for successful derivation. In this protocol, we describe two methodologies, namely metaphase spread analysis and cell sorting, for the identification of haploid human cells within parthenogenetic ESC lines. The cell sorting approach also enables the isolation of haploid cells at low percentages, as well as the maintenance of highly enriched haploid ESC lines throughout passaging. The isolation of essentially pure populations of haploid human ESCs by this protocol requires basic PSC culture expertise and can be achieved within 4-6 weeks.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nprot.2016.145DOI Listing
November 2016

Derivation and differentiation of haploid human embryonic stem cells.

Nature 2016 Apr 16;532(7597):107-11. Epub 2016 Mar 16.

The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem 91904, Israel.

Diploidy is a fundamental genetic feature in mammals, in which haploid cells normally arise only as post-meiotic germ cells that serve to ensure a diploid genome upon fertilization. Gamete manipulation has yielded haploid embryonic stem (ES) cells from several mammalian species, but haploid human ES cells have yet to be reported. Here we generated and analysed a collection of human parthenogenetic ES cell lines originating from haploid oocytes, leading to the successful isolation and maintenance of human ES cell lines with a normal haploid karyotype. Haploid human ES cells exhibited typical pluripotent stem cell characteristics, such as self-renewal capacity and a pluripotency-specific molecular signature. Moreover, we demonstrated the utility of these cells as a platform for loss-of-function genetic screening. Although haploid human ES cells resembled their diploid counterparts, they also displayed distinct properties including differential regulation of X chromosome inactivation and of genes involved in oxidative phosphorylation, alongside reduction in absolute gene expression levels and cell size. Surprisingly, we found that a haploid human genome is compatible not only with the undifferentiated pluripotent state, but also with differentiated somatic fates representing all three embryonic germ layers both in vitro and in vivo, despite a persistent dosage imbalance between the autosomes and X chromosome. We expect that haploid human ES cells will provide novel means for studying human functional genomics and development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature17408DOI Listing
April 2016

Pluripotent stem cells in disease modelling and drug discovery.

Nat Rev Mol Cell Biol 2016 Mar 28;17(3):170-82. Epub 2016 Jan 28.

The Azrieli Center for Stem Cells and Genetic Research, Institute of Life Sciences, Hebrew University of Jerusalem, Givat-Ram, Jerusalem 91904, Israel.

Experimental modelling of human disorders enables the definition of the cellular and molecular mechanisms underlying diseases and the development of therapies for treating them. The availability of human pluripotent stem cells (PSCs), which are capable of self-renewal and have the potential to differentiate into virtually any cell type, can now help to overcome the limitations of animal models for certain disorders. The ability to model human diseases using cultured PSCs has revolutionized the ways in which we study monogenic, complex and epigenetic disorders, as well as early- and late-onset diseases. Several strategies are used to generate such disease models using either embryonic stem cells (ES cells) or patient-specific induced PSCs (iPSCs), creating new possibilities for the establishment of models and their use in drug screening.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nrm.2015.27DOI Listing
March 2016

Comparable frequencies of coding mutations and loss of imprinting in human pluripotent cells derived by nuclear transfer and defined factors.

Cell Stem Cell 2014 Nov 6;15(5):634-42. Epub 2014 Nov 6.

The New York Stem Cell Foundation Research Institute, New York, NY 10032, USA; Naomi Berrie Diabetes Center & Department of Pediatrics, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA. Electronic address:

The recent finding that reprogrammed human pluripotent stem cells can be derived by nuclear transfer into human oocytes as well as by induced expression of defined factors has revitalized the debate on whether one approach might be advantageous over the other. Here we compare the genetic and epigenetic integrity of human nuclear-transfer embryonic stem cell (NT-ESC) lines and isogenic induced pluripotent stem cell (iPSC) lines, derived from the same somatic cell cultures of fetal, neonatal, and adult origin. The two cell types showed similar genome-wide gene expression and DNA methylation profiles. Importantly, NT-ESCs and iPSCs had comparable numbers of de novo coding mutations, but significantly more than parthenogenetic ESCs. As iPSCs, NT-ESCs displayed clone- and gene-specific aberrations in DNA methylation and allele-specific expression of imprinted genes. The occurrence of these genetic and epigenetic defects in both NT-ESCs and iPSCs suggests that they are inherent to reprogramming, regardless of derivation approach.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stem.2014.10.002DOI Listing
November 2014

The noncoding RNA IPW regulates the imprinted DLK1-DIO3 locus in an induced pluripotent stem cell model of Prader-Willi syndrome.

Nat Genet 2014 Jun 11;46(6):551-7. Epub 2014 May 11.

Stem Cell Unit, Department of Genetics, Institute of Life Sciences, The Hebrew University, Jerusalem, Israel.

Parental imprinting is a form of epigenetic regulation that results in parent-of-origin differential gene expression. To study Prader-Willi syndrome (PWS), a developmental imprinting disorder, we generated case-derived induced pluripotent stem cells (iPSCs) harboring distinct aberrations in the affected region on chromosome 15. In studying PWS-iPSCs and human parthenogenetic iPSCs, we unexpectedly found substantial upregulation of virtually all maternally expressed genes (MEGs) in the imprinted DLK1-DIO3 locus on chromosome 14. Subsequently, we determined that IPW, a long noncoding RNA in the critical region of the PWS locus, is a regulator of the DLK1-DIO3 region, as its overexpression in PWS and parthenogenetic iPSCs resulted in downregulation of MEGs in this locus. We further show that gene expression changes in the DLK1-DIO3 region coincide with chromatin modifications rather than DNA methylation levels. Our results suggest that a subset of PWS phenotypes may arise from dysregulation of an imprinted locus distinct from the PWS region.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ng.2968DOI Listing
June 2014

Human oocytes reprogram adult somatic nuclei of a type 1 diabetic to diploid pluripotent stem cells.

Nature 2014 Jun 28;510(7506):533-6. Epub 2014 Apr 28.

The New York Stem Cell Foundation Research Institute, New York, New York 10032, USA.

The transfer of somatic cell nuclei into oocytes can give rise to pluripotent stem cells that are consistently equivalent to embryonic stem cells, holding promise for autologous cell replacement therapy. Although methods to induce pluripotent stem cells from somatic cells by transcription factors are widely used in basic research, numerous differences between induced pluripotent stem cells and embryonic stem cells have been reported, potentially affecting their clinical use. Because of the therapeutic potential of diploid embryonic stem-cell lines derived from adult cells of diseased human subjects, we have systematically investigated the parameters affecting efficiency of blastocyst development and stem-cell derivation. Here we show that improvements to the oocyte activation protocol, including the use of both kinase and translation inhibitors, and cell culture in the presence of histone deacetylase inhibitors, promote development to the blastocyst stage. Developmental efficiency varied between oocyte donors, and was inversely related to the number of days of hormonal stimulation required for oocyte maturation, whereas the daily dose of gonadotropin or the total number of metaphase II oocytes retrieved did not affect developmental outcome. Because the use of concentrated Sendai virus for cell fusion induced an increase in intracellular calcium concentration, causing premature oocyte activation, we used diluted Sendai virus in calcium-free medium. Using this modified nuclear transfer protocol, we derived diploid pluripotent stem-cell lines from somatic cells of a newborn and, for the first time, an adult, a female with type 1 diabetes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature13287DOI Listing
June 2014

Involvement of parental imprinting in the antisense regulation of onco-miR-372-373.

Nat Commun 2013 ;4:2724

Stem Cell Unit, Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem 91904, Israel.

The monoallelic nature of imprinted genes renders them highly susceptible to genetic and epigenetic perturbations, potentially resulting in transformation and disease. Here we show, using parthenogenetic induced pluripotent stem cells, an imprinted transcript that serves as an antisense regulator of onco-miR-372-3 (named anti-miR-371-3). As miR-372-3 have been shown to have an oncogenic role in testicular germ cell tumours, we study the involvement of their antisense transcript in these cells. Our results suggest that hypermethylation, leading to loss-of-expression of the imprinted antisense transcript, contributes to tumorigenic transformation by affecting the downstream target LATS2. Finally, we provide evidence for a tumour suppressive role of anti-miR-371-3, as its overexpression in tumour cells results in cell growth arrest and apoptosis, and prevents tumour formation on injection into immunodeficient mice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms3724DOI Listing
July 2014
-->