24 results on '"Michelle Percharde"'
Search Results
2. Depletion of nuclear LINE1 RNA in mouse ESCs and embryos
- Author
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Michelle Percharde, Chih-Jen Lin, and Miguel Ramalho-Santos
- Subjects
developmental biology ,microscopy ,molecular biology ,gene expression ,in situ hybridization ,stem cells ,Science (General) ,Q1-390 - Abstract
Summary: LINE1 is the most active and abundant family of retrotransposons; it is implicated in a number of pathologies, as well as in early embryo development. We present a protocol to specifically knockdown LINE1 in mouse embryonic stem cells and embryos, including details for the nucleofection and zygote microinjection of LINE antisense oligos, followed by RNA FISH validation. This protocol can be used in development, as well as other cell types where LINE1 is believed to be expressed.For complete information on the use and execution of this protocol, please refer to Percharde et al. (2018).
- Published
- 2021
- Full Text
- View/download PDF
3. Delineating the early transcriptional specification of the mammalian trachea and esophagus
- Author
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Akela Kuwahara, Ace E Lewis, Coohleen Coombes, Fang-Shiuan Leung, Michelle Percharde, and Jeffrey O Bush
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trachea ,esophagus ,Nkx2-1 ,Sox2 ,tracheoesophageal fistula ,lung ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
The genome-scale transcriptional programs that specify the mammalian trachea and esophagus are unknown. Though NKX2-1 and SOX2 are hypothesized to be co-repressive master regulators of tracheoesophageal fates, this is untested at a whole transcriptomic scale and their downstream networks remain unidentified. By combining single-cell RNA-sequencing with bulk RNA-sequencing of Nkx2-1 mutants and NKX2-1 ChIP-sequencing in mouse embryos, we delineate the NKX2-1 transcriptional program in tracheoesophageal specification, and discover that the majority of the tracheal and esophageal transcriptome is NKX2-1 independent. To decouple the NKX2-1 transcriptional program from regulation by SOX2, we interrogate the expression of newly-identified tracheal and esophageal markers in Sox2/Nkx2-1 compound mutants. Finally, we discover that NKX2-1 binds directly to Shh and Wnt7b and regulates their expression to control mesenchymal specification to cartilage and smooth muscle, coupling epithelial identity with mesenchymal specification. These findings create a new framework for understanding early tracheoesophageal fate specification at the genome-wide level.
- Published
- 2020
- Full Text
- View/download PDF
4. Genomic Repeats Categorize Genes with Distinct Functions for Orchestrated Regulation
- Author
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J. Yuyang Lu, Wen Shao, Lei Chang, Yafei Yin, Tong Li, Hui Zhang, Yantao Hong, Michelle Percharde, Lerui Guo, Zhongyang Wu, Lichao Liu, Wei Liu, Pixi Yan, Miguel Ramalho-Santos, Yujie Sun, and Xiaohua Shen
- Subjects
Biology (General) ,QH301-705.5 - Abstract
Summary: Repetitive elements are abundantly distributed in mammalian genomes. Here, we reveal a striking association between repeat subtypes and gene function. SINE, L1, and low-complexity repeats demarcate distinct functional categories of genes and may dictate the time and level of gene expression by providing binding sites for different regulatory proteins. Importantly, imaging and sequencing analysis show that L1 repeats sequester a large set of genes with specialized functions in nucleolus- and lamina-associated inactive domains that are depleted of SINE repeats. In addition, L1 transcripts bind extensively to its DNA in embryonic stem cells (ESCs). Depletion of L1 RNA in ESCs leads to relocation of L1-enriched chromosomal segments from inactive domains to the nuclear interior and de-repression of L1-associated genes. These results demonstrate a role of L1 DNA and RNA in gene silencing and suggest a general theme of genomic repeats in orchestrating the function, regulation, and expression of their host genes. : Lu et al. report a striking association between genomic repeats and gene regulation and demonstrate a key role of L1 repeat RNA in sequestering L1-rich sequences and associated genes in inactive domains for silencing, revealing a general theme of repeat sequences in shaping gene regulatory networks within their host genome. Keywords: repetitive elements, L1, B1 or Alu, SINE, low-complexity repeats, gene function, regulation and expression, nucleolus and lamina-associated domains, L1 RNA, gene silencing
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- 2020
- Full Text
- View/download PDF
5. Global Hypertranscription in the Mouse Embryonic Germline
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Michelle Percharde, Priscilla Wong, and Miguel Ramalho-Santos
- Subjects
hypertranscription ,primordial germ cells ,germline ,Myc ,n-Myc ,l-Myc ,P-TEFb ,cell competition ,Biology (General) ,QH301-705.5 - Abstract
Primordial germ cells (PGCs) are vital for inheritance and evolution. Their transcriptional program has been extensively studied and is assumed to be well known. We report here a remarkable global upregulation of the transcriptome of mouse PGCs compared to somatic cells. Using cell-number-normalized genome-wide analyses, we uncover significant transcriptional amplification in PGCs, including mRNAs, rRNA, and transposable elements. Hypertranscription preserves tissue-specific gene expression patterns, correlates with cell size, and can still be detected in E15.5 male germ cells when proliferation has ceased. PGC hypertranscription occurs at the level of nascent transcription, is accompanied by increased translation rates, and is driven by Myc factors n-Myc and l-Myc (but not c-Myc) and by P-TEFb. This study provides a paradigm for transcriptional analyses during development and reveals a major global hyperactivity of the germline transcriptome.
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- 2017
- Full Text
- View/download PDF
6. YAP Induces Human Naive Pluripotency
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Han Qin, Miroslav Hejna, Yanxia Liu, Michelle Percharde, Mark Wossidlo, Laure Blouin, Jens Durruthy-Durruthy, Priscilla Wong, Zhongxia Qi, Jingwei Yu, Lei S. Qi, Vittorio Sebastiano, Jun S. Song, and Miguel Ramalho-Santos
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embryonic stem cells (ESCs) ,induced pluripotent stem cells (iPSCs) ,pluripotent stem cells (PSCs) ,naive pluripotency ,Hippo pathway ,Yes-associated protein (YAP) ,lysophosphatidic acid (LPA) ,Biology (General) ,QH301-705.5 - Abstract
The human naive pluripotent stem cell (PSC) state, corresponding to a pre-implantation stage of development, has been difficult to capture and sustain in vitro. We report that the Hippo pathway effector YAP is nuclearly localized in the inner cell mass of human blastocysts. Overexpression of YAP in human embryonic stem cells (ESCs) and induced PSCs (iPSCs) promotes the generation of naive PSCs. Lysophosphatidic acid (LPA) can partially substitute for YAP to generate transgene-free human naive PSCs. YAP- or LPA-induced naive PSCs have a rapid clonal growth rate, a normal karyotype, the ability to form teratomas, transcriptional similarities to human pre-implantation embryos, reduced heterochromatin levels, and other hallmarks of the naive state. YAP/LPA act in part by suppressing differentiation-inducing effects of GSK3 inhibition. CRISPR/Cas9-generated YAP−/− cells have an impaired ability to form colonies in naive but not primed conditions. These results uncover an unexpected role for YAP in the human naive state, with implications for early human embryology.
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- 2016
- Full Text
- View/download PDF
7. Nucleolar-based Dux repression is essential for embryonic two-cell stage exit
- Author
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Sheila Q. Xie, Bryony J. Leeke, Chad Whilding, Ryan T. Wagner, Ferran Garcia-Llagostera, YiXuan Low, Paul Chammas, Nathan T.-F. Cheung, Dirk Dormann, Michael T. McManus, and Michelle Percharde
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Genetics ,Developmental Biology - Abstract
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.
- Published
- 2022
8. Nucleolar-based Dux repression is essential for 2-cell stage exit
- Author
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Michelle Percharde, Ferran Garcia-Llagostera, Ryan T. Wagner, Bryony J Leeke, Michael T. McManus, Sheila Q Xie, Dirk Dormann, Nathan T-F Cheung, Chad Whilding, and Paul Chammas
- Subjects
Downregulation and upregulation ,Activator (genetics) ,Nucleolus ,RNA polymerase I ,Gene silencing ,Biology ,Gene ,Psychological repression ,Embryonic stem cell ,Cell biology - Abstract
Upon fertilisation, the mammalian embryo must switch from dependence on maternal transcripts to transcribing its own genome, and in mice involves the transient upregulation of MERVL transposons and MERVL-driven genes at the 2-cell stage. The mechanisms and requirement for MERVL and 2-cell (2C) gene upregulation are poorly understood. Moreover, this MERVL-driven transcriptional program must be rapidly shut off to allow 2C 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 2C embryos show similar immature nucleoli with altered structure and reduced rRNA output. We reveal that nucleolar disruption via blocking Pol 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 Dux silencing and leads to 2-4 cell arrest. Our findings reveal an intriguing link between rRNA synthesis, nucleolar maturation and gene repression during early development.
- Published
- 2021
9. Nucleolar-based
- Author
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Sheila Q, Xie, Bryony J, Leeke, Chad, Whilding, Ryan T, Wagner, Ferran, Garcia-Llagostera, YiXuan, Low, Paul, Chammas, Nathan T-F, Cheung, Dirk, Dormann, Michael T, McManus, and Michelle, Percharde
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Mammals ,Mice ,Genome ,RNA, Ribosomal ,Animals ,Embryonic Development ,Embryo, Mammalian ,Cell Nucleolus ,Embryonic Stem Cells - Abstract
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
- Published
- 2021
10. Maternal vitamin C regulates reprogramming of DNA methylation and germline development
- Author
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Kathryn Mesh, Michalis Agathocleous, Diana J. Laird, Marie Justine Guerquin, Kevin T. Ebata, Swetha Mahesula, Gabriel Livera, Evelyne Collignon, Estelle Wall, Stephanie P. DiTroia, Miguel Ramalho-Santos, and Michelle Percharde
- Subjects
Epigenomics ,Cell Count ,Ascorbic Acid ,Biology ,Germline ,Transcriptome ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Loss of Function Mutation ,Pregnancy ,Proto-Oncogene Proteins ,Animals ,Epigenetics ,030304 developmental biology ,0303 health sciences ,Multidisciplinary ,Methylation ,DNA Methylation ,Cell biology ,DNA-Binding Proteins ,Meiosis ,Germ Cells ,DNA demethylation ,Models, Animal ,DNA methylation ,Ascorbic Acid Deficiency ,Female ,Reprogramming ,030217 neurology & neurosurgery - Abstract
Development is often assumed to be hardwired in the genome, but several lines of evidence indicate that it is susceptible to environmental modulation with potential long-term consequences, including in mammals1,2. The embryonic germline is of particular interest because of the potential for intergenerational epigenetic effects. The mammalian germline undergoes extensive DNA demethylation3-7 that occurs in large part by passive dilution of methylation over successive cell divisions, accompanied by active DNA demethylation by TET enzymes3,8-10. TET activity has been shown to be modulated by nutrients and metabolites, such as vitamin C11-15. Here we show that maternal vitamin C is required for proper DNA demethylation and the development of female fetal germ cells in a mouse model. Maternal vitamin C deficiency does not affect overall embryonic development but leads to reduced numbers of germ cells, delayed meiosis and reduced fecundity in adult offspring. The transcriptome of germ cells from vitamin-C-deficient embryos is remarkably similar to that of embryos carrying a null mutation in Tet1. Vitamin C deficiency leads to an aberrant DNA methylation profile that includes incomplete demethylation of key regulators of meiosis and transposable elements. These findings reveal that deficiency in vitamin C during gestation partially recapitulates loss of TET1, and provide a potential intergenerational mechanism for adjusting fecundity to environmental conditions.
- Published
- 2019
11. Homotypic clustering of L1 and B1/Alu repeats compartmentalizes the 3D genome
- Author
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Ting Wang, Pixi Yan, Peizhe Wang, Jie Na, Michelle Percharde, Qi Peng, Xue Han, Yantao Hong, Yibing Tao, Wen Shao, Jing Zhang, Ke Zhang, Yujie Sun, Xiaohua Shen, Zhongyang Wu, Runze Ma, Yingping Hou, Liang Wang, Wenzhi Li, Ge Zhan, Lei Chang, Hui Zhang, Yafei Yin, Pilong Li, Zai Chang, Xianju Bi, Tong Li, J. Yuyang Lu, Miguel Ramalho-Santos, Bing Zhu, and Wei Xie
- Subjects
Euchromatin ,Retroelements ,Heterochromatin ,Alu element ,Retrotransposon ,Biology ,Chromatin structure ,Genome ,Article ,chemistry.chemical_compound ,Mice ,Transposition ,Animals ,Cluster Analysis ,Molecular Biology ,Repetitive Sequences, Nucleic Acid ,Regulation of gene expression ,Nuclear organization ,Cell Biology ,Research Highlight ,Chromatin ,Cell biology ,Long Interspersed Nucleotide Elements ,chemistry ,RNA ,DNA - Abstract
Organization of the genome into euchromatin and heterochromatin appears to be evolutionarily conserved and relatively stable during lineage differentiation. In an effort to unravel the basic principle underlying genome folding, here we focus on the genome itself and report a fundamental role for L1 (LINE1 or LINE-1) and B1/Alu retrotransposons, the most abundant subclasses of repetitive sequences, in chromatin compartmentalization. We find that homotypic clustering of L1 and B1/Alu demarcates the genome into grossly exclusive domains, and characterizes and predicts Hi-C compartments. Spatial segregation of L1-rich sequences in the nuclear and nucleolar peripheries and B1/Alu-rich sequences in the nuclear interior is conserved in mouse and human cells and occurs dynamically during the cell cycle. In addition, de novo establishment of L1 and B1 nuclear segregation is coincident with the formation of higher-order chromatin structures during early embryogenesis and appears to be critically regulated by L1 and B1 transcripts. Importantly, depletion of L1 transcripts in embryonic stem cells drastically weakens homotypic repeat contacts and compartmental strength, and disrupts the nuclear segregation of L1- or B1-rich chromosomal sequences at genome-wide and individual sites. Mechanistically, nuclear co-localization and liquid droplet formation of L1 repeat DNA and RNA with heterochromatin protein HP1α suggest a phase-separation mechanism by which L1 promotes heterochromatin compartmentalization. Taken together, we propose a genetically encoded model in which L1 and B1/Alu repeats blueprint chromatin macrostructure. Our model explains the robustness of genome folding into a common conserved core, on which dynamic gene regulation is overlaid across cells.
- Published
- 2020
12. Author response: Delineating the early transcriptional specification of the mammalian trachea and esophagus
- Author
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Coohleen Coombes, Jeffrey O. Bush, Michelle Percharde, Akela Kuwahara, Ace E. Lewis, and Fang-Shiuan Leung
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medicine.anatomical_structure ,medicine ,Anatomy ,Esophagus ,Biology - Published
- 2020
13. L1 and B1 repeats blueprint the spatial organization of chromatin
- Author
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Yantao Hong, Pilong Li, Miguel Ramalho-Santos, Jing Zhang, Ke Zhang, Yingping Hou, Wen Shao, Liang Wang, Wenzhi Li, Qi Peng, Jie Na, Zhongyang Wu, Wei Xie, Yujie Sun, Tong Li, Ting Wang, Pixi Yan, Hui Zhang, J. Yuyang Lu, Yafei Yin, Xiaohua Shen, Peizhe Wang, Michelle Percharde, Xianju Bi, Zai Chang, Xue Han, Ge Zhan, and Lei Chang
- Subjects
Heterochromatin ,Retrotransposon ,Biology ,Cell cycle ,Mutually exclusive events ,Genome ,Spatial organization ,DNA sequencing ,Chromatin ,Cell biology - Abstract
SUMMARYDespite extensive mapping of three-dimensional (3D) chromatin structures, the basic principles underlying genome folding remain unknown. Here, we report a fundamental role for L1 and B1 retrotransposons in shaping the macroscopic 3D genome structure. Homotypic clustering of B1 and L1 repeats in the nuclear interior or at the nuclear and nucleolar peripheries, respectively, segregates the genome into mutually exclusive nuclear compartments. This spatial segregation of L1 and B1 is conserved in mouse and human cells, and occurs dynamically during establishment of the 3D chromatin structure in early embryogenesis and the cell cycle. Depletion of L1 transcripts drastically disrupts the spatial distributions of L1- and B1-rich compartments. L1 transcripts are strongly associated with L1 DNA sequences and induce phase separation of the heterochromatin protein HP1α. Our results suggest that genomic repeats act as the blueprint of chromatin macrostructure, thus explaining the conserved higher-order structure of chromatin across mammalian cells.
- Published
- 2019
- Full Text
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14. Genomic Repeats Categorize Genes with Distinct Functions for Orchestrated Regulation
- Author
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Yujie Sun, Pixi Yan, Wen Shao, Xiaohua Shen, Lei Chang, Zhongyang Wu, Yafei Yin, Wei Liu, Lerui Guo, Tong Li, Hui Zhang, J. Yuyang Lu, Michelle Percharde, Yantao Hong, Miguel Ramalho-Santos, and Lichao Liu
- Subjects
0301 basic medicine ,Transcription, Genetic ,Nucleolus ,Embryonic Development ,Computational biology ,Biology ,Genome ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,0302 clinical medicine ,Gene expression ,Gene silencing ,Animals ,Humans ,Binding site ,Gene ,lcsh:QH301-705.5 ,Embryonic Stem Cells ,Repetitive Sequences, Nucleic Acid ,Nuclear Lamina ,Base Sequence ,Models, Genetic ,RNA ,Gene Expression Regulation, Developmental ,RNA-Binding Proteins ,Phosphoproteins ,Chromatin ,030104 developmental biology ,Gene Ontology ,HEK293 Cells ,chemistry ,lcsh:Biology (General) ,K562 Cells ,030217 neurology & neurosurgery ,DNA ,Cell Nucleolus - Abstract
Summary: Repetitive elements are abundantly distributed in mammalian genomes. Here, we reveal a striking association between repeat subtypes and gene function. SINE, L1, and low-complexity repeats demarcate distinct functional categories of genes and may dictate the time and level of gene expression by providing binding sites for different regulatory proteins. Importantly, imaging and sequencing analysis show that L1 repeats sequester a large set of genes with specialized functions in nucleolus- and lamina-associated inactive domains that are depleted of SINE repeats. In addition, L1 transcripts bind extensively to its DNA in embryonic stem cells (ESCs). Depletion of L1 RNA in ESCs leads to relocation of L1-enriched chromosomal segments from inactive domains to the nuclear interior and de-repression of L1-associated genes. These results demonstrate a role of L1 DNA and RNA in gene silencing and suggest a general theme of genomic repeats in orchestrating the function, regulation, and expression of their host genes. : Lu et al. report a striking association between genomic repeats and gene regulation and demonstrate a key role of L1 repeat RNA in sequestering L1-rich sequences and associated genes in inactive domains for silencing, revealing a general theme of repeat sequences in shaping gene regulatory networks within their host genome. Keywords: repetitive elements, L1, B1 or Alu, SINE, low-complexity repeats, gene function, regulation and expression, nucleolus and lamina-associated domains, L1 RNA, gene silencing
- Published
- 2019
15. What Doesn't Kill You Makes You Stronger: Transposons as Dual Players in Chromatin Regulation and Genomic Variation
- Author
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Tania Sultana, Michelle Percharde, and Miguel Ramalho-Santos
- Subjects
Transposable element ,Regulator ,Gene regulatory network ,Gene Expression ,Mutagenesis (molecular biology technique) ,Endogenous retrovirus ,Computational biology ,Biology ,Genome ,General Biochemistry, Genetics and Molecular Biology ,Evolution, Molecular ,03 medical and health sciences ,0302 clinical medicine ,Animals ,Deoxyribonuclease I ,Humans ,Gene Regulatory Networks ,030304 developmental biology ,Mammals ,0303 health sciences ,Binding Sites ,Genetic Variation ,food and beverages ,Chromatin ,DNA binding site ,Enhancer Elements, Genetic ,DNA Transposable Elements ,030217 neurology & neurosurgery - Abstract
Transposable elements (TEs) are sequences currently or historically mobile, and are present across all eukaryotic genomes. A growing interest in understanding the regulation and function of TEs has revealed seemingly dichotomous roles for these elements in evolution, development, and disease. On the one hand, many gene regulatory networks owe their organization to the spread of cis-elements and DNA binding sites through TE mobilization during evolution. On the other hand, the uncontrolled activity of transposons can generate mutations and contribute to disease, including cancer, while their increased expression may also trigger immune pathways that result in inflammation or senescence. Interestingly, TEs have recently been found to have novel essential functions during mammalian development. Here, the function and regulation of TEs are discussed, with a focus on LINE1 in mammals. It is proposed that LINE1 is a beneficial endogenous dual regulator of gene expression and genomic diversity during mammalian development, and that both of these functions may be detrimental if deregulated in disease contexts.
- Published
- 2020
16. A LINE1-Nucleolin partnership regulates early development and ES cell identity
- Author
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Michelle Percharde, Miguel Ramalho-Santos, Bo Huang, Steffen Biechele, Aydan Bulut-Karslioglu, Juan Guan, Xiaohua Shen, Chih-Jen Lin, Gabriel A. Peixoto, and Yafei Yin
- Subjects
Male ,0301 basic medicine ,Stem Cell Research - Embryonic - Non-Human ,Gene Expression ,Retrotransposon ,Tripartite Motif-Containing Protein 28 ,Inbred C57BL ,MERVL ,Medical and Health Sciences ,Mice ,Transcription (biology) ,Cell Self Renewal ,rRNA ,In Situ Hybridization ,2-cell stage ,RNA-Binding Proteins ,Cell Differentiation ,Mouse Embryonic Stem Cells ,Biological Sciences ,retrotransposons ,Up-Regulation ,Chromatin ,Cell biology ,ESCs ,Kap1 ,embryonic structures ,RNA Interference ,Female ,RNA, Long Noncoding ,Chromatin Immunoprecipitation ,TRIM28 ,1.1 Normal biological development and functioning ,Biology ,Fluorescence ,General Biochemistry, Genetics and Molecular Biology ,Article ,Cell Line ,Evolution, Molecular ,03 medical and health sciences ,Underpinning research ,Genetics ,Gene silencing ,Animals ,Dux ,Antisense ,Homeodomain Proteins ,Ribosomal ,Nucleolin ,Oligoribonucleotides ,Endogenous Retroviruses ,RNA ,hypertranscription ,LINE1 ,Phosphoproteins ,Stem Cell Research ,Embryonic stem cell ,030104 developmental biology ,DNA Transposable Elements ,Generic health relevance ,Developmental Biology - Abstract
Transposable elements represent nearly half of mammalian genomes and are generally described as parasites or ‘junk DNA’. The LINE1 retrotransposon is the most abundant class and is thought to be deleterious for cells, yet is paradoxically highly expressed during early development. Here we report that LINE1 plays essential roles in mouse embryonic stem (ES) cells and pre-implantation embryos. In ES cells, LINE1 acts as a nuclear RNA scaffold that recruits Nucleolin and Kap1/Trim28 to repress Dux, the master activator of a transcriptional program specific to the 2-cell embryo. In parallel, LINE1 RNA mediates binding of Nucleolin and Kap1 to rDNA, promoting rRNA synthesis and ES cell self-renewal. In embryos, LINE1 RNA is required for Dux silencing, synthesis of rRNA and exit from the 2-cell stage. The results reveal an essential partnership between LINE1 RNA, Nucleolin, Kap1 and peri-nucleolar chromatin in the regulation of transcription, developmental potency and ES cell self-renewal.
- Published
- 2018
17. The Transcriptionally Permissive Chromatin State of Embryonic Stem Cells Is Acutely Tuned to Translational Output
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Sergio Covarrubias, Michael T. McManus, Juan A. Oses-Prieto, Alma L. Burlingame, Miguel Ramalho-Santos, Michelle Percharde, Gregory M. Ku, Trisha A. Macrae, Aaron Diaz, and Aydan Bulut-Karslioglu
- Subjects
0301 basic medicine ,Male ,Euchromatin ,Transcription, Genetic ,translation ,Stem Cell Research - Embryonic - Non-Human ,RNA polymerase II ,Regenerative Medicine ,Medical and Health Sciences ,Mice ,Models ,Transcription (biology) ,Genome ,biology ,Protein Stability ,TOR Serine-Threonine Kinases ,Nuclear Proteins ,Cell Differentiation ,Biological Sciences ,Chromatin ,Cell biology ,Histone Code ,Histone ,Enhancer Elements, Genetic ,ribosome ,embryonic structures ,mTOR ,Molecular Medicine ,Stem Cell Research - Nonembryonic - Non-Human ,Female ,RNA Interference ,Transcription ,Enhancer Elements ,1.1 Normal biological development and functioning ,Models, Biological ,Proto-Oncogene Proteins c-myc ,03 medical and health sciences ,Genetic ,Underpinning research ,Genetics ,Animals ,Progenitor cell ,Enhancer ,Embryonic Stem Cells ,permissive chromatin ,blastocyst ,Human Genome ,Chd1 ,hypertranscription ,Cell Biology ,Biological ,Stem Cell Research ,Embryonic stem cell ,030104 developmental biology ,Blastocyst ,Protein Biosynthesis ,biology.protein ,DNA Transposable Elements ,Generic health relevance ,Developmental Biology - Abstract
A permissive chromatin environment coupled to hypertranscription drives the rapid proliferation of embryonic stem cells (ESCs) and peri-implantation embryos. We carried out a genome-wide screen to systematically dissect the regulation of the euchromatic state of ESCs. The results revealed that cellular growth pathways, most prominently translation, perpetuate the euchromatic state and hypertranscription of ESCs. Acute inhibition of translation rapidly depletes euchromatic marks in mouse ESCs and blastocysts, concurrent with delocalization of RNA polymerase II and reduction in nascent transcription. Translation inhibition promotes rewiring of chromatin accessibility, which decreases at a subset of active developmental enhancers and increases at histone genes and transposable elements. Proteome-scale analyses revealed that several euchromatin regulators are unstable proteins and continuously depend on a high translational output. We propose that this mechanistic interdependence of euchromatin, transcription, and translation sets the pace of proliferation at peri-implantation and may be employed by other stem/progenitor cells.
- Published
- 2017
18. The transcriptionally permissive chromatin state of ES cells is acutely tuned to translational output
- Author
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Michael T. McManus, Aydan Bulut-Karslioglu, Gregory M. Ku, Miguel Ramalho-Santos, Aaron Diaz, Alma L. Burlingame, Michelle Percharde, Sergio Covarrubias, Juan A. Oses-Prieto, and Trisha A. Macrae
- Subjects
0303 health sciences ,Euchromatin ,RNA polymerase II ,Biology ,Embryonic stem cell ,Molecular biology ,Chromatin ,Cell biology ,03 medical and health sciences ,0302 clinical medicine ,Histone ,Transcription (biology) ,Protein biosynthesis ,biology.protein ,Enhancer ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
SUMMARYA permissive chromatin environment coupled to hypertranscription is critical to drive the rapid proliferation of Embryonic Stem (ES) cells and peri-implantation embryos. We carried out a genome-wide screen to systematically dissect the regulation of the euchromatic state of ES cells. The results reveal that activity of cellular growth pathways, prominently protein synthesis, perpetuates the euchromatic state and hypertranscription of ES cells. Acute, mild inhibition of translation results in rapid depletion of euchromatic marks in ES cells and blastocysts, concurrent with delocalization of RNA polymerase II and reduction in nascent transcription. Remarkably, reduced translational output leads to rewiring of open chromatin within 3 hours, including decreased accessibility at a subset of active developmental enhancers and increased accessibility at histone genes and transposable elements. Using a proteome-scale analysis, we show that several euchromatin regulators are unstable proteins and thus continuously depend on a high translational output. We propose that this mechanistic interdependence of euchromatin, transcription and translation sets the pace of proliferation at peri-implantation and may be employed generally by stem/progenitor cells.
- Published
- 2017
- Full Text
- View/download PDF
19. Hypertranscription in development, stem cells, and regeneration
- Author
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Aydan Bulut-Karslioglu, Miguel Ramalho-Santos, and Michelle Percharde
- Subjects
0301 basic medicine ,Transcription, Genetic ,Zygote ,Embryonic Development ,Genomics ,Self renewal ,Biology ,Lymphocyte Activation ,General Biochemistry, Genetics and Molecular Biology ,Article ,Transcriptome ,03 medical and health sciences ,Animals ,Humans ,Regeneration ,Molecular Biology ,Regeneration (biology) ,Stem Cells ,Cell Biology ,Cell biology ,030104 developmental biology ,Biological significance ,Evolutionary biology ,Lymphocyte activation ,Stem cell ,Stem cell biology ,Developmental Biology - Abstract
Cells can globally up-regulate their transcriptome during specific transitions, a phenomenon called hypertranscription. Evidence for hypertranscription dates back over 70 years, but it has gone largely ignored in the genomics era until recently. We discuss data supporting the notion that hypertranscription is a unifying theme in embryonic development, stem cell biology, regeneration and cell competition. We review the history, methods for analysis, underlying mechanisms and biological significance of hypertranscription.
- Published
- 2016
20. Author Correction: Maternal vitamin C regulates reprogramming of DNA methylation and germline development
- Author
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Swetha Mahesula, Gabriel Livera, Stephanie P. DiTroia, Marie Justine Guerquin, Michelle Percharde, Kathryn Mesh, Michalis Agathocleous, Diana J. Laird, Evelyne Collignon, Kevin T. Ebata, Miguel Ramalho-Santos, and Estelle Wall
- Subjects
Multidisciplinary ,DNA methylation ,Biology ,Bioinformatics ,Reprogramming ,Article ,Germline ,Maternal vitamin - Abstract
Development is often assumed to be hardwired in the genome, but several lines of evidence indicate that it is susceptible to environmental modulation with potential long-term consequences, including in mammals(1,2). The embryonic germline is of particular interest because of the potential for intergenerational epigenetic effects. The mammalian germline undergoes extensive DNA demethylation(3-7) that occurs in large part by passive dilution over successive cell divisions, accompanied by active DNA demethylation via Ten-eleven translocation (Tet) enzymes(3,8-10). Tet activity has been shown to be modulated by nutrients and metabolites, including Vitamin C (VitC)(11-15). We report here that maternal VitC is required for proper DNA demethylation and development of female fetal germ cells in a mouse model. Maternal VitC deficiency does not affect overall embryonic development but leads to reduced germ cell numbers, delayed meiosis and reduced fecundity in adulthood. The transcriptome of germ cells from VitC-deficient embryos is remarkably similar to that of embryos carrying a null mutation in Tet1. VitC deficiency leads to an aberrant DNA methylation profile that includes incomplete demethylation of key regulators of meiosis and transposable elements. These findings reveal that deficiency in VitC during gestation partially recapitulates Tet1 loss and provide a potential intergenerational mechanism for adjusting fecundity to environmental conditions.
- Published
- 2019
21. The context and potential of epigenetics in oncology
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Helen M. Coley, Tim Crook, Andrew Webb, Michelle Percharde, and Juanita Lopez
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Male ,Cancer Research ,Context (language use) ,Disease ,Biology ,Bioinformatics ,Epigenesis, Genetic ,Neoplasms ,microRNA ,Clinical Studies ,medicine ,Biomarkers, Tumor ,Humans ,Epigenetics ,Epigenesis ,Genetics ,epigenetics ,Cancer ,DNA Methylation ,medicine.disease ,MicroRNAs ,Cancer cell ,DNA methylation ,oncology ,Female ,methylation - Abstract
Cancer has long been known to be a disease caused by alterations in the genetic blueprint of cells. In the past decade it has become evident that epigenetic processes have a function, at least equally important, in neoplasia. Epigenetics describes the mechanisms that result in heritable alterations in gene expression profiles without an accompanying change in DNA sequence. Genetics and epigenetics intricately interact in the pathogenesis of cancer (Esteller, 2007). In this review, we paint a broad picture of current understanding of epigenetic changes in cancer cells and reflect on the immense clinical potential of emerging knowledge of epigenetics in the diagnosis, prognostic assessment, treatment, and screening of cancer.
- Published
- 2009
22. Ncoa3 functions as an essential Esrrb coactivator to sustain embryonic stem cell self-renewal and reprogramming
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Jesús Gil, Vibhor Kumar, Malcolm G. Parker, Nadine Martin, Véronique Azuara, Michelle Percharde, Shyam Prabhakar, Jia-Chi Yeo, Huck-Hui Ng, Jia-Hui Ng, Fabrice Lavial, and Rute A. Tomaz
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Homeobox protein NANOG ,Male ,Biology ,Nuclear Receptor Coactivator 3 ,Mice ,SOX2 ,Coactivator ,Chlorocebus aethiops ,Genetics ,Animals ,Humans ,Enhancer ,Transcription factor ,reproductive and urinary physiology ,Embryonic Stem Cells ,Cell Proliferation ,Genome ,General transcription factor ,Gene Expression Regulation, Developmental ,Cellular Reprogramming ,Cell biology ,HEK293 Cells ,Receptors, Estrogen ,Nuclear receptor coactivator 3 ,embryonic structures ,COS Cells ,Female ,biological phenomena, cell phenomena, and immunity ,Reprogramming ,Developmental Biology ,Research Paper - Abstract
Embryonic stem cell (ESC) pluripotency depends on a well-characterized gene regulatory network centered on Oct4, Sox2, and Nanog. In contrast, little is known about the identity of the key coregulators and the mechanisms by which they may potentiate transcription in ESCs. Alongside core transcription factors, the orphan nuclear receptor Esrrb (estrogen-related receptor β) is vital for the maintenance of ESC identity and furthermore is uniquely associated with the basal transcription machinery. Here, we show that Ncoa3, an essential coactivator, is required to mediate Esrrb function in ESCs. Ncoa3 interacts with Esrrb via its ligand-binding domain and bridges Esrrb to RNA polymerase II complexes. Functionally, Ncoa3 is critical for both the induction and maintenance of pluripotency. Through chromatin immunoprecipitation (ChIP) sequencing and microarray experiments, we further demonstrate that Ncoa3 shares overlapping gene regulatory functions with Esrrb and cooperates genome-wide with the Oct4–Sox2–Nanog circuitry at active enhancers to up-regulate genes involved in self-renewal and pluripotency. We propose an integrated model of transcriptional and coactivator control, mediated by Ncoa3, for the maintenance of ESC self-renewal and somatic cell reprogramming.
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- 2012
23. Essential roles for the nuclear receptor coactivator Ncoa3 in pluripotency
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Véronique Azuara and Michelle Percharde
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Homeobox protein NANOG ,Pluripotent Stem Cells ,CARM1 ,Biology ,Editorials: Cell Cycle Features ,Regenerative Medicine ,Models, Biological ,Nuclear Receptor Coactivator 3 ,SOX2 ,Coactivator ,Humans ,Esrrb ,Induced pluripotent stem cell ,Enhancer ,Molecular Biology ,Genetics ,SRC-3 ,reprogramming ,Cell Biology ,Ncoa3 ,embryonic stem cells ,pluripotency ,coactivator ,Cell biology ,Receptors, Estrogen ,Nuclear receptor coactivator 3 ,Reprogramming ,Developmental Biology - Abstract
Embryonic stem cells (ESCs) are valuable tools for regenerative medicine, being capable of self-renewing in culture indefinitely while retaining their pluripotency, i.e., the ability to generate any cell of the adult organism. At the heart of these capabilities is a complex transcriptional network, which carefully guards an uncommitted state, yet permits ESCs to remain poised for differentiation. So far, many ESC transcription factors, including the core pluripotency proteins, Oct4, Sox2 and Nanog (OSN), have been identified and extensively characterized. These molecules participate in highly interrelated pathways to activate their own expression, as well as downstream self-renewal regulators, via co-recruitment to enhancer regions in the vicinity of these genes. However, we know much less about the individual roles played by each of these factors within multi-protein complexes, the identity of their potential coregulators, or how they functionally connect to the general transcription and chromatin remodeling machineries. In this context, we recently investigated the protein-protein interactions required for the function of one particular molecule, the orphan nuclear receptor Esrrb. In doing so, we uncovered novel and essential roles for the nuclear receptor coactivator Ncoa3 in pluripotent cells,1 making this the first characterization of a nuclear receptor coactivator in stem cell biology. Esrrb has recently been found to act both as a powerful enhancer of ESC self-renewal,1-4 a crucial downstream target of the Wnt/GSK3/Tcf3 signaling pathway3 and also able to substitute for Nanog in pluripotent cells.2 Additionally, an important role for Esrrb is apparent in the induction of pluripotency via somatic cell reprogramming.5,6 Despite these findings, however, nothing is known of the mechanisms by which Esrrb function is conferred in ESCs. To address this, we based our investigations on previous knowledge gained from studying estrogen-related receptors (ERRs) in somatic cells, where their activity canonically requires the recruitment of a protein coactivator to a conserved portion of their ligand-binding domain (LBD), termed the AF-2 region. We theorized that Esrrb might function similarly in ESCs and through a combination of mutagenesis and functional assays, demonstrated that Esrrb’s ability to sustain ESC self-renewal crucially resides in its LBD/AF-2 domain.1 In turn, we demonstrated that Ncoa3 is the coactivator specifically recruited to this AF-2 region and is an essential mediator of Esrrb function in pluripotent cells. Widely known as the oncogene, AIB1 in cancer or SRC-3/ACTR in somatic cells, Ncoa3 is reported to bind to many nuclear receptors as well as some transcription factors, where it induces the strong expression of specific target genes. Correspondingly in ESCs, we found that Ncoa3 is critical for controlling Esrrb-dependent activation of important self-renewal regulators such as Esrrb, Klf4, Nanog and Sox2. Depleting Ncoa3 in ESCs leads to downregulation of these genes and triggers differentiation,1 mirroring the effect of shRNA-mediated Esrrb knockdown itself. Through a combination of Ncoa3 ChIP-sequencing and DNA microarray techniques, we furthermore investigated the genome-wide relevance of the Esrrb-Ncoa3 partnership in pluripotent cells. Sites co-bound by these proteins alongside the OSN triad include ESC-associated genes as well as, interestingly, a high proportion of germ cell targets including some relevant to reprogramming events in the germline.7 Accordingly, depletion of Ncoa3 significantly hinders the generation of induced pluripotent stem cells (iPSCs) in vitro.1 Together, these observations suggest that Esrrb and Ncoa3 might also act in synergy during the formation and/or reprogramming of primordial germ cells (PGCs) in vivo. Our collective findings lead onto a key question: what is the functional role for Esrrb and Ncoa3 underlying their essential nature in the ESC network? Genome-wide, both ESC and PGC-relevant target genes are also enriched for marks of active enhancers, H3K4me1/H3K27ac and p300 recruitment, suggesting a link between the presence of Esrrb-Ncoa3 and transcriptional activation. Moreover, we discovered that Ncoa3 interacts with RNA polymerase II itself and is required for the association between Esrrb and the general transcription machinery.1 Complementing our data, a concurrent study reports that Ncoa3 also recruits the chromatin-modifying proteins CARM1 and CBP to the Nanog locus,8 thus ensuring the maintenance of active histone modifications at this gene. Overall, these results point toward a crucial role for Ncoa3 in potentiating transcription in ESCs by locally facilitating an “open” chromatin state as well as providing a scaffold upon which to recruit the basal transcription machinery itself (Fig. 1A). Loss of Esrrb/Ncoa3 upon differentiation or shRNA-mediated depletion would therefore inhibit the transcription of ESC-associated genes, consequently triggering a collapse of the network and the exit of self-renewal (Fig. 1B). Figure 1. Proposed model depicting the role of Esrrb and Ncoa3 at ESC-specific enhancers. (A) In pluripotent cells, Esrrb binds to ERR response elements (ERREs) at active enhancer regions, which also contain bound core proteins such as Nanog and ... In conclusion, these findings establish Ncoa3 as a key member of pluripotency transcriptional circuitries, emphasizing the importance of this coactivator not just in somatic and cancer cells, but also in early developmental contexts. An interesting future avenue is to characterize the upstream signaling pathways that may regulate Ncoa3 in pluripotent cells, as this molecule is a well-known target for numerous post-translational modifications. Accordingly, increased Ncoa3 stability notably correlates with GSK3 inhibition in ESCs.8 Ncoa3 modification in response to one or more external signaling pathways could therefore alter its protein stability, cellular location or even its transcriptional activity, thus serving as an elegant way to fine-tune the cellular state of ESCs and iPSCs.
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- 2013
24. YAP Induces Human Naive Pluripotency
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Jens Durruthy-Durruthy, Miguel Ramalho-Santos, Miroslav Hejna, Han Qin, Priscilla Wong, Zhongxia Qi, Yanxia Liu, Michelle Percharde, Mark Wossidlo, Jingwei Yu, Vittorio Sebastiano, Lei S. Qi, Laure Blouin, and Jun S. Song
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Male ,0301 basic medicine ,Pyridines ,Cellular differentiation ,Hippo pathway ,Glycogen Synthase Kinase 3 ,RNA interference ,Heterochromatin ,Inner cell mass ,Induced pluripotent stem cell ,lcsh:QH301-705.5 ,Wnt Signaling Pathway ,Cells, Cultured ,Genetics ,Regulation of gene expression ,Teratoma ,Wnt signaling pathway ,Cell Differentiation ,Yes-associated protein (YAP) ,Cell biology ,lysophosphatidic acid (LPA) ,embryonic stem cells (ESCs) ,Benzamides ,Female ,RNA Interference ,Pluripotent Stem Cells ,pluripotent stem cells (PSCs) ,Induced Pluripotent Stem Cells ,Karyotype ,Biology ,Article ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Humans ,Embryonic Stem Cells ,Adaptor Proteins, Signal Transducing ,Hippo signaling pathway ,naive pluripotency ,Diphenylamine ,YAP-Signaling Proteins ,Phosphoproteins ,Embryonic stem cell ,Blastocyst ,Pyrimidines ,030104 developmental biology ,lcsh:Biology (General) ,Gene Expression Regulation ,induced pluripotent stem cells (iPSCs) ,CRISPR-Cas Systems ,Lysophospholipids ,Transcription Factors - Abstract
SUMMARY The human naive pluripotent stem cell (PSC) state, corresponding to a pre-implantation stage of development, has been difficult to capture and sustain in vitro. We report that the Hippo pathway effector YAP is nuclearly localized in the inner cell mass of human blastocysts. Overexpression of YAP in human embryonic stem cells (ESCs) and induced PSCs (iPSCs) promotes the generation of naive PSCs. Lysophosphatidic acid (LPA) can partially substitute for YAP to generate transgene-free human naive PSCs. YAP- or LPA-induced naive PSCs have a rapid clonal growth rate, a normal karyotype, the ability to form teratomas, transcriptional similarities to human pre-implantation embryos, reduced heterochromatin levels, and other hallmarks of the naive state. YAP/LPA act in part by suppressing differentiation-inducing effects of GSK3 inhibition. CRISPR/Cas9-generated YAP−/− cells have an impaired ability to form colonies in naive but not primed conditions. These results uncover an unexpected role for YAP in the human naive state, with implications for early human embryology., Graphical abstract
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