scholarly journals Transcriptional repression by FACT is linked to regulation of chromatin accessibility at the promoter of ES cells

2018 ◽  
Author(s):  
Constantine Mylonas ◽  
Peter Tessarz
Keyword(s):  
Rna Polymerase Ii ◽  
Cell Fate ◽  
Transcriptional Repression ◽  
Mammalian Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Antisense Transcription ◽  
Chromatin Accessibility ◽  
Promoter Architecture ◽  
Neuronal Lineage

The conserved and essential histone chaperone FACT (Facilitates Chromatin Transcription) reorganizes nucleosomes during DNA transcription, replication and repair and ensures both, efficient elongation of RNA Pol II and nucleosome integrity. In mammalian cells, FACT is a heterodimer, consisting of SSRP1 and SUPT16. Here, we show that in contrast to yeast, FACT accumulates at the transcription start site of genes reminiscent of RNA Polymerase II profile. Depletion of FACT in mouse embryonic stem cells leads to up-regulation of pro-proliferative genes and key pluripotency factors concomitant with hyper-proliferation of mES cells. Using MNase-, ATAC-, and Nascent Elongating Transcript Sequencing (NET-seq) we show that up-regulation of genes coincides with loss of nucleosomes upstream of the TSS and concomitant increase in antisense transcription, indicating that FACT impacts the promoter architecture to regulate expression of these genes. Finally, we demonstrate a role for FACT in cell fate determination and show that FACT depletion primes ES cells for the neuronal lineage.

scholarly journals Transcriptional repression by FACT is linked to regulation of chromatin accessibility at the promoter of ES cells

2018 ◽  
Vol 1 (3) ◽  
pp. e201800085 ◽  
Author(s):  
Constantine Mylonas ◽  
Peter Tessarz
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Rna Polymerase Ii ◽  
Cell Fate ◽  
Transcription Start Site ◽  
Mammalian Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Start Site ◽  
Transcription Start

The conserved and essential histone chaperone, facilitates chromatin transcription (FACT), reorganizes nucleosomes during DNA transcription, replication, and repair and ensures both efficient elongation of RNA Pol II and nucleosome integrity. In mammalian cells, FACT is a heterodimer, consisting of SSRP1 and SUPT16. Here, we show that in contrast to yeast, FACT accumulates at the transcription start site of genes reminiscent of RNA polymerase II profile. Depletion of FACT in mouse embryonic stem cells leads to deregulation of developmental and pro-proliferative genes concomitant with hyper-proliferation of mES cells. Using MNase-seq, Assay for Transposase-Accessible Chromatin sequencing, and nascent elongating transcript sequencing, we show that up-regulation of genes coincides with loss of nucleosomes upstream of the transcription start site and concomitant increase in antisense transcription, indicating that FACT impacts the promoter architecture to regulate the expression of these genes. Finally, we demonstrate a role for FACT in cell fate determination and show that FACT depletion primes embryonic stem cells for the neuronal lineage.

scholarly journals Endogenous fluctuations of OCT4 and SOX2 bias pluripotent cell fate decisions

2018 ◽  
Author(s):  
Daniel Strebinger ◽  
Cédric Deluz ◽  
Elias T. Friman ◽  
Subashika Govindan ◽  
Andrea B. Alber ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Cell Fate ◽  
Es Cells ◽  
Embryonic Stem ◽  
Chromatin Accessibility ◽  
Directed Differentiation ◽  
Es Cell ◽  
Endogenous Fluctuations ◽  
Cell Fate Decisions ◽  
Oct4 Protein

AbstractSOX2 and OCT4 are pioneer transcription factors playing a key role in embryonic stem (ES) cell self-renewal and differentiation. However, how temporal fluctuations in their expression levels bias lineage commitment is unknown. Here we generated knock-in reporter fusion ES cell lines allowing to monitor endogenous SOX2 and OCT4 protein fluctuations in living cells and to determine their impact on mesendodermal and neuroectodermal commitment. We found that small differences in SOX2 and OCT4 levels impact cell fate commitment in G1 but not in S phase. Elevated SOX2 levels modestly increased neuroectodermal commitment and decreased mesendodermal commitment upon directed differentiation. In contrast, elevated OCT4 levels strongly biased ES cell towards both neuroectodermal and mesendodermal fates. Using ATAC-seq on ES cells gated for different endogenous SOX2 and OCT4 levels, we found that high OCT4 levels increased chromatin accessibility at differentiation-associated enhancers. This suggests that small endogenous fluctuations of pioneer transcription factors can bias cell fate decisions by concentration-dependent priming of differentiation-associated enhancers.

Human embryonic stem cells: challenges and opportunities

2006 ◽  
Vol 18 (8) ◽  
pp. 839 ◽  
Author(s):  
Steven L. Stice ◽  
Nolan L. Boyd ◽  
Sujoy K. Dhara ◽  
Brian A. Gerwe ◽  
David W. Machacek ◽  
...  
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Cell Line ◽  
Cell Fate ◽  
Neural Development ◽  
Es Cells ◽  
Embryonic Stem ◽  
Normal Karyotype ◽  
Es Cell ◽  
Cell Therapies

Human and non-human primate embryonic stem (ES) cells are invaluable resources for developmental studies, pharmaceutical research and a better understanding of human disease and replacement therapies. In 1998, subsequent to the establishment of the first monkey ES cell line in 1995, the first human ES cell line was developed. Later, three of the National Institute of Health (NIH) lines (BG01, BG02 and BG03) were derived from embryos that would have been discarded because of their poor quality. A major challenge to research in this area is maintaining the unique characteristics and a normal karyotype in the NIH-registered human ES cell lines. A normal karyotype can be maintained under certain culture conditions. In addition, a major goal in stem cell research is to direct ES cells towards a limited cell fate, with research progressing towards the derivation of a variety of cell types. We and others have built on findings in vertebrate (frog, chicken and mouse) neural development and from mouse ES cell research to derive neural stem cells from human ES cells. We have directed these derived human neural stem cells to differentiate into motoneurons using a combination of developmental cues (growth factors) that are spatially and temporally defined. These and other human ES cell derivatives will be used to screen new compounds and develop innovative cell therapies for degenerative diseases.

scholarly journals Target frequency and integration pattern for insertion and replacement vectors in embryonic stem cells

1991 ◽  
Vol 11 (9) ◽  
pp. 4509-4517
Author(s):  
P Hasty ◽  
J Rivera-Pérez ◽  
C Chang ◽  
A Bradley
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Mammalian Cells ◽  
Germ Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Homologous Sequence ◽  
Reciprocal Recombination ◽  
Replacement Vector ◽  
Insertion Vector

Gene targeting has been used to direct mutations into specific chromosomal loci in murine embryonic stem (ES) cells. The altered locus can be studied in vivo with chimeras and, if the mutated cells contribute to the germ line, in their offspring. Although homologous recombination is the basis for the widely used gene targeting techniques, to date, the mechanism of homologous recombination between a vector and the chromosomal target in mammalian cells is essentially unknown. Here we look at the nature of gene targeting in ES cells by comparing an insertion vector with replacement vectors that target hprt. We found that the insertion vector targeted up to ninefold more frequently than a replacement vector with the same length of homologous sequence. We also observed that the majority of clones targeted with replacement vectors did not recombine as predicted. Analysis of the recombinant structures showed that the external heterologous sequences were often incorporated into the target locus. This observation can be explained by either single reciprocal recombination (vector insertion) of a recircularized vector or double reciprocal recombination/gene conversion (gene replacement) of a vector concatemer. Thus, single reciprocal recombination of an insertion vector occurs 92-fold more frequently than double reciprocal recombination of a replacement vector with crossover junctions on both the long and short arms.

scholarly journals Epigenetic regulations follow cell cycle progression during differentiation of human pluripotent stem cells

2020 ◽  
Author(s):  
Pedro Madrigal ◽  
Siim Pauklin ◽  
Kim Jee Goh ◽  
Rodrigo Grandy ◽  
Anna Osnato ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Cell Fate ◽  
Cell Cycle Progression ◽  
Molecular Mechanisms ◽  
Embryonic Stem ◽  
Chromatin Accessibility ◽  
Activator Protein 1 ◽  
Cellular Division ◽  
Mapk Signalling

AbstractMost mammalian stem cells undergo cellular division during their differentiation to produce daughter cells with a new cellular identity. However, the cascade of epigenetic events and molecular mechanisms occurring between successive cell divisions upon differentiation have not yet been described in detail due to technical limitations. Here, we address this question by taking advantage of the Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) reporter to develop a culture system allowing the differentiation of human Embryonic Stem Cells (hESCs) synchronised for their cell cycle. Using this approach, we have assessed the epigenome and transcriptome dynamics during the first two divisions leading to definitive endoderm. We first observed that transcription of key markers of differentiation occurs before division suggesting that differentiation is initiated during the progression of cell cycle. Furthermore, ATAC-seq shows a major decrease in chromatin accessibility after pluripotency exit indicating that the first event of differentiation is the inhibition of alternative cell fate. In addition, using digital genomic footprinting we identified novel cell cycle-specific transcription factors with regulatory potential in endoderm specification. Of particular interest, Activator protein 1 (AP-1) controlled p38/MAPK signalling seems to be necessary for blocking endoderm shifting cell fate toward mesoderm lineage. Finally, histone modifications analyses suggest a temporal order between different marks. We can also conclude that enhancers are dynamically and rapidly established / decommissioned between different cell cycle upon differentiation. Overall, these data not only reveal key the successive interplays between epigenetic modifications during differentiation but also provide a valuable resource to investigate novel mechanisms in germ layer specification.

scholarly journals Specific Double-Stranded RNA Interference in Undifferentiated Mouse Embryonic Stem Cells

2001 ◽  
Vol 21 (22) ◽  
pp. 7807-7816 ◽  
Author(s):  
Shicheng Yang ◽  
Stephen Tutton ◽  
Eric Pierce ◽  
Kyonggeun Yoon
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Es Cells ◽  
Embryonic Stem ◽  
Transient Transfection ◽  
Interferon Response ◽  
Green Fluorescent Protein Gene ◽  
Double Stranded Rna ◽  
Rnai Activity

ABSTRACT Specific mRNA degradation mediated by double-stranded RNA (dsRNA) interference (RNAi) is a powerful way of suppressing gene expression in plants, nematodes, and fungal, insect, and protozoan systems. However, only a few cases of RNAi have been reported in mammalian systems. Here, we investigated the feasibility of the RNAi strategy in several mammalian cells by using the enhanced green fluorescent protein gene as a target, either by in situ production of dsRNA from transient transfection of a plasmid harboring a 547-bp inverted repeat or by direct transfection of dsRNA made by in vitro transcription. Several mammalian cells including differentiated embryonic stem (ES) cells did not exhibit specific RNAi in transient transfection. This long dsRNA, however, was capable of inducing a sequence-specific RNAi for the episomal and chromosomal target gene in undifferentiated ES cells. dsRNA at 8.3 nM decreased the cognate gene expression up to 70%. However, RNAi activity was not permanent because it was more pronounced in early time points and diminished 5 days after transfection. Thus, undifferentiated ES cells may lack the interferon response, similar to mouse embryos and oocytes. Regardless of their apparent RNAi activity, however, cytoplasmic extracts from mammalian cells produced a small RNA of 21 to 22 nucleotides from the long dsRNA. Our results suggest that mammalian cells may possess RNAi activity but nonspecific activation of the interferon response by longer dsRNA may mask the specific RNAi. The findings offer an opportunity to use dsRNA for inhibition of gene expression in ES cells to study differentiation.

scholarly journals Fgf and Esrrb integrate epigenetic and transcriptional networks that regulate self-renewal of trophoblast stem cells

2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Paulina A. Latos ◽  
Angela Goncalves ◽  
David Oxley ◽  
Hisham Mohammed ◽  
Ernest Turro ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Rna Polymerase Ii ◽  
Es Cells ◽  
Embryonic Stem ◽  
Transcriptional Networks ◽  
Self Renewal ◽  
Downstream Target ◽  
Trophoblast Stem

Abstract Esrrb (oestrogen-related receptor beta) is a transcription factor implicated in embryonic stem (ES) cell self-renewal, yet its knockout causes intrauterine lethality due to defects in trophoblast development. Here we show that in trophoblast stem (TS) cells, Esrrb is a downstream target of fibroblast growth factor (Fgf) signalling and is critical to drive TS cell self-renewal. In contrast to its occupancy of pluripotency-associated loci in ES cells, Esrrb sustains the stemness of TS cells by direct binding and regulation of TS cell-specific transcription factors including Elf5 and Eomes. To elucidate the mechanisms whereby Esrrb controls the expression of its targets, we characterized its TS cell-specific interactome using mass spectrometry. Unlike in ES cells, Esrrb interacts in TS cells with the histone demethylase Lsd1 and with the RNA Polymerase II-associated Integrator complex. Our findings provide new insights into both the general and context-dependent wiring of transcription factor networks in stem cells by master transcription factors.

scholarly journals Stem cell culture on polyvinyl alcohol hydrogels having different elasticity and immobilized with ECM-derived oligopeptides

2017 ◽  
Vol 37 (7) ◽  
pp. 647-660 ◽  
Author(s):  
Saradaprasan Muduli ◽  
Li-Hua Chen ◽  
Meng-Pei Li ◽  
Zhao-wen Heish ◽  
Cheng-Hui Liu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Culture ◽  
Extracellular Matrix ◽  
Stem Cell ◽  
Cell Fate ◽  
Es Cells ◽  
Embryonic Stem ◽  
Poly Vinyl Alcohol ◽  
Hematopoietic Stem ◽  
Stem Cell Culture

Abstract The physical characteristics of cell culture materials, such as their elasticity, affect stem cell fate with respect to cell proliferation and differentiation. We systematically investigated the morphologies and characteristics of several stem cell types, including human amniotic-derived stem cells, human hematopoietic stem cells, human induced pluripotent stem (iPS) cells, and embryonic stem (ES) cells on poly(vinyl alcohol) (PVA) hydrogels immobilized with and without extracellular matrix-derived oligopeptide. Human ES cells did not adhere well to soft PVA hydrogels immobilized with oligovitronectin, whereas they did adhere well to PVA hydrogel dishes with elasticities greater than 15 kPa. These results indicate that biomaterials such as PVA hydrogels should be designed to possess minimum elasticity to facilitate human ES cell attachment. PVA hydrogels immobilized with and without extracellular matrix-derived oligopeptides are excellent candidates of cell culture biomaterials for investigations into how cell culture biomaterial elasticity affects stem cell culture and differentiation.

scholarly journals A dual role for H2A.Z.1 in modulating the dynamics of RNA Polymerase II initiation and elongation

2020 ◽  
Author(s):  
Constantine Mylonas ◽  
Alexander L. Auld ◽  
Choongman Lee ◽  
Ibrahim I. Cisse ◽  
Laurie A. Boyer
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Mammalian Cells ◽  
Embryonic Stem ◽  
Super Resolution ◽  
Fine Tuning ◽  
Transcriptional Initiation ◽  
Single Molecule Level

AbstractRNAPII pausing immediately downstream of the transcription start site (TSS) is a critical rate limiting step at most metazoan genes that allows fine-tuning of gene expression in response to diverse signals1–5. During pause-release, RNA Polymerase II (RNAPII) encounters an H2A.Z.1 nucleosome6–8, yet how this variant contributes to transcription is poorly understood. Here, we use high resolution genomic approaches2,9 (NET-seq and ChIP-nexus) along with live cell super-resolution microscopy (tcPALM)10 to investigate the role of H2A.Z.1 on RNAPII dynamics in embryonic stem cells (ESCs). Using a rapid, inducible protein degron system11 combined with transcriptional initiation and elongation inhibitors, our quantitative analysis shows that H2A.Z.1 slows the release of RNAPII, impacting both RNAPII and NELF dynamics at a single molecule level. We also find that H2A.Z.1 loss has a dramatic impact on nascent transcription at stably paused, signal-dependent genes. Furthermore, we demonstrate that H2A.Z.1 inhibits re-assembly and re-initiation of the PIC to reinforce the paused state and acts as a strong additional pause signal at stably paused genes. Together, our study suggests that H2A.Z.1 fine-tunes gene expression by regulating RNAPII kinetics in mammalian cells.

scholarly journals Nucleus-cytoskeleton communication impacts on OCT4-chromatin interactions in embryonic stem cells

BMC Biology ◽  
2022 ◽  
Vol 20 (1) ◽  
Author(s):  
Juan José Romero ◽  
María Cecilia De Rossi ◽  
Camila Oses ◽  
Camila Vázquez Echegaray ◽  
Paula Verneri ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cell Fate ◽  
Opposite Effect ◽  
Es Cells ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Cytoskeleton Organization ◽  
Chromatin Interactions ◽  
Cellular Environment ◽  
Dimensional Distribution

Abstract Background The cytoskeleton is a key component of the system responsible for transmitting mechanical cues from the cellular environment to the nucleus, where they trigger downstream responses. This communication is particularly relevant in embryonic stem (ES) cells since forces can regulate cell fate and guide developmental processes. However, little is known regarding cytoskeleton organization in ES cells, and thus, relevant aspects of nuclear-cytoskeletal interactions remain elusive. Results We explored the three-dimensional distribution of the cytoskeleton in live ES cells and show that these filaments affect the shape of the nucleus. Next, we evaluated if cytoskeletal components indirectly modulate the binding of the pluripotency transcription factor OCT4 to chromatin targets. We show that actin depolymerization triggers OCT4 binding to chromatin sites whereas vimentin disruption produces the opposite effect. In contrast to actin, vimentin contributes to the preservation of OCT4-chromatin interactions and, consequently, may have a pro-stemness role. Conclusions Our results suggest roles of components of the cytoskeleton in shaping the nucleus of ES cells, influencing the interactions of the transcription factor OCT4 with the chromatin and potentially affecting pluripotency and cell fate.

Sign in / Sign up

Export Citation Format

Share Document