Relief of a repressed gene expression state in the mouse 1-cell embryo requires DNA replication

Development ◽  
1998 ◽  
Vol 125 (16) ◽  
pp. 3153-3166
Author(s):  
S. Forlani ◽  
C. Bonnerot ◽  
S. Capgras ◽  
J.F. Nicolas
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
Long Range ◽  
Embryonic Stem ◽  
Hypoxanthine Phosphoribosyl Transferase ◽  
Cell Stage ◽  
Enhancer Activity ◽  
Position Effects ◽  
Cell Proliferation And Differentiation ◽  
Cell Embryo

In the mouse, transcriptional permissiveness is established in the fertilized egg prior to the activation of zygotic genes at the 2-cell stage. Therefore, gene inactivity initiated at the end of gametogenesis results from a complex process, involving more than an inhibition of the basal transcriptional apparatus. We have examined the ability of the first intron (I1) of the human hypoxanthine phosphoribosyl transferase gene, which functions as an enhancer in embryonic stem cells, to activate a reporter gene when placed proximally to or at a distance from the HSV-tk promoter, or when integrated into the mouse genome as part of a stable transgene. In microinjected embryos, I1 functions as an enhancer sequence; however, its competence for long-range activation appears only after the late 1-cell stage and depends on the first DNA replication. Moreover, activation of microinjected transgenes from proximal enhancers occurs in the late 2-cell embryo and in the male pronucleus of 1-cell embryos blocked for DNA replication; whereas, for integrated transgenes, proximal enhancer activity is subject to position effects in the 2-cell embryo and first occurs at the 2- or 4-cell stage, but only after completion of DNA replication. Therefore, the absence of long-range activation and a non-permissive genomic state (the relief of which both depend on DNA replication), together with an inactive transcriptional apparatus, appear to converge to prevent any gene activity in the 1-cell embryo. We propose that the embryo exploits the process of DNA replication to relieve the transcriptionally repressive state that was initially established to fulfil two purposes: (1) to arrest maternal gene expression in the maturing oocyte and (2) to protect the unicellular egg and 1-cell embryo from premature differentiation. Reactivation of gene expression by DNA replication would therefore serve to coordinate cell proliferation and differentiation in the preimplantation embryo.

scholarly journals Development of enhancer-trapping and -detection vectors mediated by the Tol2 transposon in zebrafish

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6862 ◽  
Author(s):  
Shuheng Chan ◽  
Dan Shen ◽  
Yatong Sang ◽  
Saisai Wang ◽  
Yali Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Zebrafish Embryo ◽  
Trapping Efficiency ◽  
Minimal Promoter ◽  
Beta Globin ◽  
Cell Stage ◽  
Enhancer Activity ◽  
Position Effects ◽  
Basic Expression ◽  
Enhancer Trapping

Enhancers are key transcriptional drivers of gene expression. The identification of enhancers in the genome is central for understanding gene-expression programs. Although transposon-mediated enhancer trapping (ET) is a powerful approach to the identification of enhancers in zebrafish, its efficiency varies considerably. To improve the ET efficiency, we constructed Tol2-mediated ET vectors with a reporter gene (mCherry) expression box driven by four minimal promoters (Gata, Myc, Krt4 and Oct4), respectively. The ET efficiency and expression background were compared among the four promoters by zebrafish embryo injection at the one-cell stage. The results showed that the Gata minimal promoter yielded the lowest basic expression and the second-highest trapping efficiency (44.6% at 12 hpf (hour post-fertilization) and 23.1% at 72 hpf, n = 305 and n = 307). The Krt4 promoter had the highest trapping efficiency (64% at 12 hpf and 67.1% at 72 hpf, n = 302 and n = 301) and the strongest basic expression. To detect enhancer activity, chicken 5′HS4 double insulators were cloned into the two ET vectors with the Gata or Krt4 minimal promoter, flanking the mCherry expression box. The resulting detection vectors were injected into zebrafish embryos. mCherry expression driven by the Gata promoter (about 5%, n = 301) was decreased significantly compared with that observed for embryos injected with the ET vectors (23% at 72 hpf, n = 308). These results suggest that the insulators block the genome-position effects and that this vector is fit for enhancer-activity evaluation. To assess the compatibility between the enhancers and the minimal promoters, four enhancers (CNS1, Z48, Hand2 and Hs769) were cloned upstream of the Gata or Beta-globin minimal promoter in the enhancer-activity-detection vectors. The resulting recombinant vectors were assayed by zebrafish embryo injection. We found that Z48 and CNS1 responded to the Gata minimal promoter, and that Hand2 only responded to the Beta-globin minimal promoter. In contrast, Hs769 did not respond to either the Gata or Beta-globin minimal promoters. These results suggest the existence of compatibility between enhancers and minimal promoters. This study represents a systematic approach to the discovery of optional ET and enhancer-detection vectors. We are eager to provide a superior tool for understanding functional genomics.

scholarly journals Diet-dependent Natriuretic Peptide Receptor C expression in adipose tissue is mediated by PPARγ via long-range distal enhancers

2021 ◽  
Author(s):  
Fubiao Shi ◽  
Zoltan Simandi ◽  
Laszlo Nagy ◽  
Sheila Collins
Keyword(s):  
Gene Expression ◽  
Adipose Tissue ◽  
Long Range ◽  
Environmental Changes ◽  
Peroxisome Proliferator ◽  
Enhancer Activity ◽  
Peroxisome Proliferator Activated Receptor ◽  
Pparγ Agonist ◽  
Mouse Adipocytes ◽  
Sirna Knockdown

AbstractIn addition to their established role to maintain blood pressure and fluid volume, the cardiac natriuretic peptides (NPs) can stimulate adipocyte lipolysis and control the brown fat gene program of nonshivering thermogenesis. The NP “clearance” receptor C (NPRC) functions to clear NPs from the circulation via peptide internalization and degradation and thus is an important regulator of NP signaling and adipocyte metabolism. It is well appreciated that the Nprc gene is highly expressed in adipose tissue and is dynamically regulated with nutrition and environmental changes. However, the molecular basis for how Nprc gene expression is regulated is still poorly understood. Here we identified Peroxisome Proliferator-Activated Receptor gamma (PPARγ) as a transcriptional regulator of Nprc expression in mouse adipocytes. During 3T3-L1 adipocyte differentiation, levels of Nprc expression increase in parallel with PPARγ induction. Rosiglitazone, a classic PPARγ agonist, increases, while siRNA knockdown of PPARγ reduces, Nprc expression in 3T3-L1 adipocytes. We demonstrate that PPARγ controls Nprc gene expression in adipocytes through its long-range distal enhancers. Furthermore, the induction of Nprc expression in adipose tissue during high-fat diet feeding is associated with increased PPARγ enhancer activity. Our findings define PPARγ as a mediator of adipocyte Nprc gene expression and establish a new connection between PPARγ and the control of adipocyte NP signaling in obesity.

scholarly journals Zscan4c activates endogenous retrovirus MERVL and cleavage embryo genes

2019 ◽  
Author(s):  
Weiyu Zhang ◽  
Fuquan Chen ◽  
Ruiqing Chen ◽  
Dan Xie ◽  
Jiao Yang ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Embryonic Stem ◽  
Endogenous Retrovirus ◽  
Endogenous Retroviruses ◽  
Enhancer Activity ◽  
Cell Cleavage ◽  
Cell Embryo ◽  
And Function ◽  
Scan Domain

AbstractEndogenous retroviruses (ERVs) contribute to ∼10 percent of the mouse genome. They are often silenced in differentiated somatic cells but differentially expressed at various embryonic developmental stages. A minority of mouse embryonic stem cells (ESCs), like 2-cell cleavage embryos, highly express ERV MERVL. However, the role of ERVs and mechanism of their activation in these cells are still poorly understood. In this study, we investigated the regulation and function of the stage-specific expressed ERVs, with a particular focus on the totipotency marker MT2/MERVL. We show that the transcription factor Zscan4c functions as an activator of MT2/MERVL and 2-cell/4-cell embryo genes. Zinc finger domains of Zscan4c play an important role in this process. In addition, Zscan4c interacts with MT2 and regulates MT2-nearby 2-cell/4-cell genes through promoting enhancer activity of MT2. Furthermore, MT2 activation is accompanied by enhanced H3K4me1, H3K27ac, and H3K14ac deposition on MT2. Zscan4c also interacts with GBAF chromatin remodelling complex through SCAN domain to further activate MT2 enhancer activity. Taken together, we delineate a previously unrecognized regulatory axis that Zscan4c interacts with and activates MT2/MERVL loci and their nearby genes through epigenetic regulation.

scholarly journals A long-range chromatin interaction regulates SATB homeobox 1 gene expression in trophoblast stem cells

2020 ◽  
Author(s):  
Wei Yu ◽  
V. Praveen Chakravarthi ◽  
Shaon Borosha ◽  
Anamika Ratri ◽  
Khyati Dalal ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Long Range ◽  
Molecular Mechanisms ◽  
Regulatory Region ◽  
Chromatin Interaction ◽  
Enhancer Activity ◽  
Histone Marks ◽  
Developmental Gene Expression ◽  
Trophoblast Stem

ABSTRACTSATB homeobox proteins are important regulators of developmental gene expression. Among the stem cell lineages determined during early embryonic development, trophoblast stem (TS) cells exhibit robust SATB expression. Both SATB1 and SATB2 act to maintain trophoblast stem-state. However, the molecular mechanisms that regulate TS-specific Satb expression are not yet known. We identified Satb1 variant 2 as the predominant transcript in trophoblasts. Histone marks, and RNA polymerase II occupancy in TS cells indicated active state of the promoter. A novel cis-regulatory region with active histone marks was identified ∼21kbp upstream of variant 2 promoter. CRISPR/Cas9 mediated disruption of this sequence decreased Satb1 expression in TS cells and chromatin conformation capture confirmed looping of this regulatory region into the promoter. Scanning position weight matrices across the enhancer predicted two ELF5 binding sites in close vicinity of SATB1 sites, which were confirmed by chromatin immunoprecipitation. Knockdown of ELF5 downregulated Satb1 expression in TS cells and overexpression of ELF5 increased the enhancer-reporter activity. Interestingly, ELF5 interacts with SATB1 in TS cells, and the enhancer activity was upregulated following SATB overexpression. Our findings indicate that trophoblast-specific Satb1 expression is regulated by long-range chromatin looping of an enhancer that interacts with ELF5 and SATB proteins.

scholarly journals Thiol-linked alkylation for the metabolic sequencing of RNA

2017 ◽  
Author(s):  
Veronika A. Herzog ◽  
Brian Reichholf ◽  
Tobias Neumann ◽  
Philipp Rescheneder ◽  
Pooja Bhat ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Embryonic Stem ◽  
Cost Effective ◽  
Enhancer Activity ◽  
Regulatory Pathways ◽  
Nucleotide Resolution

AbstractGene expression profiling by high-throughput sequencing reveals qualitative and quantitative changes in RNA species at steady-state but obscures the intracellular dynamics of RNA transcription, processing and decay. We developed thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq), an orthogonal chemistry-based epitranscriptomics-sequencing technology that uncovers 4-thiouridine (s4U)-incorporation in RNA species at single-nucleotide resolution. In combination with well-established metabolic RNA labeling protocols and coupled to standard, low-input, high-throughput RNA sequencing methods, SLAM-seq enables rapid access to RNA polymerase II-dependent gene expression dynamics in the context of total RNA. When applied to mouse embryonic stem cells, SLAM-seq provides global and transcript-specific insights into pluripotency-associated gene expression. We validated the method by showing that the RNA-polymerase II-dependent transcriptional output scales with Oct4/Sox2/Nanog-defined enhancer activity; and we provide quantitative and mechanistic evidence for transcript-specific RNA turnover mediated by post-transcriptional gene regulatory pathways initiated by microRNAs and N6-methyladenosine. SLAM-seq facilitates the dissection of fundamental mechanisms that control gene expression in an accessible, cost-effective, and scalable manner.One Sentence Summary:Chemical nucleotide-analog derivatization provides global insights into transcriptional and post-transcriptional gene regulation

scholarly journals Formation of an Active Tissue-Specific Chromatin Domain Initiated by Epigenetic Marking at the Embryonic Stem Cell Stage

2005 ◽  
Vol 25 (5) ◽  
pp. 1804-1820 ◽  
Author(s):  
Henrietta Szutorisz ◽  
Claudia Canzonetta ◽  
Andrew Georgiou ◽  
Cheok-Man Chow ◽  
László Tora ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
B Cells ◽  
B Cell ◽  
Histone H3 ◽  
Embryonic Stem ◽  
Chromatin Domain ◽  
Cell Stage ◽  
General Transcription Factors ◽  
Regulated Gene Expression

ABSTRACT The differentiation potential of stem cells is determined by the ability of these cells to establish and maintain developmentally regulated gene expression programs that are specific to different lineages. Although transcriptionally potentiated epigenetic states of genes have been described for haematopoietic progenitors, the developmental stage at which the formation of lineage-specific gene expression domains is initiated remains unclear. In this study, we show that an intergenic cis-acting element in the mouse λ5-VpreB1 locus is marked by histone H3 acetylation and histone H3 lysine 4 methylation at a discrete site in embryonic stem (ES) cells. The epigenetic modifications spread from this site toward the VpreB1 and λ5 genes at later stages of B-cell development, and a large, active chromatin domain is established in pre-B cells when the genes are fully expressed. In early B-cell progenitors, the binding of haematopoietic factor PU.1 coincides with the expansion of the marked region, and the region becomes a center for the recruitment of general transcription factors and RNA polymerase II. In pre-B cells, E2A also binds to the locus, and general transcription factors are distributed across the active domain, including the gene promoters and the intergenic region. These results suggest that localized epigenetic marking is important for establishing the transcriptional competence of the λ5 and VpreB1 genes as early as the pluripotent ES cell stage.

scholarly journals An ERK5-KLF2 signalling module regulates early embryonic gene expression and telomere rejuvenation in stem cells

2021 ◽  
Author(s):  
Helen A Brown ◽  
Charles AC Williams ◽  
Houjiang Zhou ◽  
Diana Rios-Szwed ◽  
Rosalia Fernandez-Alonso ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Embryonic Stem ◽  
Feedback Regulation ◽  
Proteomic Profiling ◽  
Cell Stage ◽  
Negative Feedback Regulation ◽  
Regulatory Module ◽  
Prolonged Culture ◽  
Pluripotency Genes

The ERK5 MAP kinase signalling pathway drives transcription of naïve pluripotency genes in mouse Embryonic Stem Cells (mESCs). However, how ERK5 impacts on other aspects of mESC biology has not been investigated. Here, we employ quantitative proteomic profiling to identify proteins whose expression is regulated by the ERK5 pathway in mESCs. This reveals a function for ERK5 signalling in regulating dynamically expressed early embryonic 2-cell stage (2C) genes including the mESC rejuvenation factor ZSCAN4. ERK5 signalling and ZSCAN4 induction in mESCs increases telomere length, a key rejuvenative process required for prolonged culture. Mechanistically, ERK5 promotes ZSCAN4 and 2C gene expression via transcription of the KLF2 pluripotency transcription factor. Surprisingly, ERK5 also directly phosphorylates KLF2 to drive ubiquitin-dependent degradation, encoding negative-feedback regulation of 2C gene expression. In summary, our data identify a regulatory module whereby ERK5 kinase and transcriptional activities bi-directionally control KLF2 levels to pattern 2C gene transcription and a key mESC rejuvenation process.

scholarly journals Modelling human zygotic genome activation in 8C-like cells in vitro

2021 ◽  
Author(s):  
Jasmin Taubenschmid-Stowers ◽  
Maria Rostovskaya ◽  
Fatima Santos ◽  
Sebastian Ljung ◽  
Ricard Argelaguet ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Human Embryos ◽  
Marker Genes ◽  
Cell Stage ◽  
Embryonic Genome Activation ◽  
Embryonic Genome ◽  
Cell Embryo ◽  
Genome Activation

The remodelling of the epigenome and transcriptome of the fertilised oocyte to establish totipotency in the zygote and developing embryo is one of the most critical processes in mammalian embryogenesis. Zygotic or embryonic genome activation (ZGA, EGA) in the 2-cell embryo in mouse, and the 8-cell embryo in humans, constitutes the first major wave of transcription. Failure to initiate ZGA leads to developmental defects, and contributes to the high attrition rates of human pre-implantation embryos. Due to limitations in cell numbers and experimental tractability, the mechanisms that regulate human embryonic genome activation in the totipotent embryo remain poorly understood. Here we report the discovery of human 8-cell like cells (8CLCs) specifically among naive embryonic stem cells, but not primed pluripotent cells. 8CLCs express ZGA marker genes such as ZSCAN4, LEUTX and DUXA and their transcriptome closely resembles that of the 8-cell human embryo. 8-cell like cells reactivate 8-cell stage specific transposable elements such as HERVL and MLT2A1 and are characterized by upregulation of the DNA methylation regulator DPPA3. 8CLCs show reduced SOX2 protein, and can be identified based on expression of the novel ZGA-associated protein markers TPRX1 and H3.Y in vitro. Overexpression of the transcription factor DUX4 as well as spliceosome inhibition increase ZGA-like transcription and enhance TPRX1+ 8CLCs formation. Excitingly, the in vitro identified 8CLC marker proteins TPRX1 and H3.Y are also expressed in 8-cell human embryos at the time of genome activation and may thus be relevant in vivo. The discovery of 8CLCs provides a unique opportunity to model and manipulate human ZGA-like transcriptional programs in vitro, and might provide critical functional insights into one of the earliest events in human embryogenesis in vivo.

scholarly journals Splicing of enhancer-associated lincRNAs contributes to enhancer activity

2020 ◽  
Vol 3 (4) ◽  
pp. e202000663 ◽  
Author(s):  
Jennifer Y Tan ◽  
Adriano Biasini ◽  
Robert S Young ◽  
Ana C Marques
Keyword(s):  
Gene Expression ◽  
Target Gene ◽  
Gene Expression Regulation ◽  
Embryonic Stem ◽  
Chromosomal Dna ◽  
Neighboring Gene ◽  
Enhancer Activity ◽  
Efficient Processing ◽  
Enhancer Function

Transcription is common at active mammalian enhancers sometimes giving rise to stable enhancer-associated long intergenic noncoding RNAs (elincRNAs). Expression of elincRNA is associated with changes in neighboring gene product abundance and local chromosomal topology, suggesting that transcription at these loci contributes to gene expression regulation in cis. Despite the lack of evidence supporting sequence-dependent functions for most elincRNAs, splicing of these transcripts is unexpectedly common. Whether elincRNA splicing is a mere consequence of cognate enhancer activity or if it directly impacts enhancer function remains unresolved. Here, we investigate the association between elincRNA splicing and enhancer activity in mouse embryonic stem cells. We show that multi-exonic elincRNAs are enriched at conserved enhancers, and the efficient processing of elincRNAs is strongly associated with their cognate enhancer activity. This association is supported by their enrichment in enhancer-specific chromatin signatures; elevated binding of co-transcriptional regulators; increased local intra-chromosomal DNA contacts; and strengthened cis-regulation on target gene expression. Our results support the role of efficient RNA processing of enhancer-associated transcripts to cognate enhancer activity.

The relationship between cleavage, DNA replication, and gene expression in the mouse 2-cell embryo

Development ◽  
1984 ◽  
Vol 79 (1) ◽  
pp. 139-163
Author(s):  
V. N. Bolton ◽  
P. J. Oades ◽  
M. H. Johnson
Keyword(s):  
Dna Replication ◽  
Synthetic Activity ◽  
Cleavage Division ◽  
Cell Stage ◽  
Maternal Mrna ◽  
Embryonic Genome ◽  
Cell Embryo ◽  
Two Phases ◽  
The Relationship

The 2-cell stage of mouse embryogenesis is characterized by two phases of α-amanitin-sensitive polypeptide synthetic activity, which appear to mark the first major expression of the embryonic genome, as assessed by examination of in vitro translates of mRNA. Using populations of embryos synchronized to the first cleavage division, we have established that DNA replication takes place over the period 1 to 5·5 h after the first cleavage division; the two bursts of putative transcription take place before and immediately after DNA replication, and the translation products are detectable in each case within 3–4 h. In addition, we have shown that suppression of cytokinesis and the second round of DNA replication does not affect synthesis of the α-amanitin-sensitive polypeptides, and that neither DNA replication nor the loss of maternal mRNA that take place during the 2-cell stage are dependent upon synthesis of the α-amanitin-sensitive polypeptides.

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