scholarly journals Synthetic CpG islands reveal DNA sequence determinants of chromatin structure

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Elisabeth Wachter ◽  
Timo Quante ◽  
Cara Merusi ◽  
Aleksandra Arczewska ◽  
Francis Stewart ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Chromatin Structure ◽  
Transcription Initiation ◽  
De Novo ◽  
Cpg Islands ◽  
Histone H3 ◽  
Embryonic Stem ◽  
Mammalian Genome ◽  
State Bearing ◽  
Inactive Chromatin

The mammalian genome is punctuated by CpG islands (CGIs), which differ sharply from the bulk genome by being rich in G + C and the dinucleotide CpG. CGIs often include transcription initiation sites and display ‘active’ histone marks, notably histone H3 lysine 4 methylation. In embryonic stem cells (ESCs) some CGIs adopt a ‘bivalent’ chromatin state bearing simultaneous ‘active’ and ‘inactive’ chromatin marks. To determine whether CGI chromatin is developmentally programmed at specific genes or is imposed by shared features of CGI DNA, we integrated artificial CGI-like DNA sequences into the ESC genome. We found that bivalency is the default chromatin structure for CpG-rich, G + C-rich DNA. A high CpG density alone is not sufficient for this effect, as A + T-rich sequence settings invariably provoke de novo DNA methylation leading to loss of CGI signature chromatin. We conclude that both CpG-richness and G + C-richness are required for induction of signature chromatin structures at CGIs.

scholarly journals Collaboration between CpG sites is needed for stable somatic inheritance of DNA methylation states

2013 ◽  
Vol 42 (4) ◽  
pp. 2235-2244 ◽  
Author(s):  
Jan O. Haerter ◽  
Cecilia Lövkvist ◽  
Ian B. Dodd ◽  
Kim Sneppen
Keyword(s):  
Dna Sequences ◽  
De Novo ◽  
Cpg Islands ◽  
Stochastic Simulations ◽  
Systematic Sampling ◽  
Cell Cycles ◽  
Inheritance Model ◽  
Multiple Cell ◽  
Dynamic Collaboration ◽  
Methylation Patterns

Abstract Inheritance of 5-methyl cytosine modification of CpG (CG/CG) DNA sequences is needed to maintain early developmental decisions in vertebrates. The standard inheritance model treats CpGs as independent, with methylated CpGs maintained by efficient methylation of hemimethylated CpGs produced after DNA replication, and unmethylated CpGs maintained by an absence of de novo methylation. By stochastic simulations of CpG islands over multiple cell cycles and systematic sampling of reaction parameters, we show that the standard model is inconsistent with many experimental observations. In contrast, dynamic collaboration between CpGs can provide strong error-tolerant somatic inheritance of both hypermethylated and hypomethylated states of a cluster of CpGs, reproducing observed stable bimodal methylation patterns. Known recruitment of methylating enzymes by methylated CpGs could provide the necessary collaboration, but we predict that recruitment of demethylating enzymes by unmethylated CpGs strengthens inheritance and allows CpG islands to remain hypomethylated within a sea of hypermethylation.

scholarly journals Histone H3 Acetylation and H3 K4 Methylation Define Distinct Chromatin Regions Permissive for Transgene Expression

2006 ◽  
Vol 26 (17) ◽  
pp. 6357-6371 ◽  
Author(s):  
Chunhong Yan ◽  
Douglas D. Boyd
Keyword(s):  
Histone Modifications ◽  
Transgene Expression ◽  
De Novo ◽  
Histone H3 ◽  
Mammalian Genome ◽  
Chromatin Region ◽  
Histone H3 Acetylation ◽  
Transcription Activators ◽  
And Function ◽  
H3 Acetylation

ABSTRACT Histone modifications are associated with distinct transcription states and serve as heritable epigenetic markers for chromatin structure and function. While H3 K9 methylation defines condensed heterochromatin that is able to silence a nearby gene, how gene silencing within euchromatin regions is achieved remains elusive. We report here that histone H3 K4 methylation or K9/K14 acetylation defines distinct chromatin regions permissive or nonpermissive for transgene expression. A permissive chromatin region is enriched in H3 K4 methylation and H3 acetylation, while a nonpermissive region is poor in or depleted of these two histone modifications. The histone modification states of the permissive chromatin can spread to transgenic promoters. However, de novo histone H3 acetylation and H3 K4 methylation at a transgenic promoter in a nonpermissive chromatin region are stochastic, leading to variegated transgene expression. Moreover, nonpermissive chromatin progressively silences a transgene, an event that is accompanied by the reduction of H3 K4 methylation and H3 acetylation levels at the transgenic promoter. These repressive effects of nonpermissive chromatin cannot be completely countered by strong transcription activators, indicating the dominance of the chromatin effects. We therefore propose a model in which histone H3 acetylation and H3 K4 methylation localized to discrete sites in the mammalian genome mark distinct chromatin functions that dictate transgene expression or silencing.

scholarly journals Role of transcription complexes in the formation of the basal methylation pattern in early development

2018 ◽  
Vol 115 (41) ◽  
pp. 10387-10391 ◽  
Author(s):  
Razi Greenfield ◽  
Amalia Tabib ◽  
Ilana Keshet ◽  
Joshua Moss ◽  
Ofra Sabag ◽  
...  
Keyword(s):  
De Novo ◽  
Cpg Islands ◽  
Embryonic Stem ◽  
Methylation Pattern ◽  
Entire Genome ◽  
Genome Wide ◽  
Topological Pattern ◽  
Histone H3k4 ◽  
Transcription Complexes ◽  
De Novo Methylation

Following erasure in the blastocyst, the entire genome undergoes de novo methylation at the time of implantation, with CpG islands being protected from this process. This bimodal pattern is then preserved throughout development and the lifetime of the organism. Using mouse embryonic stem cells as a model system, we demonstrate that the binding of an RNA polymerase complex on DNA before de novo methylation is predictive of it being protected from this modification, and tethering experiments demonstrate that the presence of this complex is, in fact, sufficient to prevent methylation at these sites. This protection is most likely mediated by the recruitment of enzyme complexes that methylate histone H3K4 over a local region and, in this way, prevent access to the de novo methylation complex. The topological pattern of H3K4me3 that is formed while the DNA is as yet unmethylated provides a strikingly accurate template for modeling the genome-wide basal methylation pattern of the organism. These results have far-reaching consequences for understanding the relationship between RNA transcription and DNA methylation.

scholarly journals snRNA and Heterochromatin Formation Are Involved in DNA Excision during Macronuclear Development in Stichotrichous Ciliates

2005 ◽  
Vol 4 (11) ◽  
pp. 1934-1941 ◽  
Author(s):  
Stefan A. Juranek ◽  
Sina Rupprecht ◽  
Jan Postberg ◽  
Hans J. Lipps
Keyword(s):  
Dna Sequences ◽  
Chromatin Immunoprecipitation ◽  
De Novo ◽  
Histone H3 ◽  
Heterochromatin Formation ◽  
Macronuclear Development ◽  
Dna Elimination ◽  
Histone H3 Variant ◽  
Specific Sequences

ABSTRACT Several models for specific excision of micronucleus-specific DNA sequences during macronuclear development in ciliates exist. While the template-guided recombination model suggests recombination events resulting in specific DNA excision and reordering of macronucleus-destined sequences (MDS) guided by a template, there is evidence that an RNA interference-related mechanism is involved in DNA elimination in holotrichous ciliates. We describe that in the stichotrichous ciliate Stylonychia, snRNAs homologous to micronucleus-specific sequences are synthesized during macronuclear differentiation. Western and in situ analyses demonstrate that histone H3 becomes methylated at K9 de novo during macronuclear differentiation, and chromatin immunoprecipitation revealed that micronucleus-specific sequences are associated with methylated H3. To link both observations, expression of a PIWI homolog, member of the RNA-induced silencing complex, was silenced. In these cells, the methylated micronucleus-specific histone H3 variant “X” is still present in macronuclear anlagen and no K9 methylation of histone H3 is observed. We suggest that snRNA recruits chromatin-modifying enzymes to sequences to be excised. Based on our and earlier observations, we believe that this mechanism is not sufficient for specific excision of sequences and reordering of MDS in the developing macronucleus and propose a model for internal eliminated sequence excision and MDS reordering in stichotrichous ciliates.

De novo assembly and delivery to mouse cells of a 101 kb functional human gene

Genetics ◽  
2021 ◽  
Author(s):  
Leslie A Mitchell ◽  
Laura H McCulloch ◽  
Sudarshan Pinglay ◽  
Henri Berger ◽  
Nazario Bosco ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Large Scale ◽  
De Novo ◽  
Human Gene ◽  
Embryonic Stem ◽  
Building Blocks ◽  
Functional Study ◽  
Dna Assembly ◽  
Functional Evaluation ◽  
Mouse Cells

Abstract Design and large-scale synthesis of DNA has been applied to the functional study of viral and microbial genomes. New and expanded technology development is required to unlock the transformative potential of such bottom-up approaches to the study of larger mammalian genomes. Two major challenges include assembling and delivering long DNA sequences. Here we describe a workflow for de novo DNA assembly and delivery that enables functional evaluation of mammalian genes on the length scale of 100 kilobase pairs (kb). The DNA assembly step is supported by an integrated robotic workcell. We demonstrate assembly of the 101 kb human HPRT1 gene in yeast from 3 kb building blocks, precision delivery of the resulting construct to mouse embryonic stem cells, and subsequent expression of the human protein from its full-length human gene in mouse cells. This workflow provides a framework for mammalian genome writing. We envision utility in producing designer variants of human genes linked to disease and their delivery and functional analysis in cell culture or animal models.

scholarly journals A DNA signal from the Thy-1 gene defines de novo methylation patterns in embryonic stem cells.

1990 ◽  
Vol 10 (8) ◽  
pp. 4396-4400 ◽  
Author(s):  
M Szyf ◽  
G Tanigawa ◽  
P L McCarthy
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Dna Sequences ◽  
De Novo ◽  
Cpg Island ◽  
Embryonic Stem ◽  
Cpg Dinucleotides ◽  
Somatic Tissues ◽  
Methylation Patterns ◽  
De Novo Methylation

Although DNA can be extensively methylated de novo when introduced into pluripotent cells, the CpG island in the Thy-1 gene does not become methylated either in the mouse embryo or in embryonic stem cells. A 214-base-pair region near the promoter of the Thy-1 gene protects itself as well as heterologous DNA sequences from de novo methylation. We propose that this nucleotide sequence is representative of a class of important signals that limits de novo methylation in the embryo and establishes the pattern of hypomethylated CpG dinucleotides found in somatic tissues.

Transcripts and CpG islands associated with the pro-opiomelanocortin gene and other neurally expressed genes

1994 ◽  
Vol 12 (3) ◽  
pp. 365-382 ◽  
Author(s):  
M Gardiner-Garden ◽  
M Frommer
Keyword(s):  
Xenopus Laevis ◽  
Dna Sequences ◽  
Cpg Island ◽  
Mammalian Species ◽  
Cpg Islands ◽  
Embryonic Stem ◽  
Northern Blot Hybridization ◽  
Embryonal Carcinoma Cell ◽  
Transcription Start ◽  
The Third

ABSTRACT DNA sequences of vertebrate genes which code for neural or neuroendocrine peptides were analysed in terms of CpG dinucleotide distribution and G+C content. The vast majority of the genes were found to contain a region with the sequence characteristics of a CpG island surrounding the 5′ end. In mammalian species, the gene which codes for the neuroendocrine polypeptide pro-opiomelanocortin (POMC) was shown to be associated with two separate CpG islands: a 5′ CpG island which surrounds the POMC transcription start site and a 3′ CpG island which lies approximately 5 kb downstream, encompassing the third exon of POMC. Short POMC-related transcripts, known to be transcribed in the germline, were found to initiate from a promoter within the 3′ CpG island. The start sites of the short POMC-related transcripts in mouse testis were mapped to the region coding for γMSH in exon 3, in a similar location to transcription start sites identified in other mammalian POMC genes. Similar short POMC-related transcripts were identified in both the mouse F9 embryonal carcinoma cell line and mouse embryonic stem cells, suggesting that transcription initiating within the third exon may occur very early in development. No short transcripts were detected by Northern blot hybridization in either Xenopus laevis testis or oocyte poly(A)+ RNA extracts. The Xenopus laevis POMC genes, A and B, were associated with neither a 5′ nor a 3′ CpG island. Hence, the presence of a 5′ CpG island is not required for production of full-length transcripts from the Xenopus laevis POMC gene, but the presence of a 3′ CpG island may be required for transcription to occur from the third exon.

The epigenetic regulator Cfp1

2010 ◽  
Vol 1 (5-6) ◽  
pp. 325-334 ◽  
Author(s):  
David G. Skalnik
Keyword(s):  
Dna Methyltransferase ◽  
Cellular Differentiation ◽  
Cytosine Methylation ◽  
Cpg Islands ◽  
Histone H3 ◽  
Embryonic Stem ◽  
Finger Protein ◽  
Epigenetic Regulator ◽  
Genomic Locations ◽  
Cxxc Finger Protein 1

AbstractNumerous epigenetic modifications have been identified and correlated with transcriptionally active euchromatin or repressed heterochromatin and many enzymes responsible for the addition and removal of these marks have been characterized. However, less is known regarding how these enzymes are regulated and targeted to appropriate genomic locations. Mammalian CXXC finger protein 1 is an epigenetic regulator that was originally identified as a protein that binds specifically to any DNA sequence containing an unmethylated CpG dinucleotide. Mouse embryos lacking CXXC finger protein 1 die prior to gastrulation, and embryonic stem cells lacking CXXC finger protein 1 are viable but are unable to achieve cellular differentiation and lineage commitment. CXXC finger protein 1 is a regulator of both cytosine and histone methylation. It physically interacts with DNA methyltransferase 1 and facilitates maintenance cytosine methylation. Rescue studies reveal that CXXC finger protein 1 contains redundant functional domains that are sufficient to support cellular differentiation and proper levels of cytosine methylation. CXXC finger protein 1 is also a component of the Setd1 histone H3-Lys4 methyltransferase complexes and functions to target these enzymes to unmethylated CpG islands. Depletion of CXXC finger protein 1 leads to loss of histone H3-Lys4 tri-methylation at CpG islands and inappropriate drifting of this euchromatin mark into areas of hetero-chromatin. Thus, one function of CXXC finger protein 1 is to serve as an effector protein that interprets cytosine methylation patterns and facilitates crosstalk with histone-modifying enzymes.

scholarly journals 278 Jak/Stat3 SIGNALING PROMOTES SOMATIC CELL REPROGRAMMING BY EPIGENETIC REGULATION

2013 ◽  
Vol 25 (1) ◽  
pp. 287
Author(s):  
Y. Tang ◽  
Y. Luo ◽  
Z. Jiang ◽  
M. Carter ◽  
X. (Cindy) Tian
Keyword(s):  
Stem Cells ◽  
Dna Sequences ◽  
Histone Deacetylases ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Embryonic Stem ◽  
Dna Demethylation ◽  
Janus Kinase ◽  
Cell Reprogramming ◽  
Open Chromatin

Although leukemia inhibitory factor (LIF) maintains the ground state pluripotency of mouse embryonic stem cells and induced pluripotent stem cells (iPSC) by activating the Janus kinase/signal transducer and activator of transcription 3 (Jak/Stat3) pathway, the mechanism remains unclear. Stat3 has only been shown to promote complete reprogramming of epiblast and neural stem cells, and the partially reprogrammed cells (pre-iPSC). We investigated if and how Jak/Stat3 activation promotes reprogramming of terminally differentiated mouse embryonic fibroblasts (MEF). We demonstrated that activated Stat3 not only promotes but is essential for the pluripotency establishment in MEF cell reprogramming. We further demonstrated that, during reprogramming, inhibiting Jak/Stat3 activity blocks demethylation of Oct4 and Nanog regulatory DNA sequences in induced cells, which are marked by suppressed endogenous pluripotent gene expression. These are correlated with significant upregulation of DNA methyltransferase (Dnmt) 1 and histone deacetylases (HDAC) expression, as well as the increased expression of lysine-specific histone demethylase 2 and methyl-CpG binding protein 2. Inhibiting Jak/Stat3 also blocks the expression of Dnmt3L, which is correlated with the failure of retroviral transgene silencing. Furthermore, Dnmt or HDAC inhibitor but not overexpression of Nanog rescues the reprogramming arrested by Jak/Stat3 inhibition or LIF deprivation. Finally, we demonstrated that LIF/Stat3 signal also represents the prerequisite for complete reprogramming of pre-iPSC. We conclude that Jak/Stat3 activity plays a fundamental role in promoting the establishment of pluripotency at the epigenetic level, by facilitating DNA demethylation/de novo methylation and open-chromatin formation during late stage reprogramming.

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