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2022 ◽  
Author(s):  
Mélanie Pailles ◽  
Mélanie Hirlemann ◽  
Vincent Brochard ◽  
Martine Chebrout ◽  
Jean-François Oudin ◽  
...  

Abstract Early mouse development is characterized by structural and epigenetic changes at the chromatin level while cells progress towards differentiation. At blastocyst stage, the segregation of the three primordial lineages is accompanied by establishment of differential patterns of DNA methylation and post-translational modifications of histones, such as H3K27me3. In this study, we have analysed the dynamics of H3K27me3 at pericentromeric heterochromatin (PCH) during development of the mouse blastocyst, in comparison with cultured embryonic cells. We show that this histone modification is first enriched at PCH in the whole embryo and evolves into a diffuse distribution in epiblast during its specification and maturation. Concomitantly, the level of transcription from major satellite decreases. Stem cells derived from blastocyst (naïve ESCs and TSCs) do not fully maintain the H3K27me3 enrichment at PCH. Moreover, the dynamic of H3K27me3 at PCH during in vitro conversion from naïve to primed pluripotent state and during ESCs derivation suggests that the mechanisms underlying the control of this histone mark at PCH are different in embryo and in vitro. We also conclude that the non-canonical presence of H3K27me3 at PCH is a defining feature of embryonic cells in the young blastocyst before epiblast segregation.


2021 ◽  
Vol 8 ◽  
Author(s):  
Samanta Raboni ◽  
Serena Montalbano ◽  
Stephanie Stransky ◽  
Benjamin A. Garcia ◽  
Annamaria Buschini ◽  
...  

Methionine is an essential amino acid used, beyond protein synthesis, for polyamine formation and DNA/RNA/protein methylation. Cancer cells require particularly high methionine supply for their homeostasis. A successful approach for decreasing methionine concentration is based on the systemic delivery of methionine γ-lyase (MGL), with in vitro and in vivo studies demonstrating its efficacy in cancer therapy. However, the mechanisms explaining how cancer cells suffer from the absence of methionine more significantly than non-malignant cells are still unclear. We analyzed the outcome of the human colorectal adenocarcinoma cancer cell line HT29 to the exposure of MGL for up to 72 h by monitoring cell viability, proteome expression, histone post-translational modifications, and presence of spurious transcription. The rationale of this study was to verify whether reduced methionine supply would affect chromatin decondensation by changing the levels of histone methylation and therefore increasing genomic instability. MGL treatment showed a time-dependent cytotoxic effect on HT29 cancer cells, with an IC50 of 30 µg/ml, while Hs27 normal cells were less affected, with an IC50 of >460 µg/ml. Although the levels of total histone methylation were not altered, a loss of the silencing histone mark H3K9me2 was observed, as well as a decrease in H4K20me3. Since H3K9me2/3 decorate repetitive DNA elements, we proved by qRT-PCR that MGL treatment leads to an increased expression of major satellite units. Our data indicate that selected histone methylation marks may play major roles in the mechanism of methionine starvation in cancer cells, proving that MGL treatment directly impacts chromatin homeostasis.


2021 ◽  
Author(s):  
Madlen Mueller ◽  
Tara Faeh ◽  
Moritz Schaefer ◽  
Janina Luitz ◽  
Patrick Stalder ◽  
...  

In the past years, several studies reported nuclear roles for the Argonaute (AGO) proteins associating them with transcriptional activation or repression, alternative splicing and, chromatin organization. However, as most of these experiments have been conducted in human cancer cell lines, the nuclear functions of the AGO proteins in mouse early embryonic development still remains elusive. In this study, we investigated possible nuclear functions of the AGO proteins in mouse Embryonic Stem Cells (mESCs). By biochemical assays, we observed that AGO1 and AGO2 are present in a small fraction in the nucleus and even less on chromatin in mESCs. To profile the nuclear interactome of the AGO proteins, we performed immunoprecipitation followed by Mass Spectrometry and identified three novel nuclear interactors for AGO1, namely DNMT3a, HP1a;, and ATRX. These interactors are well-known proteins involved in the establishment and maintenance of heterochromatin at pericentromeric regions. Indeed, upon depletion of Ago1, we observed a specific redistribution of the heterochromatin protein HP1a; and the repressive histone mark H3K9me3, away from pericentromeric regions. Furthermore, these regions are characterized by AT-rich tandem repeats known as major satellite sequences. We demonstrated that major satellite transcripts are strongly upregulated in Ago1_KO mESCs. Interestingly, this phenotype was not caused by the loss of genome integrity at pericentromeres, as these could still form normally in Ago1_KO mESCs. Lastly, we showed that specific microRNAs loaded in AGO1, regulate the expression of the major satellite transcripts. Overall, our results demonstrate for the first time a novel role for AGO1 in regulating major satellite transcripts and localization of HP1a; and H3K9me3 at pericentromeres in mESCs.


PLoS Genetics ◽  
2021 ◽  
Vol 17 (5) ◽  
pp. e1009570
Author(s):  
Kanako Kibe ◽  
Kenjiro Shirane ◽  
Hiroaki Ohishi ◽  
Shuhei Uemura ◽  
Hidehiro Toh ◽  
...  

DNA methylation at CG sites is important for gene regulation and embryonic development. In mouse oocytes, de novo CG methylation requires preceding transcription-coupled histone mark H3K36me3 and is mediated by a DNA methyltransferase DNMT3A. DNMT3A has a PWWP domain, which recognizes H3K36me2/3, and heterozygous mutations in this domain, including D329A substitution, cause aberrant CG hypermethylation of regions marked by H3K27me3 in somatic cells, leading to a dwarfism phenotype. We herein demonstrate that D329A homozygous mice show greater CG hypermethylation and severer dwarfism. In oocytes, D329A substitution did not affect CG methylation of H3K36me2/3-marked regions, including maternally methylated imprinting control regions; rather, it caused aberrant hypermethylation in regions lacking H3K36me2/3, including H3K27me3-marked regions. Thus, the role of the PWWP domain in CG methylation seems similar in somatic cells and oocytes; however, there were cell-type-specific differences in affected regions. The major satellite repeat was also hypermethylated in mutant oocytes. Contrary to the CA hypomethylation in somatic cells, the mutation caused hypermethylation at CH sites, including CA sites. Surprisingly, oocytes expressing only the mutated protein could support embryonic and postnatal development. Our study reveals that the DNMT3A PWWP domain is important for suppressing aberrant CG hypermethylation in both somatic cells and oocytes but that D329A mutation has little impact on the developmental potential of oocytes.


2021 ◽  
Author(s):  
Vito Abbruzzese

In this study, the DNA of Rana dalmatina was digested with Asp 718I and the two bands of highly repeated DNA produced were cloned and characterised. The largest fragment (494 bp) corresponded to the entire repetitive unit of the major satellite DNA (RdS1a), while the smaller fragment of 385 bp corresponded to the major fragment of RdS1a produced by digestion. A fragment of 332 bpbcorresponding to the repetitive unit of satellite S1b (RdS1b) was instead achieved by digestion with Eco RV. RdS1b is highly homologous to the corresponding portion of the repetition of RdS1a and presents the first 36 bp repeated and inverted. This suggested that RdS1b would have been derived from satellite S1a by two distinct and subsequent events. Further, the high sequence homology and length between RdS1a and the S1a of Rana italica (RiS1a) confirmed the hypothesis that the satellite S1a is antecedent to S1b and inherited from a common ancestor. Southern blots of R. dalmatina genomic DNA digested with Asp 718I produced hybrid bands of fragments of different sizes containing in addition to the satellite S1a, also one or more copies of the S1b satellites. The only sequenced band at the moment corresponded to the repetitive unit of the satellite RdS1a + b (826 bp) deleted of the fragment Asp 718I less than RdS1a (109 bp), while the other double bands should almost certainly correspond to repetitive units of satellites RdS1a + 2b and RdS1a + 3b. Our data suggested different satellite DNA organisation in R. dalmatina, including the tandem structure of the repetitive units of the RdS1a or RdS1b. Our data also suggested the existence in R. dalmatina of at least four different types of hybrid repeating units in all the populations examined.


2021 ◽  
Author(s):  
Deepika Puri ◽  
Birgit Koschorz ◽  
Bettina Engist ◽  
Megumi Onishi-Seebacher ◽  
Devon Ryan ◽  
...  

Repeat element transcription plays a vital role in early embryonic development. Expression of repeats such as MERVL characterises mouse embryos at the 2-cell stage, and defines a 2-cell-like cell (2CLC) population in a mouse embryonic stem cell culture. Repeat element sequences contain binding sites for numerous transcription factors. We identify the forkhead domain transcription factor FOXD3 as a regulator of repeat element transcription in mouse embryonic stem cells. FOXD3 binds to and recruits the histone methyltransferase SUV39H1 to MERVL and major satellite repeats, consequentially repressing the transcription of these repeats by the establishment of the H3K9me3 heterochromatin modification. Notably, depletion of FOXD3 leads to the de-repression of MERVL and major satellite repeats as well as a subset of genes expressed in the 2-cell state, shifting the balance between the stem cell and 2 cell-like population in culture. Thus, FOXD3 acts as a negative regulator of repeat transcription, ascribing a novel function to this transcription factor.


2021 ◽  
Author(s):  
Antoine Canat ◽  
Adeline Veillet ◽  
Robert Illingworth ◽  
Emmanuelle Fabre ◽  
Pierre Therizols

AbstractDNA methylation is essential for heterochromatin formation and repression of DNA repeat transcription, both of which are essential for genome integrity. Loss of DNA methylation is associated with disease, including cancer, but is also required for development. Alternative pathways to maintain heterochromatin are thus needed to limit DNA damage accumulation. Here, we find that DAXX, an H3.3 chaperone, protects pericentromeric heterochromatin and is essential for embryonic stem cells (ESCs) maintenance in the ground-state of pluripotency. Upon DNA demethylation-mediated damage, DAXX relocalizes to pericentromeric regions, and recruits PML and SETDB1, thereby promoting heterochromatin formation. In the absence of DAXX, the 3D-architecture and physical properties of pericentric heterochromatin are disrupted, resulting in derepression of major satellite DNA. Using epigenome editing tools, we demonstrate that H3.3, and specifically H3.3K9 modification, directly contribute to maintaining pericentromeric chromatin conformation. Altogether, our data reveal that DAXX and H3.3 unite DNA damage response and heterochromatin maintenance in ESCs.


2021 ◽  
Author(s):  
Martine Chebrout ◽  
Maimouna Coura Kone ◽  
Habib U. Jan ◽  
Marie Cournut ◽  
Martine Letheule ◽  
...  

AbstractDuring the first cell cycles of the early development, the chromatin of the embryo is highly reprogrammed alongside that embryonic genome starts its own transcription. The spatial organization of the genome is a major process that contributes to regulating gene transcription in time and space, however, it is poorly studied in the context of early embryos. To study the cause and effect link between transcription and spatial organization in embryos, we focused on the ribosomal genes, that are first silent and begin to transcribe during the 2-cell stage in mouse. We demonstrated that ribosomal sequences are spatially organized in a very peculiar manner from the 2-cell to the 16-cell stage with transcription and processing of ribosomal RNAs excluding mutually. Using drugs inhibiting the RNA polymerase I, we show that this organization, totally different from somatic cells, depends on an active transcription of ribosomal genes and induces a unique chromatin environment that favors major satellite sequences transcription after the 4-cell stage.


BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Uma P. Arora ◽  
Caleigh Charlebois ◽  
Raman Akinyanju Lawal ◽  
Beth L. Dumont

Abstract Background Mammalian centromeres are satellite-rich chromatin domains that execute conserved roles in kinetochore assembly and chromosome segregation. Centromere satellites evolve rapidly between species, but little is known about population-level diversity across these loci. Results We developed a k-mer based method to quantify centromere copy number and sequence variation from whole genome sequencing data. We applied this method to diverse inbred and wild house mouse (Mus musculus) genomes to profile diversity across the core centromere (minor) satellite and the pericentromeric (major) satellite repeat. We show that minor satellite copy number varies more than 10-fold among inbred mouse strains, whereas major satellite copy numbers span a 3-fold range. In contrast to widely held assumptions about the homogeneity of mouse centromere repeats, we uncover marked satellite sequence heterogeneity within single genomes, with diversity levels across the minor satellite exceeding those at the major satellite. Analyses in wild-caught mice implicate subspecies and population origin as significant determinants of variation in satellite copy number and satellite heterogeneity. Intriguingly, we also find that wild-caught mice harbor dramatically reduced minor satellite copy number and elevated satellite sequence heterogeneity compared to inbred strains, suggesting that inbreeding may reshape centromere architecture in pronounced ways. Conclusion Taken together, our results highlight the power of k-mer based approaches for probing variation across repetitive regions, provide an initial portrait of centromere variation across Mus musculus, and lay the groundwork for future functional studies on the consequences of natural genetic variation at these essential chromatin domains.


2021 ◽  
Author(s):  
Michael Gutbrod ◽  
Benjamin Roche ◽  
Joshua Steinberg ◽  
Asad Lakhani ◽  
Kenneth Chang ◽  
...  

RNA interference is essential for transcriptional silencing and genome stability, but conservation of this role in mammals has been difficult to demonstrate. Dicer1-/- mouse embryonic stem cells have microRNA-independent proliferation defects, and we conducted a CRISPR-Cas9 screen to restore viability. We identified suppressor mutations in transcriptional activators, H3K9 methyltransferases, and chromosome segregation factors, strongly resembling Dicer suppressors in fission yeast. Suppressors rescued chromosomal defects, and reversed strand-specific transcription of major satellite repeats in Dicer1-/-. The strongest suppressors were in Brd4, and in the transcriptional elongator/histone acetyltransferase Elp3. Using viable mutants and pharmaceutical inhibitors, we demonstrate that deletion of specific residues in Brd4 rescue genome instability defects of Dicer1-/- in both mammalian cells and fission yeast, implicating Dicer in coordinating transcription and replication of satellite repeats.


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