scholarly journals The chromatin remodeler LSH controls genome-wide cytosine hydroxymethylation

2020 ◽  
Author(s):  
Maud de Dieuleveult ◽  
Martin Bizet ◽  
Laurence Colin ◽  
Emilie Calonne ◽  
Martin Bachman ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Embryonic Stem ◽  
Direct Consequence ◽  
Wild Type ◽  
Embryonic Fibroblasts ◽  
Knock Out ◽  
Tet Proteins ◽  
Genome Wide ◽  
Specific Factors ◽  
Epigenetic Repression

ABSTRACTTET proteins convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), leading to a dynamic epigenetic state of DNA that can influence transcription. While TET proteins have been associated with either epigenetic repression or activation complexes, the overall understanding of the molecular mechanisms involved in TET-mediated regulation of gene transcription still remains limited. Here, we show that TET proteins interact with lymphoid-specific helicase (LSH), a chromatin remodeling factor belonging to the SNF2 super family. Lsh knock-out leads to a significant reduction of 5-hydroxymethylation global level in mouse embryonic fibroblasts (MEFs) and in embryonic stem cells (ESC). Whole genome sequencing of 5hmC in wild-type versus Lsh knock-out MEFs and ESCs showed that in absence of Lsh, some regions of the genome gain 5hmC while others lose it, with not much effect on gene expression. We further show that 5hmC modifications upon Lsh loss is not a direct consequence of 5mC decrease, as differentially hydroxymethylated regions (DhMR) did not overlap with DMR (differentially methylated regions), underlying that these modifications occurred at different genomic loci. Altogether, our results suggest that LSH is a key regulator of 5hmC in both MEFs and ESC and that TET proteins rely on specific factors to establish genome-wide 5hmC patterns.

Dysregulation of schizophrenia-associated genes and genome-wide hypomethylation in neurons overexpressing DNMT1

Epigenomics ◽  
2021 ◽  
Author(s):  
Sonal Saxena ◽  
Sumana Choudhury ◽  
Pranay Amruth Maroju ◽  
Anuhya Anne ◽  
Lov Kumar ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Copy Number ◽  
Embryonic Stem ◽  
Wild Type ◽  
Independent Manner ◽  
Transcript Levels ◽  
Genome Wide ◽  
Methylation Patterns ◽  
Increased Activity

Aim: To study the effects of DNMT1 overexpression on transcript levels of genes dysregulated in schizophrenia and on genome-wide methylation patterns. Materials & methods: Transcriptome and DNA methylome comparisons were made between R1 (wild-type) and Dnmt1tet/tet mouse embryonic stem cells and neurons overexpressing DNMT1. Genes dysregulated in both Dnmt1tet/tet cells and schizophrenia patients were studied further. Results & conclusions: About 50% of dysregulated genes in patients also showed altered transcript levels in Tet/Tet neurons in a DNA methylation-independent manner. These neurons unexpectedly showed genome-wide hypomethylation, increased transcript levels of Tet1 and Apobec 1-3 genes and increased activity and copy number of LINE-1 elements. The observed similarities between Tet/Tet neurons and schizophrenia brain samples reinforce DNMT1 overexpression as a risk factor.

scholarly journals Dual Effect of Tamoxifen on Arterial KCa Channels Does Not Depend on the Presence of the β1 Subunit

2005 ◽  
Vol 280 (23) ◽  
pp. 21739-21747 ◽  
Author(s):  
Guillermo J. Pérez
Keyword(s):  
Molecular Mechanisms ◽  
Single Channel ◽  
Protective Role ◽  
Wild Type ◽  
Open Probability ◽  
Dual Effect ◽  
Knock Out ◽  
Kca Channels ◽  
Molecular Specificity ◽  
Excised Patches

Tamoxifen has been reported to directly activate large conductance calcium-activated potassium (KCa) channels through the KCa β1 subunit, suggesting a cardio-protective role of this compound. The present study using knock-out (KO) mice for the KCa channel β1 subunit was aimed at understanding the molecular mechanisms of the effects of tamoxifen on arterial smooth muscle KCa channels. Single channel studies were conducted in excised patches from cerebral artery myocytes from both wild-type and KO animals. The present data demonstrated that tamoxifen can inhibit arterial KCa channels due to a major decrease in channel open probability (Po), a mechanism different from the reduction in single channel amplitude reported previously and also observed in the present work. A tamoxifen-induced decrease in Po was present in arterial KCa channels from both wild-type and β1 KO animals. This inhibition was concentration-dependent and partially reversible with a half-maximal concentration constant IC50 of 2.6 μm. The effect of tamoxifen was actually dual Single channel kinetic analysis showed that tamoxifen shortens both mean closed time and mean open time; the latter is probably due to an intermediate duration voltage-independent blocking mechanism. Thus, tamoxifen block would predominate when KCa channel Po is >0.1–0.2, limiting the maximum Po, whereas a leftward shift in voltage or Ca2+ activation curves can be observed for Po values lower than those values. This dual effect of tamoxifen appears to be independent of the β1 subunit. The molecular specificity of tamoxifen, or eventually other xenoestrogen derivatives, for the KCa channel β1 subunit is uncertain.

New Role of the Regulatory Gene SOX2 in Hematopoiesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4195-4195
Author(s):  
Elena Levantini ◽  
Francesca Bertolotti ◽  
Francesco Cerisoli ◽  
Anna L. Ferri ◽  
Elisa Brescia ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Blood Cell ◽  
Molecular Mechanisms ◽  
Bone Marrow Cells ◽  
Regulatory Gene ◽  
Embryonic Stem ◽  
Mutant Mice ◽  
Wild Type ◽  
Cell Production

Abstract Several genes encoding transcription factors of different families have been implicated in the development and differentiation of multiple cell systems. The Sry-type high-mobility-group box 2 gene (Sox2) encodes a transcription factor that is expressed in very early cells such as embryonic stem cells and neural stem cells, where it plays important functional roles (Genes and Dev.17:126, 2003; Development131:3805, 2004). To investigate whether Sox2 plays a role also in blood cell production, we first analyzed its expression in murine hematopoietic cells. Results indicate that the gene is transcriptionally active at low levels in primitive progenitors. Furthermore, in order to address the functional implication of Sox2 in hematopoiesis we analyzed mature and precursor cells in mutant mice compound heterozygotes for a null Sox2 allele and for the deletion of a Sox2 5′ enhancer, as the complete inactivation of the gene in homozygosis is embryonic lethal. At the peripheral blood level we did not detect significant variations in the mutants. However analysis of bone marrow precursors in clonogenic assays showed that Sox2 knock-down mice exhibited a significant increase in the number of multipotent precursors, as compared to wild type animals. Moreover, bone marrow cells of wild type and mutant mice were analyzed for the expression of a panel of regulatory genes involved in the control of different somatic stem cells. Preliminary evidence suggests that some of these genes are modulated in the mutant cells. These observations support the view that Sox2 plays a role at early stages of blood cell production, providing further evidence that common molecular mechanisms may be involved in the regulation of several different types of multipotent cells.

scholarly journals Genetically Modified Rabbits for Cardiovascular Research

2021 ◽  
Vol 12 ◽  
Author(s):  
Jianglin Fan ◽  
Yanli Wang ◽  
Y. Eugene Chen
Keyword(s):  
Molecular Mechanisms ◽  
Genetically Modified ◽  
Embryonic Stem ◽  
Cardiovascular Research ◽  
Knock Out ◽  
Experimental Animals ◽  
Transgenic Rabbits ◽  
Long Time ◽  
Human Atherosclerosis ◽  
Genome Information

Rabbits are one of the most used experimental animals for investigating the mechanisms of human cardiovascular disease and lipid metabolism because they are phylogenetically closer to human than rodents (mice and rats). Cholesterol-fed wild-type rabbits were first used to study human atherosclerosis more than 100 years ago and are still playing an important role in cardiovascular research. Furthermore, transgenic rabbits generated by pronuclear microinjection provided another means to investigate many gene functions associated with human disease. Because of the lack of both rabbit embryonic stem cells and the genome information, for a long time, it has been a dream for scientists to obtain knockout rabbits generated by homologous recombination-based genomic manipulation as in mice. This obstacle has greatly hampered using genetically modified rabbits to disclose the molecular mechanisms of many human diseases. The advent of genome editing technologies has dramatically extended the applications of experimental animals including rabbits. In this review, we will update genetically modified rabbits, including transgenic, knock-out, and knock-in rabbits during the past decades regarding their use in cardiovascular research and point out the perspectives in future.

scholarly journals Loss of AMPKalpha1 Triggers Centrosome Amplification via PLK4 Upregulation in Mouse Embryonic Fibroblasts

2020 ◽  
Vol 21 (8) ◽  
pp. 2772
Author(s):  
Qiang Zhao ◽  
Kathleen A Coughlan ◽  
Ming-Hui Zou ◽  
Ping Song
Keyword(s):  
Molecular Mechanisms ◽  
Adenosine Monophosphate ◽  
Centrosome Amplification ◽  
Wild Type ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Cellular Energy ◽  
Chromosome Missegregation ◽  
Cell Mitosis ◽  
Metaphase Chromosome Spread

Recent evidence indicates that activation of adenosine monophosphate-activated protein kinase (AMPK), a highly conserved sensor and modulator of cellular energy and redox, regulates cell mitosis. However, the underlying molecular mechanisms for AMPKα subunit regulation of chromosome segregation remain poorly understood. This study aimed to ascertain if AMPKα1 deletion contributes to chromosome missegregation by elevating Polo-like kinase 4 (PLK4) expression. Centrosome proteins and aneuploidy were monitored in cultured mouse embryonic fibroblasts (MEFs) isolated from wild type (WT, C57BL/6J) or AMPKα1 homozygous deficient (AMPKα1−/−) mice by Western blotting and metaphase chromosome spread. Deletion of AMPKα1, the predominant AMPKα isoform in immortalized MEFs, led to centrosome amplification and chromosome missegregation, as well as the consequent aneuploidy (34–66%) and micronucleus. Furthermore, AMPKα1 null cells exhibited a significant induction of PLK4. Knockdown of nuclear factor kappa B2/p52 ameliorated the PLK4 elevation in AMPKα1-deleted MEFs. Finally, PLK4 inhibition by Centrinone reversed centrosome amplification of AMPKα1-deleted MEFs. Taken together, our results suggest that AMPKα1 plays a fundamental role in the maintenance of chromosomal integrity through the control of p52-mediated transcription of PLK4, a trigger of centriole biogenesis.

scholarly journals Insight into Nephrocan Function in Mouse Endoderm Patterning

2019 ◽  
Vol 21 (1) ◽  
pp. 8
Author(s):  
Martina Addeo ◽  
Silvia Buonaiuto ◽  
Ilaria Guerriero ◽  
Elena Amendola ◽  
Feliciano Visconte ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Specific Expression ◽  
Knock Out ◽  
New Variant ◽  
Liver And Pancreas ◽  
Regenerative Therapies

Endoderm-derived organs as liver and pancreas are potential targets for regenerative therapies, and thus, there is great interest in understanding the pathways that regulate the induction and specification of this germ layer. Currently, the knowledge of molecular mechanisms that guide the in vivo endoderm specification is restricted by the lack of early endoderm specific markers. Nephrocan (Nepn) is a gene whose expression characterizes the early stages of murine endoderm specification (E7.5–11.5) and encodes a secreted N-glycosylated protein. In the present study, we report the identification of a new transcript variant that is generated through alternative splicing. The new variant was found to have differential and tissue specific expression in the adult mouse. In order to better understand Nepn role during endoderm specification, we generated Nepn knock-out (KO) mice. Nepn−/− mice were born at Mendelian ratios and displayed no evident phenotype compared to WT mice. In addition, we produced nullizygous mouse embryonic stem cell (mESC) line lacking Nepn by applying (CRISPR)/CRISPR-associated systems 9 (Cas9) and employed a differentiation protocol toward endoderm lineage. Our in vitro results revealed that Nepn loss affects the endoderm differentiation impairing the expression of posterior foregut-associated markers.

scholarly journals Deposition of Centromeric Histone H3 Variant CENP-A/Cse4 into Chromatin Is Facilitated by Its C-Terminal Sumoylation

Genetics ◽  
2020 ◽  
Vol 214 (4) ◽  
pp. 839-854 ◽  
Author(s):  
Kentaro Ohkuni ◽  
Evelyn Suva ◽  
Wei-Chun Au ◽  
Robert L. Walker ◽  
Reuben Levy-Myers ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Histone Chaperones ◽  
Wild Type ◽  
Link Type ◽  
C Terminus ◽  
Genome Wide ◽  
Evolutionarily Conserved ◽  
Histone H3 Variant ◽  
Assembly Factor ◽  
Active Investigation

Centromeric localization of CENP-A (Cse4 in Saccharomyces cerevisiae, CID in flies, CENP-A in humans) is essential for faithful chromosome segregation. Mislocalization of overexpressed CENP-A contributes to aneuploidy in yeast, flies, and humans, and is proposed to promote tumorigenesis in human cancers. Hence, defining molecular mechanisms that promote or prevent mislocalization of CENP-A is an area of active investigation. In budding yeast, evolutionarily conserved histone chaperones Scm3 and chromatin assembly factor-1 (CAF-1) promote localization of Cse4 to centromeric and noncentromeric regions, respectively. Ubiquitin ligases, such as Psh1 and Slx5, and histone chaperones (HIR complex) regulate proteolysis of overexpressed Cse4 and prevent its mislocalization to noncentromeric regions. In this study, we have identified sumoylation sites lysine (K) 215/216 in the C terminus of Cse4, and shown that sumoylation of Cse4 K215/216 facilitates its genome-wide deposition into chromatin when overexpressed. Our results showed reduced levels of sumoylation of mutant Cse4 K215/216R/A [K changed to arginine (R) or alanine (A)] and reduced interaction of mutant Cse4 K215/216R/A with Scm3 and CAF-1 when compared to wild-type Cse4. Consistent with these results, levels of Cse4 K215/216R/A in the chromatin fraction and localization to centromeric and noncentromeric regions were reduced. Furthermore, in contrast to GAL-CSE4, which exhibits Synthetic Dosage Lethality (SDL) in psh1∆, slx5∆, and hir2∆ strains, GAL-cse4 K215/216R does not exhibit SDL in these strains. Taken together, our results show that deposition of Cse4 into chromatin is facilitated by its C-terminal sumoylation.

NF-κB Protection against Apoptosis Induced by HEMA

2004 ◽  
Vol 83 (11) ◽  
pp. 837-842 ◽  
Author(s):  
G. Spagnuolo ◽  
C. Mauro ◽  
A. Leonardi ◽  
M. Santillo ◽  
R. Paternò ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Protective Role ◽  
Oxygen Species ◽  
Wild Type ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Primary Fibroblasts ◽  
Underlying Mechanisms ◽  
Induced Apoptosis ◽  
Dose Dependent

The cytotoxicity of dental monomers has been widely investigated, but the underlying mechanisms have not been elucidated. We studied the molecular mechanisms involved in cell death induced by HEMA. In human primary fibroblasts, HEMA induced a dose-dependent apoptosis that was confirmed by the activation of caspases-8, -9, and -3. We found an increase of reactive oxygen species (ROS) and NF-κB activation after HEMA exposure. Blocking of ROS production by anti-oxidants had no direct influence on apoptosis caused by HEMA, but inhibition of NF-κB increased the fraction of apoptotic cells. Accordingly, mouse embryonic fibroblasts (MEF) from p65−/− mice were more susceptible to HEMA-induced apoptosis than were wild-type controls. Our results indicate that exposure to HEMA triggers apoptosis and that this mechanism is not directly dependent upon redox signaling. Nevertheless, ROS induction by HEMA activates NF-κB, which exerts a protective role in counteracting apoptosis.

scholarly journals Genome wide efficiency profiling reveals modulation of maintenance and de novo methylation by Tets

2020 ◽  
Author(s):  
Pascal Giehr ◽  
Charalampos Kyriakopoulos ◽  
Karl Nordström ◽  
Abduhlrahman Salhab ◽  
Fabian Müller ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Epigenetic Modification ◽  
Embryonic Stem ◽  
Regulatory Elements ◽  
Sequencing Data ◽  
Reduced Representation ◽  
Genome Wide ◽  
Global And Local

AbstractBackgroundDNA methylation is an essential epigenetic modification which is set and maintained by DNA methyl transferases (Dnmts) and removed via active and passive mechanisms involving Tet mediated oxidation. While the molecular mechanisms of these enzymes are well studied, their interplay on shaping cell specific methylomes remains less well understood. In our work we model the activities of Tets and Dnmts at single CpGs across the genome using a novel type of high resolution sequencing data.ResultsTo accurately measure 5mC and 5hmC levels at single CpGs we developed RRHPoxBS, a reduced representation hairpin oxidative bisulfite sequencing approach. Using this method we mapped the methylomes and hydroxymethylomes of wild type and Tet triple knockout mouse embryonic stem cells. These comprehensive datasets were then used to develop an extended Hidden Markov model allowing us i) to determine the symmetrical methylation and hydroxymethylation state at millions of individual CpGs, ii) infer the maintenance and de novo methylation efficiencies of Dnmts and the hydroxylation efficiencies of Tets at individual CpG positions. We find that Tets exhibit their highest activity around unmethylated regulatory elements, i.e. active promoters and enhancers. Furthermore, we find that Tets’ presence has a profound effect on the global and local maintenance and de novo methylation activities by the Dnmts, not only substantially contributing to a universal demethylation of the genome but also shaping the overall methylation landscape.ConclusionsOur analysis demonstrates that a fine tuned and locally controlled interplay between Tets and Dnmts is important to modulate de novo and maintenance activities of Dnmts across the genome. Tet activities contribute to DNA methylation patterning in the following ways: They oxidize 5mC, they locally shield DNA from accidental de novo methylation and at the same time modulate maintenance and de novo methylation efficiencies of Dnmts across the genome.

scholarly journals Global analyses of the dynamics of mammalian microRNA metabolism

2019 ◽  
Author(s):  
Elena R. Kingston ◽  
David P. Bartel
Keyword(s):  
Turnover Rate ◽  
Embryonic Stem ◽  
Cell Types ◽  
Metabolic Labeling ◽  
Embryonic Fibroblasts ◽  
Passenger Strand ◽  
Individual Mirnas ◽  
Specific Factors ◽  
Mirna Abundance ◽  
Apparent Influence

AbstractRates of production and degradation together specify microRNA (miRNA) abundance and dynamics. Here, we used approach-to-equilibrium metabolic labeling to assess these rates for 176 miRNAs in contact-inhibited mouse embryonic fibroblasts (MEFs), 182 miRNAs in dividing MEFs, and 136 miRNAs in mouse embryonic stem cells (mESCs). MicroRNA duplexes, each comprising a mature miRNA and its passenger strand, are produced at rates as fast as 114 ± 49 copies/cell per min, which exceeds rates reported for any mRNAs. These duplexes are rapidly loaded into Argonaute, with <10 min typically required for duplex loading and silencing-complex maturation. Within Argonaute, guide strands have stabilities that vary by 100-fold. Half-lives also vary globally between cell lines, with median values ranging from 6.3 to 34 h in mESCs and contact-inhibited MEFs, respectively. Moreover, relative half-lives for individual miRNAs vary between cell types, implying the influence of cell-specific factors in dictating turnover rate. The apparent influence of miRNA regions most important for targeting, together with the effect of one target on miR-7a-5p accumulation, suggest that targets fulfill this role. Analysis of the tailing and trimming of miRNA 3′ termini showed that the flux was typically greatest through the isoform tailed with a single uridine, although changes in this flux did not correspond to changes in stability, which suggested that the processes of tailing and trimming might be independent from that of decay. Together these results establish a framework for describing the dynamics and regulation of miRNAs throughout their lifecycle.

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