scholarly journals Reconstitution of eukaryotic chromosomes and manipulation of DNA N6-methyladenine alters chromatin and gene expression

2018 ◽  
Author(s):  
Leslie Y. Beh ◽  
Galia T. Debelouchina ◽  
Derek M. Clay ◽  
Robert E. Thompson ◽  
Kelsi A. Lindblad ◽  
...  
Keyword(s):  
De Novo ◽  
Nucleosome Occupancy ◽  
Dna Methyltransferases ◽  
Chromatin Organization ◽  
Sexual Cycle ◽  
List Type ◽  
Adenine Methylation ◽  
The Impact

SummaryDNA N6-adenine methylation (6mA) has recently been reported in diverse eukaryotes, spanning unicellular organisms to metazoans. Yet the functional significance of 6mA remains elusive due to its low abundance, difficulty of manipulation within native DNA, and lack of understanding of eukaryotic 6mA writers. Here, we report a novel DNA 6mA methyltransferase in ciliates, termed MTA1. The enzyme contains an MT-A70 domain but is phylogenetically distinct from all known RNA and DNA methyltransferases. Disruption of MTA1in vivoleads to the genome-wide loss of 6mA in asexually growing cells and abolishment of the consensus ApT dimethylated motif. Genes exhibit subtle changes in chromatin organization or RNA expression upon loss of 6mA, depending on their starting methylation level. Mutants fail to complete the sexual cycle, which normally coincides with a peak of MTA1 expression. Thus, MTA1 functions in a developmental stage-specific manner. We determine the impact of 6mA on chromatin organizationin vitroby reconstructing complete, full-length ciliate chromosomes harboring 6mA in native or ectopic positions. Using these synthetic chromosomes, we show that 6mA directly disfavors nucleosomesin vitroin a local, quantitative manner, independent of DNA sequence. Furthermore, the chromatin remodeler ACF can overcome this effect. Our study identifies a novel MT-A70 protein necessary for eukaryotic 6mA methylation and defines the impact of 6mA on chromatin organization using epigenetically defined synthetic chromosomes.HighlightsThe MT-A70 protein MTA1 mediates DNA N6-adenine methylation inOxytrichaMTA1 mutants exhibit subtle changes in nucleosome organization and transcriptionin vivo6mA directly disfavors nucleosome occupancy in natural and synthetic chromosomesin vitroDe novosynthesis of complete, epigenetically definedOxytrichachromosomes

scholarly journals Synthesis of a eukaryotic chromosome reveals a role for N6-methyladenine in nucleosome organization

2017 ◽  
Author(s):  
Leslie Y. Beh ◽  
Galia T. Debelouchina ◽  
Kelsi A. Lindblad ◽  
Katarzyna Kulej ◽  
Elizabeth R. Hutton ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
De Novo ◽  
Nucleosome Occupancy ◽  
List Type ◽  
Eukaryotic Chromosome ◽  
Nucleosome Organization ◽  
Eukaryotic Chromatin ◽  
Dna Substrates ◽  
The Impact

SummaryBiochemical studies of chromatin have typically used either artificial DNA templates with unnaturally high affinity for histones, or small genomic DNA fragments deprived of their cognate physical environment. It has thus been difficult to dissect chromatin structure and function within fully native DNA substrates. Here, we circumvent these limitations by exploiting the minimalist genome of the eukaryoteOxytricha trifallax, whose notably small ~3kb chromosomes mainly encode single genes. Guided by high-resolution epigenomic maps of nucleosome organization, transcription, and DNA N6-methyladenine (m6dA) locations, we reconstruct full-lengthOxytrichachromosomesin vitroand use these synthetic facsimiles to dissect the influence of m6dA and histone post-translational modifications on nucleosome organization. We show that m6dA directly disfavors nucleosomes in a quantitative manner, leading to local decreases in nucleosome occupancy that are synergistic with histone acetylation. The effect of m6dA can be partially reversed by the action of an ATP-dependent chromatin remodeler. Furthermore, erasing m6dA marks fromOxytrichachromosomes leads to proportional increases in nucleosome occupancy across the genome. This work showcasesOxytrichachromosomes as powerful yet practical models for studying eukaryotic chromatin and transcription in the context of biologically relevant DNA substrates.HighlightsDe novosynthesis of complete, epigenetically definedOxytrichachromosomesEpigenomic profiles of chromatin organization inOxytricha’sminiature chromosomesm6dA directly disfavors nucleosome occupancy in natural and synthetic chromosomesHistone acetylation and chromatin remodelers temper the impact of m6dA on chromatin

scholarly journals Distribution and vulnerability of transcriptional outputs across the genome in Myc-amplified medulloblastoma cells

2021 ◽  
Author(s):  
Rui Yang ◽  
Wenzhe Wang ◽  
Meichen Dong ◽  
Kristen Roso ◽  
Paula Greer ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Tumor Cells ◽  
Target Genes ◽  
De Novo ◽  
Inhibitory Effect ◽  
Nucleotide Synthesis ◽  
High Level ◽  
The Impact

Myc plays a central role in tumorigenesis by orchestrating the expression of genes essential to numerous cellular processes1-4. While it is well established that Myc functions by binding to its target genes to regulate their transcription5, the distribution of the transcriptional output across the human genome in Myc-amplified cancer cells, and the susceptibility of such transcriptional outputs to therapeutic interferences remain to be fully elucidated. Here, we analyze the distribution of transcriptional outputs in Myc-amplified medulloblastoma (MB) cells by profiling nascent total RNAs within a temporal context. This profiling reveals that a major portion of transcriptional action in these cells was directed at the genes fundamental to cellular infrastructure, including rRNAs and particularly those in the mitochondrial genome (mtDNA). Notably, even when Myc protein was depleted by as much as 80%, the impact on transcriptional outputs across the genome was limited, with notable reduction mostly only in genes involved in ribosomal biosynthesis, genes residing in mtDNA or encoding mitochondria-localized proteins, and those encoding histones. In contrast to the limited direct impact of Myc depletion, we found that the global transcriptional outputs were highly dependent on the activity of Inosine Monophosphate Dehydrogenases (IMPDHs), rate limiting enzymes for de novo guanine nucleotide synthesis and whose expression in tumor cells was positively correlated with Myc expression. Blockage of IMPDHs attenuated the global transcriptional outputs with a particularly strong inhibitory effect on infrastructure genes, which was accompanied by the abrogation of MB cells proliferation in vitro and in vivo. Together, our findings reveal a real time action of Myc as a transcriptional factor in tumor cells, provide new insight into the pathogenic mechanism underlying Myc-driven tumorigenesis, and support IMPDHs as a therapeutic vulnerability in cancer cells empowered by a high level of Myc oncoprotein.

scholarly journals A novel microRNA-based strategy to expand the differentiation potency of stem cells

2019 ◽  
Author(s):  
María Salazar-Roa ◽  
Marianna Trakala ◽  
Mónica Álvarez-Fernández ◽  
Fátima Valdés-Mora ◽  
Cuiqing Zhong ◽  
...  
Keyword(s):  
Stem Cells ◽  
De Novo ◽  
Cardiac Injury ◽  
Dna Methyltransferases ◽  
Short Exposure ◽  
Differentiation Potential ◽  
Differentiation Capacity ◽  
Tetraploid Complementation

SUMMARYFull differentiation potential along with self-renewal capacity is a major property of pluripotent stem cells (PSCs). However, the differentiation capacity frequently decreases during expansion of PSCs in vitro. We show here that transient exposure to a single microRNA, expressed at early stages during normal development, improves the differentiation capacity of already-established murine and human PSCs. Short exposure to miR-203 in PSCs (miPSCs) results in expanded differentiation potency as well as improved efficiency in stringent assays such as tetraploid complementation and human-mouse interspecies chimerism. Mechanistically, these effects are mediated by direct repression of de novo DNA methyltransferases Dnmt3a and Dnmt3b, leading to transient and reversible erasing of DNA methylation. As a proof of concept, miR-203 improves differentiation and maturation of PSCs into cardiomyocytes in vitro as well as cardiac regeneration in vivo, after cardiac injury. These data support the use of transient exposure to miR-203 as a general and single method to reset the epigenetic memory in PSCs, and improve their use in regenerative medicine.

scholarly journals IL-21 Receptor Blockade Shifts the Follicular T Cell Balance and Reduces De Novo Donor-Specific Antibody Generation

2021 ◽  
Vol 12 ◽  
Author(s):  
Yeqi Nian ◽  
Zhilei Xiong ◽  
Panpan Zhan ◽  
Zhen Wang ◽  
Yang Xu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Germinal Center ◽  
De Novo ◽  
Antibody Mediated Rejection ◽  
Treg Differentiation ◽  
The Impact

Donor-specific antibodies (DSAs) play a key role in chronic kidney allograft injury. Follicular T helper (Tfh) cells trigger the humoral alloimmune response and promote DSA generation, while T-follicular regulatory (Tfr) cells inhibit antibody production by suppressing Tfh and B cells. Interleukin (IL)-21 exerts a distinct effect on Tfh and Tfr. Here, we studied whether blocking IL-21R with anti-IL-21R monoclonal antibody (αIL-21R) changes the Tfh/Tfr balance and inhibits DSA generation. First, we investigated the impact of αIL-21R on CD4+ T cell proliferation and apoptosis. The results showed that αIL-21R did not have cytotoxic effects on CD4+ T cells. Next, we examined Tfh and regulatory T cells (Tregs) in an in vitro conditioned culture model. Naïve CD4+ T cells were isolated from 3-month-old C57BL/6 mice and cultured in Tfh differentiation inducing conditions in presence of αIL-21R or isotype IgG and differentiation was evaluated by CXCR5 expression, a key Tfh marker. αIL-21R significantly inhibited Tfh differentiation. In contrast, under Treg differentiation conditions, FOXP3 expression was inhibited by IL-21. Notably, αIL-21R rescued IL-21-inhibited Treg differentiation. For in vivo investigation, a fully mismatched skin transplantation model was utilized to trigger the humoral alloimmune response. Consistently, flow cytometry revealed a reduced Tfh/Tfr ratio in recipients treated with αIL-21R. Germinal center response was evaluated by flow cytometry and lectin histochemistry. We observed that αIL-21R significantly inhibited germinal center reaction. Most importantly, DSA levels after transplantation were significantly inhibited by αIL-21R at different time points. In summary, our results demonstrate that αIL-21R shifts the Tfh/Tfr balance toward DSA inhibition. Therefore, αIL-21R may be a useful therapeutic agent to prevent chronic antibody mediated rejection after organ transplantation.

scholarly journals BIOL-10. DISTRIBUTION AND VULNERABILITY OF TRANSCRIPTIONAL OUTPUTS ACROSS THE GENOME IN MYC-AMPLIFIED MEDULLOBLASTOMA CELLS

2021 ◽  
Vol 23 (Supplement_1) ◽  
pp. i5-i5
Author(s):  
Rui Yang ◽  
Wenzhe Wang ◽  
Meichen Dong ◽  
Kristen Roso ◽  
Xuhui Bao ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Target Genes ◽  
De Novo ◽  
Inhibitory Effect ◽  
Nucleotide Synthesis ◽  
Expression Of Genes ◽  
High Level ◽  
The Impact

Abstract Myc plays a central role in tumorigenesis by orchestrating the expression of genes essential to numerous cellular processes. While it is well established that Myc functions by binding to its target genes to regulate their transcription, the distribution of the transcriptional output across human genome in Myc-amplified cancer cells, and the susceptibility of such transcriptional outputs to therapeutic interferences remain to be fully elucidated. Here, we analyze the distribution of transcriptional outputs in Myc-amplified medulloblastoma (MB) cells by profiling nascent total RNAs within a temporal context. This profiling reveals a major portion of transcriptional action in these cells was directed at the genes fundamental to cellular infrastructures, including rRNAs and particularly those in the mitochondrial genome (mtDNA). Notably, even when Myc protein was depleted by as much as 80%, the impact on transcriptional outputs across the genome was limited, with notable reduction mostly in genes of involved in ribosomal biosynthesis, genes residing in mtDNA or encoding mitochondria-localized proteins, and those encoding histones. In contrast to the limited direct impact of Myc depletion, we found that the global transcriptional outputs were highly dependent on the activity of Inosine Monophosphate Dehydrogenases (IMPDHs), rate limiting enzymes for de novo guanine nucleotide synthesis and whose expression in tumor cells was positively correlated with Myc’s expression. Blockage of IMPDHs attenuated the global transcriptional outputs with a particularly strong inhibitory effect on the aforementioned infrastructure genes, which was accompanied by the abrogation of MB cell’s proliferation in vitro and in vivo. Together, our findings reveal a real time action of Myc as a transcriptional factor in tumor cells, gain new insight into the pathogenic mechanism underlying Myc-driven tumorigenesis, and support IMPDHs as a therapeutic vulnerability in MB cells empowered by a high level of Myc oncoprotein.

scholarly journals Ferredoxin 1b (Fdx1b) Is the Essential Mitochondrial Redox Partner for Cortisol Biosynthesis in Zebrafish

Endocrinology ◽  
2016 ◽  
Vol 157 (3) ◽  
pp. 1122-1134 ◽  
Author(s):  
Aliesha Griffin ◽  
Silvia Parajes ◽  
Meltem Weger ◽  
Andreas Zaucker ◽  
Angela E. Taylor ◽  
...  
Keyword(s):  
Redox Regulation ◽  
Light Adaptation ◽  
De Novo ◽  
Transcription Activator ◽  
Dark Light ◽  
Cyp Enzymes ◽  
Redox Partner ◽  
The Impact

Abstract Mitochondrial cytochrome P450 (CYP) enzymes rely on electron transfer from the redox partner ferredoxin 1 (FDX1) for catalytic activity. Key steps in steroidogenesis require mitochondrial CYP enzymes and FDX1. Over 30 ferredoxin mutations have been explored in vitro; however, no spontaneously occurring mutations have been identified in humans leaving the impact of FDX1 on steroidogenesis in the whole organism largely unknown. Zebrafish are an important model to study human steroidogenesis, because they have similar steroid products and endocrine tissues. This study aimed to characterize the influence of ferredoxin on steroidogenic capacity in vivo by using zebrafish. Zebrafish have duplicate ferredoxin paralogs: fdx1 and fdx1b. Although fdx1 was observed throughout development and in most tissues, fdx1b was expressed after development of the zebrafish interrenal gland (counterpart to the mammalian adrenal gland). Additionally, fdx1b was restricted to adult steroidogenic tissues, such as the interrenal, gonads, and brain, suggesting that fdx1b was interacting with steroidogenic CYP enzymes. By using transcription activator-like effector nucleases, we generated fdx1b mutant zebrafish lines. Larvae with genetic disruption of fdx1b were morphologically inconspicuous. However, steroid hormone analysis by liquid chromatography tandem mass spectrometry revealed fdx1b mutants failed to synthesize glucocorticoids. Additionally, these mutants had an up-regulation of the hypothalamus-pituitary-interrenal axis and showed altered dark-light adaptation, suggesting impaired cortisol signaling. Antisense morpholino knockdown confirmed Fdx1b is required for de novo cortisol biosynthesis. In summary, by using zebrafish, we generated a ferredoxin knockout model system, which demonstrates for the first time the impact of mitochondrial redox regulation on glucocorticoid biosynthesis in vivo.

scholarly journals FSMP-07. CYSTATHIONINE-Γ-LYASE DRIVES ANTIOXIDANT DEFENSE IN CYSTEINE-RESTRICTED IDH1 MUTANT ASTROCYTOMAS

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. i17-i17
Author(s):  
Andrés Cano-Galiano ◽  
Anais Oudin ◽  
Fred Fack ◽  
Maria-Francesca Allega ◽  
David Sumpton ◽  
...  
Keyword(s):  
Antioxidant Defense ◽  
De Novo ◽  
Idh1 Mutation ◽  
Wild Type ◽  
Isocitrate Dehydrogenase 1 ◽  
Astrocytic Gliomas ◽  
The Impact ◽  
Reducing Potential

Abstract Mutations in isocitrate dehydrogenase 1 or 2 (IDH1/2) define glioma subtypes and are considered primary events in gliomagenesis, impacting tumor epigenetics and metabolism. IDH enzymes are crucial for the generation of reducing potential, yet the impact of the mutation on the cellular antioxidant system is not understood. Here, we investigate how glutathione (GSH) levels are maintained in IDH1 mutant gliomas, despite an altered NADPH/NADP balance. We find that IDH1 mutant astrocytomas specifically upregulate cystathionine γ-lyase (CSE), the enzyme responsible for cysteine production upstream of GSH biosynthesis. Genetic and chemical interference with CSE in patient-derived glioma cells carrying the endogenous IDH1 mutation, sensitized tumor cells to cysteine depletion, an effect not observed in IDH1 wild-type gliomas. This correlated with reduced GSH synthesis as shown by in vitro and in vivo serine tracing and led to delayed tumor growth in mice. Thus we show that IDH1 mutant astrocytic gliomas critically rely on NADPH-independent de novo GSH synthesis to maintain the antioxidant defense, which uncovers a novel metabolic vulnerability in this dismal disease.

scholarly journals BZLF1 interacts with chromatin remodelers promoting escape from latent infections with EBV

2019 ◽  
Vol 2 (2) ◽  
pp. e201800108 ◽  
Author(s):  
Marisa Schaeffner ◽  
Paulina Mrozek-Gorska ◽  
Alexander Buschle ◽  
Anne Woellmer ◽  
Takanobu Tagawa ◽  
...  
Keyword(s):  
Chromatin Remodeling ◽  
Transcriptional Activation ◽  
Nucleosome Occupancy ◽  
Dna Methyltransferases ◽  
Viral Transcription ◽  
Sequence Motifs ◽  
Viral Dna ◽  
Accessible Chromatin

A hallmark of EBV infections is its latent phase, when all viral lytic genes are repressed. Repression results from a high nucleosome occupancy and epigenetic silencing by cellular factors such as the Polycomb repressive complex 2 (PRC2) and DNA methyltransferases that, respectively, introduce repressive histone marks and DNA methylation. The viral transcription factor BZLF1 acts as a molecular switch to induce transition from the latent to the lytic or productive phase of EBV’s life cycle. It is unknown how BZLF1 can bind to the epigenetically silenced viral DNA and whether it directly reactivates the viral genome through chromatin remodeling. We addressed these fundamental questions and found that BZLF1 binds to nucleosomal DNA motifs both in vivo and in vitro. BZLF1 co-precipitates with cellular chromatin remodeler ATPases, and the knock-down of one of them, INO80, impaired lytic reactivation and virus synthesis. In Assay for Transposase-Accessible Chromatin-seq experiments, non-accessible chromatin opens up locally when BZLF1 binds to its cognate sequence motifs in viral DNA. We conclude that BZLF1 reactivates the EBV genome by directly binding to silenced chromatin and recruiting cellular chromatin-remodeling enzymes, which implement a permissive state for lytic viral transcription. BZLF1 shares this mode of action with a limited number of cellular pioneer factors, which are instrumental in transcriptional activation, differentiation, and reprogramming in all eukaryotic cells.

scholarly journals Development and assessment of the performance of a shared ventilatory system that uses clinically available components to individualize tidal volumes

2020 ◽  
Author(s):  
David M. Hannon ◽  
Tim Jones ◽  
Jack Conolly ◽  
Conor Judge ◽  
Talha Iqbal ◽  
...  
Keyword(s):  
Invasive Mechanical Ventilation ◽  
Ventilation System ◽  
List Type ◽  
Test Lung ◽  
Minimal Impact ◽  
Wide Range ◽  
The Impact ◽  
Ventilator Settings

AbstractObjectivesTo develop and assess the performance of a system for shared ventilation that uses clinically available components to individualize tidal volumes under a variety of clinically relevant conditions.DesignEvaluation and in vitro validation study.SettingVentilator shortage during the SARS-CoV-2 global pandemic.ParticipantsThe design and validation team consisted of intensive care physicians, bioengineers, computer programmers, and representatives from the medtech sector.MethodsUsing standard clinical components, a system of shared ventilation consisting of two ventilatory limbs was assembled and connected to a single ventilator. Individual monitors for each circuit were developed using widely available equipment and open source software. System performance was determined under 2 sets of conditions. First, the effect of altering ventilator settings (Inspiratory Pressure, Respiratory rate, I:E ratio) on the tidal volumes delivered to each lung circuit was determined. Second, the impact of altering the compliance and resistance in one simulated lung circuit on the tidal volumes delivered to that lung and the second lung circuit was determined. All measurements at each setting were repeated three times to determine the variability in the system.ResultsThe system permitted accurate and reproducible titration of tidal volumes to each ‘lung circuit’ over a wide range of ventilator settings and simulated lung conditions. Alteration of ventilator inspiratory pressures stepwise from 4-20cm H2O, of respiratory rates from 6-20 breaths/minute and I:E ratio from 1:1 to 1:4 resulted in near identical tidal volumes delivered under each set of conditions to each simulated ‘lung’. Stepwise alteration of compliance and resistance in one ‘test’ lung circuit resulted in reproducible alterations in tidal volume to the ‘test’ lung, with little change to tidal volumes in the ‘control’ lung (a change of only 6% is noted). All tidal volumes delivered were highly reproducible upon repetition.ConclusionsWe demonstrate the reliability of a simple shared ventilation system assembled using commonly available clinical components that allows individual titration of tidal volumes. This system may be useful as a temporary strategy of last resort where the numbers of patients requiring invasive mechanical ventilation exceeds supply of ventilators.Article SummaryStrengths and limitations of this studyThis solution provides the ability to safely and robustly ventilate two patients simultaneously while allowing differing tidal volumes in each limb.The designed solution uses equipment readily available in most hospitals.Accurate and reproducible titration of tidal volumes to each ‘lung’ was possible over a wide range of ventilator settings.Alteration of one simulated ‘lung’ conditions had minimal impact on the tidal volumes delivered to the unaffected lungThe system relies on patients being sedated and paralysed.We have not yet tested this solution in vivo, on COVID-19 patients.

scholarly journals Disabling de novo DNA methylation in embryonic stem cells allows an illegitimate fate trajectory

2021 ◽  
Vol 118 (38) ◽  
pp. e2109475118
Author(s):  
Masaki Kinoshita ◽  
Meng Amy Li ◽  
Michael Barber ◽  
William Mansfield ◽  
Sabine Dietmann ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cell Fate ◽  
De Novo ◽  
Es Cells ◽  
Embryonic Stem ◽  
Dna Methyltransferases ◽  
Bhlh Transcription Factor ◽  
Mammalian Development

Genome remethylation is essential for mammalian development but specific reasons are unclear. Here we examined embryonic stem (ES) cell fate in the absence of de novo DNA methyltransferases. We observed that ES cells deficient for both Dnmt3a and Dnmt3b are rapidly eliminated from chimeras. On further investigation we found that in vivo and in vitro the formative pluripotency transition is derailed toward production of trophoblast. This aberrant trajectory is associated with failure to suppress activation of Ascl2. Ascl2 encodes a bHLH transcription factor expressed in the placenta. Misexpression of Ascl2 in ES cells provokes transdifferentiation to trophoblast-like cells. Conversely, Ascl2 deletion rescues formative transition of Dnmt3a/b mutants and improves contribution to chimeric epiblast. Thus, de novo DNA methylation safeguards against ectopic activation of Ascl2. However, Dnmt3a/b-deficient cells remain defective in ongoing embryogenesis. We surmise that multiple developmental transitions may be secured by DNA methylation silencing potentially disruptive genes.

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