Adenine DNA methylation, 3D genome organization, and gene expression in the parasite Trichomonas vaginalis

Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogenital tract causing infections that range from asymptomatic to highly inflammatory. Recent works have highlighted the importance of histone modifications in the regulation of transcription and parasite pathogenesis. However, the nature of DNA methylation in the parasite remains unexplored. Using a combination of immunological techniques and ultrahigh-performance liquid chromatography (UHPLC), we analyzed the abundance of DNA methylation in strains with differential pathogenicity demonstrating that N6-methyladenine (6mA), and not 5‐methylcytosine (5mC), is the main DNA methylation mark in T. vaginalis. Genome-wide distribution of 6mA reveals that this mark is enriched at intergenic regions, with a preference for certain superfamilies of DNA transposable elements. We show that 6mA in T. vaginalis is associated with silencing when present on genes. Interestingly, bioinformatics analysis revealed the presence of transcriptionally active or repressive intervals flanked by 6mA-enriched regions, and results from chromatin conformation capture (3C) experiments suggest these 6mA flanked regions are in close spatial proximity. These associations were disrupted when parasites were treated with the demethylation activator ascorbic acid. This finding revealed a role for 6mA in modulating three-dimensional (3D) chromatin structure and gene expression in this divergent member of the Excavata.Fil: Lizarraga, Ayelen. Universidad Nacional de San Martin. Instituto Tecnologico de Chascomus. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - la Plata. Instituto Tecnologico de Chascomus.; ArgentinaFil: O'Brown, Zach Klapholz. Harvard Medical School; Estados UnidosFil: Boulias, Konstantinos. Harvard Medical School; Estados UnidosFil: Roach, Lara. Harvard Medical School; Estados UnidosFil: Greer, Eric Lieberman. Harvard Medical School; Estados UnidosFil: Johnson, Patricia J.. University of California at Los Angeles; Estados UnidosFil: Strobl Mazzulla, Pablo H.. Universidad Nacional de San Martin. Instituto Tecnologico de Chascomus. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - la Plata. Instituto Tecnologico de Chascomus.; ArgentinaFil: de Miguel, Natalia. Universidad Nacional de San Martin. Instituto Tecnologico de Chascomus. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - la Plata. Instituto Tecnologico de Chascomus.; Argentin

Functional role of G9a-induced histone methylation in small heterodimer partner-mediated transcriptional repression

Site-specific modification of nucleosomal histones plays a central role in the formation of transcriptionally active and inactive chromatin structures. These modifications may serve as specific recognition motifs for chromatin proteins, which act as a signal for the adoption of the appropriate regulatory responses. Here, we show that the orphan nuclear receptor SHP (small heterodimer partner), a coregulator that inhibits the activity of several nuclear receptors, can associate with unmodified and lysine 9-methylated histone-3, but not with the acetylated protein. The naturally occurring SHP mutant (R213C), which exhibits decreased transrepression potential, interacts less avidly with K9-methylated histone 3. We demonstrate that SHP can functionally interact with histone deacetylase-1 and the G9a methyltransferase and that it is localized exclusively in nuclease-sensitive euchromatin. The results point to the involvement of a multistep mechanism in SHP-dependent transcriptional repression, which includes histone deacetylation, followed by H3-K9 methylation and stable association of SHP itself with chromatin

Μεταγραφική ρύθμιση μέσω του σηματοδοτικού μονοπατιού των χολικών οξέων

Ο κύριος τρόπος απομάκρυνσης της χοληστερόλης από τα σώμα, είναι μέσω της αποικοδόμησης σε χολικά οξέα, μια διαδικασία που λαμβάνει χώρα αποκλειστικά στο ήπαρ. Το πρώτο, και καθοριστικό για τα επόμενα, βήμα καταλύεται από το ένζυμο Υδροξυλάση-7α της χοληστερόλης (CYP7A1). Τα χολικά οξέα αποτελούν τα τελικά προϊόντα του μονοπατιού και ρυθμίζουν τη σύνθεσή τους μέσω καταστολής της μεταγραφής του γονιδίου CYP7A1. Ο προτεινόμενος μηχανισμός καταστολής περιλαμβάνει τη δράση των ορμονικών πυρηνικών υποδοχέων FXR, SHP και CPF. Τα χολικά οξέα είναι σηματοδοτικά μόρια και ενεργοποιούν τον FXR. Σύμφωνα με το μοντέλο, ο FXR επηρεάζει τη μεταγραφή του CYP7A1 μέσω ενός έμμεσου μηχανισμού που περιλαμβάνει την αύξηση της έκφρασης του γονιδίου του καταστολέα SHP. Ο SHP αλληλεπιδρά με τον CPF και παρεμποδίζει τη δράση του, η οποία είναι απαραίτητη για τη μεταγραφική ενεργοποίηση του CYP7A1. Στην παρούσα εργασία αντικείμενο μελέτης ήταν η μεταγραφική ρύθμιση του γονιδίου CYP7A1 από τη σηματοδότηση των χολικών οξέων. Βρέθηκε ότι ο προτεινόμενος μηχανισμός δε μπορεί να εξηγήσει απόλυτα την καταστολή της μεταγραφής και επομένως πρέπει να υπάρχουν εναλλακτικοί μηχανισμοί καταστολής. Στη συνέχεια, διερευνήθηκε η υπόθεση ότι τα χολικά οξέα μπορούν να επιδράσουν άμεσα στη μεταγραφή του CYP7A1 δρώντας ως συνδέτες για τον πυρηνικό υποδοχέα CPF ή SHP. Για το σκοπό αυτό, εξετάστηκε αν τα χολικά οξέα επηρεάζουν την αλληλεπίδραση in vitro μεταξύ CPF και μεταγραφικών συνενεργοποιητών και μεταξύ CPF και SHP. Από τα αποτελέσματα των πειραμάτων που έχουν γίνει έως αυτή τη στιγμή, δεν προκύπτουν ενδείξεις που να αποδεικνύουν την παραπάνω υπόθεση. Ωστόσο, πρέπει να πραγματοποιηθούν επιπλέον πειράματα για την τελική απόδειξη ή απόρριψή της. Επιπρόσθετα, στα πλαίσια της παρούσης εργασίας, μελετήθηκε η επίδραση του σηματοδοτικού μονοπατιού των χολικών οξέων στην έκφραση των γονιδίων ApoCIII και ApoA1 που κωδικοποιούν για τις αντίστοιχες απολιποπρωτεΐνες. Βρέθηκε ότι τα χολικά οξέα καταστέλλουν τη μεταγραφή του γονιδίου ApoCIII και ενεργοποιούν τη μεταγραφή του γονιδίου ApoA1. Από πρoκαταρκτικά πειράματα που έγιναν για τη διερεύνηση του μηχανισμού δράσης των χολικών οξέων, προκύπτουν ενδείξεις ότι για την καταστολή της μεταγραφής του ApoCIII εμπλέκεται ο καταστολέας SHP, ενώ η ενεργοποίηση της μεταγραφής του ApoA1 φαίνεται να είναι αποτέλεσμα ενός μηχανισμού που δεν εξαρτάται από τον FXR

The C. elegans MicroRNA mir-71 Acts in Neurons to Promote Germline-Mediated Longevity through Regulation of DAF-16/FOXO

The life span of Caenorhabditis elegans is controlled by signaling between the germline and the soma. Germ cell removal extends life span by triggering the activation of the DAF-16/FOXO transcription factor in the intestine. Here we analyze microRNA function in C. elegans aging and show that the microRNA mir-71 functions to mediate the effects of germ cell loss on life span. mir-71 is required for the life span extension caused by germline removal, and overexpression of mir-71 further extends the life span of animals lacking germ cells. mir-71 functions in the nervous system to facilitate the localization and transcriptional activity of DAF-16 in the intestine. Our findings reveal a microRNA-dependent mechanism of life span regulation by the germline and indicate that signaling among the gonad, the nervous system, and the intestine coordinates the life span of the entire organism.Howard Hughes Medical InstituteEuropean Molecular Biology Organization (Fellowship)Ellison Medical Foundation (Grant

Identification of the m6Am Methyltransferase PCIF1 Reveals the Location and Functions of m6Am in the Transcriptome

International audiencemRNAs are regulated by nucleotide modifications that influence their cellular fate. Two of the most abundant modified nucleotides are N6-methyladenosine (m6A), found within mRNAs, and N6,2'-O-dimethyladenosine (m6Am), which is found at the first transcribed nucleotide. Distinguishing these modifications in mapping studies has been difficult. Here, we identify and biochemically characterize PCIF1, the methyltransferase that generates m6Am. We find that PCIF1 binds and is dependent on the m7G cap. By depleting PCIF1, we generated transcriptome-wide maps that distinguish m6Am and m6A. We find that m6A and m6Am misannotations arise from mRNA isoforms with alternative transcription start sites (TSSs). These isoforms contain m6Am that maps to "internal" sites, increasing the likelihood of misannotation. We find that depleting PCIF1 does not substantially affect mRNA translation but is associated with reduced stability of a subset of m6Am-annotated mRNAs. The discovery of PCIF1 and our accurate mapping technique will facilitate future studies to characterize m6Am's function

Coping with darkness: The adaptive response of marine picocyanobacteria to repeated light energy deprivation

The picocyanobacteria Prochlorococcus and Synechococcus are found throughout the ocean's euphotic zone, where the daily light:dark cycle drives their physiology. Periodic deep mixing events can, however, move cells below this region, depriving them of light for extended periods of time. Here, we demonstrate that members of these genera can adapt to tolerate repeated periods of light energy deprivation. Strains kept in the dark for 3 d and then returned to the light initially required 18–26 d to resume growth, but after multiple rounds of dark exposure they began to regrow after only 1–2 d. This dark-tolerant phenotype was stable and heritable; some cultures retained the trait for over 132 generations even when grown in a standard 13:11 light:dark cycle. We found no genetic differences between the dark-tolerant and parental strains of Prochlorococcus NATL2A, indicating that an epigenetic change is likely responsible for the adaptation. To begin to explore this possibility, we asked whether DNA methylation—one potential mechanism mediating epigenetic inheritance in bacteria—occurs in Prochlorococcus. LC–MS/MS analysis showed that while DNA methylations, including 6 mA and 5 mC, are found in some other Prochlorococcus strains, there were no methylations detected in either the parental or dark-tolerant NATL2A strains. These findings suggest that Prochlorococcus utilizes a yet-to-be-determined epigenetic mechanism to adapt to the stress of extended light energy deprivation, and highlights phenotypic heterogeneity as an additional dimension of Prochlorococcus diversity

Paternal methotrexate exposure affects sperm small RNA content and causes craniofacial defects in the offspring

Folate is an essential vitamin for vertebrate embryo development. Methotrexate (MTX) is a folate antagonist that is widely prescribed for autoimmune diseases, blood and solid organ malignancies, and dermatologic diseases. Although it is highly contraindicated for pregnant women, because it is associated with an increased risk of multiple birth defects, the effect of paternal MTX exposure on their offspring has been largely unexplored. Here, we found MTX treatment of adult medaka male fish (Oryzias latipes) causes cranial cartilage defects in their offspring. Small non-coding RNA (sncRNAs) sequencing in the sperm of MTX treated males identify differential expression of a subset of tRNAs, with higher abundance for specific 5′ tRNA halves. Sperm RNA methylation analysis on MTX treated males shows that m5C is the most abundant and differential modification found in RNAs ranging in size from 50 to 90 nucleotides, predominantly tRNAs, and that it correlates with greater testicular Dnmt2 methyltransferase expression. Injection of sperm small RNA fractions from MTX-treated males into normal fertilized eggs generated cranial cartilage defects in the offspring. Overall, our data suggest that paternal MTX exposure alters sperm sncRNAs expression and modifications that may contribute to developmental defects in their offspring.Fil: Alata Jimenez, Nagif. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Castellano, Mauricio. Instituto Pasteur de Montevideo; Uruguay. Universidad de la Republica; UruguayFil: Santillan, Emilio M.. University Johns Hopkins; Estados UnidosFil: Boulias, Konstantinos. Harvard Medical School; Estados Unidos. Boston Children’s Hospital; Estados UnidosFil: Boan, Agustín Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Arias Padilla, Luisa Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Fernandino, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Greer, Eric L.. Harvard Medical School; Estados Unidos. Boston Children’s Hospital; Estados UnidosFil: Tosar, Juan P.. Instituto Pasteur de Montevideo; Uruguay. Universidad de la Republica; UruguayFil: Cochella, Luisa. University Johns Hopkins; Estados UnidosFil: Strobl Mazzulla, Pablo H.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentin

Regulation of hepatic metabolic pathways by the orphan nuclear receptor SHP

SHP (small heterodimer partner) is an important component of the feedback regulatory cascade, which controls the conversion of cholesterol to bile acids. In order to identify the bona fide molecular targets of SHP, we performed global gene expression profiling combined with chromatin immunoprecipitation assays in transgenic mice constitutively expressing SHP in the liver. We demonstrate that SHP affects genes involved in diverse biological pathways, and in particular, several key genes involved in consecutive steps of cholesterol degradation, bile acid conjugation, transport and lipogenic pathways. Sustained expression of SHP leads to the depletion of hepatic bile acid pool and a concomitant accumulation of triglycerides in the liver. The mechanism responsible for this phenotype includes SHP-mediated direct repression of downstream target genes and the bile acid sensor FXRα, and an indirect activation of PPARγ and SREBP-1c genes. We present evidence for the role of altered chromatin configurations in defining distinct gene-specific mechanisms by which SHP mediates differential transcriptional repression. The multiplicity of genes under its control suggests that SHP is a pleiotropic regulator of diverse metabolic pathways