Mice and men: Their promoter properties

Using the two largest collections of Mus musculus and Homo sapiens transcription start sites ( TSSs) determined based on CAGE tags, ditags, full- length cDNAs, and other transcript data, we describe the compositional landscape surrounding TSSs with the aim of gaining better insight into the properties of mammalian promoters. We classified TSSs into four types based on compositional properties of regions immediately surrounding them. These properties highlighted distinctive features in the extended core promoters that helped us delineate boundaries of the transcription initiation domain space for both species. The TSS types were analyzed for associations with initiating dinucleotides, CpG islands, TATA boxes, and an extensive collection of statistically significant cis- elements in mouse and human. We found that different TSS types show preferences for different sets of initiating dinucleotides and ciselements. Through Gene Ontology and eVOC categories and tissue expression libraries we linked TSS characteristics to expression. Moreover, we show a link of TSS characteristics to very specific genomic organization in an example of immune- response- related genes ( GO: 0006955). Our results shed light on the global properties of the two transcriptomes not revealed before and therefore provide the framework for better understanding of the transcriptional mechanisms in the two species, as well as a framework for development of new and more efficient promoter- and gene- finding tools.

Long-range translational coupling in single-stranded RNA bacteriophages: an evolutionary analysis

In coliphage MS2 RNA a long-distance interaction (LDI) between an internal segment of the upstream coat gene and the start region of the replicase gene prevents initiation of replicase synthesis in the absence of coat gene translation. Elongating ribosomes break up the repressor LDI and thus activate the hidden initiation site. Expression studies on partial MS2 cDNA clones identified base pairing between 1427-1433 and 1738-1744, the so-called Min Jou (MJ) interaction, as the molecular basis for the long-range coupling mechanism. Here, we examine the biological significance of this interaction for the control of replicase gene translation. The LDI was disrupted by mutations in the 3'-side and the evolutionary adaptation was monitored upon phage passaging. Two categories of pseudorevertants emerged. The first type had restored the MJ interaction but not necessarily the native sequence. The pseudorevertants of the second type acquired a compensatory substitution some 80 nt downstream of the MJ interaction that stabilizes an adjacent LDI. In one examined case we confirmed that the second site mutations had restored coat-replicase translational coupling. Our results show the importance of translational control for fitness of the phage. They also reveal that the structure that buries the replicase start extends to structure elements bordering the MJ interaction.

The metabolic and epigenetic effects of the isocitrate dehydrogenase-1 mutation in glioma

Cancer cells have been noted to have an altered metabolic phenotype for over ninety years. In the presence of oxygen, differentiated cells predominately utilise the tricarboxylic acid (TCA) cycle and oxidative phosphorylation to efficiently produce energy and the metabolites necessary for protein and lipid synthesis. However, in hypoxia, this process is altered and cells switch to a higher rate of glycolysis and lactate production to maintain their energy and metabolic needs. In cancer cells, glycolysis is maintained at a high rate, even in the presence of oxygen; a term described as “aerobic glycolysis”. Tumour cells are rapidly dividing and have a much greater need for anabolism compared to normal differentiated cells. Rapid glucose metabolism enables faster ATP production as well as a greater redistribution of carbons to nucleotide, protein, and fatty acid synthesis, thus maximising cell growth. Recently, other metabolic changes, driven by mutations in genes related to the TCA cycle, indicate an alternative role for metabolism in cancer, the “oncometabolite”. This is where a particular metabolite builds up within the cell and contributes to the tumorigenic process. One of these genes is isocitrate dehydrogenase (IDH) IDH is an enzyme that forms part of the tricarboxylic acid (TCA) cycle and converts isocitrate to α-ketoglutarate (α-KG). It exists in three isoforms; IDH1, IDH2 and IDH3 with the former present in the cytoplasm and the latter two in the mitochondria. Point mutations have been identified in the IDH1 and IDH2 genes in glioma which result in a gain of function by converting α-KG to 2-hydroxyglutarate (2HG), an oncometabolite. 2HG acts as a competitive inhibitor of the α-KG dependent dioxygenases, a superfamily of enzymes that are involved in numerous cellular processes such as DNA and histone demethylation. It was hypothesised that the IDH1 mutation would result in other metabolic changes in the cell other than 2HG production, and could potentially identify pathways which could be targeted for therapeutic treatment. In addition, 2HG can act as a potential competitive inhibitor of α-KG dependent dioxygenases, so it was hypothesised that there would be an effect on histone methylation. This may alter gene expression and provide a mechanism for tumourogenesis and potentially identify further therapeutic targets. Metabolic analysis of clinical tumour samples identified changes associated with the IDH1 mutation, which included a reduction in α-KG and an increase in GABA, in addition to the increase in 2HG. This was replicated in several cell models, where 13C labelled metabolomics was also used to identify a possible increase in metabolic flux from glutamate to GABA, as well as from α-KG to 2HG. This may provide a mechanism whereby the cell can bypass the IDH1 mutation as GABA can be metabolised to succinate in the mitochondria by GABA transaminase via the GABA shunt. JMJ histone demethylases are a subset of the α-KG dependent dioxygenases, and are involved in removing methyl groups from histone tails. Changes in histone methylation are associated with changes in gene expression depending on the site and extent of chemical modification. To identify whether the increase in 2HG and fall in α-KG was associated with inhibition of histone demethylases a histone methylation screen was used. The IDH1 mutation was associated with an increase in methylation of H3K4, which is associated with gene activation. ChiP and RNA sequencing identified an increase in H3K4me3 at the transcription start site of the GABRB3 subunit, resulting in an increase in gene expression. The GABRB3 subunit forms part of the GABA-A receptor, a chloride channel, which on activation can reduce cell proliferation. The IDH1 mutation was associated with an increase in GABA and GABRB3 subunit of the GABA-A receptor. This raises the possibility of GABA transaminase as a potential therapeutic target. Inhibition of this enzyme could reduce GABA metabolism, potentially reducing any beneficial effect of the GABA shunt in IDH1 mutant tumours, and increasing activation of the GABA-A receptor by increasing the concentration of GABA in the brain. This in turn may reduce cell proliferation, and could be achieved by using Vigabatrin, a GABA transaminase inhibitor licensed for use in epilepsy.

AnnoTALE: Bioinformatics tools for identification, annotation, and nomenclature of TALEs from Xanthomonas genomic sequences

Transcription activator-like effectors (TALEs) are virulence factors, produced by the bacterial plant-pathogen Xanthomonas, that function as gene activators inside plant cells. Although the contribution of individual TALEs to infectivity has been shown, the specific roles of most TALEs, and the overall TALE diversity in Xanthomonas spp. is not known. TALEs possess a highly repetitive DNA-binding domain, which is notoriously difficult to sequence. Here, we describe an improved method for characterizing TALE genes by the use of PacBio sequencing. We present 'AnnoTALE', a suite of applications for the analysis and annotation of TALE genes from Xanthomonas genomes, and for grouping similar TALEs into classes. Based on these classes, we propose a unified nomenclature for Xanthomonas TALEs that reveals similarities pointing to related functionalities. This new classification enables us to compare related TALEs and to identify base substitutions responsible for the evolution of TALE specificities. © 2016, Nature Publishing Group. All rights reserved.

Association of follicle stimulating hormone receptor promoter with ovarian response in IVF-ET patients

Background: Poor ovarian response phenomenon has been observed in some of the in vitro fertilization-embryo transfer patients. Some investigations found that follicle stimulating hormone receptor (FSHR) gene plays a role in the process, but no direct evidence shows the correlation between genotypes of FSHR and ovarian response. Objective: Exploring the molecular mechanism behind the mutation of FSHR promoter association with ovarian granulosa cells and poor ovarian response. Materials and Methods: This cross sectional study was performed using 158 women undergoing the controlled short program ovarian stimulation for IVF treatment. The 263 bp DNA fragments before the follicle stimulating hormone (FSH) receptor 5' initiation site were sequenced in the patients under IVF cycle, 70 of which had poor ovarian response and 88 showed normal ovarian responses. Results: With a mutation rate of 40%, 63 in 158 cases showed a 29th site G→A point mutation; among the mutated cases, the mutation rate of the poor ovarian responders was significantly higher than the normal group (60% versus 23.9%; χ2=21.450, p<0.001). Besides, the variability was also obvious in antral follicle count, and ovum pick-ups. The estradiol peak values and the number of mature eggs between the two groups had significant difference. However, there was no obvious variability (t=0.457, p=0.324) in the basic FSH values between the two groups (normal group, 7.2±2.3 U/L; mutation group, 7.1±2.0 U/L). Conclusion: The activity of FSHR promoter is significantly affected by the 29th site G→A mutation that will weaken promoter activity and result in poor response to FSH.

Initiation of transcription of rDNA in rice

Three direct repeats of 320, 340 and 238 nucleotides were detected upstream to the 5′ end of the 18S rRNA gene of an rDNA unit present on a 9.8 kb EcoRT fragment of the rice DNA. The primer extension analysis showed that the site of initiation of transcription is in the 1st repeat at an A, the 623rd nucleotide upstream to the 5′ end of the 18S rRNA gene. Different stretches of the intergenic spacer DNA linked to the Chloramphenicol acetyl transferase gene were transcribed in the intact nuclei of rice embryos. The S1 nuclease protection analysis of the transcripts using [32P]-labelled Chloramphenicol acetyl transferase gene as the probe showed the presence of multiple promoters for rDNA transcription.

Yhh1p/Cft1p directly links poly(A) site recognition and RNA polymerase II transcription termination

RNA polymerase II (pol II) transcription termination requires co‐transcriptional recognition of a functional polyadenylation signal, but the molecular mechanisms that transduce this signal to pol II remain unclear. We show that Yhh1p/Cft1p, the yeast homologue of the mammalian AAUAAA interacting protein CPSF 160, is an RNA‐binding protein and provide evidence that it participates in poly(A) site recognition. Interestingly, RNA binding is mediated by a central domain composed of predicted β‐propeller‐forming repeats, which occurs in proteins of diverse cellular functions. We also found that Yhh1p/Cft1p bound specifically to the phosphorylated C‐terminal domain (CTD) of pol II in vitro and in a two‐hybrid test in vivo. Furthermore, transcriptional run‐on analysis demonstrated that yhh1 mutants were defective in transcription termination, suggesting that Yhh1p/Cft1p functions in the coupling of transcription and 3′‐end formation. We propose that direct interactions of Yhh1p/Cft1p with both the RNA transcript and the CTD are required to communicate poly(A) site recognition to elongating pol II to initiate transcription termination.

Distinctive contributions of the ribosomal P-site elements m(2)G966, m(5)C967 and the C-terminal tail of the S9 protein in the fidelity of initiation of translation in Escherichia coli

The accuracy of pairing of the anticodon of the initiator tRNA (tRNA(fMet)) and the initiation codon of an mRNA, in the ribosomal P-site, is crucial for determining the translational reading frame. However, a direct role of any ribosomal element(s) in scrutinizing this pairing is unknown. The P-site elements, m(2)G966 (methylated by RsmD), m(5)C967 (methylated by RsmB) and the C-terminal tail of the protein S9 lie in the vicinity of tRNA(fMet). We investigated the role of these elements in initiation from various codons, namely, AUG, GUG, UUG, CUG, AUA, AUU, AUC and ACG with tRNA(CAU)(fmet) (tRNA(fMet) with CAU anticodon); CAC and CAU with tRNA(GUG)(fme); UAG with tRNA(GAU)(fMet) using in vivo and computational methods. Although RsmB deficiency did not impact initiation from most codons, RsmD deficiency increased initiation from AUA, CAC and CAU (2- to 3.6-fold). Deletion of the S9 C-terminal tail resulted in poorer initiation from UUG, GUG and CUG, but in increased initiation from CAC, CAU and UAC codons (up to 4-fold). Also, the S9 tail suppressed initiation with tRNA(CAU)(fMet)lacking the 3GC base pairs in the anticodon stem. These observations suggest distinctive roles of 966/967 methylations and the S9 tail in initiation.

Topoisomerase IIα promotes activation of RNA polymerase I transcription by facilitating pre-initiation complex formation

Type II DNA topoisomerases catalyse DNA double-strand cleavage, passage and re-ligation to effect topological changes. There is considerable interest in elucidating topoisomerase II roles, particularly as these proteins are targets for anti-cancer drugs. Here we uncover a role for topoisomerase IIa in RNA polymerase I-directed ribosomal RNA gene transcription, which drives cell growth and proliferation and is upregulated in cancer cells. Our data suggest that topoisomerase IIa is a component of the initiation-competent RNA polymerase Iß complex and interacts directly with RNA polymerase I-associated transcription factor RRN3, which targets the polymerase to promoter-bound SL1 in pre-initiation complex formation. In cells, activation of rDNA transcription is reduced by inhibition or depletion of topoisomerase II, and this is accompanied by reduced transient double-strand DNA cleavage in the rDNA-promoter region and reduced pre-initiation complex formation. We propose that topoisomerase IIa functions in RNA polymerase I transcription to produce topological changes at the rDNA promoter that facilitate efficient de novo pre-initiation complex formation.

Small ubiquitin-like modifier conjugating enzyme with active site mutation acts as dominant negative inhibitor of SUMO conjugation in arabidopsis(F).

Small ubiquitin-like modifier (SUMO) conjugation affects a broad range of processes in plants, including growth, flower initiation, pathogen defense, and responses to abiotic stress. Here, we investigate in vivo and in vitro a SUMO conjugating enzyme with a Cys to Ser change in the active site, and show that it has a dominant negative effect. In planta expression significantly perturbs normal development, leading to growth retardation, early flowering and gene expression changes. We suggest that the mutant protein can serve as a probe to investigate sumoylation, also in plants for which poor genetic infrastructure precludes analysis via loss-of-function mutants.

Identification et caractérisation des cibles transcriptionnelles de ETV6, un facteur de transcription impliqué dans la leucémie de l’enfant.

La leucémie lymphoblastique aiguë (LLA) est responsable d’environ 25% de l’ensemble des cancers pédiatriques. Chez 85% des enfants diagnostiqués, la LLA entraîne une prolifération massive et incontrôlée de lymphocytes immatures de type précurseurs B dans la moelle osseuse (LLA pré-B). Des avancées intéressantes ont été faites au cours des trente dernières années et ont mené à une augmentation de l’efficacité des traitements thérapeutiques. Plus de 80% des enfants atteints de LLA seront guéris de cette maladie. Malheureusement, ces traitements manquent de spécificité à cause du manque de connaissances sur les mécanismes moléculaires impliqués durant l’initiation et le développement de la LLA pré-B pédiatrique. En d’autres termes, nous connaissons peu de chose sur l’étiologie de cette maladie. Plus de 25% des enfants atteints de la LLA pré-B présentent la translocation chromosomique t(12;21)(p13;q22) qui implique les gènes ETV6 et AML1. Celle-ci est formée in utero et mène à l’expression de la protéine chimère transcriptionnelle ETV6-AML1, dont la présence seule ne suffit pas au développement de la LLA pré-B. Ainsi, d’autres événements génétiques sont nécessaires au développement de cette leucémie. La délétion de l’allèle résiduel de ETV6 est un événement génétique fréquemment rencontré au moment du diagnostic de la LLA pré-B t(12;21)+. Cette délétion entraîne l’inactivation complète de ETV6 dans les lymphocytes pré-B leucémiques. ETV6 est un répresseur transcriptionnel de la famille Ets. Mon hypothèse de recherche est que ETV6 agit comme gène suppresseur de tumeur dans la LLA pré-B pédiatrique. L’inactivation de ETV6 causerait une dérégulation de l’expression de ses cibles transcriptionnelles et, par le fait même, favoriserait l’initiation et le déroulement de la leucémogenèse pédiatrique. Dans le cadre de mon projet, comme peu de cibles transcriptionnelles de ETV6 sont connues, j’ai effectué des expériences d’immunoprécipitation de la chromatine et des essais luciférases qui ont permis d’identifier six nouvelles cibles transcriptionnelles: TP53 (p53 et Δ133p53), SPHK1, IL-18, PTGER4 et LUM. J’ai démontré que la régulation transcriptionnelle médiée par ETV6 requiert la présence de ses deux domaines fonctionnels: PNT (interactions protéiques) et ETS (liaison à l’ADN). Ces domaines favorisent la reconnaissance d’un site EBS consensus dans une région située près du promoteur de base. Ce mécanisme peut dépendre du promoteur régulé par ETV6, mais également du contexte cellulaire. Des études fonctionnelles réalisées sur des lymphocytes pré-B leucémiques ont permis de mesurer l’impact de la dérégulation de l’expression des cibles transcriptionnelles de ETV6 sur trois voies biologiques: la prolifération cellulaire, l’apoptose induite par un stress génotoxique et la migration cellulaire dirigée par la voie de signalisation CXCL12/CXCR4. Ceci a permis de démontrer l’implication des gènes SPHK1, IL-18 et PTGER4 durant la leucémogenèse pédiatrique. Cette étude est une des premières à suggérer le rôle de ETV6 comme gène suppresseur de tumeur dans la LLA pré-B pédiatrique. Suite à l’inactivation du répresseur transcriptionnel ETV6, l’augmentation de l’expression de ses cibles transcriptionnelles favoriserait la prolifération et la survie des lymphocytes pré-B leucémiques dans la moelle osseuse. L’identification de nouveaux gènes impliqués dans le développement de la LLA pré-B pédiatrique ouvre la porte au développement de nouveaux traitements thérapeutiques qui pourront présenter une meilleure spécificité envers l’étiologie de la maladie.