53 research outputs found
Ătude de la rĂ©gulation de l'expression de gĂšnes cibles du rĂ©cepteur aryl hydrocarbone dans des cellules cancĂ©reuses de la glande mammaire
Notre laboratoire s'intĂ©resse aux mĂ©canismes impliquĂ©s dans la rĂ©gulation de l'expression gĂ©nique et plus particuliĂšrement au rĂŽle de la chromatine dans cette rĂ©gulation. En effet, chez les eucaryotes l'ADN est compactĂ©e autour de protĂ©ines appelĂ©es histones crĂ©ant ainsi des nuclĂ©osomes lesquels forment une structure plus complexe, la chromatine. Cette derniĂšre est une barriĂšre aux processus cellulaires touchant l'ADN dont la transcription. La comprĂ©hension de la rĂ©gulation de la structure de la chromatine est essentielle pour saisir les variations de l'expression gĂ©nique. Mon projet de doctorat a portĂ© sur l'Ă©tude de la rĂ©gulation des gĂšnes cibles du rĂ©cepteur aryl hydrocarbone (AhR), CYP1A1 et CYP1B1, et plus particuliĂšrement sur le rĂŽle du variant d'histone H2A.Z dans l'expression de ces gĂšnes. AhR est un senseur molĂ©culaire auquel va [i.e. vont] se lier de nombreux polluants appartenant principalement Ă ces deux grandes familles : les hydrocarbones aromatiques halogĂšnes (HAH) et les hydrocarbones aromatiques polycycliques (PAH). En rĂ©ponse Ă la liaison de ces polluants, AhR va induire l'expression de ses gĂšnes cibles. CYP1A1 et CYP1B1 sont impliquĂ©es dans le mĂ©tabolisme de l'estradiol (E2) en 2hydroxyestradiol et 4-hydroxyestradiol respectivement. Il a Ă©tĂ© proposĂ© qu'une diminution du ratio CYP1A1/CYP1B1 soit importante pour l'initiation du cancer du sein. Au cours de mon doctorat, j'ai pu mettre en Ă©vidence un rĂŽle du variant H2A.Z dans la rĂ©gulation de l'expression de CYP1A1 et CYP1B1. J'ai aussi pu montrer que le statut de ER[alpha] dĂ©terminait l'importance de H2A.Z lors de l'induction de CYP1A1. De plus, nous avons observĂ© que la dĂ©plĂ©tion de H2A.Z induit une augmentation de la mĂ©thylation de l'ADN au promoteur de CYP1A1. En parallĂšle, nous avons confirmĂ© que ER[alpha] rĂ©prime spĂ©cifiquement l'induction de CYP1A1 sans affecter celle de CYP1B1. Nos rĂ©sultats montrent qu'en prĂ©sence de TCDD et d'E2, ER[alpha] et DNMT3B sont recrutĂ©s au promoteur de CYP1A1, ce qui conduit Ă une augmentation de la mĂ©thylation du promoteur de CYP1A1 et consĂ©quemment Ă une diminution de son induction. AhR possĂšde de nombreux ligands d'origine trĂšs variĂ©e qui peuvent ĂȘtre aussi bien toxiques que bĂ©nĂ©fiques. Nous avons choisi de comparer deux de ces ligands : le TCDD et le DIM. Au cours de ces travaux, nous avons montrĂ© que le DIM utilisĂ© Ă forte concentration (>50[mu]M) induit les gĂšnes cibles de AhR (CYP1A1 et CYP1B1) mais aussi un arrĂȘt de la croissance et la mort des cellules. Ă l'opposĂ©, le traitement avec des concentrations plus faible [i.e. faibles] de DIM (10[mu]M) induit principalement les gĂšnes cibles de ER[alpha] (TFF1 et GREB1) et la prolifĂ©ration des cellules. Nous avons aussi montrĂ© que l'activation de ER[alpha] par le DIM est due Ă l'action de la protĂ©ine kinase A (PKA). En effet, l'inhibition de la PKA ainsi que la dĂ©plĂ©tion de ER[alpha] abolissent les effets du DIM sur l'expression de GREB1 et CYPIA1 ainsi que sur la prolifĂ©ration cellulaire. En conclusion, nous avons dans un premier temps mis en Ă©vidence le rĂŽle de deux protĂ©ines, DNMT3B et H2A.Z, dans la rĂ©gulation de CYP1A1 dans les cellules MCF7. Nous avons ainsi dĂ©couvert un nouveau corĂ©presseur partenaire de ER[alpha] en DNMT3B et nous avons proposĂ© une nouvelle façon pour ER[alpha] de promouvoir la carcinogenĂšse en dĂ©rĂ©gulant le ratio CYP1A1/CYP1B1. Dans un deuxiĂšme temps, nous avons montrĂ© que la concentration de DIM utilisĂ©e dans les expĂ©riences peut conduire Ă des rĂ©sultats diamĂ©tralement opposĂ©s sur la croissance cellulaire
PCRTiler: automated design of tiled and specific PCR primer pairs
Efficiency and specificity of PCR amplification is dependent on several parameters, such as amplicon length, as well as hybridization specificity and melting temperature of primer oligonucleotides. Primer design is thus of critical importance for the success of PCR experiments, but can be a time-consuming and repetitive task, for example when large genomic regions are to be scanned for the presence of a protein of interest by chromatin immunoprecipitation experiments. We present here a webserver that allows the automated design of tiled primer pairs for any number of genomic loci. PCRTiler splits the target DNA sequences into smaller regions, and identifies candidate primers for each sub-region by running the well-known program Primer3 followed by the elimination of primers with a high cross-hybridization potential via BLAST. Tiling density and primer characteristics are specified by the user via a simple and user-friendly interface. The webserver can be accessed at http://pcrtiler.alaingervais.org:8080/PCRTiler. Additionally, users may download a standalone Java-based implementation of this software. Experimental validation of PCRTiler has demonstrated that it produces correct results. We have tiled a region of the human genome, in which 96 of 123 primer pairs worked in the first attempt, and 105 of 123 (85%) could be made to work by optimizing the conditions of the PCR assay
Concerted action of kinesins kif5b and kif13b promotes efficient secretory vesicle transport to microtubule plus ends
Intracellular transport relies on multiple kinesins, but it is poorly understood which kinesins are present on particular cargos, what their contributions are and whether they act simultaneously on the same cargo. Here, we show that Rab6-positive secretory vesicles are transported from the Golgi apparatus to the cell periphery by kinesin-1 KIF5B and kinesin-3 KIF13B, which determine the location of secretion events. KIF5B plays a dominant role, whereas KIF13B helps Rab6 vesicles to reach freshly polymerized microtubule ends, to which KIF5B binds poorly, likely because its cofactors, MAP7-family proteins, are slow in populating these ends. Sub-pixel localization demonstrated that during microtubule plus-end directed transport, both kinesins localize to the vesicle front and can be engaged on the same vesicle. When vesicles reverse direction, KIF13B relocates to the middle of the vesicle, while KIF5B shifts to the back, suggesting that KIF5B but not KIF13B undergoes a tug-of-war with a minus-end directed motor.info:eu-repo/semantics/publishe
Competition between translation initiation factor eIF5 and its mimic protein 5MP determines non-AUG initiation rate genome-wide
Citation: Tang, L., Morris, J., Wan, J., Moore, C., Fujita, Y., Gillaspie, S., ⊠Asano, K. (2017). Competition between translation initiation factor eIF5 and its mimic protein 5MP determines non-AUG initiation rate genome-wide. Nucleic Acids Research, 45(20), 11941â11953. https://doi.org/10.1093/nar/gkx808In the human genome, translation initiation from nonAUG codons plays an important role in various gene regulation programs. However, mechanisms regulating the non-AUG initiation rate remain poorly understood. Here, we show that the non-AUG initiation rate is nearly consistent under a ïŹxed nucleotide context in various human and insect cells. Yet, it ranges from <1% to nearly 100% compared to AUG translation, depending on surrounding sequences, including Kozak, and possibly additional nucleotide contexts. Mechanistically, this range of non-AUG initiation is controlled in part, by the eIF5-mimic protein (5MP). 5MP represses non-AUG translation by competing with eIF5 for the Met-tRNAi-binding factor eIF2. Consistently, eIF5 increases, whereas 5MP decreases translation of NAT1/EIF4G2/DAP5, whose sole start codon is GUG. By modulating eIF5 and 5MP1 expression in combination with ribosome proïŹling we identiïŹed a handful of previously unknown non-AUG initiation sites, some of which serve as the exclusive start codons. If the initiation rate for these codons is low, then an AUG-initiated downstream ORF prevents the generation of shorter, AUGinitiated isoforms. We propose that the homeostasis of the non-AUG translatome is maintained through balanced expression of eIF5 and 5MP
Genome-wide meta-analysis associates HLA-DQA1/DRB1 and LPA and lifestyle factors with human longevity
Genomic analysis of longevity offers the potential to illuminate the biology of human aging. Here, using genome-wide association meta-analysis of 606,059 parents' survival, we discover two regions associated with longevity (HLA-DQA1/DRB1 and LPA). We also validate previous suggestions that APOE, CHRNA3/5, CDKN2A/B, SH2B3 and FOXO3A influence longevity. Next we show that giving up smoking, educational attainment, openness to new experience and high-density lipoprotein (HDL) cholesterol levels are most positively genetically correlated with lifespan while susceptibility to coronary artery disease (CAD), cigarettes smoked per day, lung cancer, insulin resistance and body fat are most negatively correlated. We suggest that the effect of education on lifespan is principally mediated through smoking while the effect of obesity appears to act via CAD. Using instrumental variables, we suggest that an increase of one body mass index unit reduces lifespan by 7 months while 1 year of education adds 11 months to expected lifespan
Low levels of 3,3âČ-diindolylmethane activate estrogen receptor α and induce proliferation of breast cancer cells in the absence of estradiol
Chemotherapy reduces PARP1 in cancers of the ovary: implications for future clinical trials involving PARP inhibitors
Time to Switch to Second-line Antiretroviral Therapy in Children With Human Immunodeficiency Virus in Europe and Thailand.
Background: Data on durability of first-line antiretroviral therapy (ART) in children with human immunodeficiency virus (HIV) are limited. We assessed time to switch to second-line therapy in 16 European countries and Thailand. Methods: Children aged <18 years initiating combination ART (â„2 nucleoside reverse transcriptase inhibitors [NRTIs] plus nonnucleoside reverse transcriptase inhibitor [NNRTI] or boosted protease inhibitor [PI]) were included. Switch to second-line was defined as (i) change across drug class (PI to NNRTI or vice versa) or within PI class plus change of â„1 NRTI; (ii) change from single to dual PI; or (iii) addition of a new drug class. Cumulative incidence of switch was calculated with death and loss to follow-up as competing risks. Results: Of 3668 children included, median age at ART initiation was 6.1 (interquartile range (IQR), 1.7-10.5) years. Initial regimens were 32% PI based, 34% nevirapine (NVP) based, and 33% efavirenz based. Median duration of follow-up was 5.4 (IQR, 2.9-8.3) years. Cumulative incidence of switch at 5 years was 21% (95% confidence interval, 20%-23%), with significant regional variations. Median time to switch was 30 (IQR, 16-58) months; two-thirds of switches were related to treatment failure. In multivariable analysis, older age, severe immunosuppression and higher viral load (VL) at ART start, and NVP-based initial regimens were associated with increased risk of switch. Conclusions: One in 5 children switched to a second-line regimen by 5 years of ART, with two-thirds failure related. Advanced HIV, older age, and NVP-based regimens were associated with increased risk of switch
Analysis of changes to mRNA levels and CTCF occupancy upon TFII-I knockdown
CTCF is a key regulator of nuclear chromatin structure, chromatin organization and gene regulation. The impact of CTCF on transcriptional output is quite varied, ranging from repression, to transcriptional pausing and transactivation. The multifunctional nature of CTCF is mediated, in part, through differential association with protein partners having unique properties. We identified the general transcription factor TFII-I as an interacting partner of CTCF. To gain an understanding of the function of TFII-I in regulating gene expression and CTCF binding genome wide, we conducted microarray experiments following TFII-I knockdown and chromatin immunoprecipitation of CTCF followed by next generation sequencing (ChIP-seq) from the same TFII-I depleted cells. Here, we described the experimental design and the quality control and analysis that were performed on the dataset. The data is publicly available through the GEO database with accession number GSE60918. The interpretation and description of these data are included in a manuscript in revision (1)
Anticancer Properties of Phyllanthus emblica (Indian Gooseberry)
There is a wealth of information emanating from both in vitro and in vivo studies indicating fruit extract of the Phyllanthus emblica tree, commonly referred to as Indian Gooseberries, has potent anticancer properties. The bioactivity in this extract is thought to be principally mediated by polyphenols, especially tannins and flavonoids. It remains unclear how polyphenols from Phyllanthus emblica can incorporate both cancer-preventative and antitumor properties. The antioxidant function of Phyllanthus emblica can account for some of the anticancer activity, but clearly other mechanisms are equally important. Herein, we provide a brief overview of the evidence supporting anticancer activity of Indian Gooseberry extracts, suggest possible mechanisms for these actions, and provide future directions that might be taken to translate these findings clinically
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