Genomics Core FacilityPublications
Accounting for technical noise in single-cell RNA-seq experiments.
Brennecke, P., Anders, S., Kim, J.K., Kolodziejczyk, A.A., Zhang, X., Proserpio, V., Baying, B., Benes, V., Teichmann, S.A., Marioni, J.C. & Heisler, M.G.
Nat Methods. 2013 Nov;10(11):1093-5. doi: 10.1038/nmeth.2645. Epub 2013 Sep 22.
Single-cell RNA-seq can yield valuable insights about the variability within a population of seemingly homogeneous cells. We developed a quantitative statistical method to distinguish true biological variability from the high levels of technical noise in single-cell experiments. Our approach quantifies the statistical significance of observed cell-to-cell variability in expression strength on a gene-by-gene basis. We validate our approach using two independent data sets from Arabidopsis thaliana and Mus musculus.
The need for transparency and good practices in the qPCR literature.
Bustin, S.A., Benes, V., Garson, J., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M.W., Shipley, G., Wittwer, C.T., Schjerling, P., Day, P.J., Abreu, M., Aguado, B., Beaulieu, J.F., Beckers, A., Bogaert, S., Browne, J.A., Carrasco-Ramiro, F., Ceelen, L., Ciborowski, K., Cornillie, P., Coulon, S., Cuypers, A., De Brouwer, S., De Ceuninck, L., De Craene, J., De Naeyer, H., De Spiegelaere, W., Deckers, K., Dheedene, A., Durinck, K., Ferreira-Teixeira, M., Fieuw, A., Gallup, J.M., Gonzalo-Flores, S., Goossens, K., Heindryckx, F., Herring, E., Hoenicka, H., Icardi, L., Jaggi, R., Javad, F., Karampelias, M., Kibenge, F., Kibenge, M., Kumps, C., Lambertz, I., Lammens, T., Markey, A., Messiaen, P., Mets, E., Morais, S., Mudarra-Rubio, A., Nakiwala, J., Nelis, H., Olsvik, P.A., Perez-Novo, C., Plusquin, M., Remans, T., Rihani, A., Rodrigues-Santos, P., Rondou, P., Sanders, R., Schmidt-Bleek, K., Skovgaard, K., Smeets, K., Tabera, L., Toegel, S., Van Acker, T., Van den Broeck, W., Van der Meulen, J., Van Gele, M., Van Peer, G., Van Poucke, M., Van Roy, N., Vergult, S., Wauman, J., Tshuikina-Wiklander, M., Willems, E., Zaccara, S., Zeka, F. & Vandesompele, J.
Nat Methods. 2013 Oct 30;10(11):1063-7. doi: 10.1038/nmeth.2697.
Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.
Multiple epigenetic mechanisms and the piRNA pathway enforce LINE1 silencing during adult spermatogenesis.
Di Giacomo, M., Comazzetto, S., Saini, H., De Fazio, S., Carrieri, C., Morgan, M., Vasiliauskaite, L., Benes, V., Enright, A.J. & O'Carroll, D.
Mol Cell. 2013 May 23;50(4):601-8. doi: 10.1016/j.molcel.2013.04.026.
Transposons present an acute challenge to the germline, and mechanisms that repress their activity are essential for transgenerational genomic integrity. LINE1 (L1) is the most successful retrotransposon and is epigenetically repressed by CpG DNA methylation. Here, we identify two additional important mechanisms by which L1 is repressed during spermatogenesis. We demonstrate that the Piwi protein Mili and the piRNA pathway are required to posttranscriptionally silence L1 in meiotic pachytene cells even in the presence of normal L1 DNA methylation. Strikingly, in the absence of both a functional piRNA pathway and DNA methylation, L1 elements are normally repressed in mitotic stages of spermatogenesis. Accordingly, we find that the euchromatic repressive histone H3 dimethylated lysine 9 modification cosuppresses L1 expression therein. We demonstrate the existence of multiple epigenetic mechanisms that in conjunction with the piRNA pathway sequentially enforce L1 silencing and genomic stability during mitotic and meiotic stages of adult spermatogenesis.
An efficient method for genome-wide polyadenylation site mapping and RNA quantification.
Wilkening, S., Pelechano, V., Jarvelin, A.I., Tekkedil, M.M., Anders, S., Benes, V. & Steinmetz, L.M.
Nucleic Acids Res. 2013 Mar 1;41(5):e65. doi: 10.1093/nar/gks1249. Epub 2013 Jan7.
The use of alternative poly(A) sites is common and affects the post-transcriptional fate of mRNA, including its stability, subcellular localization and translation. Here, we present a method to identify poly(A) sites in a genome-wide and strand-specific manner. This method, termed 3'T-fill, initially fills in the poly(A) stretch with unlabeled dTTPs, allowing sequencing to start directly after the poly(A) tail into the 3'-untranslated regions (UTR). Our comparative analysis demonstrates that it outperforms existing protocols in quality and throughput and accurately quantifies RNA levels as only one read is produced from each transcript. We use this method to characterize the diversity of polyadenylation in Saccharomyces cerevisiae, showing that alternative RNA molecules are present even in a genetically identical cell population. Finally, we observe that overlap of convergent 3'-UTRs is frequent but sharply limited by coding regions, suggesting factors that restrict compression of the yeast genome.
A Role for Fkbp6 and the Chaperone Machinery in piRNA Amplification and Transposon Silencing.
Xiol, J., Cora, E., Koglgruber, R., Chuma, S., Subramanian, S., Hosokawa, M., Reuter, M., Yang, Z., Berninger, P., Palencia, A., Benes, V., Penninger, J., Sachidanandam, R. & Pillai, R.S.
Mol Cell. 2012 Sep 28;47(6):970-9. doi: 10.1016/j.molcel.2012.07.019. Epub 2012Aug 16.
Epigenetic silencing of transposons by Piwi-interacting RNAs (piRNAs) constitutes an RNA-based genome defense mechanism. Piwi endonuclease action amplifies the piRNA pool by generating new piRNAs from target transcripts by a poorly understood mechanism. Here, we identified mouse Fkbp6 as a factor in this biogenesis pathway delivering piRNAs to the Piwi protein Miwi2. Mice lacking Fkbp6 derepress LINE1 (L1) retrotransposon and display reduced DNA methylation due to deficient nuclear accumulation of Miwi2. Like other cochaperones, Fkbp6 associates with the molecular chaperone Hsp90 via its tetratricopeptide repeat (TPR) domain. Inhibition of the ATP-dependent Hsp90 activity in an insect cell culture model results in the accumulation of short antisense RNAs in Piwi complexes. We identify these to be byproducts of piRNA amplification that accumulate only in nuage-localized Piwi proteins. We propose that the chaperone machinery normally ejects these inhibitory RNAs, allowing turnover of Piwi complexes for their continued participation in piRNA amplification.
Genomics of DNA cytosine methylation in Escherichia coli reveals its role in stationary phase transcription.
Kahramanoglou, C., Prieto, A.I., Khedkar, S., Haase, B., Gupta, A., Benes, V., Fraser, G.M., Luscombe, N.M. & Seshasayee, A.S.
Nat Commun. 2012 Jun 6;3:886. doi: 10.1038/ncomms1878.
DNA cytosine methylation regulates gene expression in mammals. In bacteria, its role in gene expression and genome architecture is less understood. Here we perform high-throughput sequencing of bisulfite-treated genomic DNA from Escherichia coli K12 to describe, for the first time, the extent of cytosine methylation of bacterial DNA at single-base resolution. Whereas most target sites (C(m)CWGG) are fully methylated in stationary phase cells, many sites with an extended CC(m)CWGG motif are only partially methylated in exponentially growing cells. We speculate that these partially methylated sites may be selected, as these are slightly correlated with the risk of spontaneous, non-synonymous conversion of methylated cytosines to thymines. Microarray analysis in a cytosine methylation-deficient mutant of E. coli shows increased expression of the stress response sigma factor RpoS and many of its targets in stationary phase. Thus, DNA cytosine methylation is a regulator of stationary phase gene expression in E. coli.
Genome-wide RNAi screening identifies human proteins with a regulatory function in the early secretory pathway.
Simpson, J.C., Joggerst, B., Laketa, V., Verissimo, F., Cetin, C., Erfle, H., Bexiga, M.G., Singan, V.R., Heriche, J.K., Neumann, B., Mateos, A., Blake, J., Bechtel, S., Benes, V., Wiemann, S., Ellenberg, J. & Pepperkok, R.
Nat Cell Biol. 2012 Jun 3;14(7):764-74. doi: 10.1038/ncb2510.
The secretory pathway in mammalian cells has evolved to facilitate the transfer of cargo molecules to internal and cell surface membranes. Use of automated microscopy-based genome-wide RNA interference screens in cultured human cells allowed us to identify 554 proteins influencing secretion. Cloning, fluorescent-tagging and subcellular localization analysis of 179 of these proteins revealed that more than two-thirds localize to either the cytoplasm or membranes of the secretory and endocytic pathways. The depletion of 143 of them resulted in perturbations in the organization of the COPII and/or COPI vesicular coat complexes of the early secretory pathway, or the morphology of the Golgi complex. Network analyses revealed a so far unappreciated link between early secretory pathway function, small GTP-binding protein regulation, actin cytoskeleton organization and EGF-receptor-mediated signalling. This work provides an important resource for an integrative understanding of global cellular organization and regulation of the secretory pathway in mammalian cells.
Iron regulatory protein-1 and -2: transcriptome-wide definition of binding mRNAs and shaping of the cellular proteome by iron regulatory proteins.
Sanchez, M., Galy, B., Schwanhaeusser, B., Blake, J., Bahr-Ivacevic, T., Benes, V., Selbach, M., Muckenthaler, M.U. & Hentze, M.W.
Blood. 2011 Nov 24;118(22):e168-79. Epub 2011 Sep 22.
Iron regulatory proteins (IRPs) 1 and 2 are RNA-binding proteins that control cellular iron metabolism by binding to conserved RNA motifs called iron-responsive elements (IREs). The currently known IRP-binding mRNAs encode proteins involved in iron uptake, storage, and release as well as heme synthesis. To systematically define the IRE/IRP regulatory network on a transcriptome-wide scale, IRP1/IRE and IRP2/IRE messenger ribonucleoprotein complexes were immunoselected, and the mRNA composition was determined using microarrays. We identify 35 novel mRNAs that bind both IRP1 and IRP2, and we also report for the first time cellular mRNAs with exclusive specificity for IRP1 or IRP2. To further explore cellular iron metabolism at a system-wide level, we undertook proteomic analysis by pulsed stable isotope labeling by amino acids in cell culture in an iron-modulated mouse hepatic cell line and in bone marrow-derived macrophages from IRP1- and IRP2-deficient mice. This work investigates cellular iron metabolism in unprecedented depth and defines a wide network of mRNAs and proteins with iron-dependent regulation, IRP-dependent regulation, or both.