Software development and analysis of high throughput sequencing data for genomic enhancer prediction

High Throughput Sequencing technologies (HTS) are becoming the standard in genomic regulation analysis. During my thesis I developed software for the analysis of HTS data. Through collaborations with other research groups, I specialized in the analysis of ChIP-Seq short mapped reads. For instance, I collaborated in the analysis of the effect of Hog1 stress induced response in Yeast and helped in the design of a multiple promoter-alignment method using ChIP-Seq data, among other collaborations. Making use of expertise and the software developed during this time, I analyzed ENCODE datasets in order to detect active genomic enhancers. Genomic enhancers are regions in the genome known to regulate transcription levels of close by or distant genes. Mechanism of activation and silencing of enhancers is still poorly understood. Epigenomic elements, like histone modifications and transcription factors play a critical role in enhancer activity. Modeling epigenomic signals, I predicted active and silenced enhancers in two cell lines and studied their effect in splicing and transcription initiation.Las tecnologías High Throughput Sequencing (HTS) se están convirtiendo en el método standard de análisis de la regulación genómica. Durante mi tesis, he desarrollado software para el análisis de datos HTS. Mediante la colaboración con otros grupos de investigaci n, me he especializado ́ en el análisis de datos de ChIP-Seq. Por ejemplo, colaborado en el análisis del efecto de Hog1 en células de levadura afectadas por stress, colaboré en el diseño de un m ́ todo para el alineamiento m ́ ltiple de promotores usando datos de ChIP-Seq, entre otras colaboraciones. Usando el conocimiento y el software desarrollados durante este tiempo, analicé datos producidos por el proyecto ENCODE para detectar enhancers genómicos activos. Los enhancers son areas del genoma conocidas por regular la transcripción de genes cercanos y lejanos. Los mecanismos de activación y silenciamiento de enhancers son aún poco entendidos. Elementos epigenómicos, como las modificaciones de histonas y los factores de transcripción juegan un papel crucial en la actividad de enhancers. Construyendo un modelo con estas señales epigen ́ micas, predije enhancers activos y silenciados en dos lineas celulares y estudié su efecto sobre splicing y sobre la iniciacion de la transcripción

A semi-supervised approach uncovers thousands of intragenic enhancers differentially activated in human cells

Background. Transcriptional enhancers are generally known to regulate gene transcription from afar. Their activation involves a series of changes in chromatin marks and recruitment of protein factors. These enhancers may also occur inside genes, but how many may be active in human cells and their effects on the regulation of the host gene remains unclear./nResults. We describe a novel semi-supervised method based on the relative enrichment of chromatin signals between 2 conditions to predict active enhancers. We applied this method to the tumoral K562 and the normal GM12878 cell lines to predict enhancers that are differentially active in one cell type. These predictions show enhancer-like properties according to positional distribution, correlation with gene expression and production of enhancer RNAs. Using this model, we predict 10,365 and 9777 intragenic active enhancers in K562 and GM12878, respectively, and relate the differential activation of these enhancers to expression and splicing differences of the host genes./nConclusions. We propose that the activation or silencing of intragenic transcriptional enhancers modulate the regulation of the host gene by means of a local change of the chromatin and the recruitment of enhancer-related factors that may interact with the RNA directly or through the interaction with RNA binding proteins. Predicted enhancers are available at http://regulatorygenomics.upf.edu/Projects/enhancers.html.The authors would like to thank E. Furlong, Y. Barash, B. Blencowe and U. Braunschweig for useful discussions. This work was supported by grants from Plan Nacional I + D (BIO2011-23920) and Consolider (CSD2009-00080) from MINECO (Spanish Government), and by the Sandra Ibarra Foundation for Cancer (FSI 2013). JGV and BS were supported FPI grants from the MINECO (Spanish Government) BES-2009-018064 and BES-2012-052683, respectively

Pyicos: a versatile toolkit for the analysis of high-throughput sequencing data

MOTIVATION: High-throughput sequencing (HTS) has revolutionized gene regulation studies and is now fundamental for the detection of protein-DNA and protein-RNA binding, as well as for measuring RNA expression. With increasing variety and sequencing depth of HTS datasets, the need for more flexible and memory-efficient tools to analyse them is growing. RESULTS: We describe Pyicos, a powerful toolkit for the analysis of mapped reads from diverse HTS experiments: ChIP-Seq, either punctuated or broad signals, CLIP-Seq and RNA-Seq. We prove the effectiveness of Pyicos to select for significant signals and show that its accuracy is comparable and sometimes superior to that of methods specifically designed for each particular type of experiment. Pyicos facilitates the analysis of a variety of HTS datatypes through its flexibility and memory efficiency, providing a useful framework for data integration into models of regulatory genomics. AVAILABILITY: Open-source software, with tutorials and protocol files, is available at http://regulatorygenomics.upf.edu/pyicos or as a Galaxy server at http://regulatorygenomics.upf.edu/galaxyGeneralitat de Catalunya by FI grant (to S.A.); Spanish Ministry of Science (MICINN) by FPI grant (to J.G.V.); MICINN grant BIO2008-01091 (to E.E.); European Commission grant EURASNET-(LSHG-CT-2005-518238) (to E.E.)

Pyicos: a versatile toolkit for the analysis of high-throughput sequencing data

MOTIVATION: High-throughput sequencing (HTS) has revolutionized gene regulation studies and is now fundamental for the detection of protein-DNA and protein-RNA binding, as well as for measuring RNA expression. With increasing variety and sequencing depth of HTS datasets, the need for more flexible and memory-efficient tools to analyse them is growing. RESULTS: We describe Pyicos, a powerful toolkit for the analysis of mapped reads from diverse HTS experiments: ChIP-Seq, either punctuated or broad signals, CLIP-Seq and RNA-Seq. We prove the effectiveness of Pyicos to select for significant signals and show that its accuracy is comparable and sometimes superior to that of methods specifically designed for each particular type of experiment. Pyicos facilitates the analysis of a variety of HTS datatypes through its flexibility and memory efficiency, providing a useful framework for data integration into models of regulatory genomics. AVAILABILITY: Open-source software, with tutorials and protocol files, is available at http://regulatorygenomics.upf.edu/pyicos or as a Galaxy server at http://regulatorygenomics.upf.edu/galaxyGeneralitat de Catalunya by FI grant (to S.A.); Spanish Ministry of Science (MICINN) by FPI grant (to J.G.V.); MICINN grant BIO2008-01091 (to E.E.); European Commission grant EURASNET-(LSHG-CT-2005-518238) (to E.E.)

Hog1 bypasses stress-mediated down-regulation of transcription by RNA polymerase II redistribution and chromatin remodeling

Cells are subjected to dramatic changes of gene expression upon environmental changes. Stress/ncauses a general down-regulation of gene expression together with the induction of a set of stress-responsive/ngenes. The p38-related stress-activated protein kinase Hog1 is an important regulator of transcription upon/nosmostress in yeast. Genome-wide localization studies of RNA polymerase II (RNA Pol II) and Hog1 showed that stress induced major changes in RNA Pol II localization, with a shift toward stress-responsive genes relative to housekeeping genes. RNA Pol II relocalization required Hog1, which was also localized to stress-responsive loci. In addition to RNA Pol II-bound genes, Hog1 also localized to RNA polymerase III-bound genes, pointing to a wider role for Hog1 in transcriptional control than initially expected. Interestingly, an increasing association of Hog1 with stressresponsive genes was strongly correlated with chromatin remodeling and increased gene expression. Remarkably, MNase-Seq analysis showed that although chromatin structure was not significantly altered at a genome-wide level in response to stress, there was pronounced chromatin remodeling for those genes that displayed Hog1 association. Hog1 serves to bypass the general down-regulation of gene expression that occurs in response to osmostress, and does so both by targeting RNA Pol II machinery and by inducing chromatin remodeling at stressresponsive loci.MN is a recipient of an FIS fellowship. This work was supported by grants from the Spanish Government (BIO2011-23920 to EE, BIO2009-07762 and BFU2012-33503 to FP, BFU2011-26722 to EN), the Consolider Ingenio 2010/nprogramme CSD2007-0015 to FP and FP7 UNICELLSYS grant 201142, the Fundación Marcelino Botín (FMB) to FP. EN and FP are recipients of an ICREA Acadèmia (Generalitat de Catalunya)

Nucleosome-driven transcription factor binding and gene regulation

In fission yeast cells, Cds1 is the effector kinase of the DNA replication checkpoint. We previously showed that when the DNA replication checkpoint is activated, the repressor Yox1 is phosphorylated and inactivated by Cds1, resulting in activation of MluI-binding factor (MBF)-dependent transcription. This is essential to reinitiate DNA synthesis and for correct G1-to-S transition. Here we show that Cdc10, which is an essential part of the MBF core, is the target of the DNA damage checkpoint. When fission yeast cells are treated with DNA-damaging agents, Chk1 is activated and phosphorylates Cdc10 at its carboxy-terminal domain. This modification is responsible for the repression of MBF-dependent transcription through induced release of MBF from chromatin. This inactivation of MBF is important for survival of cells challenged with DNA-damaging agents. Thus Yox1 and Cdc10 couple normal cell cycle regulation in unperturbed conditions and the DNA replication and DNA damage checkpoints into a single transcriptional complex.The experimental work was supported by grants from the Spanish government (BMC 2003-02902 and 2010-15313; CSD2006-00049), the European Union (IP HEROIC), and the Catalan government (AGAUR). L.G. was a recipient of a fellowship from the International PhD program of LaCaixa; G.P.V. was a recipient of a fellowship from the Ramón y Cajal program

Nucleosome-driven transcription factor binding and gene regulation

In fission yeast cells, Cds1 is the effector kinase of the DNA replication checkpoint. We previously showed that when the DNA replication checkpoint is activated, the repressor Yox1 is phosphorylated and inactivated by Cds1, resulting in activation of MluI-binding factor (MBF)-dependent transcription. This is essential to reinitiate DNA synthesis and for correct G1-to-S transition. Here we show that Cdc10, which is an essential part of the MBF core, is the target of the DNA damage checkpoint. When fission yeast cells are treated with DNA-damaging agents, Chk1 is activated and phosphorylates Cdc10 at its carboxy-terminal domain. This modification is responsible for the repression of MBF-dependent transcription through induced release of MBF from chromatin. This inactivation of MBF is important for survival of cells challenged with DNA-damaging agents. Thus Yox1 and Cdc10 couple normal cell cycle regulation in unperturbed conditions and the DNA replication and DNA damage checkpoints into a single transcriptional complex.The experimental work was supported by grants from the Spanish government (BMC 2003-02902 and 2010-15313; CSD2006-00049), the European Union (IP HEROIC), and the Catalan government (AGAUR). L.G. was a recipient of a fellowship from the International PhD program of LaCaixa; G.P.V. was a recipient of a fellowship from the Ramón y Cajal program