Publications by authors named "Giovanna Ambrosini"

23 Publications

  • Page 1 of 1

Intraductal xenografts show lobular carcinoma cells rely on their own extracellular matrix and LOXL1.

EMBO Mol Med 2021 Mar 22;13(3):e13180. Epub 2021 Feb 22.

ISREC - Swiss Institute for Experimental Cancer Research, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

Invasive lobular carcinoma (ILC) is the most frequent special histological subtype of breast cancer, typically characterized by loss of E-cadherin. It has clinical features distinct from other estrogen receptor-positive (ER ) breast cancers but the molecular mechanisms underlying its characteristic biology are poorly understood because we lack experimental models to study them. Here, we recapitulate the human disease, including its metastatic pattern, by grafting ILC-derived breast cancer cell lines, SUM-44 PE and MDA-MB-134-VI cells, into the mouse milk ducts. Using patient-derived intraductal xenografts from lobular and non-lobular ER HER2 tumors to compare global gene expression, we identify extracellular matrix modulation as a lobular carcinoma cell-intrinsic trait. Analysis of TCGA patient datasets shows matrisome signature is enriched in lobular carcinomas with overexpression of elastin, collagens, and the collagen modifying enzyme LOXL1. Treatment with the pan LOX inhibitor BAPN and silencing of LOXL1 expression decrease tumor growth, invasion, and metastasis by disrupting ECM structure resulting in decreased ER signaling. We conclude that LOXL1 inhibition is a promising therapeutic strategy for ILC.
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http://dx.doi.org/10.15252/emmm.202013180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933935PMC
March 2021

Insights gained from a comprehensive all-against-all transcription factor binding motif benchmarking study.

Genome Biol 2020 05 11;21(1):114. Epub 2020 May 11.

School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015, Lausanne, Switzerland.

Background: Positional weight matrix (PWM) is a de facto standard model to describe transcription factor (TF) DNA binding specificities. PWMs inferred from in vivo or in vitro data are stored in many databases and used in a plethora of biological applications. This calls for comprehensive benchmarking of public PWM models with large experimental reference sets.

Results: Here we report results from all-against-all benchmarking of PWM models for DNA binding sites of human TFs on a large compilation of in vitro (HT-SELEX, PBM) and in vivo (ChIP-seq) binding data. We observe that the best performing PWM for a given TF often belongs to another TF, usually from the same family. Occasionally, binding specificity is correlated with the structural class of the DNA binding domain, indicated by good cross-family performance measures. Benchmarking-based selection of family-representative motifs is more effective than motif clustering-based approaches. Overall, there is good agreement between in vitro and in vivo performance measures. However, for some in vivo experiments, the best performing PWM is assigned to an unrelated TF, indicating a binding mode involving protein-protein cooperativity.

Conclusions: In an all-against-all setting, we compute more than 18 million performance measure values for different PWM-experiment combinations and offer these results as a public resource to the research community. The benchmarking protocols are provided via a web interface and as docker images. The methods and results from this study may help others make better use of public TF specificity models, as well as public TF binding data sets.
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http://dx.doi.org/10.1186/s13059-020-01996-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212583PMC
May 2020

The secreted protease Adamts18 links hormone action to activation of the mammary stem cell niche.

Nat Commun 2020 03 26;11(1):1571. Epub 2020 Mar 26.

Ecole Polytechnique Fédérale de Lausanne, Station 19, CH-1015, Lausanne, Switzerland.

Estrogens and progesterone control breast development and carcinogenesis via their cognate receptors expressed in a subset of luminal cells in the mammary epithelium. How they control the extracellular matrix, important to breast physiology and tumorigenesis, remains unclear. Here we report that both hormones induce the secreted protease Adamts18 in myoepithelial cells by controlling Wnt4 expression with consequent paracrine canonical Wnt signaling activation. Adamts18 is required for stem cell activation, has multiple binding partners in the basement membrane and interacts genetically with the basal membrane-specific proteoglycan, Col18a1, pointing to the basement membrane as part of the stem cell niche. In vitro, ADAMTS18 cleaves fibronectin; in vivo, Adamts18 deletion causes increased collagen deposition during puberty, which results in impaired Hippo signaling and reduced Fgfr2 expression both of which control stem cell function. Thus, Adamts18 links luminal hormone receptor signaling to basement membrane remodeling and stem cell activation.
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http://dx.doi.org/10.1038/s41467-020-15357-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7099066PMC
March 2020

Common genetic variants associated with Parkinson's disease display widespread signature of epigenetic plasticity.

Sci Rep 2019 12 5;9(1):18464. Epub 2019 Dec 5.

Department of Neurology, University Clinic Bonn, Bonn, Germany.

Parkinson disease (PD) is characterized by a pivotal progressive loss of substantia nigra dopaminergic neurons and aggregation of α-synuclein protein encoded by the SNCA gene. Genome-wide association studies identified almost 100 sequence variants linked to PD in SNCA. However, the consequences of this genetic variability are rather unclear. Herein, our analysis on selective single nucleotide polymorphisms (SNPs) which are highly associated with the PD susceptibility revealed that several SNP sites attribute to the nucleosomes and overlay with bivalent regions poised to adopt either active or repressed chromatin states. We also identified large number of transcription factor (TF) binding sites associated with these variants. In addition, we located two docking sites in the intron-1 methylation prone region of SNCA which are required for the putative interactions with DNMT1. Taken together, our analysis reflects an additional layer of epigenomic contribution for the regulation of the SNCA gene in PD.
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http://dx.doi.org/10.1038/s41598-019-54865-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895091PMC
December 2019

EPD in 2020: enhanced data visualization and extension to ncRNA promoters.

Nucleic Acids Res 2020 01;48(D1):D65-D69

Swiss Institute of Bioinformatics (SIB), CH-1015 Lausanne, Switzerland.

The Eukaryotic Promoter Database (EPD), available online at https://epd.epfl.ch, provides accurate transcription start site (TSS) information for promoters of 15 model organisms plus corresponding functional genomics data that can be viewed in a genome browser, queried or analyzed via web interfaces, or exported in standard formats (FASTA, BED, CSV) for subsequent analysis with other tools. Recent work has focused on the improvement of the EPD promoter viewers, which use the UCSC Genome Browser as visualization platform. Thousands of high-resolution tracks for CAGE, ChIP-seq and similar data have been generated and organized into public track hubs. Customized, reproducible promoter views, combining EPD-supplied tracks with native UCSC Genome Browser tracks, can be accessed from the organism summary pages or from individual promoter entries. Moreover, thanks to recent improvements and stabilization of ncRNA gene catalogs, we were able to release promoter collections for certain classes of ncRNAs from human and mouse. Furthermore, we developed automatic computational protocols to assign orphan TSS peaks to downstream genes based on paired-end (RAMPAGE) TSS mapping data, which enabled us to add nearly 9000 new entries to the human promoter collection. Since our last article in this journal, EPD was extended to five more model organisms: rhesus monkey, rat, dog, chicken and Plasmodium falciparum.
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http://dx.doi.org/10.1093/nar/gkz1014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145694PMC
January 2020

Short-lived AUF1 p42-binding mRNAs of RANKL and BCL6 have two distinct instability elements each.

PLoS One 2018 12;13(11):e0206823. Epub 2018 Nov 12.

Ecole Polytechnique Fédérale de Lausanne (EPFL), SV-Sciences de la Vie, ISREC-Swiss Institute for Experimental Cancer Research, Lausanne, Switzerland.

Regulation of mRNA stability by RNA-protein interactions contributes significantly to quantitative aspects of gene expression. We have identified potential mRNA targets of the AU-rich element binding protein AUF1. Myc-tagged AUF1 p42 was induced in mouse NIH/3T3 cells and RNA-protein complexes isolated using anti-myc tag antibody beads. Bound mRNAs were analyzed with Affymetrix microarrays. We have identified 508 potential target mRNAs that were at least 3-fold enriched compared to control cells without myc-AUF1. 22.3% of the enriched mRNAs had an AU-rich cluster in the ARED Organism database, against 16.3% of non-enriched control mRNAs. The enrichment towards AU-rich elements was also visible by AREScore with an average value of 5.2 in the enriched mRNAs versus 4.2 in the control group. Yet, numerous mRNAs were enriched without a high ARE score. The enrichment of tetrameric and pentameric sequences suggests a broad AUF1 p42-binding spectrum at short U-rich sequences flanked by A or G. Still, some enriched mRNAs were highly unstable, as those of TNFSF11 (known as RANKL), KLF10, HES1, CCNT2, SMAD6, and BCL6. We have mapped some of the instability determinants. HES1 mRNA appeared to have a coding region determinant. Detailed analysis of the RANKL and BCL6 3'UTR revealed for both that full instability required two elements, which are conserved in evolution. In RANKL mRNA both elements are AU-rich and separated by 30 bases, while in BCL6 mRNA one is AU-rich and 60 bases from a non AU-rich element that potentially forms a stem-loop structure.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0206823PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6231638PMC
April 2019

PWMScan: a fast tool for scanning entire genomes with a position-specific weight matrix.

Bioinformatics 2018 07;34(14):2483-2484

The Swiss Institute for Experimental Cancer Research (ISREC), Swiss Federal Institute of Technology Lausanne (EPFL).

Summary: Transcription factors regulate gene expression by binding to specific short DNA sequences of 5-20 bp to regulate the rate of transcription of genetic information from DNA to messenger RNA. We present PWMScan, a fast web-based tool to scan server-resident genomes for matches to a user-supplied PWM or transcription factor binding site model from a public database.

Availability And Implementation: The web server and source code are available at http://ccg.vital-it.ch/pwmscan and https://sourceforge.net/projects/pwmscan, respectively.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/bty127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6041753PMC
July 2018

MGA repository: a curated data resource for ChIP-seq and other genome annotated data.

Nucleic Acids Res 2018 01;46(D1):D175-D180

Swiss Institute of Bioinformatics (SIB), CH-1015 Lausanne, Switzerland.

The Mass Genome Annotation (MGA) repository is a resource designed to store published next generation sequencing data and other genome annotation data (such as gene start sites, SNPs, etc.) in a completely standardised format. Each sample has undergone local processing in order the meet the strict MGA format requirements. The original data source, the reformatting procedure and the biological characteristics of the samples are described in an accompanying documentation file manually edited by data curators. 10 model organisms are currently represented: Homo sapiens, Mus musculus, Danio rerio, Drosophila melanogaster, Apis mellifera, Caenorhabditis elegans, Arabidopsis thaliana, Zea mays, Saccharomyces cerevisiae and Schizosaccharomyces pombe. As of today, the resource contains over 24 000 samples. In conjunction with other tools developed by our group (the ChIP-Seq and SSA servers), it allows users to carry out a great variety of analysis task with MGA samples, such as making aggregation plots and heat maps for selected genomic regions, finding peak regions, generating custom tracks for visualizing genomic features in a UCSC genome browser window, or downloading chromatin data in a table format suitable for local processing with more advanced statistical analysis software such as R. Home page: http://ccg.vital-it.ch/mga/.
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http://dx.doi.org/10.1093/nar/gkx995DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5753388PMC
January 2018

SMiLE-seq identifies binding motifs of single and dimeric transcription factors.

Nat Methods 2017 03 16;14(3):316-322. Epub 2017 Jan 16.

Institute of Bioengineering, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

Resolving the DNA-binding specificities of transcription factors (TFs) is of critical value for understanding gene regulation. Here, we present a novel, semiautomated protein-DNA interaction characterization technology, selective microfluidics-based ligand enrichment followed by sequencing (SMiLE-seq). SMiLE-seq is neither limited by DNA bait length nor biased toward strong affinity binders; it probes the DNA-binding properties of TFs over a wide affinity range in a fast and cost-effective fashion. We validated SMiLE-seq by analyzing 58 full-length human, mouse, and Drosophila TFs from distinct structural classes. All tested TFs yielded DNA-binding models with predictive power comparable to or greater than that of other in vitro assays. De novo motif discovery on all JUN-FOS heterodimers and several nuclear receptor-TF complexes provided novel insights into partner-specific heterodimer DNA-binding preferences. We also successfully analyzed the DNA-binding properties of uncharacterized human C2H2 zinc-finger proteins and validated several using ChIP-exo.
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http://dx.doi.org/10.1038/nmeth.4143DOI Listing
March 2017

The eukaryotic promoter database in its 30th year: focus on non-vertebrate organisms.

Nucleic Acids Res 2017 01 28;45(D1):D51-D55. Epub 2016 Nov 28.

Swiss Institute of Bioinformatics (SIB), CH-1015 Lausanne, Switzerland.

We present an update of the Eukaryotic Promoter Database EPD (http://epd.vital-it.ch), more specifically on the EPDnew division, which contains comprehensive organisms-specific transcription start site (TSS) collections automatically derived from next generation sequencing (NGS) data. Thanks to the abundant release of new high-throughput transcript mapping data (CAGE, TSS-seq, GRO-cap) the database could be extended to plant and fungal species. We further report on the expansion of the mass genome annotation (MGA) repository containing promoter-relevant chromatin profiling data and on improvements for the EPD entry viewers. Finally, we present a new data access tool, ChIP-Extract, which enables computational biologists to extract diverse types of promoter-associated data in numerical table formats that are readily imported into statistical analysis platforms such as R.
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http://dx.doi.org/10.1093/nar/gkw1069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5210552PMC
January 2017

SNP2TFBS - a database of regulatory SNPs affecting predicted transcription factor binding site affinity.

Nucleic Acids Res 2017 01 28;45(D1):D139-D144. Epub 2016 Nov 28.

Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland

SNP2TFBS is a computational resource intended to support researchers investigating the molecular mechanisms underlying regulatory variation in the human genome. The database essentially consists of a collection of text files providing specific annotations for human single nucleotide polymorphisms (SNPs), namely whether they are predicted to abolish, create or change the affinity of one or several transcription factor (TF) binding sites. A SNP's effect on TF binding is estimated based on a position weight matrix (PWM) model for the binding specificity of the corresponding factor. These data files are regenerated at regular intervals by an automatic procedure that takes as input a reference genome, a comprehensive SNP catalogue and a collection of PWMs. SNP2TFBS is also accessible over a web interface, enabling users to view the information provided for an individual SNP, to extract SNPs based on various search criteria, to annotate uploaded sets of SNPs or to display statistics about the frequencies of binding sites affected by selected SNPs. Homepage: http://ccg.vital-it.ch/snp2tfbs/.
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http://dx.doi.org/10.1093/nar/gkw1064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5210548PMC
January 2017

The ChIP-Seq tools and web server: a resource for analyzing ChIP-seq and other types of genomic data.

BMC Genomics 2016 11 18;17(1):938. Epub 2016 Nov 18.

School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015, Lausanne, Switzerland.

Background: ChIP-seq and related high-throughput chromatin profilig assays generate ever increasing volumes of highly valuable biological data. To make sense out of it, biologists need versatile, efficient and user-friendly tools for access, visualization and itegrative analysis of such data.

Results: Here we present the ChIP-Seq command line tools and web server, implementing basic algorithms for ChIP-seq data analysis starting with a read alignment file. The tools are optimized for memory-efficiency and speed thus allowing for processing of large data volumes on inexpensive hardware. The web interface provides access to a large database of public data. The ChIP-Seq tools have a modular and interoperable design in that the output from one application can serve as input to another one. Complex and innovative tasks can thus be achieved by running several tools in a cascade.

Conclusions: The various ChIP-Seq command line tools and web services either complement or compare favorably to related bioinformatics resources in terms of computational efficiency, ease of access to public data and interoperability with other web-based tools. The ChIP-Seq server is accessible at http://ccg.vital-it.ch/chipseq/ .
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http://dx.doi.org/10.1186/s12864-016-3288-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5116162PMC
November 2016

Influence of Rotational Nucleosome Positioning on Transcription Start Site Selection in Animal Promoters.

PLoS Comput Biol 2016 Oct 7;12(10):e1005144. Epub 2016 Oct 7.

Swiss Institute of Bioinformatics (SIB), Lausanne, Switzerland.

The recruitment of RNA-Pol-II to the transcription start site (TSS) is an important step in gene regulation in all organisms. Core promoter elements (CPE) are conserved sequence motifs that guide Pol-II to the TSS by interacting with specific transcription factors (TFs). However, only a minority of animal promoters contains CPEs. It is still unknown how Pol-II selects the TSS in their absence. Here we present a comparative analysis of promoters' sequence composition and chromatin architecture in five eukaryotic model organisms, which shows the presence of common and unique DNA-encoded features used to organize chromatin. Analysis of Pol-II initiation patterns uncovers that, in the absence of certain CPEs, there is a strong correlation between the spread of initiation and the intensity of the 10 bp periodic signal in the nearest downstream nucleosome. Moreover, promoters' primary and secondary initiation sites show a characteristic 10 bp periodicity in the absence of CPEs. We also show that DNA natural variants in the region immediately downstream the TSS are able to affect both the nucleosome-DNA affinity and Pol-II initiation pattern. These findings support the notion that, in addition to CPEs mediated selection, sequence-induced nucleosome positioning could be a common and conserved mechanism of TSS selection in animals.
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http://dx.doi.org/10.1371/journal.pcbi.1005144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5055345PMC
October 2016

The Eukaryotic Promoter Database: expansion of EPDnew and new promoter analysis tools.

Nucleic Acids Res 2015 Jan 6;43(Database issue):D92-6. Epub 2014 Nov 6.

Swiss Institute of Bioinformatics (SIB), CH-1015 Lausanne, Switzerland Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland

We present an update of EPDNew (http://epd.vital-it.ch), a recently introduced new part of the Eukaryotic Promoter Database (EPD) which has been described in more detail in a previous NAR Database Issue. EPD is an old database of experimentally characterized eukaryotic POL II promoters, which are conceptually defined as transcription initiation sites or regions. EPDnew is a collection of automatically compiled, organism-specific promoter lists complementing the old corpus of manually compiled promoter entries of EPD. This new part is exclusively derived from next generation sequencing data from high-throughput promoter mapping experiments. We report on the recent growth of EPDnew, its extension to additional model organisms and its improved integration with other bioinformatics resources developed by our group, in particular the Signal Search Analysis and ChIP-Seq web servers.
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http://dx.doi.org/10.1093/nar/gku1111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4383928PMC
January 2015

Nuclear Factor I genomic binding associates with chromatin boundaries.

BMC Genomics 2013 Feb 12;14:99. Epub 2013 Feb 12.

Institute of Biotechnology and Center for Biotecghnology UNIL-EPFL, University of Lausanne, 1015, Lausanne, Switzerland.

Background: The Nuclear Factor I (NFI) family of DNA binding proteins (also called CCAAT box transcription factors or CTF) is involved in both DNA replication and gene expression regulation. Using chromatin immuno-precipitation and high throughput sequencing (ChIP-Seq), we performed a genome-wide mapping of NFI DNA binding sites in primary mouse embryonic fibroblasts.

Results: We found that in vivo and in vitro NFI DNA binding specificities are indistinguishable, as in vivo ChIP-Seq NFI binding sites matched predictions based on previously established position weight matrix models of its in vitro binding specificity. Combining ChIP-Seq with mRNA profiling data, we found that NFI preferentially associates with highly expressed genes that it up-regulates, while binding sites were under-represented at expressed but unregulated genes. Genomic binding also correlated with markers of transcribed genes such as histone modifications H3K4me3 and H3K36me3, even outside of annotated transcribed loci, implying NFI in the control of the deposition of these modifications. Positional correlation between + and - strand ChIP-Seq tags revealed that, in contrast to other transcription factors, NFI associates with a nucleosomal length of cleavage-resistant DNA, suggesting an interaction with positioned nucleosomes. In addition, NFI binding prominently occurred at boundaries displaying discontinuities in histone modifications specific of expressed and silent chromatin, such as loci submitted to parental allele-specific imprinted expression.

Conclusions: Our data thus suggest that NFI nucleosomal interaction may contribute to the partitioning of distinct chromatin domains and to epigenetic gene expression regulation.NFI ChIP-Seq and input control DNA data were deposited at Gene Expression Omnibus (GEO) repository under accession number GSE15844. Gene expression microarray data for mouse embryonic fibroblasts are on GEO accession number GSE15871.
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http://dx.doi.org/10.1186/1471-2164-14-99DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3610271PMC
February 2013

EPD and EPDnew, high-quality promoter resources in the next-generation sequencing era.

Nucleic Acids Res 2013 Jan 27;41(Database issue):D157-64. Epub 2012 Nov 27.

Swiss Institute of Bioinformatics (SIB), CH-1015 Lausanne, Switzerland.

The Eukaryotic Promoter Database (EPD), available online at http://epd.vital-it.ch, is a collection of experimentally defined eukaryotic POL II promoters which has been maintained for more than 25 years. A promoter is represented by a single position in the genome, typically the major transcription start site (TSS). EPD primarily serves biologists interested in analysing the motif content, chromatin structure or DNA methylation status of co-regulated promoter subsets. Initially, promoter evidence came from TSS mapping experiments targeted at single genes and published in journal articles. Today, the TSS positions provided by EPD are inferred from next-generation sequencing data distributed in electronic form. Traditionally, EPD has been a high-quality database with low coverage. The focus of recent efforts has been to reach complete gene coverage for important model organisms. To this end, we introduced a new section called EPDnew, which is automatically assembled from multiple, carefully selected input datasets. As another novelty, we started to use chromatin signatures in addition to mRNA 5'tags to locate promoters of weekly expressed genes. Regarding user interfaces, we introduced a new promoter viewer which enables users to explore promoter-defining experimental evidence in a UCSC genome browser window.
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http://dx.doi.org/10.1093/nar/gks1233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3531148PMC
January 2013

A gene-rich, transcriptionally active environment and the pre-deposition of repressive marks are predictive of susceptibility to KRAB/KAP1-mediated silencing.

BMC Genomics 2011 Jul 26;12:378. Epub 2011 Jul 26.

School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.

Background: KRAB-ZFPs (Krüppel-associated box domain-zinc finger proteins) are vertebrate-restricted transcriptional repressors encoded in the hundreds by the mouse and human genomes. They act via an essential cofactor, KAP1, which recruits effectors responsible for the formation of facultative heterochromatin. We have recently shown that KRAB/KAP1 can mediate long-range transcriptional repression through heterochromatin spreading, but also demonstrated that this process is at times countered by endogenous influences.

Method: To investigate this issue further we used an ectopic KRAB-based repressor. This system allowed us to tether KRAB/KAP1 to hundreds of euchromatic sites within genes, and to record its impact on gene expression. We then correlated this KRAB/KAP1-mediated transcriptional effect to pre-existing genomic and chromatin structures to identify specific characteristics making a gene susceptible to repression.

Results: We found that genes that were susceptible to KRAB/KAP1-mediated silencing carried higher levels of repressive histone marks both at the promoter and over the transcribed region than genes that were insensitive. In parallel, we found a high enrichment in euchromatic marks within both the close and more distant environment of these genes.

Conclusion: Together, these data indicate that high levels of gene activity in the genomic environment and the pre-deposition of repressive histone marks within a gene increase its susceptibility to KRAB/KAP1-mediated repression.
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http://dx.doi.org/10.1186/1471-2164-12-378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3199781PMC
July 2011

ZFN-site searches genomes for zinc finger nuclease target sites and off-target sites.

BMC Bioinformatics 2011 May 13;12:152. Epub 2011 May 13.

University of Iowa School of Medicine, Department of Internal Medicine, Iowa City, Iowa 52245, USA.

Background: Zinc Finger Nucleases (ZFNs) are man-made restriction enzymes useful for manipulating genomes by cleaving target DNA sequences. ZFNs allow therapeutic gene correction or creation of genetically modified model organisms. ZFN specificity is not absolute; therefore, it is essential to select ZFN target sites without similar genomic off-target sites. It is important to assay for off-target cleavage events at sites similar to the target sequence.

Results: ZFN-Site is a web interface that searches multiple genomes for ZFN off-target sites. Queries can be based on the target sequence or can be expanded using degenerate specificity to account for known ZFN binding preferences. ZFN off-target sites are outputted with links to genome browsers, facilitating off-target cleavage site screening. We verified ZFN-Site using previously published ZFN half-sites and located their target sites and their previously described off-target sites. While we have tailored this tool to ZFNs, ZFN-Site can also be used to find potential off-target sites for other nucleases, such as TALE nucleases.

Conclusions: ZFN-Site facilitates genome searches for possible ZFN cleavage sites based on user-defined stringency limits. ZFN-Site is an improvement over other methods because the FetchGWI search engine uses an indexed search of genome sequences for all ZFN target sites and possible off-target sites matching the half-sites and stringency limits. Therefore, ZFN-Site does not miss potential off-target sites.
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http://dx.doi.org/10.1186/1471-2105-12-152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3113941PMC
May 2011

Nuclear factor I revealed as family of promoter binding transcription activators.

BMC Genomics 2011 Apr 7;12:181. Epub 2011 Apr 7.

Institute of Biotechnology, University of Lausanne, Lausanne, Switzerland.

Background: Multiplex experimental assays coupled to computational predictions are being increasingly employed for the simultaneous analysis of many specimens at the genome scale, which quickly generates very large amounts of data. However, inferring valuable biological information from the comparisons of very large genomic datasets still represents an enormous challenge.

Results: As a study model, we chose the NFI/CTF family of mammalian transcription factors and we compared the results obtained from a genome-wide study of its binding sites with chromatin structure assays, gene expression microarray data, and in silico binding site predictions. We found that NFI/CTF family members preferentially bind their DNA target sites when they are located around transcription start sites when compared to control datasets generated from the random subsampling of the complete set of NFI binding sites. NFI proteins preferably associate with the upstream regions of genes that are highly expressed and that are enriched in active chromatin modifications such as H3K4me3 and H3K36me3. We postulate that this is a causal association and that NFI proteins mainly act as activators of transcription. This was documented for one member of the family (NFI-C), which revealed as a more potent gene activator than repressor in global gene expression analysis. Interestingly, we also discovered the association of NFI with the tri-methylation of lysine 9 of histone H3, a chromatin marker previously associated with the protection against silencing of telomeric genes by NFI.

Conclusion: Taken together, we illustrate approaches that can be taken to analyze large genomic data, and provide evidence that NFI family members may act in conjunction with specific chromatin modifications to activate gene expression.
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http://dx.doi.org/10.1186/1471-2164-12-181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3082249PMC
April 2011

KRAB-zinc finger proteins and KAP1 can mediate long-range transcriptional repression through heterochromatin spreading.

PLoS Genet 2010 Mar 5;6(3):e1000869. Epub 2010 Mar 5.

School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

Krüppel-associated box domain-zinc finger proteins (KRAB-ZFPs) are tetrapod-specific transcriptional repressors encoded in the hundreds by the human genome. In order to explore their as yet ill-defined impact on gene expression, we developed an ectopic repressor assay, allowing the study of KRAB-mediated transcriptional regulation at hundreds of different transcriptional units. By targeting a drug-controllable KRAB-containing repressor to gene-trapping lentiviral vectors, we demonstrate that KRAB and its corepressor KAP1 can silence promoters located several tens of kilobases (kb) away from their DNA binding sites, with an efficiency which is generally higher for promoters located within 15 kb or less. Silenced promoters exhibit a loss of histone H3-acetylation, an increase in H3 lysine 9 trimethylation (H3K9me3), and a drop in RNA Pol II recruitment, consistent with a block of transcriptional initiation following the establishment of silencing marks. Furthermore, we reveal that KRAB-mediated repression is established by the long-range spreading of H3K9me3 and heterochromatin protein 1 beta (HP1beta) between the repressor binding site and the promoter. We confirm the biological relevance of this phenomenon by documenting KAP1-dependent transcriptional repression at an endogenous KRAB-ZFP gene cluster, where KAP1 binds to the 3' end of genes and mediates propagation of H3K9me3 and HP1beta towards their 5' end. Together, our data support a model in which KRAB/KAP1 recruitment induces long-range repression through the spread of heterochromatin. This finding not only suggests auto-regulatory mechanisms in the control of KRAB-ZFP gene clusters, but also provides important cues for interpreting future genome-wide DNA binding data of KRAB-ZFPs and KAP1.
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http://dx.doi.org/10.1371/journal.pgen.1000869DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2832679PMC
March 2010

Nucleosome eviction from MHC class II promoters controls positioning of the transcription start site.

Nucleic Acids Res 2009 May 5;37(8):2514-28. Epub 2009 Mar 5.

Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, 1 rue Michel-Servet, CH-1211, Geneva, Switzerland.

Nucleosome depletion at transcription start sites (TSS) has been documented genome-wide in multiple eukaryotic organisms. However, the mechanisms that mediate this nucleosome depletion and its functional impact on transcription remain largely unknown. We have studied these issues at human MHC class II (MHCII) genes. Activation-induced nucleosome free regions (NFR) encompassing the TSS were observed at all MHCII genes. Nucleosome depletion was exceptionally strong, attaining over 250-fold, at the promoter of the prototypical HLA-DRA gene. The NFR was induced primarily by the transcription factor complex that assembles on the conserved promoter-proximal enhancer situated upstream of the TSS. Functional analyses performed in the context of native chromatin demonstrated that displacing the NFR without altering the sequence of the core promoter induced a shift in the position of the TSS. The NFR thus appears to play a critical role in transcription initiation because it directs correct TSS positioning in vivo. Our results provide support for a novel mechanism in transcription initiation whereby the position of the TSS is controlled by nucleosome eviction rather than by promoter sequence.
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http://dx.doi.org/10.1093/nar/gkp116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2677874PMC
May 2009

Indexing strategies for rapid searches of short words in genome sequences.

PLoS One 2007 Jun 27;2(6):e579. Epub 2007 Jun 27.

Ludwig Institute for Cancer Research, Bâtiment Génopode, Université de Lausanne, Lausanne, Switzerland; Swiss Institute of Bioinformatics, Bátiment Génopode, Université de Lausanne, Lausanne, Switzerland. Christian.

Searching for matches between large collections of short (14-30 nucleotides) words and sequence databases comprising full genomes or transcriptomes is a common task in biological sequence analysis. We investigated the performance of simple indexing strategies for handling such tasks and developed two programs, fetchGWI and tagger, that index either the database or the query set. Either strategy outperforms megablast for searches with more than 10,000 probes. FetchGWI is shown to be a versatile tool for rapidly searching multiple genomes, whose performance is limited in most cases by the speed of access to the filesystem. We have made publicly available a Web interface for searching the human, mouse, and several other genomes and transcriptomes with oligonucleotide queries.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0000579PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1894650PMC
June 2007

Signal search analysis server.

Nucleic Acids Res 2003 Jul;31(13):3618-20

ISREC Swiss Institute for Experimental Cancer Research, Ch. des Boveresses 155, 1066 Epalinges s/ Lausanne, VD, Switzerland.

Signal search analysis is a general method to discover and characterize sequence motifs that are positionally correlated with a functional site (e.g. a transcription or translation start site). The method has played an instrumental role in the analysis of eukaryotic promoter elements. The signal search analysis server provides access to four different computer programs as well as to a large number of precompiled functional site collections. The programs offered allow: (i) the identification of non-random sequence regions under evolutionary constraint; (ii) the detection of consensus sequence-based motifs that are over- or under-represented at a particular distance from a functional site; (iii) the analysis of the positional distribution of a consensus sequence- or weight matrix-based sequence motif around a functional site; and (iv) the optimization of a weight matrix description of a locally over-represented sequence motif. These programs can be accessed at: http://www.isrec.isb-sib.ch/ssa/.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC169017PMC
http://dx.doi.org/10.1093/nar/gkg611DOI Listing
July 2003