Determining the Three-dimensional Structure of Genomes and Genomic Domains Integrating Chromosome Conformation Capture Data and Microscopy Images

Programa de Doctorat en Biomedicina / Tesi realitzada al Centre Nacional d'Anàlisi Genòmica-Centre de Regulació Genòmica (CNAG-CRG)[eng] Microscopy and Chromosome Conformation Capture (3C) are the two main techniques for studying the three-dimensional (3D) organization of the genome. Microscopy, allowing the visualization of genomic loci in individual nuclei, pioneered the field of structural genomics and became the gold-standard for the validation of new discoveries. 3C and 3C-based techniques, identifying the number of contacts between pairs of genomic loci, have already been key to unveil the importance of the 3D genome organization in many cellular processes. Both techniques are continuously evolving pushing forward the technologies and giving rise to innovative assays that require the support of new computational methods for data collection, analysis and modeling. In this thesis, I have contributed to provide these essential computational methods to the Structural Genomics community. In Microscopy, I participated in the design and implementation of OligoFISSEQ, a novel multiplexing imaging technology to visualize multiple genomic regions in hundreds and thousands of individual cells. In 3C-based techniques, I contributed to the development of a tool for the reconstruction of the 3D organization of chromatin from highly-sparse 3C-based datasets (e.g. Promoter Capture Hi-C). Finally, I have introduced pTADbit, a novel approach for the reconstruction of the 3D Genome organization integrating both Microscopy and 3C data via the application of Machine Learning methods

Determining the Three-dimensional Structure of Genomes and Genomic Domains Integrating Chromosome Conformation Capture Data and Microscopy Images

[eng] Microscopy and Chromosome Conformation Capture (3C) are the two main techniques for studying the three-dimensional (3D) organization of the genome. Microscopy, allowing the visualization of genomic loci in individual nuclei, pioneered the field of structural genomics and became the gold-standard for the validation of new discoveries. 3C and 3C-based techniques, identifying the number of contacts between pairs of genomic loci, have already been key to unveil the importance of the 3D genome organization in many cellular processes. Both techniques are continuously evolving pushing forward the technologies and giving rise to innovative assays that require the support of new computational methods for data collection, analysis and modeling. In this thesis, I have contributed to provide these essential computational methods to the Structural Genomics community. In Microscopy, I participated in the design and implementation of OligoFISSEQ, a novel multiplexing imaging technology to visualize multiple genomic regions in hundreds and thousands of individual cells. In 3C-based techniques, I contributed to the development of a tool for the reconstruction of the 3D organization of chromatin from highly-sparse 3C-based datasets (e.g. Promoter Capture Hi-C). Finally, I have introduced pTADbit, a novel approach for the reconstruction of the 3D Genome organization integrating both Microscopy and 3C data via the application of Machine Learning methods

Restauració ràpida d’imatges en Microscòpia de Fluorescència d’Il·luminació plana amb profunditat de camp estesa mitjançant GPUs

Treball final de màster oficial fet en col·laboració amb Universitat Autònoma de Barcelona (UAB), Universitat de Barcelona (UB) i Institut de Ciències Fotòniques (ICFO)Light sheet fluorescence microscopy (LSFM) is used in many biological research experiments that require fast three-dimensional (3D) imaging up to a few volumes per second. Wavefront coding (WFC) microscopy provides LSFM with 3D real-time imaging capabilities introducing a controlled aberration that extends the depth of field (DOF) of the microscope objective. Resulting images, however, are blurred and need to be processed to recover the original sharpness in a CPU-intensive deconvolution which makes real-time visualization unattainable. We present computational tools based on GPU parallel computing to achieve real-time deconvolution and visualization of the images obtained with WFC light-sheet microscopy.La Microscopía de Fluorescencia de Iluminación plana (LSFM) se utiliza en muchos experimentos de investigación que requieren la obtención rápida de imágenes en tres dimensiones (3D) a velocidades de unos pocos volúmenes por segundo. La codificación de frente de onda (WFC) proporciona a LSFM la capacidad de obtener imágenes en 3D en tiempo real mediante la introducción de una aberración controlada que amplía la profundidad de campo (DOF) del objetivo del microscopio. Las imágenes obtenidas, no obstante, están difuminadas y necesitan de un procesado para recuperar su nitidez original en una operación de deconvolución que requiere un uso intensivo de la CPU y que impide su visualización en tiempo real. Se presentan herramientas computacionales basadas en procesamiento en paralelo en la GPU que permiten la deconvolución y visualización de imágenes obtenidas con un microscopio WFC de fluorescencia de iluminación plana.La Microscòpia de Fluorescència d’Il·luminació plana (LSFM) es fa servir en molts experiments de recerca que requereixen l’obtenció ràpida d’imatges en tres dimensions (3D) a velocitats d’uns pocs volums per segon. La codificació de front d’ona (WFC) proporciona a LSFM la capacitat d’obtenir imatges en 3D en temps real mitjançant la introducció d’una aberració controlada que amplia la profunditat de camp (DOF) de l’objectiu del microscopi. Tanmateix, les imatges obtingudes estan difuminades i necessiten d’un processat per recuperar la seva nitidesa original en una operació de deconvolució que requereix d’un ús intensiu de la CPU i que impedeix la seva visualització en temps real. Es presenten eines computacionals que permeten la deconvolució i visualització d’imatges obtingudes amb un microscopi WFC de fluorescència d’il·luminació plana

Restauració ràpida d’imatges en Microscòpia de Fluorescència d’Il·luminació plana amb profunditat de camp estesa mitjançant GPUs

Treball final de màster oficial fet en col·laboració amb Universitat Autònoma de Barcelona (UAB), Universitat de Barcelona (UB) i Institut de Ciències Fotòniques (ICFO)Light sheet fluorescence microscopy (LSFM) is used in many biological research experiments that require fast three-dimensional (3D) imaging up to a few volumes per second. Wavefront coding (WFC) microscopy provides LSFM with 3D real-time imaging capabilities introducing a controlled aberration that extends the depth of field (DOF) of the microscope objective. Resulting images, however, are blurred and need to be processed to recover the original sharpness in a CPU-intensive deconvolution which makes real-time visualization unattainable. We present computational tools based on GPU parallel computing to achieve real-time deconvolution and visualization of the images obtained with WFC light-sheet microscopy.La Microscopía de Fluorescencia de Iluminación plana (LSFM) se utiliza en muchos experimentos de investigación que requieren la obtención rápida de imágenes en tres dimensiones (3D) a velocidades de unos pocos volúmenes por segundo. La codificación de frente de onda (WFC) proporciona a LSFM la capacidad de obtener imágenes en 3D en tiempo real mediante la introducción de una aberración controlada que amplía la profundidad de campo (DOF) del objetivo del microscopio. Las imágenes obtenidas, no obstante, están difuminadas y necesitan de un procesado para recuperar su nitidez original en una operación de deconvolución que requiere un uso intensivo de la CPU y que impide su visualización en tiempo real. Se presentan herramientas computacionales basadas en procesamiento en paralelo en la GPU que permiten la deconvolución y visualización de imágenes obtenidas con un microscopio WFC de fluorescencia de iluminación plana.La Microscòpia de Fluorescència d’Il·luminació plana (LSFM) es fa servir en molts experiments de recerca que requereixen l’obtenció ràpida d’imatges en tres dimensions (3D) a velocitats d’uns pocs volums per segon. La codificació de front d’ona (WFC) proporciona a LSFM la capacitat d’obtenir imatges en 3D en temps real mitjançant la introducció d’una aberració controlada que amplia la profunditat de camp (DOF) de l’objectiu del microscopi. Tanmateix, les imatges obtingudes estan difuminades i necessiten d’un processat per recuperar la seva nitidesa original en una operació de deconvolució que requereix d’un ús intensiu de la CPU i que impedeix la seva visualització en temps real. Es presenten eines computacionals que permeten la deconvolució i visualització d’imatges obtingudes amb un microscopi WFC de fluorescència d’il·luminació plana

Binless normalization of Hi-C data provides significant interaction and difference detection independent of resolution

Chromosome conformation capture techniques, such as Hi-C, are fundamental in characterizing genome organization. These methods have revealed several genomic features, such as chromatin loops, whose disruption can have dramatic effects in gene regulation. Unfortunately, their detection is difficult; current methods require that the users choose the resolution of interaction maps based on dataset quality and sequencing depth. Here, we introduce Binless, a resolution-agnostic method that adapts to the quality and quantity of available data, to detect both interactions and differences. Binless relies on an alternate representation of Hi-C data, which leads to a more detailed classification of paired-end reads. Using a large-scale benchmark, we demonstrate that Binless is able to call interactions with higher reproducibility than other existing methods. Binless, which is freely available, can thus reliably be used to identify chromatin loops as well as for differential analysis of chromatin interaction maps.This work has been partially supported by the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ERC Synergy grant agreement 609989 (4Dgenome), the European Union’s Horizon 2020 research and innovation programme (agreement 676556) as well as the Spanish MINECO (BFU2017-85926-P). We acknowledge support of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership, the Centro de Excelencia Severo Ochoa and the CERCA Programme / Generalitat de Catalunya. We also acknowledge support of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) through the Instituto de Salud Carlos III, the Generalitat de Catalunya through Departament de Salut and Departament d’Empresa i Coneixement and the Co-financing by the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) with funds from the European Regional Development Fund (ERDF) corresponding to the 2014-2020 Smart Growth Operating Progra

3D reconstruction of genomic regions from sparse interaction data

Chromosome conformation capture (3C) technologies measure the interaction frequency between pairs of chromatin regions within the nucleus in a cell or a population of cells. Some of these 3C technologies retrieve interactions involving non-contiguous sets of loci, resulting in sparse interaction matrices. One of such 3C technologies is Promoter Capture Hi-C (pcHi-C) that is tailored to probe only interactions involving gene promoters. As such, pcHi-C provides sparse interaction matrices that are suitable to characterize short- and long-range enhancer-promoter interactions. Here, we introduce a new method to reconstruct the chromatin structural (3D) organization from sparse 3C-based datasets such as pcHi-C. Our method allows for data normalization, detection of significant interactions and reconstruction of the full 3D organization of the genomic region despite of the data sparseness. Specifically, it builds, with as low as the 2-3% of the data from the matrix, reliable 3D models of similar accuracy of those based on dense interaction matrices. Furthermore, the method is sensitive enough to detect cell-type-specific 3D organizational features such as the formation of different networks of active gene communities.This study makes use of data generated by the PCHI-C Consortium available in the EGA European Genome-Phenome Archive (National Institute for Health Research of England, UK Medical Research Council (MR/L007150/1) and UK Biotechnology and Biological Research Council (BB/J004480/1

Automatic analysis and 3D-modelling of Hi-C data using TADbit reveals structural features of the fly chromatin colors

The sequence of a genome is insufficient to understand all genomic processes carried out in the cell nucleus. To achieve this, the knowledge of its three-dimensional architecture is necessary. Advances in genomic technologies and the development of new analytical methods, such as Chromosome Conformation Capture (3C) and its derivatives, provide unprecedented insights in the spatial organization of genomes. Here we present TADbit, a computational framework to analyze and model the chromatin fiber in three dimensions. Our package takes as input the sequencing reads of 3C-based experiments and performs the following main tasks: (i) pre-process the reads, (ii) map the reads to a reference genome, (iii) filter and normalize the interaction data, (iv) analyze the resulting interaction matrices, (v) build 3D models of selected genomic domains, and (vi) analyze the resulting models to characterize their structural properties. To illustrate the use of TADbit, we automatically modeled 50 genomic domains from the fly genome revealing differential structural features of the previously defined chromatin colors, establishing a link between the conformation of the genome and the local chromatin composition. TADbit provides three-dimensional models built from 3C-based experiments, which are ready for visualization and for characterizing their relation to gene expression and epigenetic states. TADbit is an open-source Python library available for download from https://github.com/3DGenomes/tadbit.The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013) / ERC grant agreement 609989, the Spanish Ministry of Economy and Competitiveness (BFU2013-47736-P) and the Human Frontiers Science Program (RGP0044). We acknowledge support of the CERCA Programme / Generalitat de Catalunya and the Spanish Ministry of Economy and Competitiveness, 'Centro de Excelencia Severo Ochoa 2013-2017', SEV-2012-0208 to the CRG

Topoisomerase 1 facilitates nucleosome reassembly at stress genes during recovery

Chromatin remodeling is essential to allow full development of alternative gene expression programs in response to environmental changes. In fission yeast, oxidative stress triggers massive transcriptional changes including the activation of hundreds of genes, with the participation of histone modifying complexes and chromatin remodelers. DNA transcription is associated to alterations in DNA topology, and DNA topoisomerases facilitate elongation along gene bodies. Here, we test whether the DNA topoisomerase Top1 participates in the RNA polymerase II-dependent activation of the cellular response to oxidative stress. Cells lacking Top1 are resistant to H2O2 stress. The transcriptome of Δtop1 strain was not greatly affected in the absence of stress, but activation of the anti-stress gene expression program was more sustained than in wild-type cells. Top1 associated to stress open reading frames. While the nucleosomes of stress genes are partially and transiently evicted during stress, the chromatin configuration remains open for longer times in cells lacking Top1, facilitating RNA polymerase II progression. We propose that, by removing DNA tension arising from transcription, Top1 facilitates nucleosome reassembly and works in synergy with the chromatin remodeler Hrp1 as opposing forces to transcription and to Snf22 / Hrp3 opening remodelers.PGC2018-093920-B-I00 and PID2021-122837-NB-I00 to E.H., funded by MCIN/AEI/ 10.13039/501100011033 and by ‘ERDF A way of making Europe’; ‘European Union’. The Oxidative Stress and Cell Cycle group is also supported by Generalitat de Catalunya (Spain) [2017-SGR-539 and 2021-SGR-00007]; Excellence Unit «María de Maeztu» Grant CEX2018-000792-M funded by MCIN/AEI /10.13039/501100011033; E.H. is recipient of an ICREA Academia Award (Generalitat de Catalunya, Spain); R.F. was recipient of a FPI fellowship from the Ministerio de Economía y Competitividad (Spain). Funding for open access charge: Ministerio de Ciencia e Innovación, Gobierno de España [PID2021-122837-NB-I00]