Deciphering a gene regulation network in normal mouse pancreas through a multiomic integrative approach

Pancreatic acinar cells compose around 85% of the exocrine component of the pancreas, which constitutes the vast majority of the tissue. Genetically Engineered Mouse Models (GEMMs) provide evidence that pancreatic ductal adenocarcinoma (PDAC) can efficiently arise from acinar cells through a transdifferentiation process called acinar-to-ductal-metaplasia (ADM), proposing the loss of acinar cell identity as the predominant origin for PDAC. Here, we present a comprehensive multi-omic integrative approach to generate a network-based resource to interrogate the transcriptional regulation underlying acinar cell identity in wild type (WT) mouse pancreas. As a proof-of-concept, we examine the regulatory activity of several acinarexpressed transcription factors (TFs) involved in pancreas regulation and validate it by comparison with experimental ChIP-seq analysis, obtaining consistent results. We consider that this approach represents a valuable resource to perform a priori analyses that can be experimentally validated providing new knowledge to the field. Moreover, the presented methodology will be further explored to determine the optimal parameters for improving the potential in the detection of different regulatory events, and will be applied to GEMMs displaying different conditions, as well as to other organisms like human to cross-validate the results and the usefulness of our resource

Clonal structures and cell interactions in cancer

Despite sharing an identical genome, cells of higher order multicellular organisms display a large degree of phenotypic diversity. This diversity is maintained by a sophisticated regulatory machinery that integrates information from both intrinsic and extrinsic factors, ultimately coordinating the appropriate gene expression. Sequencing methods such as RNA and DNA sequencing have become indispensable tools in the pursuit to understand gene regulation. In recent years, the integration of single-cell sequencing techniques and CRISPR-based methods has ushered in a new era of genomic exploration, providing unprecedented opportunities to investigate the intricate interplay between genes, cellular processes, and disease progression. These cutting-edge advances have transformed the research landscape, enabling in-depth studies of gene regulation in single cells, and paving the way for future discoveries in both healthy and malignant tissues. While cancer has traditionally been studied as a genetic disease, it is now evident that mutations alone do not determine cancer initiation or progression. This notion is supported by two key observations: first, cancer-driving mutations do not always lead to malignancy; and second, identical mutations can yield different outcomes depending on the cell type in which they occur. Consequently, a deeper understanding of gene regulation and the various ways it is modulated is critical for deciphering the complex relationship between genetic changes and cancer initiation. In this thesis we aimed to develop novel single-cell methodologies applicable to studying biological complex systems. We have developed four techniques: CIM-seq, DNTR-seq, Smart3-ATAC, and ACTIseq, described in papers I-IV, respectively. The methods all capture additional modalities in combination with single-cell RNA-seq data, including spatial information, whole genome sequencing, accessible chromatin, and direct read out of guide RNAs. We applied these methods to investigate biological systems at the single-cell level, offering a more comprehensive understanding of cellular behavior in health and disease. Our approaches have allowed us to characterize stem cell niches and regeneration dynamics in the epithelial layer of the colon, and delve into the effects of gene dosage, quantifying how mutational changes impact transcriptional output. Furthermore, we have explored the complex landscape of gene regulation within pancreatic ductal adenocarcinomas, identifying mechanisms that enable cancer growth and proliferation. This body of work emphasizes the importance of multimodal and integrative approaches for unraveling the complexities of biological systems at a cellular level. The methods we've developed represent a significant step forward, promising to facilitate the discovery of molecular targets for cancer therapeutics

Parallel Genetics of Gene Regulatory Sequences in Caenorhabditis elegans

Wie regulatorische Sequenzen die Genexpression steuern, ist von grundlegender Bedeutung für die Erklärung von Phänotypen in Gesundheit und Krankheit. Die Funktion regulatorischer Sequenzen muss letztlich in ihrer genomischen Umgebung und in entwicklungs- oder gewebespezifischen Zusammenhängen verstanden werden. Da dies eine technische Herausforderung ist, wurden bisher nur wenige regulatorische Elemente in vivo charakterisiert. Hier verwenden wir Induktion von Cas9 und multiplexed-sgRNAs, um hunderte von Mutationen in Enhancern/Promotoren und 3′ UTRs von 16 Genen in C. elegans zu erzeugen. Wir quantifizieren die Auswirkungen von Mutationen auf Genexpression und Physiologie durch gezielte RNA- und DNA-Sequenzierung. Bei der Anwendung unseres Ansatzes auf den 3′ UTR von lin-41, bei der wir hunderte von Mutanten erzeugen, stellen wir fest, dass die beiden benachbarten Bindungsstellen für die miRNA let-7 die lin-41-Expression größtenteils unabhängig voneinander regulieren können, mit Hinweisen auf eine mögliche kompensatorische Interaktion. Schließlich verbinden wir regulatorische Genotypen mit phänotypischen Merkmalen für mehrere Gene. Unser Ansatz ermöglicht die parallele Analyse von genregulatorischen Sequenzen direkt in Tieren.How regulatory sequences control gene expression is fundamental for explaining phenotypes in health and disease. The function of regulatory sequences must ultimately be understood within their genomic environment and development- or tissue-specific contexts. Because this is technically challenging, few regulatory elements have been characterized in vivo. Here, we use inducible Cas9 and multiplexed guide RNAs to create hundreds of mutations in enhancers/promoters and 3′ UTRs of 16 genes in C. elegans. We quantify the impact of mutations on expression and physiology by targeted RNA sequencing and DNA sampling. When applying our approach to the lin-41 3′ UTR, generating hundreds of mutants, we find that the two adjacent binding sites for the miRNA let-7 can regulate lin-41 expression largely independently of each other, with indications of a compensatory interaction. Finally, we map regulatory genotypes to phenotypic traits for several genes. Our approach enables parallel analysis of gene regulatory sequences directly in animals

When needles look like hay: How to find tissue-specific enhancers in model organism genomes

AbstractA major prerequisite for the investigation of tissue-specific processes is the identification of cis-regulatory elements. No generally applicable technique is available to distinguish them from any other type of genomic non-coding sequence. Therefore, researchers often have to identify these elements by elaborate in vivo screens, testing individual regions until the right one is found.Here, based on many examples from the literature, we summarize how functional enhancers have been isolated from other elements in the genome and how they have been characterized in transgenic animals. Covering computational and experimental studies, we provide an overview of the global properties of cis-regulatory elements, like their specific interactions with promoters and target gene distances. We describe conserved non-coding elements (CNEs) and their internal structure, nucleotide composition, binding site clustering and overlap, with a special focus on developmental enhancers. Conflicting data and unresolved questions on the nature of these elements are highlighted. Our comprehensive overview of the experimental shortcuts that have been found in the different model organism communities and the new field of high-throughput assays should help during the preparation phase of a screen for enhancers. The review is accompanied by a list of general guidelines for such a project

Genome-Wide Screens for In Vivo Tinman Binding Sites Identify Cardiac Enhancers with Diverse Functional Architectures

The NK homeodomain factor Tinman is a crucial regulator of early mesoderm patterning and, together with the GATA factor Pannier and the Dorsocross T-box factors, serves as one of the key cardiogenic factors during specification and differentiation of heart cells. Although the basic framework of regulatory interactions driving heart development has been worked out, only about a dozen genes involved in heart development have been designated as direct Tinman target genes to date, and detailed information about the functional architectures of their cardiac enhancers is lacking. We have used immunoprecipitation of chromatin (ChIP) from embryos at two different stages of early cardiogenesis to obtain a global overview of the sequences bound by Tinman in vivo and their linked genes. Our data from the analysis of ~50 sequences with high Tinman occupancy show that the majority of such sequences act as enhancers in various mesodermal tissues in which Tinman is active. All of the dorsal mesodermal and cardiac enhancers, but not some of the others, require tinman function. The cardiac enhancers feature diverse arrangements of binding motifs for Tinman, Pannier, and Dorsocross. By employing these cardiac and non-cardiac enhancers in machine learning approaches, we identify a novel motif, termed CEE, as a classifier for cardiac enhancers. In vivo assays for the requirement of the binding motifs of Tinman, Pannier, and Dorsocross, as well as the CEE motifs in a set of cardiac enhancers, show that the Tinman sites are essential in all but one of the tested enhancers; although on occasion they can be functionally redundant with Dorsocross sites. The enhancers differ widely with respect to their requirement for Pannier, Dorsocross, and CEE sites, which we ascribe to their different position in the regulatory circuitry, their distinct temporal and spatial activities during cardiogenesis, and functional redundancies among different factor binding sites

A High-Throughput Chromatin Immunoprecipitation Approach Reveals Principles of Dynamic Gene Regulation in Mammals

Understanding the principles governing mammalian gene regulation has been hampered by the difficulty in measuring in vivo binding dynamics of large numbers of transcription factors (TF) to DNA. Here, we develop a high-throughput Chromatin ImmunoPrecipitation (HT-ChIP) method to systematically map protein-DNA interactions. HT-ChIP was applied to define the dynamics of DNA binding by 25 TFs and 4 chromatin marks at 4 time-points following pathogen stimulus of dendritic cells. Analyzing over 180,000 TF-DNA interactions we find that TFs vary substantially in their temporal binding landscapes. This data suggests a model for transcription regulation whereby TF networks are hierarchically organized into cell differentiation factors, factors that bind targets prior to stimulus to prime them for induction, and factors that regulate specific gene programs. Overlaying HT-ChIP data on gene-expression dynamics shows that many TF-DNA interactions are established prior to the stimuli, predominantly at immediate-early genes, and identified specific TF ensembles that coordinately regulate gene-induction