Analyzing combinatorial regulation of transcription in mammalian cells

Präzise Genregulation ist während der Entwicklung und Differenzierung eines Organismus äußerst wichtig, um die notwendige Homeostase während der Zellentwicklung und -differenzierung zu ermöglichen. Dabei bilden Interaktionen zwischen cis-wirkenden DNA-Elementen wie Promotern und Enhancern die Basis für eine abgestimmte Regulation der Transkription. In dieser Arbeit wurden proximale und weiter entfernte Regionen stromaufwärts aller Transkriptionsstartpunkte in silico betrachtet, um regulatorische Module vorherzusagen, die aus Transkriptionsfaktorkombinationen mit Bindestellen an Promotern und Enhancern bestehen. Die Anwendung auf verschiedene Genexpressionsprofile zeigte eine gewebe- und zeitspezifische Regulation der identifizierten Module in der embryonischen Entwicklung der Maus und in der menschlichen Stammzelldifferenzierung. Zusätzlich zur gewebespezifischen Regulation von Transkriptionsfaktorkombinationen am Promoter bestimmen Kombinationen von Transkriptionsfaktoren an Promotern und Enhancern zeitspezifische Regulation während des Entwicklungsprozesses. Die identifizierten regulatorischen Module zeigten eine gute Vorhersagefähigkeit, differenziell exprimierte Gene unterschiedlicher Zeitpunkte zu unterscheiden. Außerdem wurde gezeigt, dass Transkriptionsfaktorbindestellen unterschiedliche Eigenschaften an Promoter- und Enhancerregionen aufzeigen. Ein Beispiel für kombinatorische Regulation der Transkription in Säugetierzellen ist die Cholesterinbiosynthese. Die Cholesterinbiosynthese wird durch die SREBP (sterol regulatory element binding protein) Proteinfamilie kontrolliert, die die Expression von Genen regulieren, die in der Aufnahme und Synthese von Cholesterin und Lipiden involviert sind. SREBPs sind nur schwache transkriptionelle Aktivatoren und kooperieren mit anderen Transkriptionsfaktoren wie SP1 (Sp1 transcription factor) und NF-Y (nuclear transcription factor Y). Obwohl der Metabolismus der Cholesterinbiosynthese gut beschrieben ist, wird angenommen, dass viele weitere noch unbekannte Proteine an der Cholesterinhomeostase der Zelle beteiligt sind. Daher wurde in dieser Arbeit ein integrativer Ansatz verfolgt, um neue Zielgene von SREBP zu identifizieren. Dazu wurden Genexpressionsprofile von sterol-depletierten Zellen mit in silico Vorhersagen von SREBP, SP1, und NF-Y Bindestellen kombiniert. Insgesamt wurden 99 mögliche SREBP Zielgene identifiziert, von denen 21 Gene bereits im Zusammenhang mit Cholesterin beschrieben wurden und 78 Gene potentiell neue SREBP Zielgene darstellen. Zehn der potenziell neuen Zielgene wurden für eine experimentelle Valdierung ausgewählt, wovon slc2a6, c17orf59, hes6, and tmem55b niedrigere mRNA Expression nach SREBP Knockdowns zeigten und damit potentiell regulatorisch von SREBP abhängig sind. Kombinationen von Transkriptionsfaktoren sind äußerst wichtig, um sowohl Regulationsmechanismen der Transkription als auch die Funktion von Enhancern zu verstehen. Sie können neue Erkenntnisse über die gewebe- und zeitspezifische Regulation der Genexpression bringen und die Identifizierung neuer Zielgene in bestimmten Prozessen ermöglichen

How to understand the cell by breaking it: network analysis of gene perturbation screens

Modern high-throughput gene perturbation screens are key technologies at the forefront of genetic research. Combined with rich phenotypic descriptors they enable researchers to observe detailed cellular reactions to experimental perturbations on a genome-wide scale. This review surveys the current state-of-the-art in analyzing perturbation screens from a network point of view. We describe approaches to make the step from the parts list to the wiring diagram by using phenotypes for network inference and integrating them with complementary data sources. The first part of the review describes methods to analyze one- or low-dimensional phenotypes like viability or reporter activity; the second part concentrates on high-dimensional phenotypes showing global changes in cell morphology, transcriptome or proteome.Comment: Review based on ISMB 2009 tutorial; after two rounds of revisio

An enhanced CRISPR repressor for targeted mammalian gene regulation.

The RNA-guided endonuclease Cas9 can be converted into a programmable transcriptional repressor, but inefficiencies in target-gene silencing have limited its utility. Here we describe an improved Cas9 repressor based on the C-terminal fusion of a rationally designed bipartite repressor domain, KRAB-MeCP2, to nuclease-dead Cas9. We demonstrate the system's superiority in silencing coding and noncoding genes, simultaneously repressing a series of target genes, improving the results of single and dual guide RNA library screens, and enabling new architectures of synthetic genetic circuits

Principles of microRNA regulation of a human cellular signaling network

MicroRNAs (miRNAs) are endogenous 22-nucleotide RNAs, which suppress gene expression by selectively binding to the 3-noncoding region of specific message RNAs through base-pairing. Given the diversity and abundance of miRNA targets, miRNAs appear to functionally interact with various components of many cellular networks. By analyzing the interactions between miRNAs and a human cellular signaling network, we found that miRNAs predominantly target positive regulatory motifs, highly connected scaffolds and most downstream network components such as signaling transcription factors, but less frequently target negative regulatory motifs, common components of basic cellular machines and most upstream network components such as ligands. In addition, when an adaptor has potential to recruit more downstream components, these components are more frequently targeted by miRNAs. This work uncovers the principles of miRNA regulation of signal transduction networks and implies a potential function of miRNAs for facilitating robust transitions of cellular response to extracellular signals and maintaining cellular homeostasis

Selective Activation of Alternative MYC Core Promoters by Wnt-Responsive Enhancers.

In Metazoans, transcription of most genes is driven by the use of multiple alternative promoters. Although the precise regulation of alternative promoters is important for proper gene expression, the mechanisms that mediates their differential utilization remains unclear. Here, we investigate how the two alternative promoters (P1, P2) that drive MYC expression are regulated. We find that P1 and P2 can be differentially regulated across cell-types and that their selective usage is largely mediated by distal regulatory sequences. Moreover, we show that in colon carcinoma cells, Wnt-responsive enhancers preferentially upregulate transcription from the P1 promoter using reporter assays and in the context of the endogenous Wnt induction. In addition, multiple enhancer deletions using CRISPR/Cas9 corroborate the regulatory specificity of P1. Finally, we show that preferential activation between Wnt-responsive enhancers and the P1 promoter is influenced by the distinct core promoter elements that are present in the MYC promoters. Taken together, our results provide new insight into how enhancers can specifically target alternative promoters and suggest that formation of these selective interactions could allow more precise combinatorial regulation of transcription initiation

Mapping genetic interactions in cancer: a road to rational combination therapies.

The discovery of synthetic lethal interactions between poly (ADP-ribose) polymerase (PARP) inhibitors and BRCA genes, which are involved in homologous recombination, led to the approval of PARP inhibition as a monotherapy for patients with BRCA1/2-mutated breast or ovarian cancer. Studies following the initial observation of synthetic lethality demonstrated that the reach of PARP inhibitors is well beyond just BRCA1/2 mutants. Insights into the mechanisms of action of anticancer drugs are fundamental for the development of targeted monotherapies or rational combination treatments that will synergize to promote cancer cell death and overcome mechanisms of resistance. The development of targeted therapeutic agents is premised on mapping the physical and functional dependencies of mutated genes in cancer. An important part of this effort is the systematic screening of genetic interactions in a variety of cancer types. Until recently, genetic-interaction screens have relied either on the pairwise perturbations of two genes or on the perturbation of genes of interest combined with inhibition by commonly used anticancer drugs. Here, we summarize recent advances in mapping genetic interactions using targeted, genome-wide, and high-throughput genetic screens, and we discuss the therapeutic insights obtained through such screens. We further focus on factors that should be considered in order to develop a robust analysis pipeline. Finally, we discuss the integration of functional interaction data with orthogonal methods and suggest that such approaches will increase the reach of genetic-interaction screens for the development of rational combination therapies

Transcriptional Regulation: a Genomic Overview

The availability of the Arabidopsis thaliana genome sequence allows a comprehensive analysis of transcriptional regulation in plants using novel genomic approaches and methodologies. Such a genomic view of transcription first necessitates the compilation of lists of elements. Transcription factors are the most numerous of the different types of proteins involved in transcription in eukaryotes, and the Arabidopsis genome codes for more than 1,500 of them, or approximately 6% of its total number of genes. A genome-wide comparison of transcription factors across the three eukaryotic kingdoms reveals the evolutionary generation of diversity in the components of the regulatory machinery of transcription. However, as illustrated by Arabidopsis, transcription in plants follows similar basic principles and logic to those in animals and fungi. A global view and understanding of transcription at a cellular and organismal level requires the characterization of the Arabidopsis transcriptome and promoterome, as well as of the interactome, the localizome, and the phenome of the proteins involved in transcription

A stochastic and dynamical view of pluripotency in mouse embryonic stem cells

Pluripotent embryonic stem cells are of paramount importance for biomedical research thanks to their innate ability for self-renewal and differentiation into all major cell lines. The fateful decision to exit or remain in the pluripotent state is regulated by complex genetic regulatory network. Latest advances in transcriptomics have made it possible to infer basic topologies of pluripotency governing networks. The inferred network topologies, however, only encode boolean information while remaining silent about the roles of dynamics and molecular noise in gene expression. These features are widely considered essential for functional decision making. Herein we developed a framework for extending the boolean level networks into models accounting for individual genetic switches and promoter architecture which allows mechanistic interrogation of the roles of molecular noise, external signaling, and network topology. We demonstrate the pluripotent state of the network to be a broad attractor which is robust to variations of gene expression. Dynamics of exiting the pluripotent state, on the other hand, is significantly influenced by the molecular noise originating from genetic switching events which makes cells more responsive to extracellular signals. Lastly we show that steady state probability landscape can be significantly remodeled by global gene switching rates alone which can be taken as a proxy for how global epigenetic modifications exert control over stability of pluripotent states.Comment: 11 pages, 7 figure

Combinatorial Binding in Human and Mouse Embryonic Stem Cells Identifies Conserved Enhancers Active in Early Embryonic Development

Transcription factors are proteins that regulate gene expression by binding to cis-regulatory sequences such as promoters and enhancers. In embryonic stem (ES) cells, binding of the transcription factors OCT4, SOX2 and NANOG is essential to maintain the capacity of the cells to differentiate into any cell type of the developing embryo. It is known that transcription factors interact to regulate gene expression. In this study we show that combinatorial binding is strongly associated with co-localization of the transcriptional co-activator Mediator, H3K27ac and increased expression of nearby genes in embryonic stem cells. We observe that the same loci bound by Oct4, Nanog and Sox2 in ES cells frequently drive expression in early embryonic development. Comparison of mouse and human ES cells shows that less than 5% of individual binding events for OCT4, SOX2 and NANOG are shared between species. In contrast, about 15% of combinatorial binding events and even between 53% and 63% of combinatorial binding events at enhancers active in early development are conserved. Our analysis suggests that the combination of OCT4, SOX2 and NANOG binding is critical for transcription in ES cells and likely plays an important role for embryogenesis by binding at conserved early developmental enhancers. Our data suggests that the fast evolutionary rewiring of regulatory networks mainly affects individual binding events, whereas “gene regulatory hotspots” which are bound by multiple factors and active in multiple tissues throughout early development are under stronger evolutionary constraints