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Regulation from a distance: long-range control of gene expression in development and disease

Event

Starts:

October
222012

09:00

Ends:

October
232012

17:00

Location

The Royal Society, London, 6-9 Carlton House Terrace, London, SW1Y 5AG

Overview

Expression driven in the developing mouse by the wild-type cat-derived sonic hedgehog ZRS limb enhancer (Image courtesy of Dr Laura Lettice)

Scientific discussion meeting organised by Professor Wendy Bickmore and Professor Veronica van Heyningen FRS

Event details

The correct regulation of gene expression underpins normal development and differentiation, and is frequently perturbed in disease. Whilst events controlling transcription around gene promoters are well understood, how far-distant enhancers direct spatial and temporal
control of transcription is less clear. Our aim is to integrate experimental and computational approaches, in multiple model systems, to define the mechanisms of enhancer action. This meeting will bring together experts in the field to discuss their research and ideas and key challenges for the future.

Biographies of the organisers and speakers are available below and you can also download the programme (PDF). Recorded audio of the presentations will be available on this page after the event and papers will be published in a future issue of Philosophical Transactions B.

Attending this event

This event is intended for researchers in relevant fields and is free to attend. There are a limited number of places and registration is essential. An optional lunch is offered and should be booked during registration (all major credit cards accepted).

Participants are also encouraged to attend the related satellite meeting Regulation of gene expression from a distance: exploring mechanisms which immediately follows this event.

Enquiries: Contact the events team.

Schedule of talks

Session 1: Enhancers and development

5 talks Show detail Hide detail

Chair

Professor Douglas Epstein, University of Pennsylvania, USA

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Remote control of Shh gene expression in the limb bud

Professor Robert Hill, MRC IGMM, University of Edinburgh, Scotland

Abstract

Multi-species conserved non-coding elements occur in the vertebrate genome and are clustered in the vicinity of developmentally regulated genes. Many act as cis-regulators of transcription and may reside at long distances from the genes they regulate. The relationship of conserved sequence to encoded regulatory information and indeed, the mechanism by which these contribute to long-range transcriptional regulation is not well understood. The ZRS, a highly conserved cis-regulator, is a paradigm for long-range gene regulation acting over ~1Mb to control spatiotemporal expression of Shh in the limb bud. In addition mutations in this regulator account for a number of limb abnormalities which include polydactyly, tibial hypoplasia and syndactyly. We describe the modular nature of this developmental regulator and show that a number of activities are encoded by this enhancer. Restriction of the expression pattern in the limb can, at least in part, be attributed to distinct binding sites in highly conserved domains that lie in the ZRS. Members of two groups of ETS transcription factors mediate a differential effect on Shh expression, defining the parameters of the expression pattern. Occupancy at multiple GABP/ETS1 sites regulates the position of the ZPA boundary, whereas ETV4/ETV5 binding restricts expression outside the ZPA. In addition analyses over longer sequence stretches dissect the ZRS into two distinct activities; one that regulates spatiotemporal activity and one that controls the long-range activity. Spatiotemporal activity is encoded within an element which functions efficiently only from a close range; whereas, long range activity is encoded by a second element which transmits the spatiotemporal activity over a large genomic distance. These two encoded regulatory activities integrate to control the number of digits and morphologically, ensure a stable limb phenotype based on a pattern of five digits.

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Charting the genome regulatory architecture with transposons

Dr Francois Spitz, European Molecular Biology Laboratory, Germany

Abstract

Vertebrate genomes are characterized by the presence of cis-regulatory elements located at great distances from the genes they control. Genomic rearrangements found in humans suggest that the specific organization of large loci is not random, but contributes importantly to implement the specific activities of these remote enhancers. To determine the organization of the mammalian genome and identify elements and genomic parameters that define enhancer regulatory activities, we have developed an in vivo approach, building on the controlled mobilization of a Sleeping Beauty transposon to distribute a regulatory sensor throughout the mouse genome. Analysis of a large genome-wide collection of insertions revealed principles of the genome regulatory architecture. Furthermore, the properties of Sleeping Beauty, in combination with in vivo chromosomal engineering, allows investigation of the fine-scale structure of loci of interests, shedding light on how remote enhancers may control target gene expression.

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High Throughput Enhancer Assessment In Vivo

De Len Pennacchio, Lawrence Berkeley National Laboratory, USA

Abstract

The paucity of a defined collection of mammalian transcriptional enhancers has largely precluded both our ability to develop computational methods for predicting additional tissue-specific enhancers in the human genome and to assess such sequences for their role in human disease. In ongoing studies, we are leveraging extreme evolutionary sequence conservation as well as next generation ChIP-Sequencing to identify putative gene regulatory elements and are characterizing their in vivo enhancer activity in a transgenic mouse assay. To date we have tested over 2000 such sequences in animals, and observed that >1000 function reproducibly as tissue-specific enhancers of gene expression. As a community resource, we have established a database to visualize and query the activity of these enhancer sequences (http://enhancer.lbl.gov/) and continue to generate additional in vivo enhancer data for >300 sequences per year. In recent studies directly from human tissues, we show that the conservation of enhancers across mammals varies widely depending on the specific tissue of examination. In particular, enhancers of the nervous system have high levels of evolutionary constraint while enhancers of the heart are largely not conserved. These findings highlight the importance of enhancer identification directly from human tissues for certain organs and hence disease states. In addition, this growing set of enhancers with in vivo-defined activities provides a molecular toolbox that can be used to experimentally target gene expression to organs and tissues in animals and constitutes a starting point for studying the role of regulatory elements in human disease.

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Transcriptional enhancers in development and variation of the human face

Professor Joanna Wysocka, Stanford School of Medicine, USA

Abstract

The face is at the center of our identity: it is the feature that best distinguishes an individual, while also connecting each of us to our broader ethnic ancestry and to the genetic inheritance from our parents, often evident in familial resemblances. And yet we know very little about the genetic basis of human facial variation. Nonetheless, a growing number of reports documents a link between enhancer mutations and complex human diseases. Moreover, evidence from model organisms begins to emerge that genetic variation in cis-regulatory elements underlies much of morphological evolution and diversity. Although human craniofacial development is extremely complex, the central bauplan of facial morphology is established early in embryogenesis by the neural crest cells and their derivatives. We recently developed an in vitro model of human neural crest formation and used it to epigenomically annotate enhancer repertoire if this unique cell type. I will discuss our hypothesis that allelic sequence variants of neural crest enhancers regulate normal-range variation of craniofacial features and confer susceptibility for malformations.

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Session 2: Human disease

5 talks Show detail Hide detail

Chair

Professor Martha Bulyk, Brigham & Women’s Hospital and Harvard Medical School, USA

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A novel approach for characterising chromosomal interactions throughout the genome

Professor Douglas Higgs FRS, University of Oxford, UK

Abstract

Cis-acting elements (promoters, enhancers, silencers, locus control regions, boundary elements) can often be identified via their conserved, non-coding DNA sequences. In addition, when active, they may have characteristic chromatin signatures and are bound by transcription factors and polymerase II. Such features can now be identified across the entire genome by chromatin immunoprecipitation (using ChIP on chip and ChIPseq). However, cis-elements (e.g. promoters and enhancers) may be located 100s or even 1000s kb apart and therefore it is often not clear which regulatory sequence (e.g.enhancer) interacts with which promoter or additional regulatory element. The chromosome conformation capture (3C) technique was designed to analyse physical interactions between specific, previously characterised, widely separated DNA elements and it has been shown that such interactions are an inherent feature of their function. We have adapted the 3C protocol and developed a method which is capable of identifying all DNA elements interacting with a selected sequence (e.g. promoter) without any prior knowledge of these elements. To validate this approach, we have initially applied this method to the human  globin locus in which the interacting cis-elements have been previously characterised in detail. Using this modified chromosome conformation capture technique we can simultaneously identify the known regulatory elements with high resolution and have shown that they occur in a tissue-specific manner. The method can be applied to the entire genome and is easily analysed using tiled micro-arrays or by high-throughput sequencing which provides additional information on the nature of individual interactions.

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Regulatory genomics in human populations

Professor Emmanouil Dermitzakis, University of Geneva, Switzerland

Abstract

Molecular phenotypes are important phenotypes that informs about genetic and environmental effects on cellular state. The elucidation of the genetics of gene expression and other cellular phenotypes are highly informative of the impact of genetic variants in the cell and the subsequent consequences in the organism. In this talk I will discuss recent advances in three key areas of the analysis of the genomics of gene expression and cellular phenotypes in human populations and multiple tissues and how this assists in the interpretation of regulatory networks and human disease variants. I will also discuss how the recent advances in next generation sequencing and functional genomics are bringing closer our hopes for personalized medicine.

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Connecting non-protein coding risk loci with their target genes

Professor Matthew Freedman, Harvard Medical School, USA

Abstract

A fundamental goal of human genetics is to uncover the relationship between genotype and phenotype. To date, genome wide association studies (GWAS) have identified thousands of alleles associated with hundreds of traits. In stark contrast to Mendelian disorders, the majority of trait-associated loci are located outside of known protein coding areas. This observation poses the next set of challenges for follow-up and fine mapping of trait-associated alleles: (i) what gene is the allele is acting through? and (ii) what is the actual trait causing polymorphism(s)?. For Mendelian disorders, answers to both of these questions are usually revealed through sequencing the coding regions of candidate genes. The genetic code provides the necessary insight and ability to readily interpret DNA sequence changes and how they impact amino acids. Since there is no analogous code for non-protein coding regions, identifying the genes and causal allele(s) underlying complex diseases presents key challenges. The focus of our group is to develop strategies to address these topics.

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Functional connectivity between human regulatory regions

Professor John Stamatoyannopoulos, University of Washington, USA

Abstract

The human genome is densely populated with enhancers and other distal regulatory elements, yet little is currently known about their functional scope. Reverse genetics in an isogenic setting coupled with epigenome profiling offers a powerful paradigm for elucidating the functional network of any cis-regulatory region, and is now enabled through the application of designer nucleases. Applying this approach to the β-globin Locus Control Region (LCR) on Chr11 reveals this classical regulatory element to be functionally connected to over 1,000 predominantly erythroid-specific promoters and distal regulatory elements genome-wide. Genome-scale analysis of chromatin contacts reveals that the affected elements interact both with the LCR and with one another, and share common patterns of transcription factor occupancy. The results reveal unexpected and potent functional synchrony among large numbers of regulatory DNA regions distributed across the human genome.

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Session 3: Enhancers - evolution and identification

5 talks Show detail Hide detail

Chair

Professor Patricia Simpson FRS, University of Cambridge, UK

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Mapping the genetic and genomic basis of evolutionary change in vertebrates

Professor David Kingsley, HHMI and Stanford University School of Medicine, USA

Abstract

The relative contribution of coding and regulatory mutations to adaptive evolution has been difficult to assess, particularly in non-model organisms subject to a full range of fitness constraints in the wild. Recent improvements in genetic and genomic methods are making it possible to map key regions controlling adaptive traits in threespine stickleback fish, and to identify the type of mutations that underlie interesting evolutionary differences seen in nature. Patterns learned from initial case histories can now be extended to the entire genome, using large-scale genotyping and sequencing of many different populations that have evolved similar traits in response to similar environments. Recent studies show that regulatory changes make up the predominant category of mutation that underlies repeated adaptive evolution in threespine sticklebacks. Remarkably similar patterns are seen in genome-wide studies of adaptive loci in both sticklebacks and humans, showing the importance of regulatory differences for a detailed understanding of the molecular basis of evolutionary change in vertebrates.

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Computational identification and sequence analysis of enhancers

Dr Ivan Ovcharenko, National Institutes of Health, USA

Abstract

Transcriptional activation is commonly assumed to rely on ubiquitous promoters establishing the basal level of expression and interacting with tissue-specific enhancers responsible for temporal and cellular expression fine-tuning. We previously developed a computational approach to model the motif structure of tissue-specific enhancers and demonstrated its accuracy in predicting heart and hindbrain enhancers, for which 60% and 90% of predictions, respectively, have been validated in vivo. To address the lack of tissue-specificity in promoters, we applied the developed motif analysis approach to the sequence of promoters of tissue-specific genes. Unexpectedly, a strong tissue-specificity signal has been observed in promoters of genes expressed in some tissues, including heart and liver. To validate the identified signal as a general mark of tissue-specificity, we used it to predict distant tissue-specific enhancers. These predictions were notably overrepresented in loci of concordantly expressed genes (6-fold enrichment in liver predictions, for example; p-value < 1e-20). When tested in in vivo enhancer assays, 60% of the liver predictions have been confirmed as positive enhancers. Our results suggest dichotomy in promoter use in different regulatory programs.

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Large scale switch in Hox genes regulation during limb development

Professor Denis Duboule, University of Geneva and EPFL Lausanne, Switzerland

Abstract

The emergence and evolution of limbs was an essential step in the success of vertebrates. Amongst the key players, Hoxd genes are coordinately regulated during the development of both the proximal (arm and forearm) and distal (digits) regions. In both contexts, these genes help organize growth and patterns. We examined the associated long-range transcriptional regulation by probing their 3D organization and chromatin status in developing limbs, at both early and late stages, combined with scanning deletion approaches in vivo. During digit development, we show that the active part of the gene cluster contacts several regulatory islands, located within the centromeric gene desert, which all contribute either quantitatively or qualitatively to Hox gene transcription in future digits. This novel type of ‘regulatory archipelago’ may underlie both the great morphological flexibility in the shape and number of digits as well as their resilience to drastic variations. In contrast, during arm and forearm development, a partially overlapping set of genes is regulated via the telomeric gene desert, as determined again by conformation capture, chromatin landscapes and transgenic as well as genetic approaches. Therefore, completely different regulatory strategies have evolved to build these two parts of our developing appendages. The evolutionary relevance of this two-steps strategy will be discussed as well as the switch between these various regulatory modalities.

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The spatial organisation of the enhancers – is what you ‘C’ what you see?

Professor Wendy Bickmore, University of Edinburgh, UK

Abstract

Our understanding of chromatin states beyond the level of the nucleosome is rudimentary. Until quite recently there has been little exploration of how higher-order chromatin structure might contribute to the regulation of gene expression, but it is hard to envisage how distant enhancers can regulate expression from their target genes located many hundreds of thousands of base pairs away, without invoking chromosome folding. Using specific developmentally regulated loci in the mouse I will discuss how chromatin compaction and chromatin looping can act to regulate the spatial and temporal activation of genes during mouse development. I will compare different molecular and cytological methods that can probe higher order chromatin structure and will discuss whether these methods both point to a consensus view of enhancer function or not. I will also present evidence for a new histone modification that marks some active enhancers in embryonic stem cells.

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Session 4: Mechanisms of action

4 talks Show detail Hide detail

Chair

Dr Edith Heard, Institut Curie, France

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Transcription regulation in the 3D nucleus

Professor Wouter de Laat, Hubrecht Institute, The Netherlands

Abstract

Developmental gene regulation in mammals is often controlled by remote regulatory DNA sequences. ChIP-seq and other functional genomics profiles indicate the genome is full of potential regulatory DNA sequences. In order to understand how they are wired to genes, detailed 3D genome maps are required. I will present our work that aims to develop improved versions of 3C technology in order to understand how gene expression is controlled in the 3D nucleus.

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Deciphering cis-regulatory control of inflammatory cells

Dr Gioacchino Natoli, European Institute of Oncology, Italy

Abstract

Cell identity is determined by a complex and dynamic interplay between cell-intrinsic, lineage-restricted developmental pathways on the one hand, and cell-extrinsic micro-environmental signals on the other. In this context, macrophages represent a paradigmatic cell population whose functional specialization in vivo reflects the impact of the local microenvironment on the intrinsic differentiation program, leading to a variety of specialized macrophage types in different tissues and conditions. Players and mechanisms controlling macrophage plasticity and at the same time enforcing macrophage identity will be discussed.

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Topological domains: identification and functional implications in gene regulation

Dr Bing Ren, Ludwig Institute for Cancer Research UCSD, USA

Abstract

The structural organization of the genome has a fundamental role in its function. The transcription regulatory process, for example, involves higher order chromatin structures, where transcriptional activation is frequently mediated through long range looping interactions between promoters and enhancers and accompanied by dynamic chromatin movement in the nucleus. As such, knowledge of the higher order chromatin structure is essential for a full understanding of transcriptional control and other nuclear processes. Recently, we have investigated the 3D organization of the human and mouse genomes with the Hi-C technique, and found that these genomes are partitioned into large, megabase-sized local chromatin interaction domains, which we term “topological domains”. Here, I will discuss the functional implications of structural feature in gene regulation, and present evidence that topological domains may be involved in long-range control of gene expression by distal enhancer sequences.

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Regulation from a distance: long-range control of gene expression in development and disease The Royal Society, London 6-9 Carlton House Terrace London SW1Y 5AG UK