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Image courtesy of Dr Laura Lettice (details discussed in Lettice et al, Hum Mol Genet 17: 978-985, 2008). Expression driven in the developing mouse by the wild-type cat-derived sonic hedgehog ZRS limb enhancer.
Satellite meeting organised by Professor Wendy Bickmore and Professor Veronica van Heyningen FRS
How do distant regulatory elements (enhancers) control spatial and temporal patterns of expression of target genes? This meeting aims to integrate experimental and computational approaches that explore mechanisms of quantitative control of gene expression by enhancers in different model systems. Bringing together researchers from multiple disciplines will provoke new avenues of investigation into long-range gene regulation.
Biographies of the organisers and speakers are available below. Audio recordings are freely available and the programme can be downloaded here.
This meeting was preceded by the related Discussion meeting Regulation from a distance: long-range control of gene expression in development and disease 22 - 23 October 2012.
Enquiries: Contact the events team
Professor Wendy Bickmore, University of Edinburgh, UK
Wendy Bickmore is the Head of the Chromosomes and Gene Expression Section at the MRC Human Genetics Unit, MRC IGMM at the University of Edinburgh. Her scientific work is based on the principle being that genes do not function in isolation from their chromosomal and nuclear context. She has pioneered research into the spatial and temporal organisation of the human and mouse genomes, and the implications of this for gene regulation and genome function. Her experimental approaches combine cell biology with genomics, genetics and biochemistry. Wendy is an EMBO member, and a Fellow of both the Royal Society of Edinburgh and the Academy of Medical Sciences.
Professor Veronica Van Heyningen FRS, University of Edinburgh, UK
Veronica van Heyningen, formerly Head of the Medical and Developmental Genetics Section, is a Group Leader at the MRC Human Genetics Unit, MRC IGMM at the University of Edinburgh. The group’s work with human developmental disease brought insight into the mechanisms of cis-regulatory control, revealing the long control regions flanking many developmental regulator genes. Studies of extra-genic chromosomal rearrangements and non-coding region mutations prompted work, in mouse and zebrafish model systems, on enhancer interactions and transcription factor binding specificity. Genome-wide target predictions for PAX6 have revealed network complexity and the precision of target-sequence specificity. Veronica was appointed a CBE for services to science, is an EMBO Member and a Fellow of the Royal Society of Edinburgh, the Academy of Medical Sciences and of the Royal Society.
Professor Nicholas Hastie CBE FRS, Medical Research Council, UKChair
Biography not yet available
Professor Denis Duboule, University of Geneva and EPFL Lausanne, SwitzerlandThe evolution of global enhancers
Denis Duboule earned his PhD in Biology in 1984. He is currently Professor of Developmental genetics and genomics at the Federal Institute of Technology in Lausanne and Chairman of the department of Genetics and Evolution of the University of Geneva, Switzerland. Since 2001, he is also the director of the Swiss national research centre ‘Frontiers in Genetics’. Duboule has a longstanding interest in the function and regulation of Hox genes, a family of genes responsible for the organization and evolution of animal body plans. These genes have been a paradigm to understand embryonic patterning, in developmental, evolutionary and pathological contexts. He is an elected member of several academies and has received many awards, amongst which the Louis-Jeantet Prize for Medicine in 1998. He has four children, bikes a Look KX and plays a Gibson 355 SV stereo.
In this paper, I will discuss potential mechanisms underlying the emergence of regulatory landscapes and their subsequent evolutionary recruitment in various developmental contexts. The long-range control of Hox genes during the development of the limbs and the external genitals will be used as an example, as well as the regulation of these genes during gut development.
The gastrointestinal tract is composed of a series of morphological sub-divisions, specialized in various functions associated with food breakdown and nutrients absorbtion. In mammals, the diet can considerably differ from one species to the other, depending on particular environmental adaptations. To digest the cellulose associated with vegetarian components, mammals belonging to the so-called hindgut fermenters, rely upon the caecum, a specialized gastrointestinal organ branching from the intestinal tract at the transition between the small and the large intestines. The molecular mechanisms underlying both the positioning, the budding and the extension of the caecum, which in some species can be longer than the gut itself, only starts to be understood, and was shown to require the concerted expression of several Hox genes, in particular members of the HoxA and HoxD clusters.
To try and understand how these genes are controlled during the morphogenesis of the caecum, we isolated and characterized the regulatory elements necessary for the transcription of Hoxd genes during the budding of this structure. We observe that an unusual number of enhancer sequences, with related specificities, are located within a gene desert flanking the HoxD cluster on its telomeric side. We also document the presence, within this large DNA interval, of a long non-coding RNA, speficically expressed in the growing caecum, which is involved in cis in the transcriptional regulation of Hoxd genes in this organ. We propose a model for the acquisition of multiple enhancer sequences with comparable activities, based on the existence of a regulatory structure that facilitates successive evolutionary recruitments of such sequences.
Co-authors:Saskia Delpretti, Thomas Montavon, Nicolas Lonfat, Ecole Polytechnique Fédérale, Switzerland
Professor Wouter de Laat, Hubrecht Institute, The NetherlandsThe pluripotent 3D genome
Wouter de Laat studied biology at the Utrecht University Utrecht. During his PhD (1998) at the Erasmus University Rotterdam (Prof. Jan Hoeijmakers) he investigated the molecular mechanism of nucleotide excision repair. He identified one of the nucleases and characterized the interplay between repair factors at the site of the lesion. As a postdoc, he joined the group of Prof. Frank Grosveld to work on beta-globin gene activation. In 2000 he received a career grant (Vidi) to work on long-range gene activation. His group used 3C technology, and later developed 4C technology, to demonstrate chromatin loops between genes and enhancers and to uncover long-range DNA contacts within and between chromosomes. In 2008 he received an ERC Young Investigator Grant. In September 2008 de Laat moved his group to the Hubrecht Institute, where he continued his work on genome structure and function. In January 2009 he was appointed professor in Biomedical Genomics at the University Medical Center Utrecht.
Pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), have the unique capacity to differentiate into any somatic cell type. Microscopically, PSCs show minimal clustering of facultative heterochromatic regions such as centromeres and appear to have a more open chromatin structure, which is thought to reflect their ability to activate large portions of the genome upon differentiation. Yet, the three-dimensional organization of the pluripotent genome is not well understood. Here, we applied 4C technology to compare structural features of somatic and pluripotent genomes. In contrast to somatic cells, in PSCs, long-range contacts between inactive chromatin domains are nearly absent, however, active chromatin contacts do occur. Upon differentiation, cells lose the PSC-specific topology and adopt a somatic chromatin architecture. Conversely, the structural specifics of the pluripotent genome are regained during iPSC reprogramming. Finally, we describe a unique contact network of pluripotency genes centered around binding sites of the pluripotency factors Nanog, Oct 4 and Sox2. Collectively, our work reveals a PSC-specific DNA interaction network, which, we speculate, enhances the robustness of pluripotency.
Dr Panos Deloukas, Wellcome Trust Sanger Institute, UK Maps of open chromatin from association signals to function
Panos Deloukas is Senior Group Leader ‘Genetics of Complex Traits in Humans’, department of Human Genetics, Wellcome Trust Sanger Institute. He is Fellow of the European Society of Cardiology and honorary member of the TwinsUnit at KCL, UK. He obtained his BSc in chemistry from the Aristotelian University of Thessaloniki, Greece (1986), his MSc in microbiology from the University ParisVII, France (1987) and his PhD in molecular biology from the Biozentrum, University of Basel, Switzerland (1992). He worked as post-doctoral fellow at Hoffmann-La Roche in Basel, Switzerland and joined the Sanger Centre in 1994.
He led the sequencing of chromosomes 10 and 20 in the Human Genome Project. He went on to study common sequence variation (HapMap project) and since 2005 is investigating the molecular basis of common disease and variable response to drugs focusing on coronary artery disease. He has been a founder member of the Wellcome Trust Case-Control Consortium and co-Chair of the Immunochip and the CardiogramPlus consortia; is member of several global consortia (GIANT, GLGC, IBPC-GWAS, IWPC). He has authored and co-authored more than 220 papers in peer-reviewed journals.
Genome-wide association (GWA) studies have been very successful in identifying genetic loci associated with complex traits, including disease. Many GWA signals map outside protein-coding regions suggesting that the underlying functional variants may influence phenotype through regulation of gene expression.
We used FAIRE (formaldehyde-assisted isolation of regulatory elements) coupled with sequencing to map nucleosome-depleted regions (NDRs), which mark active regulatory elements, in primary human megakaryocytes, erythroblasts and monocytes. Our data suggest that (i) cell type-specific NDRs can guide the identification of regulatory variants and (ii) sequence variants associated with the corresponding platelet and erythrocyte traits are enriched in NDRs in a cell type-dependent manner. As a proof-of-concept, we investigated the molecular mechanism of the 7q22.3 platelet volume locus.
We identified a megakaryocyte-specific NDR harbouring the index SNP which differentially binds the transcription factor EVI1 and affects PIK3CG gene expression in platelets. Gene expression profiling of Pik3cg knockout mice indicated that PIK3CG is associated with gene pathways with an established role in platelet function.
Finally, we applied FAIRE maps in characterizing two low-frequency SNPs at the RBM8A locus, which is involved in thrombocytopenia with absent radii (TAR), a rare congenital malformation syndrome.
Dr Eileen Furlong, EMBL, GermanyDeciphering the cis-regulatory code
Eileen Furlong received her PhD at University College Dublin, Ireland and did her post-doctoral research at Stanford University, California. She has been at group leader at the European Molecular Biology Laboratory (EMBL) since Oct 2002. Since January 2009 she is a senior scientist and joint chair of the Genome Biology department at EMBL, Heidelberg.
Her work has been instrumental in developing and applying genomic approaches to multicellular developing embryos. This includes developing an automated transgenic embryo sorter, tiling arrays and protocols for ChIP-chip experiments and a new method to study cell-type specific changes in regulatory state during metazoan development.
Her group’s research interests focus on understanding how transcriptional networks drive developmental progression using Drosophila mesoderm specification as a model system. For this purpose, the group combines genomic, genetic and bioinformatics approaches to uncover basic principles of transcription and to gain predictive insights into cell fate decisions.
Embryonic development is controlled by precise patterns of temporal and spatial gene expression, which in turn are controlled by the activity of transcription factors converging on cis-regulatory modules (CRMs) or enhancer elements. The location and even combinatorial occupancy of CRMs can be experimentally measured using ChIP-seq at specific stages of development, at high-resolution. A current major challenge however, is to understand their function in terms of spatio-temporal enhancer activity. We have recently developed a new method to obtain cell-type specific signatures of chromatin state or transcription factor occupancy within the context of a developing multicellular embryo (Batch Tissue Specific ChIP: BiTS-ChIP). Using this method, we have measured chromatin modifications and RNA polymerase II occupancy in a specific tissue type at developmental stages before and after cell fate specification. The data revealed heterogeneous combinations of chromatin marks linked to active enhancers. Using a Bayesian network, we show that chromatin state is sufficient to predict, not just the location, but activity state of regulatory elements, accurately distinguishing between enhancers in an active versus inactive state. The model also revealed that Pol II occupancy and H3K79me3, a modification associated with Pol II elongation, are highly predictive for the precise timing of enhancer activity. Pol II occupancy is tightly correlated with both the timing and location of transcription factor occupancy suggesting that it may be recruited there by transcription factors. We are currently investigating how this relates to enhancer activation and three-dimensional looping interactions with promoter elements.
Professor Anne Ferguson-Smith, University of Cambridge, UKChair
Anne C Ferguson-Smith is Professor of Developmental Genetics in the Department of Physiology Development and Neuroscience at the University of Cambridge, and Wellcome Trust Senior Investigator. For over 20 years, her research has focused on genomic imprinting and epigenetic mechanisms in mammalian development. Current work is based on three main research themes – functional genomics and epigenomics, stem cells and the epigenetic programme, and the relationship between development environment and disease. Anne is an EMBO Member and a Fellow of the Academy of Medical Sciences.
Professor Mike Levine, University of California Berkeley, USADynamic use of enhancers in development
Professor Levine’s lab has studied gene regulation in the early Drosophila embryo for nearly 30 years. During this time they conducted a complete analysis of the eve stripe 2 enhancer, obtained early evidence that homeobox proteins function as sequence-specific transcription factors, and proposed a gradient threshold model for dorsal-ventral patterning. During the past few years they have used a combination of whole-genome methods and quantitative imaging assays to investigate mechanisms of transcription precision, such as synchronous activation of gene expression across embryonic tissues.
Mike Levine has been a Professor of Genetics at UC Berkeley since 1996. Prior to that he held faculty positions at Columbia University and UCSD. He was a Visiting Professor of Zoology at the University of Zurich from 1999-2000. He received the Molecular Biology Award from the National Academy of Sciences in 1996 and the Wilbur Cross Medal from Yale University in 2009. He was elected into the American Academy of Arts and Sciences in 1996 and the National Academy of Sciences in 1998.
We are interested in mechanisms of transcriptional precision, namely, how are complex patterns of gene expression reproducibly deployed in the different embryos of a population? Whole-genome assays have identified two potential mechanisms of precision, “shadow” enhancers and paused RNA polymerase (paused Pol II). I will present evidence that the snail shadow enhancer helps ensure a normal pattern of activation within the presumptive mesoderm of embryos grown at elevated temperatures. Paused Pol II appears to foster synchronous activation of gene expression within the different cells of a tissue. Replacing the paused snail promoter with a nonpaused promoter results in stochastic activation of snail expression and a curious bistable mutant phenotype: 20% of the embryos exhibit normal invagination of the mesoderm during gastrulation, while 80% fail to gastrulate and exhibit the sna- mutant phenotype. I will attempt to explain the basis for this gastrulation bistability.
Dr Sarah Teichmann, LMB, UKGene expression genomics
Sarah Teichmann obtained a Bachelor’s degree in Biochemistry in 1996 from Cambridge University, and a PhD degree in Computational Genomics in 1999 working at the MRC Laboratory of Molecular Biology in Cambridge, England. She was a Beit postdoctoral fellow from 2000-2001 at University College London. Since October 2001 she has been a group leader at the MRC Laboratory of Molecular Biology and fellow at Trinity College, Cambridge. She studies the evolution and assembly of protein complexes, and the dynamics of transcriptional regulatory networks. Her group aims to tackle these questions through integrated computational and experimental systems biology approaches. Her work has been recognized by numerous awards, including EMBO Young Investigator (2003), Lister Research Prize (2010), Colworth Medal (2010) and Royal Society Crick Lecture (2012).
Gene expression levels are subject to gross regulation as well as fine-tuning. Internal and external sources of noise are superimposed on top of transcriptional control mechanisms. Data mining of transcriptomic and epigenomic measurements yield insights into general principles of regulation of gene expression.
Control of expression levels is exerted through a combination of transcription factors against a background of repression by nucleosomal histones. We have analyzed to what extent the intrinsic DNA binding preferences of yeast TFs and histones play a role in determining nucleosome occupancy, in addition to nonintrinsic factors such as the enzymatic activity of chromatin remodelers (Charoensawan et al., Mol. Cell, 2012).
Transcription factor and epigenetic control in animal cells gives rise to two major expression levels, which vary by roughly one to two orders of magnitude. This gives rise to bimodal distributions of gene expression levels in cell populations (Hebenstreit et al., Mol Sys Biol., 2011). Analysis of histone modifications by ChIP-seq indicates that activating modifications such as H3K9/14ac and H3K4me3 are involved in this ‘digital’ expression switch (Hebenstreit et al., Nucleic Acids Res, 2011).
These findings have broad implications for the analysis RNA-seq and ChIP-seq data, and for the understanding of the regulation of gene expression in eukaryotic cells.
Dr Jonathan Chubb, University College London, UKExploring gene expression at the single cell level
Dr Jonathan Chubb studied for his PhD (Ras signaling, endocytosis and cell motility) at UCL from 1995-1999 with Robert Insall. Post-doc he spent 2000-2003 with Wendy Bickmore at the MRC Human Genetics Unit and 2003-2005 with Robert Singer Albert Einstein College of Medicine, New York. He spent 2005-2011 at the College of Life Sciences, University of Dundee and is currently Reader at MRC LMCB and Cell and Developmental Biology, University College London.
Transcription is not adequately described by the smooth, seamless process one infers from standard measures of gene expression. When visualized in individual living cells, transcription occurs in bursts or pulses, with periods of activity separated by long irregular intervals. Discontinuous activity has strong implications for our understanding of transcriptional mechanism and is considered a source of expression diversity driving cell differentiation in a number of clinical and developmental contexts. We use a combination of imaging, modeling and molecular genetic approaches to define the how the pulsing process is regulated, and its implications for cell and developmental biology.
Co-authors: Adam Corrigan, Tetsuya Muramoto, Danielle Cannon, University College London, UK
Dr Johann Elf, Uppsala University, SwedenTranscription factor search kinetics explored at the level of single molecules
Johan Elf got his PhD in Uppsala with Professor Måns Ehrenberg on a theoretical thesis on fluxes and fluctuations in metabolic networks.
He moved on for a postdoc at Harvard with Professor Sunney Xie, where he developed an imaging assay to study gene regulation at level of individual transcription factor molecules in living cells. In 2007 he established his own lab in Uppsala where he combined single molecule imaging in living cells with the development of theory and algorithms for intracellular reaction diffusion processes. His main research focus is on how gene regulation works at the level of individual molecules. Dr Elf is a Fellow of the Swedish Royal Academy of Sciences and an EMBO young investigator. Dr Elf was awarded the Academy’s Göran Gustafsson prize in molecular biology 2010.
Transcription factors (TFs) are proteins that regulate the expression of genes by binding sequence-specific sites on the chromosome. It has been proposed that to find these sites fast and accurately, TFs combine one-dimensional (1D) sliding on DNA with 3D diffusion in the cytoplasm. This facilitated diffusion mechanism has been demonstrated in vitro, but it has not been shown experimentally to be exploited in living cells. We have developed a single-molecule assay that allows us to investigate the sliding process in living bacteria. We show that the lac repressor slides ~50 base pairs on chromosomal DNA and that sliding can be obstructed by other DNA-bound proteins near the operator. Furthermore, the repressor frequently (>90%) slides over its natural lacO1 operator several times before binding. This suggests a trade-off between rapid search on nonspecific sequences and fast binding at the specific sequence.
Professor Constanze Bonifer, University of Birmingham, UKChair
Constanze Bonifer studied Biology at the University of Cologne and did her PhD in Biochemistry and Molecular Biology at the Centre for Molecular Biology, University of Heidelberg. Her postdoctoral training was at the Karolinska Institute, Stockholm, and the National Institute for Medical Research in London. 1990 she became Assistant Professor at University of Freiburg. 1997 she went back to the UK as Group Leader at the Molecular Medicine Unit, University of Leeds. She became a Reader in 2000, a full Professor in 2004 and in 2006 she was appointed Head of Section of Experimental Haematology at the Leeds Institute of Molecular Medicine. Since August 2011, Constanze holds a Chair of Experimental Haematology in the School of Cancer Sciences at the University of Birmingham. For more than 20 years she has been engaged in research in the field of gene regulation in the hematopoietic system and her group is particularly interested in how transcription factors program chromatin in development and in leukaemia. Highlights of her work include the identification of chromatin priming mechanisms in early progenitor cells, one of the first studies of epigenetic consequences of nuclear oncoprotein binding in AML and recently the discovery that aberrantly activated repeat elements can drive the expression of oncogenes in lymphoma.
Dr Roee Amit, Technion-Israel Institute of Technology, IsraelDesign rules for bacterial enhancers
Dr Amit received his undergraduate degree in Applied and Engineering Physics from Cornell University in 1994. After a short a stint in the IDF, he carried out his graduate studies on the interactions of proteins and DNA at the single molecule level in the Weizmann Institute of Science under the supervision of Professor Joel Stavans, completing his PhD in 2004. In 2006, he moved to Caltech, where he specialized in Synthetic Biology working as a post-doctoral scholar in collaboration with Professor Scott Fraser, Frances Arnold, and Rob Phillips. In 2011, Dr Amit returned to Israel with a Senior Lecturer (Assistant Professor) appointment to start the Technion's first Synthetic Biology research lab. Dr Amit's group is focused on constructing "Synthetic Enhancer Circuits" for a variety of applications and research objectives. The group's primary purpose is to decipher basic enhancer design principles using a variety of microscopy, molecular engineering, and theoretical approaches, including new synthetic biology tools developed in the lab.
Enhancers refer to functional regulatory elements located a large genomic distance from the basal promoter, and which contain several clustered TF binding sites. In bacteria, enhancers can be divided into three distinct and irreducible modules: a driver module which is absolutely essential for transcription initiation, an expression modulation region which either up or down regulates expression, and a poised promoter which integrates all the inputs. In the bacterial examples the driver module loops and contacts the poised promoter to initiate expression, while the TFs that are bound to the expression modulation region either promote or inhibit the formation of the loop by altering the ability of DNA to bend.
In order to obtain a more quantitative understanding for the regulatory output of bacterial enhancers, we constructed libraries of synthetic bacterial enhancers, where only one module at a time is systematically varied. We showed that our enhancers' regulatory response depend for the most part on the values four control parameters or design rules that can be read-off directly from the enhancer DNA sequence. As a result, our studies have allowed us to formulate a set of thermodynamics models that can qualitatively predict the regulatory output of unrelated naturally occurring bacterial enhancers.
Dr Gordon Hager, National Cancer Institute, NIH, USAComplex protein dynamics at eukaryotic regulatory elements
Dr Hager received his PhD in genetics at the University of Washington in the lab of Ben Hall. He pursued postdoctoral studies with Dick Epstein at the Institut de Biologie Moleculaire in Geneva and with Dr William Rutter at the University of California-San Francisco. He carried out the first molecular cloning of retroviruses at the NIH and described the first identification of steroid responsive regulatory elements. He also was first to introduce the concept of chromatin modification as an important component of nuclear receptor action. In 2000, Dr. Hager reported the first observation of transcription factor binding to specific regulatory elements in living cells, and discovered the phenomenon of rapid exchange of regulatory proteins with binding sites in the genome. He recently found that this dynamic mechanism of receptor/genome interaction is critically involved in the physiological action of nuclear receptors. He is currently Chief of the Laboratory of Receptor Biology and Gene Expression, and Chair of the Center of Excellence in Chromosome Biology at the NCI. His program interests include the role of chromatin structure in gene regulation, mechanisms of nuclear receptor action, genome-wide organization of regulatory elements, and the architecture of active genes in the interphase nucleus.
Rapid developments in the genome wide characterization of promoter structures and regulatory sites suggest a near complete identification of these elements in the near future. Access to regulatory elements is dramatically restricted by chromatin organization, and modification of the nucleoprotein structure to allow factor binding is emerging as a key feature of cell selective gene regulation (Biddie et al, 2011; Siersbaek et al, 2011; Voss et al, 2011). These processes are often modeled as transitions between relatively static states, with activity lifetimes of minutes or hours. In fact, many regulatory processes are highly dynamic, often with oscillatory or cyclical components operating on multiple time scales. We will discuss an integrated dynamic model for chromatin transitions leading to modulated states of gene expression.
Biddie, et al (2011). Molecular Cell 43, 145-155.Siersbaek et al (2011). EMBO J. 30, 1459-1472.Voss et al (2011). Cell 146, 544-554.
Dr Martha L Bulyk, Brigham & Women's Hospital and Harvard Medical School, USATranscription factors and DNA regulatory elements
Dr Bulyk received dual undergraduate degrees in Biology and in Mathematics from MIT in 1993. She received her PhD in Biophysics in 2001 from Harvard University, where she worked in Dr George Church’s group and pursued research on transcription factors and their DNA binding sites. Shortly thereafter, she began as an Assistant Professor at Harvard. Currently she is Associate Professor in the Division of Genetics in the Department of Medicine at Brigham & Women's Hospital and Harvard Medical School. She also holds a secondary appointment in the Department of Pathology at Brigham & Women's Hospital, is an Associate Member of the Broad [pronunciation: \brōd\] Institute of MIT and Harvard, and is an Associate Member of the Dana Farber Cancer Institute’s Center for Cancer Systems Biology.
In 2005 Dr Bulyk was named one of the TR35, MIT Technology Review’s annual competition to select the top 35 young innovators under the age of 35, and in 2007 she was named in Genome Technology’s annual selection of “Tomorrow’s PIs”. Dr Bulyk has published over 60 papers, and her group is currently focused on studies of transcription factors and DNA regulatory elements, using a variety of experimental and computational approaches, including new technologies they have developed.
The interactions between sequence-specific transcription factors (TFs) and their DNA binding sites are an integral part of the gene regulatory networks within cells. My group developed highly parallel in vitro microarray technology, termed protein binding microarrays (PBMs), for the characterization of the sequence specificities of DNA-protein interactions at high resolution. Using PBMs, we have determined the DNA binding specificities of hundreds of TFs from a wide range of species. More recently we have used the PBM technology to investigate TF heterodimers and higher order complexes. The PBM data have permitted us to identify novel TFs and their DNA binding sequence preferences, predict the target genes and condition-specific regulatory roles of TFs, predict and analyze tissue-specific transcriptional enhancers, investigate functional divergence of paralogous TFs within a TF family, investigate the molecular determinants of TF-DNA recognition specificity, and distinguish direct versus indirect TF-DNA interactions in vivo. Notably, not all DNA binding sites of a TF function equally. Further analyses of TFs and cis regulatory elements are likely to reveal features of cis regulatory sequences that are important in gene regulation.
Dr Gerd Blobel, The Children’s Hospital of Philadelphia, USAControlling long-range genomic interactions to reprogram the β-globin locus
Dr Blobel received his MD from the Medical School, Heidelberg, Germany in 1986, and his PhD from the Rockefeller University, NY, USA in 1991 (Molecular Oncology). From 1997-2003 he was Assistant Professor at the Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, from 2003-2008 Associate Professor of Pediatrics at the University of Pennsylvania and since 2008 has been Professor of Pediatrics at the same institution.
He cites 5 publications relevant to higher scale chromatin organisation:
Distal enhancers physically contact gene promoters to confer efficient transcription. Such long-range chromosomal interactions are dynamically formed and dissipated concurrent with transcriptional activity at complex gene loci. In erythroid cells, the locus control region (LCR) switches its interactions between the β-globin genes during development, concomitant with the switch in globin gene expression. Recently, we have employed artificial zinc finger (ZF) proteins to target a looping factor called Ldb1 to chromatin to promote a long-range interaction between the LCR and the adult β-globin gene, resulting in high level β-globin transcription. Here we examined whether a developmentally silenced embryonic globin gene can be reactivated in adult erythroblasts by re-direction of the LCR. Ldb1 was fused to a ZF protein (ZF-Lbd1) designed to bind to the embryonic globin promoter and stably introduced into an adult erythroid cell line. Strikingly, expression of ZF-Ldb1 re-activated embryonic globin transcription to 15%- 20% of total β-globin production. Since elevated expression ofthe fetal globin genesis beneficial to patients with sickle cell anemia, we are now investigating whether forced LCR-promoter looping can be employed to re-activate fetal globin genes in primary adult human erythroid cells.
Co-authors:Jeremy W Rupon, Hongxin Wang, The Children’s Hospital of Philadelphia, USAWulan Deng, The Children’s Hospital of Philadelphia and University of Pennsylvania, USAAndreas Reik, Philip D Gregory, Sangamo BioSciences Inc, USA
Dr Duncan Odom, University of Cambridge, Cancer Research UK, and Wellcome Trust Sanger Institute, UKChair
Duncan Odom obtained his PhD from Caltech in 2001 in Bio-Inorganic Chemistry, and then changed fields to pursue a postdoc in genetics and genomics with Rick Young at the Whitehead Institute at MIT, where his work transcriptional regulatory networks in human tissues.
More recently, his laboratory and its collaborators at the University of Cambridge have begun using interspecies experimental data to explore how regulatory networks evolve. He was awarded an ERC Starting Grant (2008) and is an EMBO Young Investigator (2010).
Dr Rupali Patwardhan, University of Washington, USA Massively parallel functional dissection of mammalian enhancers
Rupali Patwardhan is a graduate student with Dr Jay Shendure in the Department of Genome Sciences at the University of Washington in Seattle, USA. She is currently working towards developing massively parallel methods to characterize the functional impact of mutations in elements that control gene expression, and more generally, is interested in developing scalable technologies and algorithms that will help better predict the impact of variants in the human genome. Before joining the graduate program at UW, she received a Bachelor of Engineering degree from University of Mumbai (Bombay), India, and a Masters in Bioinformatics from Indiana University, Bloomington.
Massively parallel sequencing has accelerated the cataloging of cis-regulatory elements in mammalian genomes, and recent studies have highlighted the importance of regulatory variants in disease. Although diverse methods exist to predict the functional consequences of genomic variants that alter protein sequence, it remains challenging to estimate the functional effects of variation in cisregulatory elements in a high-throughput fashion. We developed a massively parallel method for interrogating the architecture of mammalian cis-regulatory elements at single nucleotide resolution. A complex population of sequence variants of a regulatory element, e.g. an enhancer, is cost-effectively synthesized, linked in cis to synthetic tags embedded in a transcriptional unit, and then subjected to a single, highly multiplexed reporter assay. Massively parallel sequencing of the transcribed synthetic tags is used to assess the relative activities of their associated variants. Application of this method to three mammalian enhancers produced a comprehensive profile of the functional effects of all possible single nucleotide variants in these enhancers. This method can similarly be applied to several types of transcriptional regulatory elements including promoters and repressors, and can be used to uncover the underlying architecture of these elements as well as to learn the effect size distribution of mutations in regulatory regions.
Dr Robert-Jan Palstra, ErasmusMC, The NetherlandsHERC2 rs12913832 modulates human pigmentation by attenuating chromatin-loop formation between a long-range enhancer and the OCA2 promoter
Robert-Jan Palstra obtained his BSc in 1995 from the Hogeschool Rotterdam en Omstreken. After several years of research abroad (Switzerland, Ethiopia and The United Kingdom) he moved to the lab of Prof. Frank Grosveld. Here he obtained his PhD studying the spatial organization of the b-globin locus under supervision of Dr. Wouter de Laat. The spatial organisation of the β-globin locus was investigated using novel 3C technology and it was demonstrated that transcription factors can play an essential role in the three-dimensional organisation of gene loci. These were the first studies elucidating the spatial organisation of a mammalian gene locus, thereby demonstrating how cis-regulatory elements communicate with genes.
After his PhD he continued to study long-range gene regulation at the ErasmusMC department of Cell biology. In 2007 he received a VENI grant from the Netherlands Organisation for Scientific Research (NWO) to study the molecular basis of long-range b-globin expression.
His current research interests include:
Pigmentation of skin, eye and hair reflects some of the most evident common phenotypes in humans. Several candidate genes for human pigmentation are identified, and the SNP rs12913832 has strong statistical association with human pigmentation. It is located within an intron of the non-pigment gene HERC2, 21 kb upstream of the pigment gene OCA2, and the region surrounding rs12913832 is highly conserved among animal species. We demonstrate that the HERC2 rs12913832 region functions as an enhancer regulating OCA2 transcription. In darkly pigmented human melanocytes carrying the rs12913832 T-allele, we detected binding of key transcription factors to the HERC2 rs12913832 enhancer, and a long-range chromatin loop between this enhancer and the OCA2 promoter which leads to elevated OCA2 expression. In contrast, in lightly pigmented melanocytes carrying the rs12913832 C-allele, chromatin-loop formation, transcription factor recruitment and OCA2 expression are all reduced. This study provides the key mechanistic insight that allele-dependent differences in chromatin-loop formation (i.e. structural differences in the folding of gene loci) results in differences in allelic gene expression that affects common phenotypic traits. This concept is highly relevant for future studies aiming to unveil the functional basis of genetically-determined phenotypes including diseases.
Dr Peter Scacheri, Case Western, USAVariant enhancer loci in cancer
Peter Scacheri is an Assistant Professor in the Department of Genetics and Genome Sciences at Case Western Reserve University, and a member of the Case Comprehensive Cancer Center. He received his undergraduate training at Gettysburg College, his PhD in Biochemistry and Molecular Genetics from the University of Pittsburgh, and his postdoctoral training at the National Human Genome Research Institute. He is a genomicist known for his studies on the role of gene regulatory elements in human disease.
Cancer is a disease involving gene expression aberrations that result in altered cell identity and function. Studies have largely focused on coding sequences and promoter regions, despite the fact that distal regulatory elements play a central role in controlling gene expression patterns. We utilized the epigenetic mark H3K4me1 to globally analyze gains or losses of enhancer activity in primary colon cancer lines relative to normal colon crypt. We identified thousands of variant enhancer loci (VELs) that are shared at high frequency across independent colon cancer samples. These common VELs define a signature that is highly predictive of the in vivo colon cancer transcriptome. Furthermore, VELs are significantly enriched in haplotype blocks containing colon cancer genetic risk variants, implicating these genomic regions in colon cancer pathogenesis. Our data demonstrate that reproducible changes in the epigenome at enhancer elements drive a specific transcriptional program to promote colon carcinogenesis. Our study also illustrates a novel concept in epigenomics.
Specifically, that epigenomic enhancer profiling and VEL mapping can cut through the complexity of heterogeneous gene expression profiles to pinpoint genes that are key mediators of cancer phenotype.
Prize lecture 18 June
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