Regulation of gene expression from a distance: exploring mechanisms

Professor Nicholas Hastie CBE FRS, Medical Research Council, UK

Professor Nicholas Hastie CBE FRS, Medical Research Council, UK

The evolution of global enhancers

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

Abstract

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

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The pluripotent 3D genome

Professor Wouter de Laat, Hubrecht Institute, The Netherlands

Abstract

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.

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Maps of open chromatin from association signals to function

Dr Panos Deloukas, Wellcome Trust Sanger Institute, UK

Abstract

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.

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Deciphering the cis-regulatory code

Dr Eileen Furlong, EMBL, Germany

Abstract

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.

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Chair

Professor Anne Ferguson-Smith, University of Cambridge, UK

Dynamic use of enhancers in development

Professor Mike Levine, University of California Berkeley, USA

Abstract

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.

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Gene expression genomics

Dr Sarah Teichmann, LMB, UK

Abstract

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.

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Exploring gene expression at the single cell level

Dr Jonathan Chubb, University College London, UK

Abstract

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

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Transcription factor search kinetics explored at the level of single molecules

Dr Johann Elf, Uppsala University, Sweden

Abstract

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.

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Chair

Professor Constanze Bonifer, University of Birmingham, UK

Design rules for bacterial enhancers

Dr Roee Amit, Technion-Israel Institute of Technology, Israel

Abstract

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.

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Complex protein dynamics at eukaryotic regulatory elements

Dr Gordon Hager, National Cancer Institute, NIH, USA

Abstract

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.

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Transcription factors and DNA regulatory elements

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

Abstract

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.

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Controlling long-range genomic interactions to reprogram the β-globin locus

Dr Gerd Blobel, The Children’s Hospital of Philadelphia, USA

Abstract

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, USA
Wulan Deng, The Children’s Hospital of Philadelphia and University of Pennsylvania, USA
Andreas Reik, Philip D Gregory, Sangamo BioSciences Inc, USA

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Dr Duncan Odom, University of Cambridge, Cancer Research UK, and Wellcome Trust Sanger Institute, UK

Dr Duncan Odom, University of Cambridge, Cancer Research UK, and Wellcome Trust Sanger Institute, UK

Massively parallel functional dissection of mammalian enhancers

Dr Rupali Patwardhan, University of Washington, USA

Abstract

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.

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HERC2 rs12913832 modulates human pigmentation by attenuating chromatin-loop formation between a long-range enhancer and the OCA2 promoter

Dr Robert-Jan Palstra, ErasmusMC, The Netherlands

Abstract

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.

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Variant enhancer loci in cancer

Dr Peter Scacheri, Case Western, USA

Abstract

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.

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