4D Nucleome Opportunity Pool Initiative announces:
Transformative Collaborative Project Awards
The Transformative Collaborative Project Award (TCPA) provides research support for up to 24 months to enable eligible investigators to pursue novel research directions or develop new technologies and tools that will further help the 4DN Program to achieve its stated goals. Applicants are asked to use the TCPA to build unique trans- or interdisciplinary teams that are focused on finding solutions to complex or challenging biological problems relevant to the 4DN mission and to propose research or tool-development projects that complement existing 4DN efforts. All non-4DN applicants who receive a TCPA will become 4DN Investigators for the duration of the TCPA project and will be expected to abide by all guidelines and policies established by the network.
Elucidating the role of nascent RNA in enhancer-promoter communication and three-dimensional genome organization
PI: Karen Adelman, Co-PI: Joanna Wysocka
Harvard Medical School
There is a clear relationship between nuclear genome organization and the regulation of gene expression, and a great deal of effort has been dedicated to defining the effects of genome architecture on gene activity. Herein, we propose to complement this work by approaching the relationship from the other direction. We will ask: how does nascent RNA synthesis from enhancers and promoters impact genome organization? Our specific hypothesis is that nascent RNA plays a central role in stabilizing transient promoter-enhancer contacts that occur within topologically-associated domains (TADs), and thus facilitates the communication between distal cis-regulatory regions. This idea is supported by recent demonstrations that nascent RNA can serve as a platform for the recruitment and retention of transcription factors and epigenetic modifiers. Accordingly, several studies have suggested roles for specific enhancer RNAs in the establishment or maintenance of three-dimensional (3D) enhancer-promoter loops and associated gene activity. However, a full appreciation for the role of nascent RNA synthesis on
genome structure will require a more rigorous, coordinated and comprehensive approach than has been carried out to date. We therefore propose a collaborative project within the framework of the 4D Nucleome network to address how enhancer and gene transcription impact chromosome organization.
Establishing the 3D chromatin architectural organization of the zebrafish embryonic genome
PI: Brad Cairns, Co-PI: Antonio Giraldez
University of Utah
Three-dimensional (3D) genome organization is important for proper gene regulation, and impacts development and disease. Topological Associating Domains (TADs) comprise the basic unit of organization within the genome, with sharp boundaries characterized by highly transcribed housekeeping genes, short interspersed nuclear elements (SINE) or sites for the DNA-binding protein CCCTC-binding factor (CTCF)
which interacts with Cohesin. This proposal addresses several major unanswered questions and explores new opportunities in the field, including: i) When does 3D genome architecture (TADs and their boundaries) begin to form during early vertebrate embryogenesis? ii) Is genome-wide transcriptional activation in the developing embryo required for the establishment of TADs? iii) How dynamic are individual TADs at the single cell level and what is their effect on gene expression and nuclear architecture? Importantly, our combination of genomics with super-resolution imaging bridges and connects two major disciplines in the 4D Nucleome community.
Our central goal is to determine when and how higher-order 3D chromatin architecture is first established in early embryos, how is it remodeled during developmental reprograming and what is their impact on transcription. This proposal is significant for several reasons. First, from a standpoint of nuclear architecture, it addresses how TADs are formed over time and what is their effect on genome
activation and nuclear architecture. Second, it addresses how positional information in the nucleus plays a role in the fundamental step of transcription in vivo. Third, within the context of the 4D Nucleome initiative and a technological standpoint, the zebrafish embryo is a powerful system for probing different technologies that
that can be further applied across the 4D Nucleome initiatives directly relevant to ES cells, mouse embryos and differentiated tissues to understand how nuclear architecture is established in vivo. From a developmental biology and fertility standpoint, we address fundamental mechanisms of initiation of gene expression after fertilization - a universal transition across animals.
In our proposal, we provide preliminary evidence supporting the following hypothesis: TADs and their boundary elements are fully established only after the onset of genome-wide transcriptional activation, to refine embryonic transcription. The experiments described below will define the true relationship of TADs
to transcription, illuminating the fundamental mechanisms responsible for assembling the 4D nuclear architecture during embryogenesis when cells become totipotent. Together, our laboratories will combine advanced genomics techniques (Hi-C, PLAC-seq, others), super-resolution/EM microscopy methods and innovative genetic and molecular approaches in zebrafish embryos, toward addressing these questions.
Multi-Contact Conformation Capture: uncovering regulatory hubs and mutually exclusive topologies
PI: Wouter de Laat, Co-PI: Jeroen de Ridder
Current C strategies measure 2-way contacts between pairs of sites. The results demonstrate that, in a population of cells, individual genomic sites often contact more than one other distal site. For example, many genes appear in contact with multiple dispersed regulatory DNA elements and vice versa, many regulatory DNA elements appear to contact each other as well as multiple genes. It is often proposed that such contacts occur simultaneously in so-called ‘hubs’. Yet, cell population-based pair-wise contact matrices do not allow making statements about cooperative interactions versus mutually-exclusive interactions. To understand the nature (and mere existence) of chromatin hubs, we need to know how the multitude of genes and regulatory sequences coordinate their action in 3D space. To do this in a meaningful manner, high-throughput strategies must be developed alongside with robust statistical methods and intuitive visualization tools for the detection, analysis and interpretation of multi-way DNA contacts. For this, we have invested in and propose to further develop and apply Multi-Contact Chromatin Capture that exploits Oxford Nanopore Technologies (ONT) long single molecule sequencing technology. Using Multi-Contact HiC (MC-HiC) we will generate genome-wide multi-way contact maps of >100x coverage for at least two of the 4DN reference cell lines (GM12878 and Castx129 ESCs). In addition, we will employ our targeted strategy, Multi-Contact 4C (MC-4C), for in depth analysis of the most intricate spatial regulatory
networks. Our work is expected to greatly enhance our understanding of how the multitude of regulatory sequences and genes spatially coordinate their action on individual alleles. It should also deliver valuable new molecular biology and computational tools for studying these under-explored dimensions of genome folding and functioning.
Tethered nuclease strategies for in situ mapping of 3D nuclear organization
PI: Steve Henikoff, Co-PIs: Kamran Ahmad, Jay Shendure, William Noble
Fred Hutchinson Cancer Research Center
The 4D Nucleome project focuses on describing the 3D organization within the nucleus, with
the ultimate goal of understanding this organization in mechanistic terms. Our project uses novel methods for epigenomic profiling that detect 3D contact sites without cross-linking and with much higher resolution than current technologies. We recently introduced a novel strategy for chromatin profiling called CUT&RUN (Cleavage Under Targets & Release Using Nuclease), in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. The method yields precise transcription factor (TF) profiles, yet is simple to perform and is inherently robust, with extremely low backgrounds requiring ~1/10 th the sequencing depth of chromatin immunoprecipitation (ChIP). CUT&RUN binding and cleavage occurs in situ, allowing for both quantitative high-resolution chromatin mapping and probing of the 3D chromatin environment. Together with our new native ChIP-seq protocol, we distinguish direct “anchor” sites of a
chromatin-bound protein from contacting sites without fixation, a kind of inference has not been possible with current methods of interrogating 3D chromatin architecture. We have two Aims: First, we will annotate the genome at high density for CTCF and cohesin sites, distinguishing between anchor and contact sites in 4D Nucleome cell lines. Second, we will continue development of a new replacement technology for 3D contact mapping, using CUT&RUN as a “front-end” for proximity ligation (CUT&PASTE – Cleave Under Targets & Polish And Splice Touching Ends), building on our novel sci-HiC protocol for high-resolution TF-specific 3D interaction mapping. By annotating more contact sites in genomes and assigning the directionality of contacts, we move towards a mechanistic model of how topology within the nucleus is organized. Participation in the 4DN program would provide the opportunity to compare and integrate our CUT&RUN/PASTE datasets with 4DN datasets on common cell lines and differentiating tissues, ideally positioning consortium investigators to adopt our alternative strategy for their own 4DN efforts.
Inference and Validation of Chromosomes 3D Structure via Statistical Shape Analysis of Elastic Curves Models
PI: Nicola Neretti, Co-PIs: Anuj Srivastava, Chao-Ting Wu
How to best infer, and compare across experimental conditions, the 3-dimensional (3D)
structure of chromosomes by using data from chromosome conformation capture (3C) techniques is still an open question. Although several methods have been proposed, these have not been systematically validated at the single cell level. In this grant application, we propose to frame these three problems, namely inference, comparison and validation, within the statistical framework of functional and shape analysis. The problem of shape analysis of three dimensional (3D) curves has seen major advances in recent years, bringing together tools from differential geometry and statistics, and reaching efficient computational solutions. These ideas, based on Riemannian frameworks, utilize elastic Riemannian metrics on representation spaces of curves and computing geodesic deformations for quantifying shape differences. Elastic implies that the problem of registration of points across curves is solved simultaneously with the quantification of their shape differences. Different parts of curves are
stretched and compressed to optimally match geometric features (e.g. bends and corners) across curves, in a fully automated way. The chosen metrics allow the re-parameterization or registration groups to act on curves in an isometric manner, an important requirement for shape analysis, thus enabling an elastic comparison of shapes. From a more practical point of view, the use of certain square root transformations flattens elastic metrics into Euclidean metric and standard Hilbert structure (L2), so that past efficient algorithms from functional data analysis become applicable. This leads to precise tools for comparing, matching, summarizing, modeling, and testing shapes of Euclidean curves. We propose to adopt tools from elastic shape analysis of 3D curves for estimation and analysis of 3D chromosome structures from 3C technologies, and to integrate them with high-resolution microscopy
for validation and study of variability of structures across individual cells.
The Cell Nucleus under Stress
PI: Karla Neugebauer, Co-PI: Joan Steitz
Environmental stress alters the organization and function of the cell nucleus. Stress induces dramatic shifts in the transcriptional activity and nuclear positioning of genes, suggesting extensive remodeling of the genome upon heat and osmotic shock, UV damage, viral infection, hypoxia, etc. Moreover, membraneless nuclear bodies, such as nucleoli and Cajal bodies (CBs) – nucleated by low complexity (LC) proteins at specific chromosomal loci – are disrupted. These loci cluster in nuclear bodies in unstressed cells, raising the possibility that stress
induces widespread changes in chromosome topology. Despite this evidence, a systematic investigation of how stress changes the 4D nuclear landscape has not been undertaken.
Our labs are expert in the investigation of nuclear bodies and of long non-coding RNAs (lncRNAs). The Neugebauer lab has shown that nuclear bodies form at active sites of transcription and that LC proteins, such as coilin in CBs, can be ChIPped at gene loci and crosslinked to the corresponding RNAs in vivo. Thus, our methods use DNA and RNA sequencing to pinpoint genomic location(s) of nuclear bodies at molecular resolution. The
Steitz lab has recently discovered thousands of novel lncRNAs produced by transcriptional read-through of protein-coding genes. These DoGs (Downstream of Gene transcripts) –induced by osmotic stress via the IP3 receptor pathway – are up to 45kb in length and >2000 in number in human cells. Also induced by heat and oxidative stress, DoGs thereby define transcriptionally active “intergenic” regions that likely contribute to and/or result from changes in the 4D organization of the genome under stress. Using heat, osmotic and oxidative stress as models, we propose to identify changes in 1) chromosome topology through Hi-C, 4C, and ChIA-PET, 2) regions of genome activity through Transient Transcriptome (TT)-Seq, ChIP-Seq and development of TT-CLIP to monitor LC protein interactions with DNA and RNA at high spatial and temporal resolution, and 3) imaging of nuclear bodies and clustered genomic loci using super-resolution fluorescence microscopy. The functional significance of nuclear bodies and DoGs will be tested during stress and recovery. A unifying theme is that LC proteins and lncRNAs are unconventional molecular species with emerging roles in genome organization. Our preliminary data show the induction of DoGs and disruption of CBs over time in human and mouse cells. Intriguingly, the CB component SMN – an LC protein implicated in transcription termination – remains in nuclear bodies upon osmotic stress only, suggesting differential functions in stress responses. All experiments will be conducted in human tissue culture cells to maximize compatibility with other 4DN projects. Our project contributes novelty and innovation to the 4DN Initiative through: the use of RNA and DNA sequencing data to identify genomic regions of interest, our commitment to uncovering comprehensive changes in genome organization due to stress, our interest in a novel class of lncRNAs (DoGs), and our ability to bridge sequencing and imaging approaches.
Dynamic 3D folding of the mammalian genome: molecular determinants and impact on gene expression in vivo
PI: Francois Spitz, Co-PIs: Leonid Mirny, Thomas Gregor
Institute Pasteur, Paris, France
For many genes cis-acting elements located hundreds of kilobases away from the promoter region provide critical regulatory information, as exemplified by the dramatic consequences of mutations of such elements in humans. The 3D folding of the genome plays an essential role in regulating the communication between distant elements, and therefore contributes to the robustness and specificity of the transcriptional programs that control cell fate and function. Therefore understanding the molecular basis of the processes that organize the 3D arrangement of the genome and how they impact enhancer-promoter interactions resulting in specific gene expression levels will be of particular importance. Here we propose to identify cis-acting elements and chromatin features that physically organize the 3D conformation of genomic loci in order to fine-tune gene expression. For this purpose, we will develop a cross-disciplinary approach using advanced chromosomal engineering tools, live-imaging with high spatial and temporal resolution as well as computational modeling. We will generate high-resolution Hi-C maps of genomic regions in a large collection of mouse strains carrying local chromosomal rearrangements impacting defined genomic loci or mutations in key regulators of chromatin conformation. We will use these large, coherent datasets to improve predictive models of chromatin folding, and identify regions and elements orchestrating the specific conformations adopted by a locus. We will develop and perform live-cell imaging of enhancer-promoter interactions in different contexts, and use biophysical modelling of this process to
identify the connections between structural dynamics and gene expression changes. Altogether, these detailed, functional analyses should provide novel insights into the molecular
mechanisms that guide the 3D folding of the genome in different structures and their role in
determining gene expression. We will conduct most of our studies in vivo, in the biological context where these different structures modulate enhancer-promoter interactions to control gene expression and cellular phenotypes. Consequently, our data will directly provide insights into the molecular and physiological consequences associated with altered 3D organization observed in human patients at the corresponding loci. Furthermore, we expect that our improved models of chromatin folding and enhancer-promoter communication will allow to better interpret current 3D maps and will open the way to better evaluate the consequences of structural variants that could be identified in human patients.
Development of TrAC-loop, a novel technique to detect genome-wide chromatin interactions
PI: Keji Zhao, Co-PIs: Gangqing Hu, Binbin Lai, Qingsong Tang
Most current techniques for genome-wide analysis of chromatin interactions are based on the
chromosome conformation capture (3C) technique. Because the traditional Hi-C technique uses a 6-bp restriction enzyme to cut chromatin, which usually finds one cleavage site every 5000bp in the genome, it does not have sufficient resolution for identification of enhancer-promoter interactions. Significant improvement has been achieved by the in situ Hi-C protocol, which uses a 4-bp cutter enzyme and reaches a resolution of about 1kb, thus enabling identification of specific enhancer-promoter interactions at high-resolution. However, this method requires a costly sequencing depth of 5 to 10 billions of paired-end tags (PETs) per library, which prohibits its application to a large number of samples. Other 3C-based techniques have been developed to focus on interactions at selected regions by capture Hi-C or tethered through specific proteins by ChIA-PET have helped to increase resolution by focusing on potential regulatory regions of the genome. However, a recent study compared
chromatin interactions detected by fluorescence in situ hybridization (FISH) and 3C-based assays and found a high degree of discrepancy between the two techniques, suggesting that cross-validation of interaction data using different strategies is critical.
We propose to develop a novel technique, TrAC-loop, for Transposition-mediated Analysis of
Chromatin loops in the genome. The method does not require SDS-mediated partial decondensation of chromatin, restriction enzyme digestion, or proximity-based ligation of chromatin fragments. TrAC-loop directly captures interacting chromatin regions by Tn5-mediated transposition of a bivalent ME (mosaic end) linker without disrupting the nuclear structure. Thus, this novel strategy avoids potential artifacts derived from SDS-mediated partial decondensation of chromatin, restriction enzyme digestion and proximity-based re-ligation of chromatin fragments that are used in the 3C assays. Our strategy represents a shift in paradigm. All other genome-wide methods of mapping chromatin interactions use 3C-based techniques, whereas ours use transposition. Although transposition has been used to tag
chromatin, this is the first time it is used to join chromatin segments.
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