Periodic Reporting for period 3 - DeCRyPT (Deciphering Cis-Regulatory Principles of Transcriptional regulation: Combining large-scale genetics and genomics to dissect functional principles of genome regulation during embryonic development)
Okres sprawozdawczy: 2022-01-01 do 2023-06-30
Understanding how genomic information is organised and interpreted to give rise to robust patterns of gene expression is a long-standing problem with direct implications for development, evolution and disease. Despite recent advances in locating regulatory elements in animal genomes, there is a general lack of functional data on elements in their endogenous setting – the bulk of our current knowledge comes from reporter assays examining elements out of context, giving insights on sufficiency but not necessity. The functional requirement of very few individual enhancers, and other elements, has been assessed by deletion, with even less known about how the action of multiple elements is integrated. To function, these elements must come together in a specific three-dimensional chromatin context that facilitates precise and robust regulation of gene expression. Understanding how this is achieved is a current major challenge in genome biology, and is the overarching goal of DeCRyPT.
DeCRyPT has three complementary aims to functionally dissect gene regulatory landscapes in their endogenous context during embryogenesis, using the fruit-fly Drosophila as a model system. Aim 1 uses natural sequence variation as a perturbation tool to dissect functional regulatory landscapes. Aim 2 takes a complementary approach to dissect functional regulatory domains through systematic genomic deletions of cis-regulatory elements to dissect their role in gene expression and genome topology. Aim 3 will alter the regulatory context in which enhancers function by reshaping chromatin topology to generate new regulatory environments for developmental genes. These Aims will provide unique functional insights, enabling us to move from correlation to causation in our understanding of genome regulation.
DeCRyPT has three complementary aims to functionally dissect gene regulatory landscapes in their endogenous context during embryogenesis, using the fruit-fly Drosophila as a model system. Aim 1 uses natural sequence variation as a perturbation tool to dissect functional regulatory landscapes. Aim 2 takes a complementary approach to dissect functional regulatory domains through systematic genomic deletions of cis-regulatory elements to dissect their role in gene expression and genome topology. Aim 3 will alter the regulatory context in which enhancers function by reshaping chromatin topology to generate new regulatory environments for developmental genes. These Aims will provide unique functional insights, enabling us to move from correlation to causation in our understanding of genome regulation.
Aim 1 uses natural genetic sequence variation as a perturbation tool to dissect functional regulatory landscapes, by measuring expression quantitative trait loci (eQTL). To expand our previous eQTL study, we used the Global Diversity Lines (GDLs), in collaboration with Andrew Clark’s lab; a panel of D. melanogaster inbred lines isolated from five continents. We performed RNA-Seq on 80 GDL samples at 10-12 hr of embryogenesis. For eQTL analyses, a linear mixed model, LIMIX, was used to account for population structure. We separately tested for eQTL linked to gene expression (gene-eQTL) and exon coverage (exon-eQTL), which provide new insights into functional variants impacting transcriptional regulation. The second goal of Aim 1 is to develop a new computational framework to more directly associate regulatory elements to their target genes. Our new multi-variant mtSet framework reduces multiple testing by an order of magnitude, thereby substantially increasing sensitivity. Due to this enhanced sensitivity of mtSet we could discovery many more eQTL with more complex properties, which we are currently functionally validating.
To identify trans eQTL, we made use of our previously generated data from F1 hybrid embryos to develop a computational framework that uses combined haplotype tests to distinguish between cis and trans genetic effects - recently published in Genome Research (PMID: 33310749).
Aim 2 dissects functional regulatory domains through systematic genomic deletions in embryos. For this we are developing a deletion strategy to achieve multiple targeted deletions in a single multi-enhancer locus. Despite substantial delays due to the complete closure of labs at the host institute for 3 months (March-May 2020) followed by only partial occupancy for over a year since then, we have made substantial progress in this ambitious aim. We hae developed a system to obtain flies with multiplex sgRNAs, which can be easily screened with two.
In addition to generating the fly lines to make the deletions (described above), we are optimizing a method for a miniaturised multiplexed Hi-C approach to determine the impact of deletions on chromatin topology (sub-aim of aim 2). We used balancer chromosomes as a proof of principle for performing Hi-C analysis on rearranged chromosomes and determining the effect on gene expression, which we recently published in Nature Genetics (PMID: 31308546).
Aim 3 will alter the regulatory context in which enhancers function by reshaping the topological associated domains (TADs) in which enhancer reside. To do this, ectopic boundaries will be inserted within large TADs to reveal the molecular requirements to establish a new TAD boundary. Despite delays due to COVID lock-down, we have made great progress in this direction with the successful generation of many insertion lines. Interestingly, some of these are homozygous viable, while others are viable, and some only have an effect in one orientation and not another. We have collected staged embryos on all lines and assessed the impact on chromatin topology (TAD compaction), which has revealed very interesting results.
To identify trans eQTL, we made use of our previously generated data from F1 hybrid embryos to develop a computational framework that uses combined haplotype tests to distinguish between cis and trans genetic effects - recently published in Genome Research (PMID: 33310749).
Aim 2 dissects functional regulatory domains through systematic genomic deletions in embryos. For this we are developing a deletion strategy to achieve multiple targeted deletions in a single multi-enhancer locus. Despite substantial delays due to the complete closure of labs at the host institute for 3 months (March-May 2020) followed by only partial occupancy for over a year since then, we have made substantial progress in this ambitious aim. We hae developed a system to obtain flies with multiplex sgRNAs, which can be easily screened with two.
In addition to generating the fly lines to make the deletions (described above), we are optimizing a method for a miniaturised multiplexed Hi-C approach to determine the impact of deletions on chromatin topology (sub-aim of aim 2). We used balancer chromosomes as a proof of principle for performing Hi-C analysis on rearranged chromosomes and determining the effect on gene expression, which we recently published in Nature Genetics (PMID: 31308546).
Aim 3 will alter the regulatory context in which enhancers function by reshaping the topological associated domains (TADs) in which enhancer reside. To do this, ectopic boundaries will be inserted within large TADs to reveal the molecular requirements to establish a new TAD boundary. Despite delays due to COVID lock-down, we have made great progress in this direction with the successful generation of many insertion lines. Interestingly, some of these are homozygous viable, while others are viable, and some only have an effect in one orientation and not another. We have collected staged embryos on all lines and assessed the impact on chromatin topology (TAD compaction), which has revealed very interesting results.
This project will dissect functionality within the non-coding genome in the context of embryonic development at a scale and complexity that has not been assessed before. More importantly, all the work packages are addressed in vivo investigating the direct biological outcomes at the level of the organism. To achieve this, new methods are needed beyond the current state of the art to: (1) More directly link genetic variation in regulatory regions to the genes they regulate. (2) To delete regulatory regions in embryos at large scale (aim 2). (3) To quantify the effect of changes in regulatory regions on chromatin 3D topology in a highly multiplexed manner. (4) To model the sequence requirements for boundary formation. While there is clear evidence that TAD boundaries are formed by loop extrusion that is blocked or slowed down at CTCF bound sites, CTCF binds to thousands of other regions in the genome that do not form boundaries, indicating that there must be other features required to form an active boundary. In line with this, loss-of-function mutations of CTCF are viable in many species including Drosophila (previous work from my lab), while other species with TAD like structures don’t even have a CTCF in their genome.
Expected results at the end of the project:
The results from aim 1 will use the high resolution of populations genetics in Drosophilds combined with multivariant QTL models to generate a functional map of genetic perturbations that disrupt embryonic gene expression. This is highly complementary to aim 2, which will yield large-scale deletions of regulatory elements in embryos, allowing us to dissect the direct functional relationship between cis-regulatory genome architecture and gene expression in vivo. We recently showed that over a third of enhancer ‘contacts’ span distances greater than 100kb (Ghavi-Helm, Nature 2014), suggesting that long-range regulation may be very common in this small genome. This approach will identify functional regulatory domains, and through deletion will also reduce the size and arrangement of regulatory domains. The results from Aim 3 will perturb regulatory landscapes by changing their topology. The resulting data will reveal new insights into the regulation of gene expression at multiple levels, including revealing information on what it takes to make a boundary and how that impacts enhancer function, gene expression and embryonic development.
Expected results at the end of the project:
The results from aim 1 will use the high resolution of populations genetics in Drosophilds combined with multivariant QTL models to generate a functional map of genetic perturbations that disrupt embryonic gene expression. This is highly complementary to aim 2, which will yield large-scale deletions of regulatory elements in embryos, allowing us to dissect the direct functional relationship between cis-regulatory genome architecture and gene expression in vivo. We recently showed that over a third of enhancer ‘contacts’ span distances greater than 100kb (Ghavi-Helm, Nature 2014), suggesting that long-range regulation may be very common in this small genome. This approach will identify functional regulatory domains, and through deletion will also reduce the size and arrangement of regulatory domains. The results from Aim 3 will perturb regulatory landscapes by changing their topology. The resulting data will reveal new insights into the regulation of gene expression at multiple levels, including revealing information on what it takes to make a boundary and how that impacts enhancer function, gene expression and embryonic development.