Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - 3DQuant (Understanding long-range transcriptional regulation in the context of the 3D genome organization)

Teaser

Enhancers are DNA sequences elements that in metazoans modulate transcription of a target promoter from (often very large) genomic distances1-3. Such type of long-range regulation is thought to arise from the formation of chromosome loops enabling physical interactions between...

Summary

Enhancers are DNA sequences elements that in metazoans modulate transcription of a target promoter from (often very large) genomic distances1-3. Such type of long-range regulation is thought to arise from the formation of chromosome loops enabling physical interactions between an enhancer and its cognate promoter3,4. However, how specific patterns physical interactions between enhancers and promoters are established, and how regulatory information is relayed is still unclear.
Recent studies have shown that the structure of mammalian chromosomes is partitioned into consecutive self-associating domains called Topologically Associating Domains (TADs)5-7. TADs not only constitute a unit of chromosome folding but, as shown by many functional studies, coincide with regulatory domains where enhancer function is constrained to certain sets of promoters8,9. Studying how the chromatin fiber folds within the nucleus and how structures such as TADs influence gene regulation has become the focus of many research groups not only for its relevance in fundamental scientific research but also because it relates to the genetic disorders10,11. Several studies have indeed shown that defects in chromatin structure that impact enhancer-promoter interactions are responsible for the onset of some genetic disorders. Yet how TADs are able to modulate enhancer action, and thus long-range transcription, is still unknown and quantitative studies dissecting the importance of this structure for the enhancer function are still missing.
Our main goal was to determine the quantitative relationship between the three-dimensional (3D) chromatin architecture (and namely the presence of TADs) and promoter activity to unravel the mechanism of long-range transcriptional modulation mediated by enhancers. Our first objective was to establish a mouse embryonic stem cell (mESC) system enabling to measure promoter-enhancer physical interactions and gene activity in parallel and in a quantitative manner, in an environment devoid of additional regulatory or structural interactions. By using this system our main goals were to assess how TADs influence the action of the enhancer on its cognate promoter as well as to explore how fluctuations in enhancer-promoter interactions impact cell-to-cell and temporal transcriptional variability.

References:
1. Visel A, et al. Nature 457:854-858 (2009)
2. Visel A, et al. Nature 461:199-205 (2009)
3. Lettice LA, et al. Hum Mol Genet 12:1725-1735 (2003)
4. Sanyal A, et al. Nature 489:109-113 (2012)
5. Dixon JR, et al. Nature 485:376-380 (2012)
6. Nora EP, et al. Nature 485:381-385 (2012)
7. Sexton T, et al. Cell 148:458-472 (2012)
8. Shen Y, et al. Nature 488:116-120 (2012)
9. Symmons O, et al. Genome Res 24:390-400 (2014)
10. Lupianez DG, et al. Cell 161:1012-1025 (2015)
11. Kragesteen BK, et al. Nat Genet 50:1463-1473 (2018)

Work performed

Our research strategy was to de novo engineer a genomic region in mESCs, constituted by a selected enhancer and its cognate promoter driving the expression of a reporter gene in a designated TAD where the enhancer can be mobilized at many various distances from the promoter. This system gives the possibility to study a single enhancer-promoter pair in the context of the 3D chromatin organization in the absence of confounding effects due to overlapping regulatory or structural interactions and enables quantitative measurements of promoter activity as a function of enhancer location and their mutual contact probability.
By using next-generation sequencing-based experiments that allow to analyze chromatin states (ChIP-seq) and chromosome conformation (Hi-C) on a genome-wide level, we selected a chromosomal locus with low level of genomic activity without being in a repressive heterochromatic environment, and where the chromatin fiber has a simple 3D structure.
We then successfully cloned targeting vectors required to generate an engineered locus that allows enhancer mobilization around a fluorescent reporter gene used as a readout for promoter activity. By testing these targeting vectors in transient transfection experiments, we measured weak basal transcription levels arising from the promoter and an increase in transcription when the enhancer was placed in close proximity to the promoter. This argues in favor of our hypothesis that once integrated in the genome, the reporter would be more transcribed in cells where the enhancer is located next or frequently occurs in 3D proximity to the promoter compared to cells carrying distal insertions of the enhancer. We therefore knocked-in the targeting vector in mESC and by measuring the expression levels of the reporter gene, we could rapidly isolate by FACS sort clonal lines where the enhancer is mobilized around the promoter. These proof-of-principle experiments demonstrated that our design and experimental set-up is suitable for a quantitative study aiming to understand the mechanism that link chromatin structure and long-range transcriptional regulation. Although we recently realized that our initial knock-in mESC lines carried multiple tandem copies of the targeting vector, we are currently generating novel knock-in lines with an optimized version of the plasmid. As soon as mESC lines with single-copy integrations will be available, they will be used to quantitatively assess how the 3D chromatin structure influences enhancer action by measuring in parallel promoter-enhancer interactions and gene activity using a combination of 3C-based technologies and single-cell methods such as quantitative RNA-DNA FISH, and live-cell imaging of transcription.

Final results

Despite its relevance in many research areas, we still poorly understand if and how chromatin architecture is able to control enhancer action on its target promoter. To date, most of the studies aimed to dissect enhancer-promoter communication are mainly of qualitative in nature and do not consider potential confounding effect deriving from regulatory elements or interactions present around the selected elements.
Conversely, our experimental system allows to study a single enhancer and promoter pair in the context of simple 3D chromatin architectures in a quantitative manner study. Based on our proof-of principle experiments, we are able to rapidly generated hundreds of knock-in lines where the enhancer is located at different distances from the cognate promoter. Measuring enhancer-promoter interactions and gene activity in these cell lines will lead to an unprecedented amount of quantitative data. These will be beneficial to understand the basic mechanisms of long-range transcriptional regulation and will be not only valuable for scientists working on gene regulation but also relevant to understand the principles by which chromatin architecture can influence the onset of some genetic disorders.