Periodic Reporting for period 1 - RE_LOCATE (Location, location, location: the (re-)positioning of regulatory elements in the mammalian genome.)
Periodo di rendicontazione: 2022-09-01 al 2025-02-28
Regulation of gene expression underlies the enormous diversity of cell types, and the responses of cells to myriad signals. Correct regulation of gene expression is thus critical, and defects in this process underlie many human disorders.
To a large extent, gene expression patterns are driven by regulatory DNA sequences that surround the genes and control their transcriptional activity. These regulatory elements include promoters (positioned at the start of each gene) and enhancers, which are often located tens to hundreds of kb away from the gene that they regulate. How such apparently haphazard linear arrangements can result in specific gene regulation is a major puzzle. To unravel this logic, it is necessary to systematically alter the positions of enhancers, promoters and other regulatory elements and study the effect on gene activity. So far, no efficient method has been available for this purpose. Our aim is to develop and apply RElocate, a scalable, broadly applicable technology to transplant selected DNA elements to hundreds of alternative positions within a ~2 Mb region, and track the functional consequences.
We will employ RElocate in combination with a high-throughput combinatorial reporter assay to systematically study how enhancers "choose" and activate the correct target promoter(s). Furthermore, we will adapt RElocate to precisely map how enhancers and promoters contact and communicate with each other. Finally, we will use the method to fine-map the repressive/activating chromatin landscape of selected regions at high resolution, and elucidate how enhancers and promoters may respond differently when inserted throughout this landscape.
This work will reveal how the ordering and spacing of regulatory elements along the genome contributes to optimal gene regulation, and will yield a powerful perturbation tool with many applications in genome biology and human genetics.
To a large extent, gene expression patterns are driven by regulatory DNA sequences that surround the genes and control their transcriptional activity. These regulatory elements include promoters (positioned at the start of each gene) and enhancers, which are often located tens to hundreds of kb away from the gene that they regulate. How such apparently haphazard linear arrangements can result in specific gene regulation is a major puzzle. To unravel this logic, it is necessary to systematically alter the positions of enhancers, promoters and other regulatory elements and study the effect on gene activity. So far, no efficient method has been available for this purpose. Our aim is to develop and apply RElocate, a scalable, broadly applicable technology to transplant selected DNA elements to hundreds of alternative positions within a ~2 Mb region, and track the functional consequences.
We will employ RElocate in combination with a high-throughput combinatorial reporter assay to systematically study how enhancers "choose" and activate the correct target promoter(s). Furthermore, we will adapt RElocate to precisely map how enhancers and promoters contact and communicate with each other. Finally, we will use the method to fine-map the repressive/activating chromatin landscape of selected regions at high resolution, and elucidate how enhancers and promoters may respond differently when inserted throughout this landscape.
This work will reveal how the ordering and spacing of regulatory elements along the genome contributes to optimal gene regulation, and will yield a powerful perturbation tool with many applications in genome biology and human genetics.
We successfully established and optimized the RElocate technology to systematically relocate DNA sequence elements to >1,000 alternative positions in a locus. This provides unprecedented and highly detailed functional maps of a genomic locus.
We have leveraged this technology to map the functional landscape of the mouse Sox2 gene, which is controlled by a strong enhancer that is located ~100 kb away. The Sox2 locus has been well-studied, but it has remained poorly understood how the functional interaction between the enhancer and promoter is shaped by the context of the genome locus as a whole. By systematic relocation of the Sox2 promoter throughout its native locus – in several instances in combination with specific genetic alterations – we obtained striking new mechanistic insights. We found that the ‘realm of activity’ of the enhancer shows an intricate landscape of peaks and valleys and is sharply confined. The Sox2 gene seems to be located in a ‘sweet spot’ within this realm. Hence, its location ~100 kb away from the enhancer is in fact highly optimal for its activation. Surprisingly, the Sox2 gene is not merely a passive respondent to the enhancer; rather, it strongly limits the activity and the realm of the enhancer, suppressing activation of a second promoter anywhere in the locus. We also obtained evidence that the three-dimensional folding of the chromosome facilitates communication between the enhancer and the gene, and that the machinery that helps to establish this folding (named cohesin) helps in this process, working its way from the enhancer towards the promoter.
We are currently applying the RElocate technology to another locus, which is very gene-dense. In this locus we systematically relocate a strong enhancer and monitor the effects on all genes in the locus. This will reveal (i) over which distance the enhancer can act, and (ii) whether some genes are intrinsically more responsive to the enhancer.
Furthermore, we have begun to dissect the "compatibility" rules that determine how promoters respond to their chromatin environment, including nearby enhancers. For this we designed a set of 60 reporters that each are controlled by only a single transcription factor (TF). We integrate a subset of these reporters into thousands of genomic locations (either by RElocate or by our TRIP technology) and compare their responsiveness to a variety of nearby features. Because each reporter is driven by only a single TF, this should reveal how individual TFs can dictate the response of promoters to regulatory elements and chromatin packaging.
We have leveraged this technology to map the functional landscape of the mouse Sox2 gene, which is controlled by a strong enhancer that is located ~100 kb away. The Sox2 locus has been well-studied, but it has remained poorly understood how the functional interaction between the enhancer and promoter is shaped by the context of the genome locus as a whole. By systematic relocation of the Sox2 promoter throughout its native locus – in several instances in combination with specific genetic alterations – we obtained striking new mechanistic insights. We found that the ‘realm of activity’ of the enhancer shows an intricate landscape of peaks and valleys and is sharply confined. The Sox2 gene seems to be located in a ‘sweet spot’ within this realm. Hence, its location ~100 kb away from the enhancer is in fact highly optimal for its activation. Surprisingly, the Sox2 gene is not merely a passive respondent to the enhancer; rather, it strongly limits the activity and the realm of the enhancer, suppressing activation of a second promoter anywhere in the locus. We also obtained evidence that the three-dimensional folding of the chromosome facilitates communication between the enhancer and the gene, and that the machinery that helps to establish this folding (named cohesin) helps in this process, working its way from the enhancer towards the promoter.
We are currently applying the RElocate technology to another locus, which is very gene-dense. In this locus we systematically relocate a strong enhancer and monitor the effects on all genes in the locus. This will reveal (i) over which distance the enhancer can act, and (ii) whether some genes are intrinsically more responsive to the enhancer.
Furthermore, we have begun to dissect the "compatibility" rules that determine how promoters respond to their chromatin environment, including nearby enhancers. For this we designed a set of 60 reporters that each are controlled by only a single transcription factor (TF). We integrate a subset of these reporters into thousands of genomic locations (either by RElocate or by our TRIP technology) and compare their responsiveness to a variety of nearby features. Because each reporter is driven by only a single TF, this should reveal how individual TFs can dictate the response of promoters to regulatory elements and chromatin packaging.
Over the past two decades, thousands of papers have focused on the descriptive mapping of the epigenome and 3D genome. The goal of these efforts has been to understand the regulation of the genome, often focused on specific genes or genomic loci. However, epigenome/3D maps are biochemical correlates, not functional maps: it has remained very difficult to identify regulatory elements with high confidence, and even more difficult to infer the functional interactions between these elements. Our high-density hopping approach generates for the first time high-resolution functional maps of a genomic locus, from which causal inferences can be made regarding the link between genomic location and the function of regulatory elements, and the interplay between these elements. We will now apply this methodology further to study other genomic loci, and integrate the results with other experimental data to gain new and generalisable insights into the fundamental mechanisms of gene regulation.