Characterization of the spatiotemporal architecture of the genome and its relevance to biological function

A variety of cis-regulatory elements govern the spatial organization of chromatin. The exact cause and consequence of this organization with respect to transcriptional activity remains elusive. Here we present an approach based on multi-color imaging to discriminate the specificity of various cis-regulatory elements in establishing and maintaining discrete chromosomal configurations at the single cell level. Specifically, targeting a locus with multiple enhancers driving expression in distinct spatial domains, we simultaneously measure the position of these elements inside and outside of their domains of control. We observe the median distance between an enhancer and active promoter is approximately 160 nm. Interestingly, distances between enhancer-promoter pairs are larger (170-220 nm) in transcribing domains in which the tagged enhancer is not actively driving transcription. This is in contrast to inactive domains which all demonstrate a relatively compact organization with median distances ranging from 120-160 nm, suggesting that transcription causes a local decompaction in conjunction with sustained enhancer-specific proximity. The magnitude of these distances is too large, however, to imply that there is sustained direct contact. To further characterize the dynamics of specific enhancer-promoter interactions we designed a system to track both the position and activity of genes and their regulatory elements in vivo. We are able to observe insulator-mediated enhancer-promoter interactions across genomic separations ranging from 47 kb to 568 kb. Surprisingly, we also observed unassisted enhancer-promoter interactions at genomic distances of > 70 kb which cease to exist at over 140 kb. Leveraging the dynamic information present in this data, we observed complex and dynamic interactions between transcription, spatial configurations, and their respective temporal stability, with potential functional consequences for the course of development

Transcription-dependent spatial organization of a gene locus

There is growing appreciation that gene function is connected to the dynamic structure of the chromosome. Here we explore the interplay between three-dimensional structure and transcriptional activity at the single cell level. We show that inactive loci are spatially more compact than active ones, and that within active loci the enhancer driving transcription is closest to the promoter. On the other hand, even this shortest distance is too long to support direct physical contact between the enhancer-promoter pair when the locus is transcriptionally active. Artificial manipulation of genomic separations between enhancers and the promoter produces changes in physical distance and transcriptional activity, recapitulating the correlation seen in wild-type embryos, but disruption of topological domain boundaries has no effect. Our results suggest a complex interdependence between transcription and the spatial organization of cis-regulatory elements

Stochastic motion and transcriptional dynamics of pairs of distal DNA loci on a compacted chromosome

Chromosomes in the eukaryotic nucleus are highly compacted. However, for many functional processes, including transcription initiation, the 3D pair-wise motion of distal chromosomal elements, such as enhancers and promoters, is essential and necessitates dynamic fluidity. Therefore, the interplay of chromosome organization and dynamics is crucial for gene regulation. Here, we use a live imaging assay to simultaneously measure the positions of pairs of enhancers and promoters and their transcriptional output in the developing fly embryo while systematically varying the genomic separation between these two DNA loci. Our analysis reveals a combination of a compact globular organization and fast subdiffusive dynamics. These combined features cause an anomalous scaling of polymer relaxation times with genomic separation and lead to long-ranged correlations compared to existing polymer models. This scaling implies that encounter times of DNA loci are much less dependent on genomic separation than predicted by existing polymer models, with potentially significant consequences for eukaryotic gene expression

Dynamic interplay between enhancer–promoter topology and gene activity

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