Large distances separate coregulated genes in living Drosophila embryos

Abstract

Transcriptional enhancers are short segments of DNA that switch genes on and off in response to a variety of cellular signals. Many enhancers map quite far from their target genes, on the order of tens or even hundreds of kilobases. There is extensive evidence that remote enhancers are brought into proximity with their target promoters via long-range looping interactions. However, the exact physical distances of these enhancer-promoter interactions remain uncertain. Here, we employ high-resolution imaging of living Drosophila embryos to visualize the distances separating linked genes that are coregulated by a shared enhancer. Cotransvection assays (linked genes on separate homologs) suggest a surprisingly large distance during transcriptional activity: at least 100-200 nm. Similar distances were observed when a shared enhancer was placed into close proximity with linked reporter genes in cis. These observations are consistent with the occurrence of "transcription hubs," whereby clusters (or condensates) of multiple RNA polymerase II complexes and associated cofactors are periodically recruited to active promoters. The dynamics of this process might be responsible for rapid fluctuations in the distances separating the transcription of coregulated reporter genes during transvection. We propose that enhancer-promoter communication depends on a combination of classical looping and linking models.

Keywords: Drosophila embryos; enhancers; live-imaging; transcription; transvection.

Fig. 1.

Large distances separate coregulated genes in trans. (A) A schematic of the transvection assay. On the top allele, there is a gyspy insulator, the sna shadow enhancer, and a PP7-lacZ reporter gene. On the bottom allele, there is a gypsy insulator and an MS2-lacZ reporter gene. (B) Stills of 2 representative nuclei taken from maximum projected Airyscan-processed images. MS2 foci were visualized by MCP-GFP (green), PP7 foci were visualized by mCherry-PCP (red), and nuclei were visualized using His2Av-eBFP2 (blue). (Scale bar, 500 nm.) (C) Distribution of xy distances separating MS2 and PP7 foci during transvection in nuclear cycle 14 (n = 2,441). Bin widths plotted were 10 nm.

Fig. 2.

Distal and proximal configurations of 2 cis-linked reporter genes yield unique distance distributions. (A and B) Schematics for 2 configurations with 2 cis-linked reporter genes. The sna shadow enhancer is regulating MS2-yellow and PP7-yellow reporter genes with either distal eve promoters (A) or proximal sna promoters (B). The promoters are separated by either ∼16.5 kb (A) or ∼3.5 kb (B). (C and D) Stills of representative nuclei with either the distal (C) or proximal (D) configuration taken from maximum projected Airyscan-processed images. MS2 foci were visualized by MCP-GFP (green), PP7 foci were visualized by mCherry-PCP (red), and nuclei were visualized using His2Av-eBFP2 (blue). (Scale bar, 500 nm.) (E) Distribution of xy distances separating MS2 and PP7 foci with the distal (blue; n = 2,098) or proximal configuration in nuclear cycle 14 (red; n = 2,016). Bin widths plotted were 10 nm. These 2 distance distributions were determined to be significantly different (P = 2.0 × 10⁻⁸⁵, Mann–Whitney U test). (F) Boxplot showing distribution of fluorescence intensity of MS2 foci between proximal (red; n = 832) or distal configuration (blue; n = 1,066). The box represents the lower (25%) and upper (75%) quantile and the solid line represents the median. Whiskers cover up to the 10th and 90th percentile of each distribution. P value was calculated using a Mann–Whitney U test.

Fig. 3.

Distance dynamics between coregulated genes during transvection. (A–D, Left) Stills of representative nuclei during transvection taken from maximum projected Airyscan-processed images. MS2 foci were visualized by MCP-GFP (green), PP7 foci were visualized by mCherry-PCP (red), and nuclei were visualized using His2Av-eBFP2 (blue). (Scale bar, 500 nm.) (A–D, Right) xy distance trajectories between MS2 and PP7 foci over time. Time points that are indicated by blue correspond to a still image shown at Left.

Fig. 4.

Molecular crowding as the source for the distances separating coregulated genes in different configurations. (A–C) In each assay, we propose that clustering and aggregation of Pol II (purple), transcription factors (orange), and other components of the general transcription machinery (e.g., PIC components, Mediator; yellow) leads to the separation seen between coregulated reporter genes. With the cis-linked assays, recruitment of fewer such molecules (e.g., Pol II, Mediator) might explain the smaller distance observed between reporter genes in the distal configuration (B) compared with the proximal configuration (C).