Genome organization controls transcriptional dynamics during development

Abstract

Past studies offer contradictory claims for the role of genome organization in the regulation of gene activity. Here, we show through high-resolution chromosome conformation analysis that the Drosophila genome is organized by two independent classes of regulatory sequences, tethering elements and insulators. Quantitative live imaging and targeted genome editing demonstrate that this two-tiered organization is critical for the precise temporal dynamics of Hox gene transcription during development. Tethering elements mediate long-range enhancer-promoter interactions and foster fast activation kinetics. Conversely, the boundaries of topologically associating domains (TADs) prevent spurious interactions with enhancers and silencers located in neighboring TADs. These two levels of genome organization operate independently of one another to ensure precision of transcriptional dynamics and the reliability of complex patterning processes.

Fig. 1.. Hierarchical genome organization: Boundaries and focal contacts.

(A) ANT-C organization (Dfd-Antp interval). The following are shown from top to bottom: Micro-C contact map showing TADs and focal contacts (arrows); Hox genes (black); other genes (gray); regulatory elements; and chromatin immunoprecipitation (ChIP) data for Trl and CP190. (B) Tethers and boundaries are physically distinct. (C) Epigenetic signatures of tethers and boundaries. DNase, deoxyribonuclease I. (D) Fraction of contacts connecting gene promoters to “orphan” DTEs and a histogram of loop spans (black, all loops). (E) Enhancer activity, by functional class (***p < 10⁻⁷ versus Zld peaks, Bonferroni-corrected chi-square test; n.s., not significant). (F) Image of a live embryo showing transcription of Dfd, Antp (cyan), and Scr (yellow, image enhanced), with nuclei in purple.

Fig. 2.. Tethering elements foster enhancer-promoter interactions and control activation kinetics.

(A) Micro-C for Scr DTE mutant embryos. (The triangle indicates the location of the deletion.) Virtual 4C (v4C) shows decreased interactions of the EE enhancer with the promoter upon DTE deletion (arrow) and increased interactions with regions beyond the DTE (asterisk). The focal contact persists in ΔScrEE embryos (inset). (B) Live measurements of endogenous Scr transcription show delayed activation in ΔScrDTE embryos. A-P, anterior-posterior; FU, fluorescence units; N, number of embryos; shading, ±SEM).

Fig. 3.. Insulators prevent regulatory interference and promote transcriptional precision.

(A) Micro-C and Dfd transcription measurements for ΔDfd3′ insulator mutant embryos. The triangle indicates the location of the deletion. (B) Micro-C and Scr transcription measurements for ΔSF1 (and ΔSF2) embryos. The focal contact persists (arrows). (C) Scr transcription in ΔScr3′ and ΔAntp3′ embryos. (D) Antp transcription in ΔAntp3′ embryos. (E) Interaction landscape of the Scr promoter upon disruption of the ftz TAD (see Micro-C above). (F) Reporter assay showing silencing by the AE1 enhancer within the Scr expression domain (dashed box). In all panels, shading indicates ±SEM.

Fig. 4.. Genome organization controls transcriptional dynamics and developmental patterning.

(A) Representative images of sex combs from adult males. (Numbers indicate tooth counts.) (B) Correlation of transcriptional output and tooth count (inset, locus map; red bar, sex comb enhancer; error bars, ±SEM). (C) Organization of the Scr locus: Tethers foster specific enhancer-promoter interactions, whereas boundaries prevent regulatory interference between TADs.