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Genomic context mediates position-dependent enhancer function at the Sonic hedgehog locus

ABSTRACT: Enhancers can regulate their target promoters over large genomic distances for which spatial proximity between these elements is assumed to be necessary. Topologically Associating Domains (TADs) are thought to provide such a spatial environment for regulatory domains harboring multiple enhancers driving complex expression of developmental genes. However, how enhancer position within the TAD affects its function in vivo is not known. Here, we address this question by generating a series of genome-engineered mouse lines in which we systematically reposition the limb enhancer of Sonic hedgehog (Shh), ZRS, to various positions within and outside of its TAD to analyze positional effects on gene expression, chromatin folding, and limb phenotype. We find that the enhancer activates Shh only when located within the same TAD, however, its activity varies across intra-TAD positions. ZRS-driven Shh expression drops rapidly with increasing distance from the promoter but eventually plateaus irrespective of linear distance. Moreover, engineering of 3D enhancer-promoter contacts through integration of different CTCF sites reveals that increased contact frequencies do not directly translate into increased transcriptional output and may cause undesired consequences in vivo due to detrimental effects on Shh expression in other tissues. Finally, we show a selective ability of the ZRS enhancer to activate non-target genes in ectopic TADs. Our work highlights the importance of genomic context within TADs for enhancer function and our limited understanding of the interplay between regulatory sequences within these domains.

ORGANISM(S): Mus musculus

PROVIDER: GSE223757 | GEO | 2023/08/01

REPOSITORIES: GEO

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A Preformed Chromatin Architecture Ensures Robust Shh Transcription during Limb Development [Capture Hi-C]

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Project description:Limb-specific expression of Shh is regulated by the long-range (~one megabasepair distant) ZRS enhancer. In the mouse, murine limb bud restricted spatiotemporal expression of Shh occurs from ~E10 until E11.5 at the distal posterior margin is essential for the correct formation of the autopod. Here, we have analyzed the higher-order chromatin conformation of Shh in expressing and non-expressing tissues, both by fluorescence in situ hybridization (FISH) and by chromosome conformation capture (5C). Conventional and super-resolution light microscopy identified significantly elevated frequences of Shh/ZRS co-localization only in the Shh expressing regions of the limb bud consistent with the formation of an enhancer-promoter loop. However, Shh-ZRS spatial distances were consistently shorter than intervening distances to a neural enhancer in all tissues and developmental stages analyzed – regardless of Shh expression. 5C also identified a topologically associating domain (TAD) over the Shh-ZRS genomic region and enriched interactions between Shh and ZRS, but in the head, body and limb buds of E11.5 embryos, so also not linked to Shh expression. We show that gene-enhancer (Shh/ZRS) co-localization correlates with the spatiotemporal domain of limb bud-specific Shh expression, but that close Shh/ZRS proximity in the nucleus occurs regardless of whether the gene or enhancer is active. We suggest that this constrained chromatin configuration optimises the opportunity for the active enhancer to locate and instigate Shh expression.

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2019-05-13 | GSE123388 | GEO

Developmentally regulated Shh expression is robust to TAD perturbations

Project description:Topologically Associating Domains (TADs) have been proposed to both guide and constrain enhancer activity. Shh is located within a TAD known to contain all its enhancers. To investigate the importance of chromatin conformation and TAD integrity on developmental gene regulation, we have manipulated the Shh TAD – creating internal deletions, deleting CTCF sites, and deleting and inverting sequences at TAD boundaries. Chromosome conformation capture and fluorescence in situ hybridisation assays were used to investigate the changes in chromatin conformation that result from these manipulations. Our data suggest that these substantial alterations in TAD structure has no readily detectable effect on Shh expression patterns during development – except where enhancers are deleted - and results in no detectable phenotypes. Only in the case of a larger deletion of one TAD boundary could ectopic influence of the Shh limb enhancer be detected on a gene (Mnx1) in the neighbouring TAD. Our data suggests that, contrary to expectations, the developmental regulation of Shh expression is remarkably robust to TAD perturbations.

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Chromatin conformation of the Shh locus in limbs.

Project description:Expression of Shh in the limb is dependent on a distant enhancer located 850kb away from the gene promoter. We performed chromatin conformation capture (4C-Seq) analysis of the Shh locus in E11.5 mouse limbs so as to better understand the relationship between this regulatory interaction and the structural organisation of the locus. The experiments were performed on wild-type animals as well as on animals carrying an engineered chromosomal inversion (INV(6-C2) (INV6)) . The different viewpoints used (Shh, ZRS, Nom1, Rbm33, Rnf32) enable to characterize the 3D organisation of the locus in the different contexts. The inversion INV6 disrupts the normal topological organisation of the Shh locus in a compact 3D-domain and prevents the physical and regulatory interaction between Shh and its limb enhancer, even though the genomic distance separating the two elements is reduced compared to WT.

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