Biology subjects

Ghavi-Helm, Y.

Publications and source records attributed to Ghavi-Helm, Y..

6 recordsLinked to original sources

bioRxiv2025

Promoter-proximal gatekeepers restrict pleiotropic enhancer inputs to achieve tissue specificity

The spatiotemporal expression of developmental genes is regulated by the interplay of two key regulatory elements: core promoters, generally assumed to support only basal transcription, and enhancers, which enable their tissue- and stage-specific activation. Here, we show that spatiotemporal specificity can also be encoded within the core promoter. Using the Drosophila twist E3 enhancer as a model, we found that E3 is pleiotropic and activates four functionally unrelated genes. Despite receiving the same enhancer input, each target gene displays distinct and non-overlapping expression patterns. We demonstrate that the selective activation of each target gene is encoded within their promoter regions. Core promoters act as "gatekeepers" that restrict enhancer input into precise tissue- and stage-specific transcription, while proximal-promoter elements facilitate enhancer responsiveness. We propose that promoters function as active interpreters rather than passive recipients of enhancer signals, providing a critical but under-appreciated layer of regulatory specificity within complex gene expression programs.

genetics↗

bioRxiv2024

Spatial reconstruction of single-cell enhancer activity in a multicellular organism

Enhancers play an essential role in developmental processes by orchestrating the spatial and temporal regulation of gene expression. However, mapping the location of these regulatory elements in the genome and precisely characterizing their spatial and temporal activity remain important challenges. Here we introduce a novel in vivo and in silico method for spatial single-cell enhancer-reporter assays (spatial-scERA) designed to reconstruct the spatial activity of multiple candidate enhancer regions in parallel in a multicellular organism. Spatial-scERA integrates massively parallel reporter assays coupled with single-cell RNA sequencing (scRNA-seq) and spatial reconstruction using optimal transport, to map cell-type-specific enhancer activity at the single-cell level on a 3D virtual representation of the sample. We evaluated spatial-scERA in stage 6 Drosophila embryos using 25 candidate enhancers (including 19 uncharacterized regions), and validated the robustness of our predicted reconstructions by comparing them to microscopy images generated by in situ hybridization. Remarkably, spatial-scERA faithfully reconstructed the spatial activity of these enhancers, even when the enhancer-reporter construct was expressed in as few as 10 cells. Our results demonstrate the importance of integrating transcriptomic and spatial data for the accurate prediction of enhancer activity patterns in complex multicellular samples. Indeed, we found that chromatin modifications and open chromatin regions are often poor predictors of enhancer activity. Moreover, spatial data can often be essential for the accurate annotation of scRNA-seq clusters. Finally, we showed that spatial-scERA could be a powerful tool to link enhancers with their potential target genes. Overall, spatial-scERA provides a scalable approach to map spatio-temporal enhancer activity at single-cell resolution without the need for imaging or a priori knowledge of embryology and can be applied to any multicellular organism amenable to transgenesis.

genomics↗

bioRxiv2023

Dual-topology of collagen XV and tenascin C acts in concert to guide and shape developing motor axons

During development, motor axons are guided towards their muscle target by various extrinsic cues including extracellular matrix (ECM) proteins those identities remain poorly documented. Using single-cell RNA-sequencing of differentiating slow muscle progenitors (SMP) in zebrafish, we charaterized the SMP as a major source of ECM proteins that were computationally predicted to form a basement membrane-like structure tailored for motor axon guidance. Multiple in vivo and in vitro approaches further revealed that motor axon shape and growth relies on the timely expression of the attractive cue Collagen XV-B (ColXV-B) that locally provides motor axons with a permissive soft microenvironment and separately organizes the repulsive cue Tenascin C into a unique functional dual topology. Bioprinted micropatterns mimicking their unique topology provide compelling evidence that it represents a sufficient condition to elicit directional motor axon growth. Our study provides the first evidence that ECM topology and stiffness critically influence motor axon navigation in vertebrates with potential applications in regenerative medicine for peripheral nerve injury.

developmental biology↗

bioRxiv2022

Enhancer-promoter interactions form independently of genomic distance and are functional across TAD boundaries

Developmental enhancers are essential regulatory elements that drive precise spatio-temporal gene expression patterns. They do so by interacting with the promoter of their target genes, often across large genomic distances, in a highly specific manner. However, it is unclear how such specificity can be achieved. While several studies have suggested that Topologically Associating Domains (TADs)1-3 facilitate and constrain enhancer-promoter interactions, the role of TAD boundaries in effectively restricting enhancer-promoter interactions is heavily debated. Here we show that enhancers can establish long-range interactions across TAD boundaries and even between different chromosomes. Moreover, some of these interactions are functional in vivo, illustrating their functional importance. Using the twist locus in Drosophila embryos, we systematically relocated one of its enhancers to different regulatory contexts and distances from the twist promoter. We found that the twist promoter can engage in functional enhancer-promoter interactions across a TAD boundary and that distal interactions are sometimes favored over proximal ones. Our results demonstrate that TAD boundaries are not sufficient to constrain enhancer-promoter interactions and that the formation of long-range interactions is not solely driven by distance. These observations suggest that other general mechanisms must exist to establish and maintain specific enhancer-promoter interactions across large distances.

genomics↗

bioRxiv2020

m6A RNA methylation regulates promoter proximal pausing of RNA Polymerase II

RNA Polymerase II (RNAP II) pausing is essential to precisely control gene expression and is critical for development of metazoans. Here, we show that the m6A RNA modification regulates promoter-proximal RNAP II pausing. The m6A methyltransferase complex (MTC), with the nuclear reader Ythdc1, are recruited to gene promoters. Depleting the m6A MTC leads to a decrease in RNAP II pause release and in Ser2P occupancy on the gene body, and affects nascent RNA transcription. Tethering Mettl3 to a heterologous gene promoter is sufficient to increase RNAP II pause release, an effect that relies on its m6A catalytic domain. Collectively, our data reveal an important link between RNAP II pausing and the m6A RNA modification, thus adding another layer to m6A-mediated gene regulation.

molecular biology↗

bioRxiv2017

The Drosophila Dosage Compensation Complex activates target genes by chromosome looping within the active compartment

X chromosome dosage compensation in Drosophila requires chromosome-wide coordination of gene activation. The male-specific-lethal dosage compensation complex (DCC) identifies X chromosomal High Affinity Sites (HAS) from which it boosts transcription. A sub-class of HAS, PionX sites, represent first contacts on the X. Here, we explored the chromosomal interactions of representative PionX sites by high-resolution 4C and determined the global chromosome conformation by Hi-C in sex-sorted embryos. Male and female X chromosomes display similar nuclear architecture, concordant with clustered, constitutively active genes. PionX sites, like HAS, are evenly distributed in the active compartment and engage in short- and long-range interactions beyond compartment boundaries. By de novo induction of DCC in female cells, we monitored the extent of activation surrounding PionX sites. This revealed a remarkable range of DCC action not only in linear proximity, but also at megabase distance if close in space, suggesting that DCC profits from pre-existing chromosome folding to activate genes.

genomics↗