Cis-regulatory chromatin loops arise before TADs and gene activation, and are independent of cell fate during development

During development, naïve cells gradually acquire distinct cell fates, through sophisticated mechanisms of precise spatio-temporal gene regulation. Acquisition of cell fate is thought to rely on the specific interaction of remote cis-regulatory modules (e.g. enhancers, silencers) (CRM) and target promoters. However, the precise interplay between chromatin structure and gene expression is still unclear, particularly in single cells within multicellular developing organisms. Here we employ Hi-M, a single-cell spatial genomics approach, to systematically detect CRM-promoter looping interactions within topological associating domains (TADs) during Drosophila development. By comparing cis-regulatory loops in alternate cell types, we show that physical proximity does not necessarily instruct transcriptional states. Moreover, multi-way analyses revealed the existence of local interactions between multiple remote CRMs to form hubs. We found that loops and CRM hubs are established early during development, prior to the emergence of TADs. Moreover, CRM hubs are formed via the action of the pioneer transcription factor Zelda and precede transcriptional activation. Our approach offers a new perspective on the role of CRM-promoter interactions in defining transcriptional activation and repression states, as well as distinct cell types.
Nature Genetics (2021)

2021, Apr 1. doi: 10.1038/s41588-021-00816-z. Epub ahead of print. PMID: 33795867.

Cis-regulatory chromatin loops arise before TADs and gene activation, and are independent of cell fate during early Drosophila development.

Sergio Martin Espinola, Markus Götz, Maelle Bellec, Olivier Messina, Jean-Bernard Fiche, Christophe Houbron, Matthieu Dejean, Ingolf Reim, Andrés M. Cardozo Gizzi, Mounia Lagha & Marcello Nollmann.

We developed an imaging-based multi-way detection method, to reveal hubs with multiple #enhancers in a multicellular organism, in two genomic regions harboring developmental genes. 3/9


Next, we looked at chromatin organization and transcriptional activation simultaneously in three different cell-types of the developing embryo: mesoderm, neuroectoderm and dorsal ectoderm progenitors. 4/9
Surprisingly, loops between cis-regulatory elements as well as hubs are very similar between the three alternate cell fates, despite their different transcriptional activation patterns! Thus loops don’t correlate with transcriptional status. 5/9
Are these cis-regulatory interactions arising during early development? In fact, cis-regulatory topologies are already present in naive pluripotent nuclei, prior to the emergence of TADs and to the onset of gene expression! 6/9
Finally, we found that formation of cis-regulatory loops and hubs requires the pioneering activity of Zelda. 7/9