Exploring the role of transcription in shaping genome topology during embryogenesis

Within the nucleus, chromatin is organised in the three-dimensional space at different scales, including compartments, topologically-associating domains (TADs), and chromatin loops. There is mounting evidence that transcription might play a role in shaping genome topology across different scales and contexts, particularly for insulation at TAD boundaries. However, it is currently not clear if the insulation activity is due to the process of transcription elongation or the loading of a transcriptional bubble (PIC loading) or a function of the RNA molecule itself. To investigate this during embryogenesis, I applied an optogenetic nuclear depletion system to perturb either transcription initiation or Pol II pausing in developing embryos. To inhibit transcription initiation, I targeted motif 1-binding protein (M1BP), which constitutively binds to the core promoter of housekeeping genes throughout embryogenesis and is enriched at TAD boundaries. My results show that, before zygotic genome activation, M1BP is already associated with insulation at a subset of target promoters, in the absence of Pol II. M1BP appears to be retained mitotically and may function as a mitotic bookmarking factor. Acute M1BP depletion during ZGA impaired the formation of the pre-initiation complex (PIC) at a subset of genes, and caused an increase in interaction frequency within the active compartment in Hi-C, suggesting that formation of a PIC does play a role in chromatin topology. To interfere with Pol II pausing, I targeted the pausing/elongation factor SPT5. SPT5 depletion resulted in more transcriptional defects at late embryonic stages compared to early, and appeared to cause both Pol II pause release and reduced processivity, with a differential effect based on gene size and basal expression levels. I will soon explore the topological changes associated with this phenotype. Gene expression is regulated by enhancers, which can work within and between TADs to regulate their target genes. While enhancer sufficiency has been tested at scale in transgenic assays, their necessity within their endogenous context, and interactions with other enhancers has not. To asses this, I am deleting all enhancers, both individually and in different combinations, in a model locus to test their effect on gene expression and tissue formation both at steady state and upon developmental stress. Preliminary results indicate that enhancers that appear redundant are often needed under stress conditions to maintain transcriptional and phenotypic robustness.