Molecular mechanisms of TAD boundary formation

International audience

English. Over the last decade, it is increasingly appreciated that the 3D organization of the genome in the cell nucleus contributes to gene regulation, genome stability and chromosome inheritance. During interphase, chromosomes are partitioned into large active and inactive compartments and smaller Topologically Associating Domains (TADs) with embedded chromatin loops between promoters and enhancers. TADs appear to be structural units of gene regulation, as they often correlate with domains of enhancer action. It is therefore of great interest to understand the molecular mechanisms by which TADs and their boundaries form. Transcription has been found to play roles in boundary formation is some organisms. This led us to study the role of transcription in TAD formation. Using a combination of cells synchronized in the cell cycle and the use of treatments that block transcription we show that establishment of compartments, TADs and CTCF-CTCF loops occurs in early G1 and in a transcription independent manner. There is now extensive evidence that the transcription factor CTCF is playing a fundamental role in TAD boundary formation. However, which other cis and trans-factors are involved, and what processes lead to domain formation is not known. To identify other cis-factors that might be crucial for TAD formation, we use CRISPR/Cas9-mediated genome engineering in human fibroblasts to dissect TAD boundaries at their endogenous locations. We show that TAD boundaries can be robust, even when 25Kb deletions are generated at CTCF sites present at boundaries. These results point to additional processes beside CTCF binding that can impose boundary activity.