Engineering chromatin contacts to interrogate the role of TADs in transcriptional control

STORTZ, MARTIN; KEIKHOSRAVI, ADIB; PEGORARO, GIANLUCA; MISTELI, TOM

Penn State University

Simposio; 39th Summer Symposium in Molecular Biology: Chromatin and Epigenetic Regulation of Transcription; 2023

Pennsylvania State University

Chromatin folding gives rise to topologically associated domains (TADs). These domains comprise self-interacting, continuous genome regions formed by loop extrusion, and they are demarcated by binding of CTCF protein at their boundaries. It has been suggested that the TAD structure facilitates intra-TAD interactions between genes and regulatory elements, while reducing interactions with extra-TAD regions. As such, the 3D genome organization would influence gene activation. However, several studies have challenged this role for TADs showing limited effects of TADs perturbations on gene regulation. Since most of the current knowledge about TAD function has come from studies that correlate changes in genome structure with changes in transcription activation, often performed in population-based bulk analyses, we sought to develop single-cell methods to manipulate genome organization and then assess functional consequences on transcription regulation. For this purpose, we are developing optogenetic tools to induce contacts between specific genome regions ―particularly TAD boundaries― in a controlled fashion and study their influence on transcriptional bursting. Our approach combines the use of the nuclease-dead Cas9 (dCas9) or the Tet repressor (TetR) as a targeting moiety and CRY2, which forms light-induced clusters to crosslink target regions in TADs. As proof of principle, we verified that the TetR-CRY2 fusion construct forms a light-induced cluster at a TetO array. We also tested the ability of the dCas9-CRY2 construct to induce specific chromatin contacts by recruiting it to two repetitive loci separated by 1.2 Mb with guide RNAs targeting the repeats and activating CRY2 with light. We demonstrate by DNA-FISH a significative increase in the contacts between these two loci, suggesting the potential value of this tool to interrogate the functional role of TADs. These experiments are the foundation for further probing the role of TAD structure on gene expression.