Engineering Genome Organization to Explore TAD Function in Living Cells

STORTZ, MARTIN; MISTELI, TOM

Victoria

Simposio; Keystone Symposia - Chromatin Architecture in Development and Human Health; 2023

Keystone Symposia

Transcription takes place in a highly organized genome. The chromatin fiber folds into loops that give rise to topologically associated domains (TADs). These structures are self-interacting, continuous genome regions (~105-106 bp) formed by a loop extrusion process and their boundaries are demarcated by binding of the CTCF protein. The prevailing model suggests that TAD structure requires interactions between boundaries and that TADs function to facilitate interactions between genes and regulatory elements within the same TAD, while the boundaries reduce interactions with adjacent regions, thus creating local regulatory hubs. However, numerous observations on local and genome-wide perturbations of TADs often show only limited effects on gene expression. A major limitation of most of these analyses is their correlative nature. There is a need for new technologies to nimbly manipulate genome organization and address the cause-effect relationship of TADs structure and transcription. We are developing optogenetic tools to induce interactions between specific genome regions ―particularly TAD boundaries― in a controlled fashion to study their influence on transcriptional bursting. Our approach is based on the use of the nuclease-dead Cas9 (dCas9) or the Tet repressor (TetR) as a targeting moiety and CRY2, an Arabidopsis protein that forms homotypic condensates in response to blue light as a means to spatially constrain and crosslink target regions. We have generated an optimized CRY2 variant that combines mutations in two different regions of the protein and exhibits a high propensity to form condensates in a light-sensitive manner. As proof of principle, we verified that TetR-CRY2 forms a light-induced condensate at a TetO array. In line with local condensate formation, FRAP assays revealed reduced exchange of molecules between the array and the nucleoplasm after light exposition. These results suggest that the system recruits TetR-CRY2 molecules through CRY2/CRY2 interactions upon light-stimulation and that these interactions are relatively stable. Our preliminary results suggest that this optogenetic system may be suitable to induce stable interactions between neighboring loci. Coupling this system with detection of endogenous gene transcription by live-cell imaging may help to understand the functional role of TADs.