During the previous grant cycle we demonstrated that non-coding transcription orchestrates chromatin folding and compartmentalization to specify T cell fate. Briefly, we identified a non-coding RNA named ThymoD (thymocyte differentiation factor) located 20 kbp upstream of the Bcl11b enhancer. ThymoD- deficient mice displayed a defect at the onset of T cell development and developed lymphoid malignancies. We found ThymoD acts to reposition the Bcl11b enhancer from the lamina into the nuclear interior. The repositioning brings the Bcl11b enhancer and promoter into a single loop domain to permit efficient enhancer-promoter communication and activation of Bcl11b expression. This process involves multiple steps: (i) CTCF occupancy across the ThymoD locus and the Bcl11b promoter region, (ii) activation of cohesin-dependent looping to juxtapose the enhancer and promoter into a single loop domain that is anchored by CTCF sites in the ThymoD locus and the Bcl11b promoter region, (iii) repositioning the enhancer from a heterochromatic to an euchromatic environment and (iv) activation of Bcl11b expression and specification of T cell fate. Here we propose to continue these studies. We would describe in molecular terms how ThymoD orchestrates T cell fate. Specifically, we would examine whether and how CTCF occupancy and cohesin-dependent looping orchestrates chromatin folding and compartmentalization to bring the enhancer and promoter regions into a single loop domain. We would track Bcl11b locus topology during cohesin loss and cohesin recovery at high spatial and temporal resolution. We would examine how cohesin mediated loop extrusion directs compartmentalization, assembles the Bcl11b loop domain and induces a T-lineage specific program of gene expression. Taken together, these studies would reveal how non-coding transcription induced loop extrusion directs enhancer-promoter communication with great specificity to specify T cell fate.
The proposal described here is aimed to examine how non-coding transcription acts to orchestrate the generation of immune cells and to suppress the development of leukemia and lymphoma. The study would provide detailed insight into how non-coding transcription modulates nuclear architecture to activate the expression of genes encoding for key developmental regulators and tumor suppressors and how abnormalities in the expression of these non-coding regions leads to immune deficiencies and the development of lymphoid malignancies.
