Friday, May 9, 2025
5/9/2025
Dissecting enhancer-promoter looping and gene induction dynamics in differentiation - Project Summary/Abstract The precise activation of transcription in specific tissues and developmental stages is regulated by enhancer and promoter interactions (EPIs), whose alterations cause developmental defects and cancer. Indeed, it is critical to understand the mechanism by which enhancers are responsible for tissue- and time-specific gene activation. Despite the importance of EPIs, the mechanisms by which EPIs are formed are still largely unknown, and available models are still debated. In particular, the central open questions can be listed as follows: 1) Is contact between enhancer and promoter necessary, or is proximity enough? 2) Are the EPIs stable or dynamic? To address these knowledge gaps, in his K99 Aim1, Dr. Gabriele will set up a super-resolution 3D live-cell imaging (SRLCI) system to visualize the role of EPIs in transcription activation in a prototype gene. He will focus Aim1 on Foxg1, which is expressed after differentiating pluripotent cells to cortical neurons. Also, in neuronal lineages, the Foxg1 promoter displays a long-range interaction with an enhancer region, absent in pluripotency, thus making Foxg1 the ideal candidate to answer the study EPIs during differentiation. Notably, Dr. Gabriele has previously established an SRLCI methodology to study chromatin looping and found that these structures are rare and transitory. Here, he will dedicate the previous methodology to studying EPIs. In Aim2 (R00 phase), he will build on his SRLCI work of Aim1 to investigate the role of chromatin regulators and histone modification in regulating EPIs and establishing and maintaining transcription after cell differentiation. Then, to measure to what extent the identified EPIs mechanism generalizes to all the EPIs, in Aim3 (between K99 and R00) he will employ sequencing methods to identify all the newly formed EPIs that result in transcription activation while differentiating mouse embryonic stem cells to cortical neurons. Dr. Gabriele’s long-term goal is to achieve independence as a principal investigator to dedicate his study to understanding the physiological and pathological mechanisms involved in the regulation of cell identity. For this purpose, he will be supported by his mentor and Scientific Advisory Committee. During the K99 phase, he will be trained in the experimental processing and analysis of single-cell genomics techniques such as SHARE- seq and Micro-C, and 3D polymer simulations. Moreover, he will improve his imaging processing analysis and deepen his bioinformatics knowledge. In addition, during the K99 phase, Dr. Gabriele will improve his scientific writing, outreach, mentoring and teaching, and management skills with a focus on diversity, equity, and inclusion values. Completing the K99 research and training will significantly facilitate Dr. Gabriele’s transition to independence and success as an independent investigator and mentor.
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