Dissecting enhancer-promoter looping and gene induction dynamics in differentiation

项目摘要/摘要 转录在特定组织和发育阶段的精确激活是由增强子和 启动子相互作用(EPI)，其改变会导致发育缺陷和癌症。事实上，至关重要的是 了解增强剂负责组织和时间特异性基因激活的机制。 尽管EPI很重要，但EPI的形成机制在很大程度上仍不为人所知 可用的型号仍然存在争议。特别是，中心开放问题可以列出如下：1)是联系人 在增强子和启动子之间是必需的，还是足够接近？2)Epis是稳定的还是动态的？至 解决这些知识空白，在他的K99 Aim1中，Gabriele博士将建立一种超分辨率3D活细胞成像 (SRLCI)系统，以可视化EPIS在原型基因转录激活中的作用。他将专注于Aim1 在FOXG1上，多能细胞分化为皮质神经元后表达。此外，在神经元谱系中， FOXG1启动子表现出与增强子区域的远程相互作用，缺乏多效性，因此 使FOXG1成为差异化过程中回答研究事件的理想候选者。值得注意的是，加布里埃尔博士 之前建立了一种SRLCI方法来研究染色质环，并发现这些结构是 罕见的和短暂的。在这里，他将把以前的方法论献给研究EPIS。 在AIM2(R00阶段)，他将在Aim1的SRLCI工作的基础上研究染色质的作用 调控EPI和建立和维持细胞后转录的调节剂和组蛋白修饰 差异化。 然后，为了测量所识别的EPIS机制在多大程度上适用于Aim3中的所有EPI (在K99和R00之间)他将使用测序方法来识别所有新形成的EPI，从而导致 小鼠胚胎干细胞分化为皮质神经元时的转录激活。 加布里埃勒博士的长期目标是作为一名首席研究员实现独立，致力于他的研究 了解细胞特性调节的生理和病理机制。为 为此，他将得到他的导师和科学咨询委员会的支持。在K99阶段，他 将接受单细胞基因组学技术的实验处理和分析方面的培训，如Share- SEQ和Micro-C，以及3D聚合物模拟。此外，他还将提高他的成像处理分析和 加深他的生物信息学知识。 此外，在K99阶段，加布里埃尔博士将提高他的科学写作、外展、指导和 教学和管理技能，注重多样性、公平性和包容性价值观。完成K99 研究和培训将极大地促进加布里埃勒博士向独立和作为一名 独立的调查者和导师。

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.