Long G4 regions (LG4s) in the human genome constitute functional enhancers that coordinate neighboring gene expressions

启动子是基因上游的DNA区域，其中蛋白质结合以通过产生基因序列的RNA拷贝来启动（开启）基因的转录。启动子可能非常复杂，通常与称为增强子的其他DNA区域一起工作，以确保特异性，稳健的基因转录。幸运的是，尽管它们很复杂，但启动子很容易识别，因为它们通常对应于基因开始之前的100- 1，000个碱基对（bp）。这使得在过去的几十年中，大量的启动子的详细表征。然而，增强子表征已被证明更具挑战性。与启动子一样，增强子通常是短的，100- 1，000 bp的DNA序列，显著调节基因转录，但与启动子不同，增强子可以在距离其影响的基因多达100万bp处找到。增强子如何在如此长的距离内发挥作用？基因组DNA可以弯曲，使增强子和启动子彼此靠近以启动转录。有趣的是，许多试图将基因组突变与各种性状和/或疾病联系起来的研究发现，超过90%的疾病相关突变位于基因组的非编码部分，更引人注目的是，这些突变中的大多数位于基因组中被认为是增强子而不是启动子的区域。尽管它们的重要性，仍然只有一个非常有限的能力来定义增强子序列，其作用机制和靶向决定因素的主要问题仍然没有答案。了解是什么驱动增强子：启动子相互作用可以显着提高我们解决许多社会挑战的能力（例如疾病，通过基因调控提高作物对不利环境条件的抗性等）。该项目还为学生（包括当地代表性不足的少数民族学生）提供了独特的跨学科培训，结合分子生物学和计算遗传学/生物信息学。更好地了解基因启动子和基因远端增强子之间的相互作用无疑将导致对基因转录的基本控制和疾病中涉及的调控突变的作用的新见解。然而，从广义上讲，增强子如何靶向并将信息传递到其同源启动子的基本机制仍然是我们知识中的关键空白。本提案中概述的工作将直接解决长期存在的问题，即增强子通常位于很远的地方（数百个酶对），如何与细胞核中的特定启动子接触。值得注意的是，增强子通常显著富含G4-能力序列，并且有趣的是，由G4-增强子调节的启动子同样不成比例地富含G4-能力序列。由于杂合G4结构可以在不同的DNA链之间一起形成，这些观察结果表明直接的G4：G4相互作用可以有助于增强子：启动子靶向。这项工作概述了这项建议是重要的，因为它将采用一系列的遗传操作，生化测定，和新的NGS-IP策略，直接表征G4 DNA的作用增强子：：启动子靶向和产生新的方法，将用于定义特定的增强子：：启动子interactions.This奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

A promoter is a region of DNA upstream of a gene where proteins bind to initiate transcription of (turn on) a gene by producing an RNA copy of the gene sequence. Promoters can be very complex and typically work together with other DNA regions known as enhancers to ensure specific, robust gene transcription. Fortunately, despite their complexity, promoters are easy to identify because they generally correspond to the 100-1,000 base pairs (bp) just before the start of a gene. This has allowed detailed characterization of a massive number of promoters over the past several decades. However, enhancer characterizations have proven much more challenging. Like promoters, enhancers are typically short, 100-1,000 bp DNA sequences that markedly regulate gene transcription but, unlike promoters, an enhancer can be found up to 1 million bp away from the gene it influences. How do enhancers work across such large distances? Genomic DNA can bend, bringing enhancers and promoters close to one another to initiate transcription. Interestingly, numerous studies attempting to link genomic mutations with various traits and/or diseases have found that more than 90% of disease-associated mutations lie within the noncoding portions of the genome, and even more strikingly, most of these mutations lie within regions of the genome thought to function as enhancers rather than promoters. Despite their importance, there is still only a very limited ability to define enhancer sequences, and major questions about their mechanisms of action and targeting determinants remain unanswered. Understanding what drives enhancer:promoter interaction can significantly improve our ability to address a number of societal challenges (e.g. disease, improving crop resistance to adverse environmental conditions via gene modulation, etc.). This project also provides unique interdisciplinary training for students (including local underrepresented minority students) combining molecular biology and computational genetics/bioinformatics.A better understanding of the interplay between gene promoters and gene-distal enhancers will unquestionably lead to fundamental new insights into the basic control of gene transcription and the action of regulatory mutations involved in disease. However, the fundamental mechanisms responsible for how enhancers target and transfer information to their cognate promoters, broadly speaking, remain key gaps in our knowledge. The work outlined in this proposal will directly address the long-standing question of how enhancers, which are often located far (hundreds of kilobase pairs) away, make contact with specific promoters in the context of the nucleus. Notably, enhancers are often significantly enriched with G4-capable sequences and, interestingly, the promoters regulated by G4-enhancers are likewise disproportionately enriched with G4-capable sequences. As hybrid G4 structures can be formed between distinct DNA strands, together, these observations suggest that direct G4:G4 interactions can contribute to enhancer:promoter targeting. The work outlined in this proposal is significant in that it will employ an array of genetic manipulations, biochemical assays, and novel NGS-IP strategies to directly characterize the role of G4 DNA in enhancer::promoter targeting and generate novel methodologies that will be of use in defining specific enhancer::promoter interactions.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.