Mechanisms of transcription regulation by CREB, with a focus on disordered domains

转录因子是一种蛋白质，它调节细胞内基因的表达，通常是通过接近DNA来实现的。许多转录因子本质上是无序的蛋白质，这意味着它们没有定形，这使得对它们的结构研究变得困难。无序转录因子bZIP家族是第二大转录因子家族，利用其bZIP结构域将DNA以二聚体的形式结合在一起，就像一双筷子一样，穿过主要的凹槽。CREB(cAMP调节元件(CRE)结合蛋白)是一种错乱的bZIP转录因子，在癌症中过表达，帮助调节许多生理过程，如生物钟。因此，CREB是研究为什么无序在转录因子中很重要的理想蛋白质，也是非常生理相关的。最近，Shamma实验室研究了使用bZIP结构域结构进行目标搜索的性质。在这个项目中，我们将使用全长CREB来揭示CREB序列的其余部分在这一过程中的作用，以及一般的转录调控。无序是如何促进转录因子活性的，当无序受到影响时会发生什么？它们是作为单体还是二聚体与DNA结合并寻找它们的靶点(CRE DNA，对于CREB)？CREB伙伴在转录调控中扮演什么角色？为了回答这些问题，我们将研究不同条件下DNA-CREB的相互作用，例如通过突变CREB，使用停流或荧光各向异性等生物物理技术，并通过荧光显微镜和基于细胞的分析来证实结果。

Transcription factors are proteins which regulate the expression of genes within our cells, often by being in close proximity to the DNA. Many transcription factors are intrinsically disordered proteins, meaning they have no setshape, which has made them difficult to study structurally. The bZIP family of disordered transcription factors, the second largest transcription factor family, binds the DNA as dimers like "a pair of chopsticks" through the major grooves using their bZIP domain. CREB (cAMP regulatory element (CRE) binding protein) is a disordered bZIP transcription factor of special interest, being overexpressed in cancers and helping regulate many physiological processes like the circadian clock. Hence, CREB is an ideal protein to study why disorder is important in transcription factors, being also very physiologically relevant. Recently, Shammas lab have investigated the nature of target search using bZIP domain constructs. In this project, we would use full-length CREB to reveal the role of the rest of the CREB sequence in this process, and transcriptional regulation in general. How does disorder contribute to transcription factor activity, and what happens when disorder is affected? Do they bind the DNA and search for their target site (CRE DNA, for CREB) as a monomer or a dimer? What roles do CREB partners play in the regulation of transcription? To answer these questions, we would study the DNA-CREB interaction under various conditions, for instance by mutating CREB, using biophysical techniques like stopped-flow or fluorescence anisotropy, confirming the results by using fluorescence microscopy and cell-based assays.