Mechanisms of cell cycle-dependent transcriptional regulation by MuvB complexes

差异基因表达在细胞周期调控中起着核心作用。许多编码通过S、G2和M过渡的调控因子的基因在静止细胞和细胞周期早期阶段被抑制，但当细胞进入细胞周期时被激活。这一过程的失调可导致细胞缺陷和致癌转化。虽然RB/E2F复合物对S期基因的调控已被广泛研究，但调控G2/M基因转录的机制仍有待深入探索。拟议的项目旨在阐明这些机制。我们已经证明，CHR启动子元件在G2/M基因的调控中发挥核心作用，并且它们与MuvB复合物DREAM、MMB和FOXM1-MuvB相互作用。这三种MuvB复合物都共享由LIN9、LIN37、LIN52、LIN54和RBBP4组成的MuvB核心。在不同的细胞周期阶段，MuvB核心与特定的蛋白质相互作用。因此，这些复合物在G0/G1中可以作为抑制因子，在G2/M中可以作为激活因子。抑制复合体DREAM由MuvB核心以及E2F4、DP1和p130蛋白组成。在细胞周期的进程中，E2F4、DP1和p130被B-MYB取代，随后被FOXM1取代。这些激活复合物分别被称为MYB-MuvB （MMB）和FOXM1-MuvB。迄今为止，对MuvB复合组分与特定DNA结合位点的相互作用还知之甚少。MuvB复合物含有四种可能与DNA结合的蛋白质：E2F4/DP1可以与E2F位点结合，Lin54与CHR元件结合。B-MYB和FOXM1分别与Myb或叉头结合位点相互作用。然而，我们的计算分析表明，只有E2F和CHR元件在MuvB靶基因中强烈富集。因此，提出的项目的一个目的是阐明不同的MuvB复合物如何确切地与DNA结合。为此，我将过表达dna结合缺陷的MuvB复合物组分，并通过全基因组ChIP和mRNA表达分析检测特异性dna结合域的缺失是否会影响MuvB复合物的结合和基因调控。此外，我将研究MuvB复合物如何通过染色质修饰调节靶基因的活性。利用TALEN和CRISPR/Cas9方法，我将删除MuvB复合体结合位点，研究这些突变在不同细胞周期阶段对募集染色质修饰酶、染色质结构和基因表达的影响。通过这些实验，将有可能首次在染色体背景下研究CHR元件及其相互作用蛋白的功能。所提出的分析数据将回答关于DREAM、MMB和FOXM1-MuvB转录调控机制的基本问题。这些结果将有助于更好地理解控制细胞周期的中心机制。

Differential gene expression plays a central role in the regulation of the cell cycle. Many genes coding for regulators of transition through S, G2 and M are repressed in quiescent cells and during early cell cycle phases, but are activated when a cell progresses through the cell cycle. Dysregulation of this process can lead to cellular defects and oncogenic transformation. While the control of S phase genes through RB/E2F complexes has been investigated extensively, the mechanisms regulating transcription of G2/M genes still await detailed exploration. The proposed project aims at elucidating these mechanisms. We have already shown that CHR promoter elements play a central role in the regulation of G2/M genes and that they interact with the MuvB complexes DREAM, MMB, and FOXM1-MuvB. These three MuvB complexes all share the MuvB core consisting of LIN9, LIN37, LIN52, LIN54, and RBBP4. During different cell cycle phases, the MuvB core interacts with specific proteins. Thus, the complexes can function as repressors in G0/G1 as well as activators in G2/M. The repressing complex DREAM is composed of the MuvB core as well as of E2F4, DP1 and p130 proteins. During progression of the cell cycle, E2F4, DP1 and p130 are replaced by B-MYB and later by FOXM1. These activating complexes are referred to as MYB-MuvB (MMB) and FOXM1-MuvB, respectively. To date, it is largely unknown which MuvB complex components interact with specific DNA binding sites. MuvB complexes contain four proteins that can potentially bind to DNA: E2F4/DP1 can bind to E2F sites and Lin54 to CHR elements. B-MYB and FOXM1 were shown to interact with Myb or forkhead binding sites, respectively. However, our computational analyses have shown that only E2F and CHR elements are strongly enriched in MuvB target genes. Thus, one aim of the proposed project is to elucidate how exactly the different MuvB complexes bind to DNA. To this end, I will over-express DNA-binding-deficient MuvB complex components and test with genome-wide ChIP and mRNA expression analyses whether loss of specific DNA-binding domains has an influence on binding of the MuvB complexes and gene regulation. Furthermore, I will investigate how MuvB complexes regulate the activity of target genes by chromatin modifications. With TALEN and CRISPR/Cas9 approaches, I will delete MuvB complex binding sites and investigate the impact of these mutations on recruiting chromatin-modifying enzymes, chromatin structure and gene expression in different cell cycle phases. With these experiments, it will be possible for the first time to study the functions of CHR elements and their interacting proteins in a chromosomal context. Data from the proposed analyses will answer essential questions on the mechanisms of transcriptional regulation by DREAM, MMB, and FOXM1-MuvB. The results will contribute to a better understanding of central mechanisms controlling the cell cycle.