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重要作用。许多编码S、G2和M过渡调节因子的基因在静止期细胞和细胞周期早期受到抑制，但当细胞通过细胞周期时被激活。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期基因转录的机制仍有待详细探索。拟议的项目旨在阐明这些机制。我们已经表明，G2/M基因的调控中起着核心作用的启动子元件，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LIN 9、LIN 37、LIN 52、LIN 54和RBBP 4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，该复合物可以在G0/G1中作为阻遏物以及在G2/M中作为激活物。阻遏复合物DREAM由MuvB核心以及E2F4、DP1和p130蛋白组成。在细胞周期的进展过程中，E2F4，DP1和p130被B-MYB取代，随后被FOXM1取代。这些活化复合物分别被称为MYB-MuvB（MMB）和FOXM1-MuvB。到目前为止，它在很大程度上是未知的MuvB复合物组分与特定的DNA结合位点相互作用。MuvB复合物含有四种可能与DNA结合的蛋白质：E2F4/DP 1可以与E2F位点结合，Lin54可以与DNA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E2F元件强烈富集MuvB靶基因。因此，拟议项目的一个目的是阐明不同的MuvB复合物如何与DNA结合。为此，我将过度表达DNA结合缺陷MuvB复合物组分，并使用全基因组ChIP和mRNA表达分析测试特定DNA结合结构域的丢失是否对MuvB复合物的结合和基因调控产生影响。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。通过TALEN和CRISPR/Cas9方法，我将删除MuvB复合物结合位点，并研究这些突变对不同细胞周期阶段的染色质修饰酶、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转录因子及其相互作用蛋白质的功能。来自拟议分析的数据将回答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了解控制细胞周期的中枢机制。

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.