Transcriptional control via CHR promoter elements: signaling proteins in cell cycle regulation

控制细胞分裂对预防癌症的发生很重要。一个中心机制代表了转录的调控。我们和其他人已经描述了基于MuvB核心复合体的转录因子家族的功能。这个家族控制着270多个基因，这些基因通常在细胞周期调节中起作用。DREAM， MMB， FOXM1-MMB和FOXM1-MuvB复合物通过结合CHR（细胞周期基因同源区）启动子元件来调节转录。为了实现不同的功能，MuvB核心交换其协会合作伙伴。当MuvB结合E2F/RB家族成员时，DREAM复合体就形成了。DREAM在G0和早期细胞周期中起抑制作用。一旦MuvB将其关联伙伴转换为致癌转录因子B-MYB或FOXM1，形成MMB、FOXM1-MMB或FOXM1-MuvB复合物，MuvB就会在细胞周期后期从抑制因子转变为转录激活因子。这种MuvB/CHR系统的重要性已经得到了很好的证实，但是对信号蛋白触发四种MuvB复合物之间切换的机制的理解仍然有限。此外，在MuvB/CHR系统中，蛋白质是如何被招募来引起抑制或激活的，这在很大程度上是未知的。此外，MuvB复合物修饰染色质和连接基础转录机制的机制仍未得到充分了解。本项目旨在鉴定目前未知的通过CHR位点间接结合DNA的蛋白质，并通过CHR元件确定它们在信号传导中的功能。总体目标是通过MuvB/CHR系统描述新发现的因子在控制细胞周期中的作用。为此，我们将通过RIME/MS和APEX /MS方法鉴定与MuvB复合物相关的蛋白质。我们将测试这些因素是否需要CHR位点进行间接DNA结合。新鉴定的蛋白质与MuvB组分的相互作用将被检查。这些结果将阐明MuvB复合物上游信号传导的机制细节。我们将在敲低和敲除实验中研究新发现的因子的缺失如何促进chrr调控启动子的转录。此外，这些蛋白对整个细胞周期调节的影响将在敲除/敲除后变得明显。在RNA-seq和ChIP-seq实验中，我们将定义这些蛋白的靶基因及其通过转录对细胞周期调控的总体影响。综上所述，我们将确定在MuvB/CHR系统中作为转录因子、辅助因子或信号蛋白的未知因子。我们将描述这些因素如何形成控制细胞分裂的信号通路的未知链接。除了提供对基本生物学过程的洞察，我们的结果也将对理解癌症的发展产生影响。

The control of cell division is important to prevent the development of cancer. A central mechanism represents the regulation of transcription. We and others have described the functions of a transcription factor family based on the MuvB core complex. This family controls more than 270 genes which typically play a role in cell cycle regulation. The DREAM, MMB, FOXM1-MMB and FOXM1-MuvB complexes regulate transcription through binding to the CHR (Cell Cycle Genes Homology Region) promoter element. To carry out different functions, the MuvB core exchanges its association partners. When MuvB binds members of the E2F/RB family, the DREAM complex is formed. DREAM functions as a repressor in G0 and the early cell cycle. MuvB changes its function from a repressor to a transcriptional activator during the later cell cycle once MuvB switches its association partners to the oncogenic transcription factors B-MYB or FOXM1 to form the MMB, FOXM1-MMB or FOXM1-MuvB complexes. The importance of this MuvB/CHR system is well established, but understanding of the mechanisms by which signaling proteins trigger the switch between the four MuvB complexes are still limited. Also, it is largely unknown how proteins are recruited to either cause repression or activation in the MuvB/CHR system. Furthermore, the mechanisms by which MuvB complexes modify chromatin and connect to the basal transcriptional machinery remain insufficiently understood. This proposed project aims at identifying currently unknown proteins binding DNA indirectly through CHR sites and at defining their function in signaling through CHR elements. The overall aim is to delineate the role of the newly identified factors in controlling the cell cycle through the MuvB/CHR system. To this end, we will identify proteins that are associated with the MuvB complex by the RIME/MS and APEX /MS methods. We will test whether these factors require the CHR site for indirect DNA binding. The interaction of the newly identified proteins with MuvB components will be examined. These results will elucidate the mechanistic details of signaling upstream of the MuvB complex. We will investigate in knockdown and knockout experiments how loss of the newly identified factors contributes to transcription of CHR-regulated promoters. Furthermore, the impact of these proteins on overall cell cycle regulation will become evident upon knockdown/knockout. In RNA-seq and ChIP-seq experiments we will define the target genes of these proteins and their overall impact on cell cycle regulation through transcription. Taken together, we will identify unknown factors that act as transcription factors, cofactors or signaling proteins as the next level of regulation in the MuvB/CHR system. We will delineate how these factors form yet unknown links to signaling pathways controlling cell division. Besides providing insight into fundamental biological processes, our results will also have implications for understanding the development of cancer.