Mechanisms of cell cycle-dependent transcriptional regulation by MuvB complexes

• Applicant: Professor Dr. Kurt Engeland, since 1/2023
• Subject Area: Cell Biology ; Biochemistry
• Term: from 2015 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 277046125

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.

• DFG Programme: Research Grants
• Ehemaliger Antragsteller: Dr. Gerd Müller, until 12/2022