Functional Analysis of the Role of MCM3 in Gene Regulation

MCM3的基因調控功能研究

Student thesis: Doctoral Thesis

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Award date8 Jun 2020

Abstract

Minichromosome maintenance component 3 (MCM3) is one of the six MCM proteins that forms heterohexamer (MCM2-7) and is well known for its function in DNA replication. As a DNA replicative helicase complex, the MCM2-7 hexamer binds to replication origin at the beginning of the G1 phase and is then activated by recruiting CDC45 and GINS to form the catalytic CMG helicase at G1/S transition to initiate chromosomal DNA replication. Although the function of MCM proteins regarding DNA replication has been well characterized in the past 30 years, there are still mysteries to be solved. The phenomenon of the “MCM paradox,” which refers to the excess amount of MCM proteins loaded on chromatin compared to replication origins, indicates that MCM proteins may have additional functions other than DNA replication. A previous study identified MCM3 as a candidate for maintaining the gene silencing of X chromosome inactivation (XCI), indicating its potential role in gene regulation. In this project, I first proved the subcellular colocalization of MCM3 with heterochromatin in both mouse and human cells. Moreover, RNA sequencing (RNAseq) with MCM3 depletion resulted in significant gene expression changes in mouse embryonic fibroblasts (MEFs), revealing the role of MCM3 in gene regulation. Additionally, we performed immunoprecipitation coupled to mass spectrometry (IPMS) and identified NONO and PSPC1 as two novel protein partners of MCM3. These two proteins form heterodimer and are both chromatin remodelers that has been shown participate in various aspects of gene regulation. I further demonstrated the direct interaction between MCM3 and PSPC1, but not NONO by in vitro binding assay. Besides, common genes that are differentially expressed following MCM3, NONO or PSPC1 knockdown were identified, suggesting that these genes may be cooperatively regulated by MCM3, NONO and PSPC1. Furthermore, to gain more insights into the mechanisms of MCM3’s role in gene regulation, genomic localizations of MCM3, NONO or PSPC1 were mapped using Cleavage Under Targets and Release Using Nuclease (CUT&RUN) sequencing. The peak distribution analysis of the CUT&RUN signals indicates that MCM3, NONO and PSPC1 are enriched in promoter regions. Moreover, MCM3, NONO and PSPC1 are found colocalized at their common target gene loci. The RNAi-based knockdown following CUT&RUN proved the specificities of the bindings of MCM3, NONO and PSPC1 and revealed that these three proteins are interdependent with each other on some of the target genes. My findings describe the gene regulatory role of the well-studied component of DNA replicative helicase, MCM3, which broaden our knowledge of the role of MCM proteins and provide additional explanation for the “MCM paradox.”