Elucidating the Functional Significance of the Lrwd1-ORC Interaction
DescriptionDNA replication is one of the most important fundamental biological processes. A cell needs to replicate its DNA before it divides into two daughter cells. In bacteria, the DNA replication origin (oriC) is governed by AT-rich DNA sequence whereas in lower eukaryotes such as yeast, the origins of DNA replication are specified by a cis-acting element called Autonomously Replicating Sequences (ARS). The Origin Recognition Complex (ORC; Orc1-6) recognizes and binds ARS and it subsequently recruits other replication licensing factors to ARS for the initiation of DNA replication in S phase. However, in metazoans, there is no consensus DNA sequence for the specification of replication origins. How are these origins defined and specified remains a big question to be addressed. Lrwd1 is a nuclear protein comprises of two domains; the N-terminal LRR domain and the C-terminal WD40 domain. It is highly conserved among higher eukaryotes but does not exist in yeast and bacteria. We have previously demonstrated that Lrwd1 is a novel ORC binding protein which interacts with ORC via the WD40 domain. Remarkably, we also reported that the WD40 domain of Lrwd1 recognizes histone marks H3K9me3, H3K27me3 and H4K20me3 in vitro. To further understand the function of Lrwd1 we have generated Lrwd1 knockout mice. Lrwd1-/-mice displayed dwarfism phenotypes which partly mimic Meier-Gorlin Syndrome (MGS) patients with mutations on genes encoding replication licensing factors (e.g. ORC1/ORC4 /ORC6/CDT1/CDC6). In addition, Lrwd1-/-MEF cells displayed cell proliferation defects. Moreover, our chromatin immunoprecipitation (ChIP) results showed the binding of Orc2 to the HoxB1 replication origin is reduced in Lrwd1-/-cells. Furthermore, bioinformatics analysis revealed the overlapping of replication origins with H3K9me3 enriched regions in mouse cells. These findings collectively highlight the possibility that Lrwd1 may play a novel and important role in designating some of the DNA replication origins in mammalian cells through the H3K9/K27/H4K20me3 -Lrwd1-ORC axis. We hypothesize that Lrwd1 may have a novel role in specifying H3K9/K27/H4K20me3 associated DNA replication origins in metazoans by recruiting ORC via its WD40 domain. In this proposal we aim to fully characterize the nature of Lrwd1-ORC interaction and the functional significance and biological relevance of this Lrwd1-ORC interplay in mammals. Our objectives are to: 1. Define the genomic localization of Lrwd1 2. Genome-wide examination of the recruitment of ORC to replication origins in Lrwd1-/-cells 3. Characterize the interactions of Lrwd1 with ORC subunit(s) and histone mark(s) 4. Investigate the biological relevance of the Lrwd1-ORC interaction on cell cycle progression, cell proliferation and specification of replication origins By completing this project, we will uncover the nature and the biological relevance of the K9/K27/K20me3-Lrwd1-ORC interactions and might make a major breakthrough in understanding how DNA replication origins are specified in mammalian cells. Furthermore, the knowledge gained from this work might provide molecular diagnosis for MGS patients without mutation on the known genes (ORC1/ORC4/ORC6/CDC6/CTD1) encoding DNA replication licensing machinery proteins. ?
|Effective start/end date||1/01/16 → …|
- Lrwd1,Origin Recognition Complex,DNA replication origins,Meier-Gorlin Syndrome (MGS),