Elucidating the Functional Significance of the Lrwd1-ORC Interaction

DNA replication is one of the most important fundamental biological processes. A cell needs toreplicate its DNA before it divides into two daughter cells. In bacteria, the DNA replicationorigin (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 AutonomouslyReplicating Sequences (ARS). The Origin Recognition Complex (ORC; Orc1-6) recognizes andbinds ARS and it subsequently recruits other replication licensing factors to ARS for theinitiation of DNA replication in S phase. However, in metazoans, there is no consensus DNAsequence for the specification of replication origins. How are these origins defined and specifiedremains a big question to be addressed.Lrwd1 is a nuclear protein comprises of two domains; the N-terminal LRR domain and the C-terminalWD40 domain. It is highly conserved among higher eukaryotes but does not exist inyeast and bacteria. We have previously demonstrated that Lrwd1 is a novel ORC binding proteinwhich interacts with ORC via the WD40 domain. Remarkably, we also reported that the WD40domain of Lrwd1 recognizes histone marks H3K9me3, H3K27me3 and H4K20me3 in vitro. Tofurther 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 theHoxB1 replication origin is reduced in Lrwd1-/-cells. Furthermore, bioinformatics analysisrevealed 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 importantrole in designating some of the DNA replication origins in mammalian cells through theH3K9/K27/H4K20me3 -Lrwd1-ORC axis.We hypothesize that Lrwd1 may have a novel role in specifying H3K9/K27/H4K20me3associated DNA replication origins in metazoans by recruiting ORC via its WD40 domain. Inthis proposal we aim to fully characterize the nature of Lrwd1-ORC interaction and thefunctional significance and biological relevance of this Lrwd1-ORC interplay in mammals.Our objectives are to:1. Define the genomic localization of Lrwd12. Genome-wide examination of the recruitment of ORC to replication origins in Lrwd1-/-cells3. 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 originsBy completing this project, we will uncover the nature and the biological relevance of theK9/K27/K20me3-Lrwd1-ORC interactions and might make a major breakthrough inunderstanding how DNA replication origins are specified in mammalian cells. Furthermore, theknowledge gained from this work might provide molecular diagnosis for MGS patients withoutmutation on the known genes (ORC1/ORC4/ORC6/CDC6/CTD1) encoding DNA replicationlicensing machinery proteins.?