Functional characterization of LRWD1 as a regulator of chromatin replication

Abstract

In eukaryotes chromatin function and organisation is defined and mediated by the recognition of DNA and histone modifications, possibly in combinatorial patterns. Cell cycle progression, chromatin organisation and DNA replication are linked and depend on one another. However, the mechanism by which these biological processes are inter-regulated is yet far from clear.

The origin recognition complex (ORC) is responsible for the initiation of DNA replication and plays an important role in cell cycle progression. Through nucleosome affinity purifications, LRWD1 (Leucine rich repeat and WD repeat domain containing 1 protein) has been identified as a novel ORC interacting protein. Since, LRWD1 has been shown to act as an adaptor that mediate ORC binding to chromatin. We have characterised the function of LRWD1 from different angles by investigating how LRWD1 might be regulated by phoshorylation, by uncovering interacting partners of LRWD1 and by studying how LRWD1 interacts with differentially modified nucleosomes.

We report here evidence that phosphorylation of LRWD1 might be involved in regulating the localisation of the protein in the cell potentially by altering a nuclear localisation sequence (NLS). Indeed, stable cell lines expressing phospho-mutants of LRWD1 show different distribution of LRWD1 depending on the phosphorylation status. Furthermore, proliferation defects were observed in the LRWD1 mutant cell. Investigation of LRWD1 interacting proteins in HeLa S3 cells by immunoprecipitation and mass-spectrometry revealed the cohesin subunit SMC3 as an LRWD1-interacting protein. Interestingly, LRWD1 binding sites in the genome, identified by chromatin immunoprecipitation linked to high throughput sequencing (ChIP-seq) positively correlate with SMC3 binding sites. Further analysis revealed that LRWD1 binding sites are enriched at High-C contact points within topologically associating-domains and, interestingly, preferentially localise to the base of chromatin loops in a published 3C study of the CFTR locus. The data so far suggest that LRWD1 could potentially be involved in controlling DNA replication through chromatin architecture.

To determine whether LRWD1 preferentially recruits ORC to differently modified nucleosomes, we expressed and purified recombinant LRWD1, ORC 2-5 and assembled modified nucleosomes and performed in vitro nucleosomes pulldowns. We were able to confirm that LRWD1 preferentially binds to repressive chromatin marks (heterochromatin) and that the binding is independent of ORC.