Iestyn Whitehouse: Overview
We now understand that genomes are composed of many functionally different segments of chromatin. Given that there is such diversity in chromatin structures, a fundamental question is: how is chromatin established and maintained within a cell? This question is perhaps most significant during S-phase when chromatin is disassembled and reassembled on the new daughter genomes. Our published work has shown that chromatin assembly is intrinsically coupled with DNA replication, and that this coupling facilitates the rapid assembly and organization of chromatin on nascent DNA. Our ongoing work will provide a basic framework for how chromatin structures are established following DNA replication.
Despite the importance of DNA replication, numerous aspects of this process are still poorly understood. One fundamental question is: how do replication forks efficiently progress through chromatin? Understanding this question is complicated by the diversity of chromatin structure and the heterogeneity of replication reactions. We have developed and are continuing to develop new technology to overcome these issues in order to map different aspects of replication in cells. A second outstanding question is: what specifies the location of replication origins in metazoan cells? Though a general association of replication origins with “open” chromatin and with higher order chromatin structure has been noted, the exact determinants of origins remain undefined. Our recent work in C. elegans directly addresses this issue. We have generated the first series of high-resolution maps of active replication origins during multiple stages of embryogenesis. Our maps have allowed us to uncover a remarkable interdependence of chromatin structures, gene transcription, and DNA replication. These findings set the stage for us to explore how fundamental aspects of chromosome biology interact to generate specific transcription and replication outcomes that are needed in different stages of development.
Exploring chromatin structure and DNA replication in C. elegans embryos.
We are defining how replication origins are specified in metazoan cells. We have recently uncovered a striking overlap between replication origins and chromatin features and we are exploring what features of chromatin structure are necessary and sufficient for origins to form. In addition, we are testing if replication origins are propagated through generations by the inheritance of “epigenetic” chromatin states.
We are interested to learn how chromatin structure is established and maintained in developing C. elegans embryos. For this project we are testing the relationship between germline transcription and embryonic chromatin states; we are exploring the idea that embryonic chromatin structure is templated from the germline and we are trying to uncover the mechanisms by which this may occur.
Developing new genomics tools for the analysis of DNA replication and chromatin structure.
Most genomics methods that study DNA replication rely on population-based assays; while these are powerful, they miss infrequent or stochastic events that occur within the mixture of cells. Such rare events, such as fork stall or collapse, are highly biologically significant, but we understand little of where and when they may occur across a genome. To overcome these limitations we are developing new genome-wide approaches to capture DNA replication with single event readouts. We aim to use these new methods to map how individual replication forks progress through the genome.
Chromatin organization is complex and we have incomplete understanding of how chromatin is organized with the nucleus. We believe that new assays are needed and we are developing methods to capture and map the relative locations of complex chromatin structures and domains within a cell. We are especially interested to learn whether DNA replication and gene transcription occurs in clustered entities known as factories.