UHRF1 as a coordinator of chromatin replication

The ~2 metres of DNA found in each of our cells can only be fitted into the nucleus through significant compaction, which is done through interactions with nucleosomes. The compacted DNA/nucleosomes, known as chromatin, is regulated through epigenetic modifications: posttranslational modifications on histone proteins as well as the DNA itself. Every time a cell divides, it has to first duplicate its genome. To do this, the chromatin must be dismantled before the replication fork, and reassembled after replication fork passage. Faithful replication of the genome and epigenome is key for normal cell proliferation, cell identity and healthy organism growth. The restoration of chromatin features is directly coupled to the process of DNA replication. Immediately behind the DNA replication fork a number of processes must happen: - DNA synthesis is completed through joining together of the short Okazaki fragments (OF) synthesised on the lagging strand, - the parental histones are transferred and deposited onto both daughter strands and newly synthesised histones incorporated, - newly synthesised unmethylated DNA strands are appropriately methylated, - the daughter strands are held together by cohesion molecules, so that they can be separated later in mitosis. We currently lack understanding of how these “behind-the-fork” processes are coordinated in time and space to efficiently deliver chromatin replication. Using the cell-free Xenopus laevis egg extract system, we have discovered that the UHRF1 ubiquitin ligase, which is known to be essential for maintenance of DNA methylation during DNA replication, is also important for execution of other aspects of chromatin replication, specifically the maturation of OF and new histone deposition. We have also shown that in human somatic cells UHRF1 has an essential, yet uncharacterised, role in S-phase progression. Our hypothesis is that UHRF1 acts as a coordinator of chromatin replication processes, so by understanding how UHRF1 acts in these processes we will shed light on their cooperation “behind-the-fork”. The Xenopus laevis egg extract system is an early embryonic system and allows for studies of synchronous DNA replication in the absence of transcription. This reductionist system allows studies of the direct replication-related consequences of UHRF1 depletion without the confounding effects of hypomethylation. Importantly, DNA replication in egg extracts is regulated very similarly to somatic cells; many fundamental discoveries within the DNA replication field were first made using this system. Through a combination of analyses in Xenopus egg extracts and in human immortalised cell lines, in which we can rapidly degrade degron-tagged UHRF1, we aim to understand: (i) what the consequences are of UHRF1 depletion, and characterise its functions during DNA replication independently of DNA methylation activity. (ii) how UHRF1 regulates OF-maturation and chromatin re-establishment, to shed light on the mechanism of UHRF1 function and the coordination between these “behind-the-fork” processes, and (iii) the differences in chromatin reconstitution dynamics between leading and lagging strand of replication fork and whether UHRF1 activity is needed equally on both strands. UHRF1 is overexpressed and serves as an oncogenic driver in multiple cancers, with significant potential as a therapeutic target and/or cancer biomarker. Our project, although aimed at understanding of fundamental rules of chromatin replication, will help to realise this therapeutic potential of UHRF1 by characterising its functions which are essential for cell proliferation and affect human disease development and healthy aging.