Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.733217
Title: An in vitro biochemical investigation into the conformation, binding and E3-ubiquitin ligase activity of mammalian UHRF1 with reconstituted chromatin
Author: Foster, Benjamin
ISNI:       0000 0004 6496 8029
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2017
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Abstract:
In the eukaryotic genome, DNA and histone modifications regulate chromatin function and mediate basic processes such as gene transcription, DNA repair and DNA replication. Maintaining chromatin modifications after DNA replication is essential for chromatin homeostasis, especially for regions of the genome that need to be kept silenced such as repetitive elements. The maintenance DNA methyltransferase, DNMT1, is responsible for ensuring that cytosine methylation at CpG dinucleotides, and thus proper transcriptional programmes, are propagated to the daughter cells. DNMT1 is specifically recruited to newly replicated, hemi-methylated DNA and the E3-ubiquitin ligase UHRF1 (Ubiquitin-like containing PHD- and RING-finger domains protein 1) plays a critical role for this. The mechanisms of the recruitment of DNMT1 to chromatin via UHRF1 are currently an area of active investigation. Several studies using modified nucleosomes, histone peptides and DNA oligonucleotides have identified UHRF1 to bind to hemi-methylated CpG dinucleotides and to histone H3 di- or tri-methylated at Lys-9. Since UHRF1 was also found to interact with DNMT1, it was postulated that UHRF1 acts as an adapter that directly recruits DNMT1 to newly replicated DNA. Additionally, it has recently been reported that the E3-ubiquitin ligase activity of its C-terminal RING-finger is required for the recruitment of DNMT1 to replication forks. Ubiquitylation of either K18 or K23 on histone H3 that is recognised by a ubiquitin-interacting motif within DNMT1 appears to be critical for DNMT1 targeting but the recruitment mechanism has so far not been completely elucidated. This study has investigated the binding and E3-ubiquitin ligase activity of UHRF1 in the context of physiologically relevant chromatin substrates. Using a fully reconstituted system, the chromatin binding and enzymatic activity of UHRF1 and how this is linked to its intra-molecular arrangement have been elucidated. In the context of modified nucleosome substrates, we observe an increase in binding of recombinant UHRF1 in the presence of hemi-methylated DNA whilst with histone H3K9me2/3, only a small increase in binding is detected. We also provide evidence that binding to nucleosome core particles is enhanced by a basic region between the SRA-domain and the RING-finger. This so called polybasic region or PBR has previously been implicated in the regulation of UHRF1 binding to H3K9me2/3 marks. Our findings therefore suggest that binding of UHRF1 to physiological chromatin substrates is more complex than previously thought. In-solution crosslinking/mass spectrometry experiments using the full-length protein confirm that UHRF1 exhibits complex intra-molecular contacts that can potentially regulate its interaction with chromatin or other factors. In addition to reported contacts between the PBR with the Tandem-Tudor domain and between the PHD-finger and the SRA-domain, the UBL-domain also makes extensive contacts to other regions within UHRF1. These appear to be weak and dynamic. Crucially, removal of the UBL-domain does not affect nucleosome binding but does result in a strong reduction in UHRF1 E3-ubiquitin ligase activity. Further experiments suggest that the UBL-domain is involved in establishing the enzyme/substrate complex between the E2-conjugating enzyme and the chromatin substrate and in stimulating the transfer of ubiquitin from the E2~Ub complex to histone H3. In summary, by combining a crosslinking/mass spectrometry approach to interrogate the intra-molecular arrangement of UHRF1 with fully reconstituted enzyme and chromatin-binding assays using physiologically relevant substrates, we have identified a function for the UBL-domain of UHRF1. Our results suggest that the UBL is highly flexible in solution and that it forms transient contacts with other parts of UHRF1 and the E2-conjugating enzyme that are required for the formation of the E2/E3/substrate complex in allosterically activating ubiquitin transfer from the E2~Ub to the histone target substrate. These findings assign, for the first time, a function for the UBL-domain and pave the way for further investigation of the involvement of this domain in the physiological role of UHRF1.
Supervisor: Bartke, Till ; DiMaggio, Peter Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.733217  DOI:
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