Concerted collaboration between transcription factors and chromatin is important for accurate transcription. Significance of it can be illustrated by frequent mutations in transcription factors (e.g. P53) and chromatin proteins (e.g. TET2) in cancer, where gene expression is deregulated and cells acquire range of phenotypes, which manifest as survival benefit for the mutant cells, but are lethal to the whole organism.
Our group is working on understanding the roles of DNA modifications. Two most abundant DNA modifications - 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are produced respectively by DNA methyltransferases and TET family of oxygenases. It has been broadly recognized that these modifications are found altered in tumour cells, but the exact mechanism how they impact function of the chromatin resulting in deregulated transcription of selective group of genes is not known. The project aims to understand the main rules, which govern 5mC and 5hmC roles in transcription in different cell types. The focus will be in delineating cancer-specific and immune cell-specific mechanisms, which could be employed for therapeutic interventions.
The engagement with the project will provide training in broad range of chromatin biology experimental techniques including DNA modification mapping by sequencing (bisulfite sequencing, TAPS), global DNA modification quantitation by mass-spectrometry, ChIP-seq, ATAC-seq, RNA polymerase occupancy using precision run-on sequencing (PRO-seq) and others. We provide opportunities for training in computational analysis of next generation datasets. In addition to genomics techniques, we have expertise in numerous molecular biology techniques including protein purification, FACS and microscopy. The student will be embedded in the vibrant research group, which investigates different aspects of DNA modifications, resulting in additional expose to the knowledge of mutational mechanisms and metabolism of modified nucleotides.
Project reference number: 1066
|Skirmantas Kriaucionis||Oxford Ludwig Institute||Oxford University, Old Road Campus Research Building||GBRfirstname.lastname@example.org|
|Colin Goding||Oxford Ludwig Institute||Oxford University, Old Road Campus Research Building||GBRemail@example.com|
Despite the importance of epigenetic regulation in neurological disorders, little is known about neuronal chromatin. Cerebellar Purkinje neurons have large and euchromatic nuclei, whereas granule cell nuclei are small and have a more typical heterochromatin distribution. While comparing the abundance of 5-methylcytosine in Purkinje and granule cell nuclei, we detected the presence of an unusual DNA nucleotide. Using thin-layer chromatography, high-pressure liquid chromatography, and mass spectrometry, we identified the nucleotide as 5-hydroxymethyl-2'-deoxycytidine (hmdC). hmdC constitutes 0.6% of total nucleotides in Purkinje cells, 0.2% in granule cells, and is not present in cancer cell lines. hmdC is a constituent of nuclear DNA that is highly abundant in the brain, suggesting a role in epigenetic control of neuronal function. Hide abstract
CpG dinucleotides are the main mutational hot-spot in most cancers. The characteristic elevated C>T mutation rate in CpG sites has been related to 5-methylcytosine (5mC), an epigenetically modified base which resides in CpGs and plays a role in transcription silencing. In brain nearly a third of 5mCs have recently been found to exist in the form of 5-hydroxymethylcytosine (5hmC), yet the effect of 5hmC on mutational processes is still poorly understood. Here we show that 5hmC is associated with an up to 53% decrease in the frequency of C>T mutations in a CpG context compared to 5mC. Tissue specific 5hmC patterns in brain, kidney and blood correlate with lower regional CpG>T mutation frequency in cancers originating in the respective tissues. Together our data reveal global and opposing effects of the two most common cytosine modifications on the frequency of cancer causing somatic mutations in different cell types. Hide abstract
Transitions of cytosine to thymine in CpG dinucleotides are the most frequent type of mutations observed in cancer. This increased mutability is commonly explained by the presence of 5-methylcytosine (5mC) and its spontaneous hydrolytic deamination into thymine. Here, we describe observations that question whether spontaneous deamination alone causes the elevated mutagenicity of 5mC. Tumours with somatic mutations in DNA mismatch-repair genes or in the proofreading domain of DNA polymerase ε (Pol ε) exhibit more 5mC to T transitions than would be expected, given the kinetics of hydrolytic deamination. This enrichment is asymmetrical around replication origins with a preference for the leading strand template, in particular in methylated cytosines flanked by guanines (GCG). Notably, GCG to GTG mutations also exhibit strand asymmetry in mismatch-repair and Pol ε wild-type tumours. Together, these findings suggest that mis-incorporation of A opposite 5mC during replication of the leading strand might be a contributing factor in the mutagenesis of methylated cytosine. Hide abstract