Understanding transcription mediated methylation of intragenic CpG islands in health and disease

1. Project outline describing the scientific rationale of the project (max 4,000 characters incl. spaces and returns)
DNA methylation is an epigenetic modification that affects gene expression without altering gene sequences. In mammals, DNA methylation occurs preferentially at cytosines (C) within CpG dinucleotides, that is Cs that are followed by a guanine (G) on the same DNA strand. Regions with a high content of CpG dinucleotides known as CpG islands or CGIs, are frequently found at gene promoters and, surprisingly, remain devoid of DNA methylation. In normal development, CGI methylation is observed in specialised situations (e.g. imprinting, X-inactivation and at some tissue specific genes). Aberrant methylation of CGIs can lead to human disease in both inherited (e.g. alpha thalassaemia, Lynch syndrome, etc) and acquired forms (e.g. cancer). The molecular mechanisms responsible for driving establishment of methylation at intragenic CGIs and for its maintenance in health and disease situations are not thoroughly understood.

We were the first to show that transcription through the alpha globin (HBA2) CGI causes its methylation and silencing in a patient with an inherited form of anaemia (alpha thalassaemia) and we have developed a mouse embryonic stem cell (mESC) line (2A3) that recapitulate this phenomenon (Tufarelli et al., 2003 Nat Gen). Using this system, we have recently demonstrated that establishment of methylation at CGIs traversed by transcription (intragenic CGIs) occurs upon differentiation and is catalysed by the de novo DNA methyltransferase DNMT3B (Jeziorska et al., 2017 PNAS). Our data indicate that transcription is necessary for methylation of the HBA2 CGI, but, in contrast to what happens at other transcribed regions of the genome (Baubec et al., 2015 Nature), for transcribed CGIs to become methylated the mESCs need to undergo differentiation. This property can be exploited to dissect the molecular mechanisms underlying this phenomenon.

This project aims to use the mES differentiating system to:
1. Identify the component of the DNA methylation establishment complex:
Stable 2A3 cells carrying an inducible dCas9-APEX construct that can be induced at different stages of differentiation will be generated and targeted to the proximity of the HBA2 CGI using specific guide RNAs. Biotinylated proteins will be isolated using streptavidin beads and identified by LC-MS/MS. Validation of complexes will be performed by ChIP and Co-IPs.

2. Determine if DNMT3B can rescue the methylation phenotype in DNMT3B KO cells and which interacting partner is necessary for the rescue.
Using CRISPR/Cas9, a recombination mediated cassette exchange (RMCE) will be targeted to a region of mouse chr 11 that we have shown to be a neutral environment (Jeziorska et al., 2017 PNAS). Wild type DNMT3B or constructs carrying deletions of specific domains or mutations will be inserted in this locus by RMCE and tested for the rescue of the methylation at the HBA2 CGI. Co-IPs will be performed to test for the integrity of the complex identified in aim 1. Terminal differentiation will be used to test whether any of the mutations affects the switch from establishment to maintenance of methylation.

3. Identify the intragenic CGIs methylated by DNMT3B at a genome wide level
Using a similar approach to that used in Jeziorska et al for human ESC, publicly available transcription, methylation and histone modification profiling datasets for undifferentiated and differentiated mESCs will be compared to identify those intragenic CGIs that become methylated upon differentiation. Custom datasets from the DNMT3B knock out and rescue clones from aim 2 will also be analysed and the results compared to that of wild type cells to identify those genes that rely solely on DNMT3B for their regulation and whether any of them are associated with human disease.

Timeline of the project
Techniques that will be undertaken during the project

- mESCs culture and differentiation. mESC manipulation, stable transformation (dCas9-APEX), locus specific targeting by CRISPR/Cas9 and Recombination mediated cassette exchange (RMCE)

- DNA methylation (bisulphite sequencing; methylated DNA immunoprecipitations; methylation sensitive PCR) and histone modification profiling by chromatin immunoprecipitations

- Proteins purification and LC-MS/MS; Co-IP and ChIP

- Bioinformatic analyses of custom and publicly available transcription, methylation and ChIP datasets

BBSRC Strategic Research Priority

Molecules, Cells and Systems
• Gene Expression

Available to UK/EU applicants only

Application information
https://www2.le.ac.uk/research-degrees/doctoral-training-partnerships/bbsrc

Closing date 6 January 2019