Identification, validation and characterisation of i-motif promoter elements in vertebrate genomes

This 3-year self-funded PhD will be based in the School of Biological Sciences and will be supervised by Dr Garry Scarlett, Professor Matt Guille and Dr Fiona Myers.

Genetic regulatory networks provide the key models underpinning our understanding of development, disease and treatment strategies. Within these networks the regulatory “nodes” are transcription factors (TFs): DNA binding proteins that turn genes on or off.

The interactions between transcription factors and regulatory DNA elements to which they bind are therefore critical for our understanding of basic and applied biomedicine – and cataloguing and studying these is the role of the ENCODE project.

These elements are normally a defined base sequence which the TF ‘reads’ in a process known as direct read-out. There are some cases however, where the TF recognises not the sequence itself but the structure that the DNA adopts in a process known as indirect read-out. This is well-established in prokaryotes but less-recognised in eukaryotic cells.

There's growing evidence for indirect read-out mechanisms controlling a selection of eukaryotic genes, and examples of DNA structures are increasingly shown to be involved in transcriptional regulation include A-form, G-quartets, Z-form and recently i-motifs. I-motifs have very recently been shown to exist in vivo but none of these DNA structure-based TF binding sites is currently included in ENCODE, leaving an important knowledge gap for those preparing and using genetic regulatory networks.

This project involves identification, validation and characterization of i-motif promoter elements in the Xenopus genome. We use this model system due to the rapid throughput for gain and loss of function studies together with the ease and speed of making transgenic frogs.

With our collaborators, we have already bioinformatically identified a number of putative i-motif promoter sequences, using bespoke advanced bioinformatic sequence motif analysis. The successful candidate will use the first tranche of these sequences for validation and characterisation. We will initially test oligonucleotides corresponding to these sequences for their ability to bind embryo proteins in EMSA assays and confirm they are i-motifs using a specific antibody.

Sequences that are confirmed as both i-motifs and able to bind specific proteins in the embryo will be tested for their gene control activity. The successful candidate will test this in the intact genome by inserting normal and mutated i-motifs into artificial genes transgenically in “landing site” frogs. In parallel to these experiments, we will use the i-motif specific antibody to immunoprecipitate chromatin for next generation sequencing (ChIP-Seq).

This experimental strategy for identifying i-motif containing promoters will complement the bioinformatic and biochemical approach, and this project will involve the latest biochemistry and molecular biology technologies, such as: vertebrate transgenesis; chromatin immunoprecipitation; bioinformatics and a variety of DNA-protein assays.

The work will be conducted within the biophysics laboratories of the School of Biological Sciences which has a well-established track-record in protein-nucleic acid research, is equipped with excellent facilities and hosts the Wellcome Trust/BBSRC-funded European Xenopus Resource Centre.

Fees and funding

Visit the research subject area page for fees and funding information for this project.

Funding availability: Self-funded PhD students only. 

PhD full-time and part-time courses are eligible for the UK Government Doctoral Loan (UK and EU students only).

How to apply

Please contact Dr Garry Scarlett (Garry.Scarlett@port.ac.uk) to discuss your interest before you apply, quoting the project code.

Please note, to be considered for this self-funded PhD opportunity you must quote project code BIOL4760219 when applying.