Dissecting transcription block in haematological malignancies

Dr Athar Aziz (a.aziz@salford.ac.uk)

Every gene in mammalian cells is tightly regulated by the conformation of chromatin in human genome. Commonly regulated gene sets define the fate of any given cell. Mechanisms ofregulating transcriptionoccurs at multiple levels, including covalent modifications of histone tails that in turn affectaccessibility, commonly known as epigenetic modifications.

The focus of my lab is to dissect the mechanisms of gene set regulation in normal and malignant haematopoietic cells at the transcriptional level. We have developed a transcription activator-like effector (TALE) - zinc fingerprotein based system – that has been used previously for genome editing – for binding specific regions of the human genome.

Using TALE coupled with protein tags we plan to purify the genomic elements along with epigenetic factors binding at those particular elements at a given stage of differentiation and in the cells derived from acute leukaemia as model malignancy. TALEs will be designed to bind the specific promoter elements in genes important not only in control of haematopoietic stem cells fate choices but also known to play role in acute leukaemia including MYC, HOXA9, BLC2, CDX2, RARa and CSF-1R.

Using tags as bait we plan to immuno-precipitate the cross-linked genomic DNA along with the epigenetic element bound to that promoter. The precipitated material will be analysed by western blot to confirm the precipitation efficiency and purified immuno-precipitated genomic material will be analysed using mass-spectrometry to define the epigenetic elements bound to a given region. Differences in the bound and unbound proteins on a given genomic locus define the ‘active’ or ‘passive’ state of that particular genetic locus.

This work is mainly going to be carried out in cell lines because of the number of cells required to purify a small region of genome compared to rest of the genome. These findings will then be confirmed in primary blast cells from AML patients using virally induced TALEs expression.

The student will spend the first year generating the optimal TALEs for promoter elements of the above mentioned targets using piggyback plasmids. In the second year, we expect to purify and analyse by mass-spectrometry the genomic loci and in the last year of PhD we expect to validate the qualitative and quantitative data generated.

The techniques that will mainly be used in this project include cloning, chromatin-immuno precipitation (ChIP), and mass-spectrometry (MALDI-TOF), therefore, this project will help to define the epigenetic landscape of a given genomic locus that plays a key role in acute myeloid leukaemia.