Identification of DNA elements that maintain epigenetic memory in T cells

We are investigating the underlying mechanisms that allow a gene that has been activated once before to be more easily activated a second time. These mechanisms involves epigenetics, as opposed to genetics, whereby the chromosomal structure but not the DNA sequence is altered to create an active conformation. This is in fact the mechanism that makes it possible for people to be vaccinated against infections, and which explain why people become immune once they have been infected once by a specific disease. Immunity is maintained by white blood cells and we have shown these cells maintain an imprint within their genes when they have been activated once by an immune response. We have shown that activated cells acquire stable structural changes within their genes that allow these genes to be reactivated more efficiently when they are activated for the second time. Our aim is to identify the specific molecules in the cell that make this process possible by binding to and then staying associated with genes. We will do this by first identifying all of the sites in the cell where structural changes are introduced, and then proceed to identifying which specific molecules are bound there. We will also investigate the importance of the structural modifications by (i) deleting DNA elements from the genome that support the maintenance of active chromosomal conformations, and (ii) by removing soluble factors from inside cells that bind to genes to create the active chromosomal structures.

We will (1) Identify transcription factors and chromatin modifiers bound to primed DHSs in previously activated CD4+ve T cells. We will perform in vivo footprinting and a bioinformatics analysis to identify motifs in the T blast-specific DHSs using CD4+ve T cell populations from IL-3/GM-CSF transgenic mice that contain 130 kb of the human locus. Cells will include (i) naive splenic CD44loT cells, (ii) splenic CD44hi cells that include memory cells and recently activated cells, and (iii) actively proliferating T blast cells maintained in culture with IL-2. (2) Global mapping of DHSs, histone methylation and factors in previously activated T cells. Genome wide analyses will be performed on naive CD4+ve CD44lo and on actively proliferating T blast cells, by Illumina sequencing of: (a) short DNA fragments purified from DNaseI treated cells to identify all DHSs. (b) DNA from Me2H3K4, Me3H3K4 and Me3H3K27 ChIP assays. (c) ChIP of the top 2 candidate transcription factors bound to primed DHSs. (3) Perform a bioinformatics analysis to identify specific transcription factor motifs that maintain stably acquired DHSs in previously activated and memory T cells. We will identify binding sites, employing motif databases such as TRANSFAC and Jaspar. We will also analyse the regions de novo and perform an unbiased search to discover potentially novel regulatory motifs. (4) Functions of primed DHSs in memory T cells and T blast cells. (a) Deletion analysis of primed DHSs in transgenic mice. We will create 2 lines of transgenic mice to delete specific DHSs with Me2K4H3 modifications and assay them alongside pre-existing lines, using naive CD4+ve CD44lo and CD4+ve T blast cells after either 2 or 4 hours of stimulation. (b) Validate the roles of specific transcription factors by siRNA- knock-down. We will use shRNAs directed against factors bound to primed DHSs in proliferating T blast cells and select GFP+ve transfected cells for analysis as above.

Our work will have a tremendous impact not only on our immediate research field but also beyond. The studies proposed here will lead to a better understanding the transcriptional basis of T cell memory, and therefore have the potential to benefit all future therapeutic approaches utilizing T cells. To achieve maximum impact we plan the following activities: (i) We will make our system-wide data sets publicly available. This will benefit anybody who studies normal or aberrant T cell development in academia, industry or the clinic. (ii) One significant potential outcome of our work is the identification of transcription factor combinations that regulate T cell memory modules. This will benefit anybody who is interested in manipulating memory T cells. We will make our expertise available to members from industry and academia who wish to explore this possibility. (iii) Our work will enhance the skills base in the UK. Future advances in biology and medicine will depend on building a skills base consisting of researchers which will be capable of thinking both in molecular terms as well as in system-wide terms, and the postdoc working on this grant will be trained to do precisely that.