Retrotransposon transcription drives epigenetic regulation of key genes involved in human innate immunity

Retrotransposons are genetic elements that have populated mammalian genomes via a copy-and-paste mechanism to such an extent that they comprise at least 50% of the human genome. Although erroneously dubbed 'junk DNA' by some researchers in the past, retrotransposons in fact helped the human genome evolve and are now recognized to affect the function of thousands of genes when they occur in close proximity to those genes. It has also been noted that retrotransposons can insert themselves within genes and cause numerous diseases, such as breast cancer and haemophilia, and that the complement of retrotransposons differs substantially between individuals. Finally, there is strong evidence that retrotransposon function is controlled by a further layer of 'epigenetic' control that also affects the function of genes. The proposed research aims to identify as many instances as possible of retrotransposons that regulate genes and to do this research upon genes that are known to be involved in human immunity and disease response. The model cells that will be used are macrophages. These large white blood cells defend the body from pathogens, comprise 10-15% of the total cells in most of our organs and can be stimulated in a laboratory setting to evoke a response nearly identical to what occurs in the body. As such, macrophages provide an excellent model to study the regulation of genes involved in the immune response, including instances where that control is due to retrotransposons. The intended results will be of both academic and biomedical benefit. In regards to the former, the study will shed light on how retrotransposons control the function of nearby genes, how many of these events occur in humans, how genetic variation between individuals affects each event and how important retrotransposons are overall in supporting innate immunity in humans. In regards to the latter, the study will aim to identify pairs of retrotransposons and nearby genes where the retrotransposon can be targeted for suppression or activation. By such means, the research will catalogue genes crucial to the immune response in humans which can be affected indirectly by targeting their controlling retrotransposons, with the ultimate aim of providing tangible improvements in human health.

This project seeks to explore the mechanisms by which retrotransposon transcription can regulate proximal genes via chromatin control. To verify this mechanism in an important human model, and to quantify variation in this model across a given population, peripheral blood monocytes will be collected from two pools of ten individuals each and differentiated to human monocyte derived macrophages (HMDMs). These HMDMs will then be treated with lipopolysaccharide (LPS) to simulate an immune response. For each of the three cell states (monocyte, HMDM, HMDM+LPS), and each of the two pools, we will use high-throughput, sequence tag technologies (CAGE and RNA-seq) combined with Solexa sequencing to produce genome-wide surveys of transcription. We will then perform chromatin immunoprecipitation followed by sequencing (ChIP-seq) for two marks of histone methylation (H3K9me2 and H3K9me3) and one mark of histone acetylation (H3K9ac) to produce genome-wide epigenetic maps. Through a systems biology approach, we will overlay these data sets upon the human genome and identify instances of strongly co-expressed retrotransposon/gene pairs where differential expression is accompanied by substantial chromatin modifications. We will then focus on those retrotransposon/gene pairs where the gene has a known immunological function. For each candidate retrotransposon/gene pair, validation will include RT-PCR to confirm expression of both elements, Sanger sequencing of the retrotransposon transcript to examine its structure, sequence and expression conservation analyses with rodent and pig to probe the evolutionary history of the retrotransposon/gene pair and an enhancer blocker assay for those retrotransposons that demarcate a chromatin boundary. Finally, we will construct gene networks underlying the human immune response to indicate the global role for each regulatory retrotransposon and rationalise these networks in terms of genetic variation across the sampled population.

Biomedical research and human health One of the biggest questions of the post-genome era is how can Science bridge the gap between broad-scale 'omics' research (genomics, transcriptomics, epigenomics etc) and the highly specific needs of biomedical researchers? To generate Gigabases of sequencing is a reasonable achievement, but this data must be translated to practical outcomes to realise its full benefit. This project will at an absolute minimum create detailed maps of transcriptional activity, gene expression and chromatin structure across the human genome using a model of cell differentiation highly relevant to human health. The short read data generated by the project will be made publically available via the Gene Expression Omnibus at NCBI, in effect creating a reference resource for any researcher interested in monocyte or macrophage biology. Moreover, the project will aim to publish a definitive list of genes regulated by retrotransposon expression and also indicate the mechanism of this control. In effect, we will produce leads for further clinical investigation which can improve diagnosis and treatment of immunological disorders, an outcome of direct benefit to human health and quality of life. Expertise in the United Kingdom Considering that each of the four available next generation sequencing platforms (manufactured by Illumina, Applied Biosystems, Roche and Helicos) were all developed in the United States, it is unsurprising that expertise in the analysis and experimental design surrounding these technologies is also concentrated in the United States. If the United Kingdom is to maintain its reputation as a leader in the genome sciences, it must fund infrastructure supporting next generation sequencing (as is the case at the Wellcome Trust Sanger Institute) and also support high profile research where these sequencing technologies are simply a means to answer meaningful biological questions. Through collaboration with the Genome Network Project, an international effort based in Japan and also including researchers from Australia, the United States and Europe, this project will bring expertise and ideas to the United Kingdom whilst highlighting the quality of research being undertaken here. It will also increase the number of researchers with skills in next generation sequencing and analysis who are based in the United Kingdom.