Role of chromatin dynamics in regulation of intestinal innate immunity gene expression

In the intestine, a single layer of cells mediates the uptake of nutrients and creates a niche for beneficial microbes while at the same time it provides a barrier against invasion by pathogenic organisms. Important aspects of this defensive function are processes of innate immunity, which summarize various cell intrinsic mechanisms by which cells fend off pathogens. Examples for such processes are the secretion of anti-bacterial peptides and hydrolytic enzymes, detoxification processes and autophagy. If the barrier function of the intestine is impaired, this can lead to deadly infections but also devastating chronic, inflammatory diseases, such as Crohn's disease and ulcerative colitis. How the expression of factors involved in innate immunity processes is regulated and orchestrated is an important question with huge relevance to our understanding of health and disease.

In this application we will contribute towards this by building on exciting findings from our labs that link the packaging of the genome to the regulation of innate immunity genes in the intestine. We found that deletion in intestinal epithelial cells of an enzyme that regulates the packaging of the genome (a chromatin remodeling factor) renders mice more resistant to experimentally induced colitis. This unprecedented finding appears to be linked to the fact that the mice without this factor over-express many genes involved in innate immunity even without any challenge. Thus, this factor appears to orchestrate the regulation of many genes involved in innate immunity. Furthermore, in the absence of this factor we find changes in the packaging of many gene regulatory elements closely linked to genes, including many genes involved in innate immunity. We hypothesize that these changes facilitate the expression of these genes and may represent a general mechanism by which cells adapt to stress.
We wish to test these ideas and examine if the changes we observe are linked to the colitis response. This work will reveal potential new diagnostic markers and therapeutic targets for intestinal inflammatory conditions, while providing insights into very fundamental gene regulatory processes.

How chromatin dynamics in the intestinal epithelium orchestrates innate immunity is an important question but little is known about it. Our hypothesis is that chromatin dynamics, especially changes of histone variant H2AZ occupancy over CpG islands, regulate innate immunity gene expression in intestinal epithelium cells. We propose that histone H2AZ occupancy relates to cellular stress and the innate immunity response, e.g., during exposure to microbes, may reflect such a stress.

To test this, we will exploit a very well established colitis model in the mouse, Dextran Sodium Sulfate (DSS) mediated colitis, and focus on the role of two chromatin remodeling factors that promote (EP400) and counteract (Smarcad1) histone H2AZ occupancy. We will examine an exciting new histone modification, crotonylation, which we find is affected by chromatin remodeling and may be linked to H2AZ occupancy and possible the microbiota.

We will use chromatin immunoprecipitation coupled to high-throughput sequencing (ChIPseq) approaches to map histone H2AZ, histone crotonylation and chromatin remodeling factor occupancy during normal and colitis conditions and link these occupancies to a detailed analysis of the transcriptome, including the noncoding transcriptome. We will systematically examine CpG island chromatin using mass spectrometry approaches under all these conditions.

Our work will test if chromatin remodeling factors are involved in coordinating an innate immunity response. Together, data generated in this proposal will paint a detailed picture how the transcriptome in colon epithelial cells is affected during colitis and how chromatin dynamics prime these cells to respond to an insult.

More than 200 000 patients are though to suffer of Crohn's disease or ulcerative colitis in the UK currently (www.crohnsandcolitis.org.uk), but the numbers of patients is rising worldwide, potentially linked to changes in life style and nutrition. Inflammatory bowel diseases are often devastating and can currently only be treated, but not cured. Novel approaches and ways of thinking with regards to these diseases have the potential to bring new advances in diagnosis and treatment. Little is known how genome-regulatory processes such as chromatin dynamics impact these conditions, despite the emerging view that epigenetic processes may have an important influence in disease occurrence. It is remarkable that several chromatin remodeling factors, including those that are the subject of this proposal, are linked to Crohn's disease by genome-wide association studies (see: www.gwascentral.org/). Our work aims to benefit patients of intestinal inflammatory diseases. Our research may provide novel diagnostic markers and therapeutic targets. We find that deletion of chromatin remodeling factor Smarcad1 renders mice relatively resistant to colitis. In the future, small molecule inhibitors of this or other factors that regulate for example H2AZ function may help improve the conditions for patients of inflammatory bowel diseases. The recent discovery of 'histone mimics' such as the BET inhibitors (I-BET) as promising new drugs to combat cancer and inflammation show that such ideas are not far-fetched. Our observation that up-regulation of osteocalcins is linked to this resistance is very exciting and may link to novel therapeutic avenues. We will test if an up-regulation of features we connect to colitis in this proposal such as histone variants H2AZ, histone crotonylation and others can be identified in tissue slices using immuno-histochemistry and thus may have potential diagnostic value.

Our research will highlight the molecular requirements for a healthy intestine during steady state conditions, identify novel genes involved in tissue homeostasis, preventing intestinal disease and could elucidate the genetic predisposition to intestinal challenges, e.g., through pathogenic bacteria. All of which has important health benefits for the general public, who will benefit from a better understanding of the mechanisms of intestinal immunity and homeostasis. We hope to engage the general public, and specifically students and teachers, with our scientific research about the link between genome regulation and intestinal health. The potential health implications for patients and the elderly suffering conditions affecting the gut, such as inflammatory bowel disease or autoimmune diseases, including coeliac disease, are innumerable. Furthermore, understanding the molecular basis for disorders of the gut will allow health care providers and policy makers to make more informed decisions, both when treating patients and setting clinical policy.

The outcome of the research will be of interest to the R&D sector, both within the UK and internationally. Identifying the genetic disposition to intestinal diseases and new mouse models could lead to the development of new drugs or novel diagnostic approaches. As well as the obvious patient benefits, this will enhance the commercial impact and competitiveness of the investigators and their industrial partners. The proposed research will provide large data sets representing gene expression (transcriptomes, RNA-seq) and chromatin states (ChIP-seq) in various cell types, including intestinal stem cells, which will be made freely available following publication. This data will benefit researchers in academic and business sectors. For more information on specific collaborations please refer to the Academic Beneficiaries section.

The grant will employ a junior postdoctoral researcher and support advanced training in a diverse range of areas. We will also train internship students and other sceintists.