REGULATORY CASCADES UNDERLYING KIDNEY DEVELOPMENT, HOMEOSTASIS AND DISEASE

Summary
The study of genes which when mutated in human disease is leading to new insights into disease mechanism, fundamental biology and potential new therapeutic avenues.
One such gene is the Wilms' tumour gene, Wt1. Mutations in this gene may lead to childhood kidney cancer, reproductive problems and to live threatening paediatric and adult kidney disease as well as, occasionally diaphragmatic hernia and heart disease. It is expected that an understanding of the mechanisms by which these diseases arise could lead to clinical benefit. Our studies and those of others in mice have shown that Wt1 is essential for the development of the kidneys, gonads, spleen and coronary vessels. Furthermore in adult mice Wt1 continued to be required for maintenance of multiple organs, including the kidney, spleen, fat and bone. The Wt1 gene encodes multiple related proteins that have special features that allow them to bind to DNA in chromosomes. Here Wt1 activates or repression of key regulators of tissue differentiation and maintenance. However, this is only part of the story. Work over many years, mainly from my laboratory has shown that Wt1 not only binds DNA in the nucleus but also interacts with RNA and with regulatory complexes that produce mature mRNA and regulate the levels of mRNA and protein synthesis. We hypothesise that Wt1 is a novel molecule that may regulate gene expression all the way from mRNA synthesis in the nucleus, to its translation on polysomes and might help couple the various steps in the cascade for specific mRNAs. In this proposal we aim to use the latest genome wide approaches to identify the key Wt1 target genes and interacting RNAs and to dissect the role of Wt1 in regulating the production, processing, transport, turnover and translation of these mRNAs. These studies should provide key insights into gene regulatory networks while shedding light on the mechanisms underpinning development, homeostasis and disease. In the longer term it is hoped the information obtained will lead to new treatments for serious illness.

We propose to use a combination of genome wide approaches to identify WT1 target genes and mRNAs and to determine whether loss or gain of WT1 function influences the transcription, splicing, turnover and translation of these targets. We will determine whether the 2 major WT1 isoforms differing by only 3 amino acids inserted between 2 zinc fingers bind to different or the same targets and function at different steps in the regulatory cascade. We will focus on 2 kidney stages most relevant to human disease that arise through WT1 mutation- the fetal metanephric mesenchyme and adult podocytes. We will identify WT1 binding target genes and RNAs in these two cell types using ChIP seq and CLASH respectively. This will also map the binding sites and identify consensus binding sequences within the RNA. We will then determine whether WT1 regulates these genes and mRNAs and at what levels, including transcription, splicing, transport, translation and turnover. These studies will be carried out in cell lines or tissues having gained or lost WT1 function or its specific isoforms. To measure transcription rates of target genes in these cells we will use polII ChIP seq. To evaluate splicing, transport and target RNA turnover RNA seq will be carried out on fractionated nuclei and cytoplasm from these cells. Translation rates of target mRNAs will be determined using ribosome profiling. Bioinformatics analysis will tell us: what target genes and mRNAs are regulated by WT1 in mesenchyme and podocytes. At what steps does WT1 regulate the expression of its target genes- transcription, splicing, transport, translation, turnover? Does it chaperone some genes throughout the whole regulatory cascade? What is the role of the two major isoforms? What is the role of ncRNA WT1 targets? What are the biological pathways being regulated and how does this inform on the normal biology and diseases of the kidney? Functional experiments will be carried out to validate the findings.

Beneficiaries
In the short term it is doubtful the findings arising from this study will benefit any group outside academia. In the longer term the work could lead to clinical benefit.