Mechanism and regulation of RNA-directed chromatin modification.

It has recently been discovered that the human genome encodes a surprisingly small number of proteins, but produces an amazingly large amount of RNA; in fact most of the genome is transcribed into RNA that does not encode protein. The functions of most of these non-coding (nc)RNAs are still unknown, however known roles include regulating gene expression via chromatin modification. Indeed, changes in ncRNA levels are associated with many diseases, suggesting they may have widespread roles in genome regulation. Understanding how ncRNAs regulate gene expression can therefore help us understand a whole range of human diseases.

In my research I will use fission yeast as a model system to study ncRNA-directed chromatin modification. I will determine the functions of four new proteins I have identified as being required for RNA-directed chromatin modification; understanding the roles of these proteins will help us to understand how the process works. I will also perform genetic screens to identify proteins that inhibit the pathway, to understand how it is regulated. Finally I will study different targets of RNA-directed chromatin modification, to understand its biological functions. The results of these studies in yeast will greatly aid our understanding of medically-important related pathways in humans.

An understanding of genome regulation is essential for understanding disease. Recent studies have revealed that non-coding (nc)RNAs have widespread roles in genome regulation, including transcriptional silencing via chromatin modification. Despite this, the molecular mechanisms underlying such RNA-directed chromatin modification (RdCM) remain poorly understood.



My aim is to understand the mechanism, regulation and roles of RdCM, using the fission yeast Schizosaccharomyces pombe as a model system. Heterochromatin formation at fission yeast centromeres is an excellent paradigm for RdCM in general. Chromatin modification is known to require non-coding centromeric RNAs, processed by the RNAi intereference (RNAi) pathway. However, the mechanism by which RNAi recruits chromatin modifiers has remained obscure.



My first objective is to elucidate the mechanism of RdCM by determining the functions of four novel proteins I have identified as being involved in RdCM. One of these proteins, Stc1, plays a key role in linking RNAi to chromatin modification; I will characterise the functions of Stc1 to determine the molecular mechanism by which Stc1 mediates RNAi-dependent chromatin modification. I will also determine the roles of the other three proteins in facilitating RdCM.



My second objective is to identify and characterise negative regulators of RdCM. Ectopic chromatin modification can down-regulate essential genes, a cause of cancer. Proper regulation is therefore critical, but despite this, little is known about regulators of RdCM. I will perform genetic screens to identify proteins that negatively regulate RdCM, and determine their mode of action.



My third objective is to characterise alternative roles of RdCM. Thus far studies in fission yeast have focussed on the role of RdCM in maintaining constitutive heterochromatin. However, preliminary data suggests that, as in higher eukaryotes, RNAi/RdCM also regulates retroelements and certain genes. I will determine the mechanisms and roles of ncRNA-mediated regulation at these loci, and examine how these pathways may contribute to stress responses.



These studies will address specific questions about the mechanism and regulation of RdCM, and broaden the scope of fission yeast as a model for ncRNA-mediated genome regulation. The characterisation of these pathways in yeast and identification of homologous components in other species will form a basis for future studies aimed at understanding analogous pathways in humans. Changes in ncRNAs and defects in RdCM have been linked to numerous diseases, so understanding the mechanisms and regulation of RdCM will help us to understand many diseases associated with mis-regulation of gene expression in humans.