Small RNA pathways during ES cell differentiation and X inactivation – RNAES

The recent discovery of the RNA interference (RNAi) process and its effector molecules ' small RNAs of 21 - 40 nt - has led to a revolution in our comprehension of gene regulation in many organisms (plants, invertebrates and vertebrates) (Fire et al., 1998; Baulcombe, 2004; Lau et al., 2006; Katiyar-Agarwal et al., 2007). Three major classes of small RNAs have been defined in mammals: microRNAs (miRNAs), endogenous small interfering RNAs (endo-siRNAs) and Piwi-interacting RNAs (piRNAs) (for review: Golden et al., 2008). This project proposes to investigate the dynamics and roles of developmentally regulated small RNAs in the mouse, using embryonic stem (ES) cells as a model system. More specifically we would like to determine the role of certain developmentally regulated small RNAs, as well as the role of small RNAs in epigenetic processes such as X inactivation. The proposed research project is based on the recent findings of a very fruitful collaboration between the Heard and Voinnet labs, where male and female differentiating mouse ES cells were used to study sex-specific differences and dynamic changes in small RNA populations (Ciaudo et al, in prep). ES cells represent a powerful tissue culture system for mouse developmental processes. Several recent reports have revealed the important role that miRNAs such as the miR-290 cluster play in preventing differentiation of ES cells (Houbaviy et al., 2005; Benetti et al., 2008; Sinkkonen et al., 2008). However no studies have so far investigated small RNA patterns in male and female ES cells during early differentiation. Our recent work represents the first such study and has allowed us to make several novel findings that we would like to explore further in the context of this proposal. Although miRNAs are implicated in several developmental processes, their dynamics have been difficult to follow during mouse embryogenesis. Our study in ES cells identified new classes of miRNAs that show defined profiles during ES cell differentiation, as well as sex-specificity. For example, this allowed us to identify miR-302 as a potentially male germ-line specific miRNA, the exact function of which we propose to investigate further in the project proposed here. We also detected siRNA-like sequences in specific regions of the X chromosome in differentiating female but not male ES cells, suggesting the potential implication of RNAi in the spread of X inactivation (Chow et al, in prep). Recent investigations have pointed to a potential link between X inactivation and the RNAi pathway at the level of Xist and Tsix, although the molecular basis of this remains unclear (Ogawa et al., 2008). We propose to investigate how different types of small RNAs (miRNAs, siRNAs') could be involved in different steps of X inactivation, and other developmental processes, using available wild type and mutant ES cells and mice, combined with deep sequencing of small RNAs, epigenomic mapping and single cell imaging techniques. The two laboratories involved provide a unique combination of expertise in small RNA pathways (O. Voinnet) and X-chromosome inactivation (E. Heard), and the project proposed should thus open up some exciting new avenues in this emerging field of research.