Small RNAs as determinants of epigenetic states
We are interested in two general questions: biogenesis and function of small non-coding RNAs.
Biogenesis of piRNA
Processing of piRNAs differs from that of other known classes of small RNAs. It was shown piRNA are produced independently of Dicer, the nuclease that generates siRNAs and microRNAs from double-stranded substrates; however, the proteins that are responsible for producing piRNAs are only partially understood.
Our investigations of piRNA biogenesis led us to the ping-pong model that proposes amplification of piRNAs in a cycle that depends on the nuclease activity of Piwi proteins themselves. One of the central mysteries of repeat silencing in both mammals and flies is how repeats are distinguished from genes and selectively silenced. We are investigating the nature of the determinants that make a particular sequence a target of the Piwi pathway. We are using biochemical purification of Piwi-piRNA complexes and genetic approaches to identify proteins involved in piRNA biogenesis.
Functions of the Piwi pathway and piRNA-guided de novo DNA methylation
We showed that the piRNA pathway is linked to de novo DNA methylation in the mouse germline. One of the three murine Piwi proteins is specifically found in germ cell nuclei during the critical window when de novo methylation patterns are established. We also showed that Piwi proteins at that developmental timepoint are associated with piRNAs that target several classes of transposable elements. The same transposons are de-repressed and their genomic sequences lose methylation in Piwi-deficient mice. The discovery that piRNAs may guide DNA methylation in germ cells is an important finding for several reasons. First, it provides a new paradigm for how small RNAs can affect gene expression. Second, it explains how a subset of sequences are tagged for de novo methylation. How methylation sites are defined remains a central mystery of epigenetics. An important goal of my lab is to define the pathway by which piRNAs guide de novo DNA methylation. We also study whether the piRNA pathway can be re-programmed to new targets and can be used to manipulate DNA methylation patterns in somatic cells.
It is clear that germ cells, somatic stem cells and probably cancer stem cells possess unique pathways for small RNA-mediated silencing. Our long-term goal is to understand how diverse RNA silencing mechanisms are integrated with other pathways in context of development and pathology. Eventually, the knowledge gained from the investigation of silencing mechanisms in stem and germ cells will help us to understand the unique biology of these cells and will impact our general understanding of gene regulation and how it is altered in disease.