Molecular analysis of the mechanisms linking co-transcriptional RNA processing with chromatin silencing

We have been studying the molecular pathways controlling flowering time in plants and this has led us into the study of a particular pathway, the autonomous pathway, which accelerates flowering by repressing a specific gene encoding a floral repressor. This pathway turns out to regulate many targets in the nucleus and not just genes regulating flowering time. A component of the autonomous pathway is a protein that binds RNA called FCA. FCA regulates the gene encoding a floral repressor, not through post-transcriptional mechanisms as suggested by its RNA-binding properties, but through a process where the transcription of the target is affected. FCA requires the activity of a second protein (FLD) that modifies the proteins (histones) intimately packaged with the DNA. We will undertake experiments to unravel how the sequence of events in gene silencing involving these proteins. This system is an excellent one to dissect a general and important question in molecular biology - how non-coding RNA drives chromatin changes. We will address how a particular RNA is selected for FCA binding and how this triggers the chromatin modification activity and thus the silencing of the locus. We will analyse one transcript in detail and then explore the generality of our findings. This work has the potential to reveal fundamental concepts important to gene regulation in many organisms.

The project will exploit our recent work demonstrating that FCA, an Arabidopsis protein containing RNA-binding domains (RRM) mediates repression of a floral repressor gene FLC through a transcriptional silencing mechanism. Our aim will be to dissect how RNA metabolism drives chromatin changes, an important question in many areas of biology. Chromatin immunoprecipitation experiments will be used to explore the relationship between RNA processing and recruitment of FCA to FLC. We will characterize in an unbiased way all the FLC sense and antisense transcripts in different genotypes and define the RNA species to which FCA binds in vivo. In order to establish the generality of our findings we will also explore the genome-wide RNA features recognized by FCA and work collaboratively with Dr H. Chang (Stanford University) who is exploring how non-coding RNAs drive chromatin changes at mammalian HOX loci. Chromatin immunoprecipitation will be used to explore the requirements for FLD association to FLC chromatin and determine if the association of the chromatin regulator, FLD, to its target FLC is dependent on the RNA metabolic activities of FCA and FY. Working collaboratively with Prof. Y. Shi (Harvard Medical School - who first demonstrated histone demethylases exist) we will test the generality of RRM protein recruitment of histone demethylases to their targets by analysing the role of the most homologous human RRM proteins to FCA, on LSD1 targets in human cells. Lastly, in order to mechanistically understand the connection between chromatin modification activity and factors involved in RNA processing we will use tandem affinity purification to identify FLD associated proteins and RNA in vivo.