Final Report Summary - VRNEPIGEN (Maintenance of cellular memory by the Arabidopsis VRN1 protein)
We have mapped VRN1 binding sites across the FLC locus using a high-resolution Chromatin immuno-precipitation (ChIP) technique optimised in the host lab. As stated above previous in situ analysis showed VRN1 binds all five chromosomes suggesting a wide binding profile. Indeed moderate levels of VRN1 could be found across the FLC locus and surrounding sequences. In addition, we detected peaks of VRN1 binding at some nucleosome free regions. Upon FLC silencing by vernalisation the binding profile of VRN1 remained unchanged albeit total levels dropped, implying that the physical closing of FLC upon vernalisation limits VRN1 binding. Lower VRN1 binding indeed coincided with the repressive histone mark H3K27me3, which is deposited by the PcG machinery and accumulates at FLC upon vernalisation.
An unexpected finding in vrn1 plants was a lower total nucleosome level, which we subsequently have shown to be caused by a relatively higher turnover rate of nucleosomes at the chromatin. Using a combination of ChIP and Fluorescence recovery after photobleaching (FRAP) experiments we have also shown that increased nucleosome turnover results in excessive usage of variant histone proteins throughout the genome (a finding never reported before and highly interesting for understanding fundamental aspects of chromatin dynamics). Using a genetic and molecular approach we have also shown that aberrant histone variant usage is at least partially causative for the vernalisation phenotype in vrn1 plants. Collectively our data have shed new light on the dynamics of chromatin and how disturbance of balanced nucleosome turnover can have profound effects on gene regulation. The impact on the general field of chromatin biology will be substantial and therefore this project has contributed to a further strengthening of the EU as a world leading collective in chromatin research. Beside the impact on fundamental research applicability of research on flowering time has been proven economically important and profitable before. In that perspective this project has added another piece of useful information allowing us to better understand and eventually manipulate crop flowering behaviour and help securing food production in the future.