Cellular and developmental control of Arabidopsis DNA replicationFunction of ORC1 and transposons

Laburpena

Plant stem cells are organized into meristems that actively divide throughout the lifespan of the plant to produce new organs. Once cells stop proliferating they frequently enter the endocycle program, which in plants is usually linked to the differentiation process. Reliable genome duplication helps maintaining genomic stability of meristematic and endocycling cells. This process starts at discrete sites called origins of DNA replication (ORIs), marked by a set of proteins that form the pre-replication complex. We focused our analysis on the largest subunit of the origin recognition complex (ORC1), which plays a key role in ORI licensing. In addition, ORC1 is tightly controlled to avoid re-replication problems in a species-specific manner. We have combined confocal microscopy techniques with analysis of translational fusion constructs and mutant plants to determine that the function, dynamics and regulation of the two Arabidopsis ORC1 proteins are different during root organogenesis. While ORC1a plays a role in the maintenance of heterochromatin in endocycling cells, ORC1b functions in ORI licensing during proliferation. Our analysis shows that ORC1b associates with euchromatin and heterochromatin in proliferating cells. The protein bounds to chromatin since G2 until the G1/S transition. Upon S-phase entry the E3-ubiquitin ligase SCFFBL17 recognizes ORC1b triggering its degradation. Plants lacking ORC1b are hypersensitive to aphidicolin, most likely due to having less licensed ORIs. On the contrary, ORC1a is only present in endocycling cells and preferentially associates with heterochromatin. Although ORC1a is only present at the G-phase of the endocycle, its degradation is independent of the ubiquitin proteasome pathway. The mark H3K27me1 is severely diminished at chromocenters of endocycling cells of plants lacking ORC1a pointing to a role of the protein in the establishment of the heterochromatin in this group of cells. In addition, we focused on defining the features associated with ORIs in heterochromatin. Arabidopsis ORIs preferentially colocalize with genes, but in pericentromeric gene-poor domains, a large proportion associate with transposable elements (TEs). ORI-TEs colocalize almost exclusively with retrotransposons, in particular of the Gypsy family. Opposite to ORI-genes, ORI-TE activity occurs independently of TE expression. In addition, ORI-TEs maintain high levels of the repressed heterochromatin marks H3K9me2 and H3K27me1. We have found a specific chromatin signature of ORI-TEs, defined by GC-rich heterochromatin. Importantly, TEs with active ORIs contain a local GC content higher than the TEs lacking them. Our results lead us to conclude that ORI colocalization with retrotransposons is determined by a specific chromatin landscape and their transposition mechanism based on transcription.