Genome-wide identification of RETINOBLASTOMA RELATED 1 binding sites in Arabidopsis reveals novel DNA damage regulators.

Retinoblastoma (pRb) is a multifunctional regulator, which was likely present in the last common ancestor of all eukaryotes. The Arabidopsis pRb homolog RETINOBLASTOMA RELATED 1 (RBR1), similar to its animal counterparts, controls not only cell proliferation but is also implicated in developmental decisions, stress responses and maintenance of genome integrity. Although most functions of pRb-type proteins involve chromatin association, a genome-wide understanding of RBR1 binding sites in Arabidopsis is still missing. Here, we present a plant chromatin immunoprecipitation protocol optimized for genome-wide studies of indirectly DNA-bound proteins like RBR1. Our analysis revealed binding of Arabidopsis RBR1 to approximately 1000 genes and roughly 500 transposable elements, preferentially MITES. The RBR1-decorated genes broadly overlap with previously identified targets of two major transcription factors controlling the cell cycle, i.e. E2F and MYB3R3 and represent a robust inventory of RBR1-targets in dividing cells. Consistently, enriched motifs in the RBR1-marked domains include sequences related to the E2F consensus site and the MSA-core element bound by MYB3R transcription factors. Following up a key role of RBR1 in DNA damage response, we performed a meta-analysis combining the information about the RBR1-binding sites with genome-wide expression studies under DNA stress. As a result, we present the identification and mutant characterization of three novel genes required for growth upon genotoxic stress.