Proteínas Polycomb RING1A/RING1B y estrés replicativo

Resumo

Polycomb proteins RING1A and RING1B are the E3 ligases of Polycomb Repressive Complex 1, responsible of H2A monoubiquitilation in lysine 119. This modification participate in transcriptional repression mediated by Polycomb group proteins needed for embryonic development and linage specification in cellular differentiation. By using mouse embryonic fibroblast and human cellular model of conditionally inactive E3 ligases (RING1A and RING1B), we characterize a new function of RING1 proteins in DNA replication and in DNA damage response to replicative stress. In unperturbed cells, RING1 proteins deficiency causes slow elongation and fork stalling during DNA replication that is associated with the accumulation of mid and late S-phase cells. Signs of replicative stress and colocalisation of double-strand breaks with pericentromeric heterocromatic (PCH) domains were enriched in cells at mid S-phase, the stage at which PCH is replicated. Altered replication was rescued by targeted monoubiquitylation of PCH through methyl-CpG binding domain protein 1. We describe a defect in replication stress signaling by the checkpoint kinase ATR, and in fork stabilization pathway mediated by FANCD2. This alterations could be related to the novel interactions described in this work between RING1B and replisome components that participates in ATR activation, as well as with FANCD2. Moreover, the acute senescence associated with the depletion of RING1 proteins, which is mediated by p21 (also known as CDKN1a) upregulation, could be uncoupled from a response to DNA damage. We identify a dual RING1B function in promote heterochromatin replication dependent of RING1B ubiquitilation capacity and a relevant involvement in replicative stress signaling and response through a H2A ubiquitilation independent mechanism by which RING1B presence, even inactive, participates in ATR activation and replication fork stabilization. All together, in this work we provide mechanisms that allow us to extend RING1 proteins function from transcriptional repression to DNA replication and DNA damage response.