Specialized Polymerase involved in Unchallenged Replication: Requisite for duplication of heterochromatin regions during cellular differentiation and consequence on spontaneous mutagenesis – SPUR

Duplication of the genome prior to cell division occurs in a spatially and temporally organized manner. The temporal order of replication (replication timing) reflects the higher order organization of the genome. During the first half of the S-phase, euchromatic regions are replicated followed by facultative heterochromatin during mid S-phase and finally constitutive heterochromatin regions in the second half of the S-phase. In the nucleus, euchromatin is localized in the interior while constitutive heterochromatin is rather located at the periphery.
The spatio-temporal program of genome replication changes throughout development and cellular differentiation and has been correlated with changes in chromatin dynamics, histone marks, and nuclear architecture indicating that genome reorganization is associated with epigenetic and gene expression changes. Abnormal replication timing has also been reported in many diseases, including cancer.
A growing body of evidence indicates that the replication-timing program strongly influences the spatial distribution of mutagenic events such that certain regions of the genome that are replicated in late S-phase present increased spontaneous mutagenesis compared to surrounding regions replicated in early S-phase. This has been observed during the evolution of species as well as during the evolution of cancer. In addition, a recent report showed that the dominant determinant of regional mutation rate variation is chromatin organization, with mutation rates elevated in more heterochromatin-like domains and repressed in more open chromatin. Although different hypotheses have been advanced, the cause of this increasing gradient of point mutation rates with later replication timing and the underlying mechanisms remain elusive. Although no mutagenic profile has been reported so far from pluripotent cells undergoing differentiation, one can expect that genome reorganization influences the mutagenic landscape.
Obvious and important questions emerge from these observations: how and why elevated mutation rates are associated with heterochromatin-like domains which are replicated in late S-phase? What can be the “cost” for the cell when global genome reorganization takes place after differentiation/dedifferentiation (e.g. pre-cancerous cells) or reprogramming (e.g. iPS cells) if late-replicating regions containing point mutations become early and are actively transcribed?
Point mutations arising in the genome are mainly caused by a special class of DNA polymerases (TLS polymerases) that support replication directly past template lesions (or unusual DNA secondary structures) that cannot be negotiated by the replicative high-fidelity polymerases. However, these specialized polymerases can be highly error-prone on undamaged DNA. An emerging concept proposes that these enzymes may also function during the unchallenged S-phase. On the basis of solid preliminary results, we assume that the essential error-prone DNA polymerase zeta (Pol zeta) is required to replicate through condensed chromatin regions and could be involved in the gradient of spontaneous mutagenesis.
Therefore, this ambitious SPUR project aims to decipher the involvement of Pol zeta??and its catalytic subunit Rev3) in the regulation of the spatio-temporal program of DNA replication during embryonic stem cell differentiation and comprehensively evaluate the role of this error-prone polymerase in the point mutation frequencies which increase in late-replicating regions.