A Surveillance Mechanism Ensures Repair of DNA Lesions during Zygotic Reprogramming

Abstract

SummarySexual reproduction culminates in a totipotent zygote with the potential to produce a whole organism. Sperm chromatin reorganization and epigenetic reprogramming that alter DNA and histone modifications generate a totipotent embryo. Active DNA demethylation of the paternal genome has been proposed to involve base excision and DNA repair-based mechanisms. The nature and consequence of DNA lesions generated during reprogramming are not known. Using mouse genetics and chemical biology, we discovered that Tet3-dependent zygotic reprogramming generates paternal DNA lesions that are monitored by a surveillance mechanism. In vivo structure-function rescue assays revealed that cohesin-dependent repair of paternal DNA lesions prevents activation of a Chk1-dependent checkpoint that delays mitotic entry. Culturing conditions affect checkpoint stringency, which has implications for human in vitro fertilization. We propose the zygotic checkpoint senses DNA lesions generated during paternal DNA demethylation and ensures reprogrammed loci are repaired before mitosis to prevent chromosome fragmentation, embryo loss, and infertility.

A Mechanism of Cohesin-Dependent Loop Extrusion Organizes Zygotic Genome Architecture

“…We used a genetic knockout approach based on (Tg)Zp3-Cre that deletes conditional alleles during the weeks of oocyte growth and generates maternal knockout zygotes after fertilization (Figure 2A). We have previously shown that Scc1 protein is efficiently depleted and sister chromatid cohesion fails to be established in Scc1 ∆(m)/+(p) zygotes (hereafter referred to as Scc1 ∆ according to the maternal allele) (Figure S2A, Ladstätter & Tachibana-Konwalski, 2016). Since sister chromatid cohesion is maintained by Rec8-cohesin in oocytes (Tachibana-Konwalski et al, 2010;Burkhardt et al, 2016), Scc1 depletion has no effect on chromosome segregation prior to fertilization and therefore a clean Scc1-cohesin knockout zygote is generated.…”

Efficient Zygotic Genome Editing via RAD51-Enhanced Interhomolog Repair

“…Because genotyping analyses of one-cell stage embryos are difficult to interpret due to failed PCR amplification of alleles harboring a Cas9-induced DSB, we chose to test the timing of zygotic IHR via immunocytochemistry (ICC) for RAD51 in uninjected, RAD51-injected, Cas9-injected (with Chd2 -targeting crRNA), and Cas9- and RAD51-injected zygotes at three stages of the cell cycle: G1,, G2 (post S-phase), and M-phase (see Methods ). Previous studies have shown that RAD51 staining is weak and/or diffuse in wildtype cells, but that strong RAD51 puncta appear in response to DSBs 23 . In agreement with our genotyping data and the hypothesis that IHR occurs after S-phase, we observed the appearance of RAD51 puncta solely during G2 ( Fig.…”

A mechanism of cohesin‐dependent loop extrusion organizes zygotic genome architecture

“…In vitro fertilization after in vitro maturation was performed as described before (Ladstätter & Tachibana-Konwalski, 2016). Oocytes from 2-to 5-month-old females were isolated by puncturing of ovaries with hypodermic needles in the presence of 0.2 mM IBMX, 20% FBS (Gibco), and 6 mg/ml fetuin (Sigma-Aldrich).…”

“…In contrast, the paternal genome is contributed from compacted sperm chromatin that is extensively remodeled upon fertilization, as protamines are evicted and naïve nucleosomal chromatin is established (Rodman et al , ). The two genomes are reprogrammed as separate nuclei with distinct epigenetic signatures at the zygote stage (Mayer et al , ; Oswald et al , ; van der Heijden et al , ; Torres‐Padilla et al , ; Ladstätter & Tachibana‐Konwalski, ). With the exception of imprinted loci, differences in chromatin states are presumably eventually equalized to facilitate the major zygotic genome activation (ZGA), which occurs at the two‐cell stage in mice (Aoki et al , ; Hamatani et al , ; Inoue et al , ).…”