Transcription–replication conflicts in primordial germ cells necessitate the Fanconi anemia pathway to safeguard genome stability

Abstract

Preserving a high degree of genome integrity and stability in germ cells is of utmost importance for reproduction and species propagation. However, the regulatory mechanisms of maintaining genome stability in the developing primordial germ cells (PGCs), in which rapid proliferation is coupled with global hypertranscription, remain largely unknown. Here, we find that mouse PGCs encounter a constitutively high frequency of transcription–replication conflicts (TRCs), which lead to R-loop accumulation and impose endogenous replication stress on PGCs. We further demonstrate that the Fanconi anemia (FA) pathway is activated by TRCs and has a central role in the coordination between replication and transcription in the rapidly proliferating PGCs, as disabling the FA pathway leads to TRC and R-loop accumulation, replication fork destabilization, increased DNA damage, dramatic loss of mitotically dividing mouse PGCs, and consequent sterility of both sexes. Overall, our findings uncover the unique source and resolving mechanism of endogenous replication stress during PGC proliferation, provide a biological explanation for reproductive defects in individuals with FA, and improve our understanding of the monitoring strategies for genome stability during germ cell development.

DNA repair protein FANCD2 has both ubiquitination-dependent and ubiquitination-independent functions during germ cell development

“…For the MT mouse, p.K559R (AAG to AGG) was introduced ( Fig. 1 C ) to generate the ubiquitination-defective FANCD2 MT protein ( 20 ).

Figure 1 The FA pathway was inactive in KO and MT mice.

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“…Actively proliferating PGCs are coupled with global transcriptional upregulation ( 30 , 31 ). We have recently reported that PGCs are faced with a high frequency of TRCs and the FA pathway counteracts TRC-induced genome instability, thus enabling the rapid proliferation of PGCs ( 20 ). To characterize the ubiquitination-dependent and ubiquitination-independent functions of FANCD2 in resolving TRCs in PGCs, a proximity ligation assay (PLA) using antibodies against proliferating cell nuclear antigen ( an indicator of the replication machinery) and Pol II ( an indicator of the transcriptional machinery) was performed to detect TRCs in WT, KO, and MT PGCs at E11.5.…”

“…After arriving at the genital ridge, PGCs undergo rapid proliferation ( 31 , 39 ). Our previous study showed that rapidly proliferating PGCs encounter high frequencies of TRCs and that the activated FA pathway protects PGCs from TRC-induced endogenous replication stress by reducing R-loop accumulation and stabilizing stalled RFs, thus ensuring the establishment of the reproductive reserve, which indicated the significant role of FANCD2 ubiquitination in PGC expansion ( 20 ). In the present study, we found that loss of PGCs in both Fancd2 KO and MT embryos caused a significant reduction in the reproductive reserve.…”

“…For KO mice, the gRNAs (gRNA1: GGC GAA AAA TAT TCT TAG TAG GG, gRNA2: GGT GGT TAG ATG ACA TTG AAG GG) for the Fancd2 gene and Cas9 mRNA were mixed and injected into the cytoplasm of fertilized eggs to generate targeted KO mice. For MT mice, the gRNA (CAG TGC TAG AGA GCT GCT TCC GG) to the mouse Fancd2 gene, the donor oligo containing p.K559R (AAG to AGG) missense mutation and p.R558= (CGG to CGA) synonymous mutation, and Cas9 mRNA were mixed and injected into the cytoplasm of fertilized eggs to generate MT mice ( 20 ). F0 founder mice were identified by PCR and bred with WT mice to acquire F1 heterozygous animals.…”

“…FA patients and FA mutant mice are infertile or only rarely fertile ( 15 , 16 , 17 , 18 , 19 ). Our recent study showed that the FA pathway counteracts TRC-induced endogenous replication stress, thus safeguarding genome stability and maintaining the rapid proliferation of PGCs ( 20 ). Combined with the findings in FA mutant mice, the drastic reduction in germ cells could be mainly attributed to PGC deficiency during embryonic development ( 21 ).…”