We report here the transcriptional responses in Saccharomyces cerevisiae to deletion of the RNH201 gene encoding the catalytic subunit of RNase H2. Deleting RNH201 alters RNA expression of 349 genes by ≥1.5-fold (q-value <0.01), of which 123 are upregulated and 226 are downregulated. Differentially expressed genes (DEGs) include those involved in stress responses and genome maintenance, consistent with a role for RNase H2 in removing ribonucleotides incorporated into DNA during replication. Upregulated genes include several that encode subunits of RNA polymerases I and III, and genes involved in ribosomal RNA processing, ribosomal biogenesis and tRNA modification and processing, supporting a role for RNase H2 in resolving R-loops formed during transcription of rRNA and tRNA genes. A role in R-loop resolution is further suggested by a higher average GC-content proximal to the transcription start site of downregulated as compared to upregulated genes. Several DEGs are involved in telomere maintenance, supporting a role for RNase H2 in resolving RNA-DNA hybrids formed at telomeres. A large number of DEGs encode nucleases, helicases and genes involved in response to dsRNA viruses, observations that could be relevant to the nucleic acid species that elicit an innate immune response in RNase H2-defective humans.

Mutations in both p53 and BRCA2 are commonly seen together in human tumors suggesting that the loss of both genes enhances tumor development. To elucidate this interaction in an animal model, mice lacking the carboxy terminal domain of Brca2 were crossed with p53 heterozygous mice. Females from this intercross were then irradiated with an acute dose of 5 Gy ionizing radiation at 5 weeks of age and compared to nonirradiated controls. We found decreased survival and timing of tumor onsets, and significantly higher overall tumor incidences and prevalence of particular tumors, including stomach tumors and squamous cell carcinomas, associated with the homozygous loss of Brca2, independent of p53 status. The addition of a p53 mutation had a further impact on overall survival, incidence of osteosarcomas and stomach tumors, and tumor latency. The spectrum of tumors observed for this Brca2 germline mouse model suggest that it faithfully recapitulates some human disease phenotypes associated with BRCA2 loss. In addition, these findings include extensive in vivo data demonstrating that germline Brca2 and p53 mutations cooperatively affect animal survivals, tumor susceptibilities, and tumor onsets.

Estrogen insensitivity syndrome (EIS) arises from rare mutations in estrogen receptor-α (ERα, encoded by ESR1 gene) resulting in the inability of estrogen to exert its biological effects. Due to its rarity, mutations in ESR1 gene and the underlying molecular mechanisms of EIS have not been thoroughly studied. Here, we investigate known ESR1 mutants, Q375H and R394H, associated with EIS patients using in vitro and in vivo systems. Comparison of the transcriptome and deoxyribonucleic acid methylome from stable cell lines of both Q375H and R394H clinical mutants shows a differential profile compared with wild-type ERα, resulting in loss of estrogen responsiveness. Molecular dynamic simulation shows that both ESR1 mutations change the ERα conformation of the ligand-receptor complexes. Furthermore, we generated a mouse model Esr1-Q harboring the human mutation using CRISPR/Cas9 genome editing. Female and male Esr1-Q mice are infertile and have similar phenotypes to αERKO mice. Overall phenotypes of the Esr1-Q mice correspond to those observed in the patient with Q375H. Finally, we explore the effects of a synthetic progestogen and a gonadotropin-releasing hormone inhibitor in the Esr1-Q mice for potentially reversing the impaired female reproductive tract function. These findings provide an important basis for understanding the molecular mechanistic consequences associated with EIS.

Estrogen insensitivity syndrome (EIS) or estrogen resistance is very rare and most commonly caused by a mutation in ERα that results in the inability of estrogen to exert its biological effects. However, mutations in ESR1 gene and the underlying molecular mechanisms have not been thoroughly studied. We investigated the structural conformation, whole transcriptome, and DNA methylome for the ERα natural mutants, ESR1 Q375H and R394H that associated with EIS patients. We indicate that Q375H located in the coactivator binding pocket, while R394H located in the ligand binding pocket of the ERα ligand binding domain. Both mutants have changed the ERα structure conformation. We also demonstrate that both mutants differentially mediated whole transcriptome and DNA methylome aberrations in the genome. These mutants result in losing alteration to the estrogen-response genes predominantly when compared to WT ERα. To investigate the biological characterization of this natural mutation in vivo , a mouse model was generated harboring the human mutation, Esr1 -Q379H ( Esr1 -Q), using CRISPR-/Cas9 and both male and female mice were analyzed. Our preliminary examinations show that the female and male Esr1 -Q and αERKO mice were consistently more obese than the wild type (WT) mice. Female Esr1 -Q mice have hemorrhagic cystic ovaries, rudimentary mammary ducts and hypoplastic uteri. In addition, the Esr1 -Q mice have elevated levels of luteinizing hormone (LH) and nearly all examined phenotypes mirror those observed in αERKO mice. To test the responsiveness to estrogen in the Esr1 -Q mice, a three-day bioassay with diethylstilbestrol (DES, a synthetic estrogen) was performed. WT uterine weight increased in the DES group when compared to controls. However, Esr1 -Q uterine weight did not change with DES treatment, suggesting that this natural mutant lost ERα function. The female patient was also non-responsive to a high-dose estrogen treatment. The Esr1 -Q male mice are infertile and show increased seminal vesicle weights as well as seminiferous tubule disruption in the testes. These findings provide an important basis for understanding the molecular and cellular mechanism of EIS and the mouse model offers a potential new way to study rare genetic receptor mutations in humans with hopes of developing a viable therapeutic approach.