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The precise and simultaneous acquisition of multiple beneficial alleles in the genome is in great demand for the development of elite pig breeders. Cytidine base editors (CBEs) that convert C:G to T:A have emerged as powerful tools for single-nucleotide replacement. Whether CBEs can effectively mediate C-to-T substitution at multiple sites/loci for trait improvement by direct zygote injection has not been verified in large animals. Here, we determined the editing efficiency of four CBE variants in porcine embryonic fibroblast cells and embryos. The findings showed that hA3A-BE3-Y130F and hA3A-eBE-Y130F consistently resulted in increased base-editing efficiency and low toxic effects in embryonic development. Further, we verified that using a one-step approach, direct zygote microinjection of the CBE system can generate pigs harboring multiple point mutations. Our process resulted in a stop codon in CD163and myostatin (MSTN) and introduced a beneficial allele in insulin-like growth factor-2 (IGF2). The pigs showed disrupted expression of CD163and MSTNand increased expression of IGF2, which significantly improved growth performance and infectious disease resistance. Our approach allows immediate introduction of multiple mutations in transgene-free animals to comprehensively improve economic traits through direct embryo microinjection, providing a potential new route to produce elite pig breeders.

RNF20, an E3 ligase critical for monoubiquitination of histone H2B at lysine 120 (H2Bub), has been implicated in the regulation of various cellar processes; however, its physiological roles in adipocytes remain poorly characterized. Here, we report that the adipocyte-specific knockout of Rnf20(ASKO) in mice led to progressive fat loss, organomegaly and hyperinsulinemia. Despite signs of hyperinsulinemia, normal insulin sensitivity and improved glucose tolerance were observed in the young and aged CD-fed ASKO mice. In addition, high-fat diet-fed ASKO mice developed severe liver steatosis. Moreover, we observed that the ASKO mice were extremely sensitive to a cold environment due to decreased expression levels of brown adipose tissue (BAT) selective genes, including uncoupling protein 1 (Ucp1), and impaired mitochondrial functions. Significantly decreased levels of peroxisome proliferator-activated receptor gamma (Pparγ) were observed in the gonadal white adipose tissues (gWAT) from the ASKO mice, suggesting that Rnf20regulates adipogenesis, at least in part, through Pparγ. Rosiglitazone-treated ASKO mice exhibited increased fat mass compared to that of the non-treated ASKO mice. Collectively, our results illustrate the critical role of RNF20 in control of white and brown adipose tissue development and physiological function.

Congenital hypothyroidism (CH) is one of the most prevalent endocrine diseases, for which the underlying mechanisms remain unknown; it is often accompanied by anemia and immunodeficiency in patients. Here, we created a severe CH model together with anemia and T lymphopenia to mimic the clinical features of hypothyroid patients by ethylnitrosourea (ENU) mutagenesis in Bama miniature pigs. A novel recessive c.1226A>G transition of the dual oxidase 2 (DUOX2) gene was identified as the causative mutation. This mutation hindered the production of hydrogen peroxide (H2O2) and thus contributed to thyroid hormone (TH) synthesis failure. Transcriptome sequencing analysis of the thymuses showed that Krüppel-like factor 9 (KLF9) was predominantly downregulated in hypothyroid mutants. KLF9was verified to be directly regulated by TH in a TH receptor (TR)-dependent manner both in vivo and in vitro. Furthermore, knockdown of klf9in zebrafish embryos impaired hematopoietic development including erythroid maturation and T lymphopoiesis. Our findings suggest that the TR-KLF9 axis is responsible for the hematopoietic dysfunction and might be exploited for the development of novel therapeutic interventions for thyroid diseases.

Congenital hypothyroidism (CH) is one of the most prevalent endocrine diseases, for which the underlying mechanisms remain unknown; it is often accompanied by anemia and immunodeficiency in patients. Here, we created a severe CH model together with anemia and T lymphopenia to mimic the clinical features of hypothyroid patients by ethylnitrosourea (ENU) mutagenesis in Bama miniature pigs. A novel recessive c.1226A>G transition of the dual oxidase 2 (DUOX2) gene was identified as the causative mutation. This mutation hindered the production of hydrogen peroxide (H2O2) and thus contributed to thyroid hormone (TH) synthesis failure. Transcriptome sequencing analysis of the thymuses showed that Krüppel-like factor 9 (KLF9) was predominantly downregulated in hypothyroid mutants. KLF9 was verified to be directly regulated by TH in a TH receptor (TR)-dependent manner both in vivo and in vitro. Furthermore, knockdown of klf9 in zebrafish embryos impaired hematopoietic development including erythroid maturation and T lymphopoiesis. Our findings suggest that the TR-KLF9 axis is responsible for the hematopoietic dysfunction and might be exploited for the development of novel therapeutic interventions for thyroid diseases.

ABSTRACTRNF20/Bre1 mediated H2B ubiquitination (H2Bub) has various physiologic functions. Recently, we found that H2Bub participates in meiotic recombination by promoting chromatin relaxation during meiosis. We then analyzed the phylogenetic relationships among the E3 ligase for H2Bub, its E2 Rad6 and their partner WW domain-containing adaptor with a coiled-coil (WAC) or Lge1, and found that the molecular mechanism underlying H2Bub is evolutionarily conserved from yeast to mammals. However, RNF20 has diverse physiologic functions in different organisms, which might be caused by the evolutionary divergency of their domain/motif architectures. In the current extra view, we not only elucidate the evolutionarily conserved molecular mechanism underlying H2Bub, but also discuss the diverse physiologic functions of RNF20 during meiosis.

Background: Pigs are important animals for agricultural and biomedical research, and improvement is needed for use of the assisted reproductive technologies. Determining underlying mechanisms of epigenetic reprogramming in the early stage of preimplantation embryos derived from in vitro fertilization (IVF), parthenogenesis, and androgenesis will not only contribute to assisted reproductive technologies of pigs but also will shed light into early human development. However, the reprogramming of three-dimensional architecture of chromatin in this process in pigs is poorly understood. Results: We generate three-dimensional chromatin profiles for pig somatic cells, IVF, parthenogenesis, and androgenesis preimplantation embryos. We find that the chromosomes in the pig preimplantation embryos are enriched for superdomains, which are more rare in mice. However, p(s) curves, compartments, and topologically associated domains (TADs) are largely conserved in somatic cells and are gradually established during preimplantation embryogenesis in both mammals. In the uniparental pig embryos, the establishment of chromatin architecture is highly asynchronized at all levels from IVF embryos, and a remarkably strong decompartmentalization is observed during zygotic genome activation (ZGA). Finally, chromosomes originating from oocytes always establish TADs faster than chromosomes originating from sperm, both before and during ZGA. Conclusions: Our data highlight a potential unique 3D chromatin pattern of enriched superdomains in pig preimplantation embryos, an unusual decompartmentalization process during ZGA in the uniparental embryos, and an asynchronized TAD reprogramming between maternal and paternal genomes, implying a severe dysregulation of ZGA in the uniparental embryos in pigs.

Pigs share many similarities with humans in terms of anatomy, physiology and genetics, and have long been recognized as important experimental animals in biomedical research. Using an N-ethyl-N-nitrosourea (ENU) mutagenesis screen, we previously identified a large number of pig mutants, which could be further established as human disease models. However, the identification of causative mutations in large animals with great heterogeneity remains a challenging endeavor. Here, we select one pig mutant, showing congenital nude skin and thyroid deficiency in a recessive inheritance pattern. We were able to efficiently map the causative mutation using family-based genome-wide association studies combined with whole-exome sequencing and a small sample size. A loss-of-function variant (c.1226 A>G) that resulted in a highly conserved amino acid substitution (D409G) was identified in the DUOX2 gene. This mutation, located within an exonic splicing enhancer motif, caused aberrant splicing of DUOX2 transcripts and resulted in lower H2O2 production, which might cause a severe defect in thyroid hormone production. Our findings suggest that exome sequencing is an efficient way to map causative mutations and that DUOX2D409G/D409G mutant pigs could be a potential large animal model for human congenital hypothyroidism.

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