Role of histone variants H2BC1 and H2AZ.2 in H2AK119ub nucleosome organization and Polycomb gene silencing.

Ubiquitination of histone H2A at lysine 119 residue (H2AK119ub) plays critical roles in a wide range of physiological processes, including Polycomb gene silencing 1,2 , replication 3-5 , DNA damage repair 6-10 , X inactivation 11,12 , and heterochromatin organization 13,14 . However, the underlying mechanism and structural basis of H2AK119ub remains largely elusive. In this study, we report that H2AK119ub nucleosomes have a unique composition, containing histone variants H2BC1 and H2AZ.2, and importantly, this composition is required for H2AK119ub and Polycomb gene silencing. Using the UAB domain of RSF1, we purified H2AK119ub nucleosomes to a sufficient amount and purity. Mass spectrometry analyses revealed that H2AK119ub nucleosomes contain the histone variants H2BC1 and H2AZ.2. A cryo-EM study resolved the structure of native H2AK119ub nucleosomes to a 2.6A resolution, confirming H2BC1 in one subgroup of H2AK119ub nucleosomes. Tandem GST-UAB pulldown, Flag-H2AZ.2, and HA-H2BC1 immunoprecipitation revealed that H2AK119ub nucleosomes could be separated into distinct subgroups, suggesting their composition heterogeneity and potential dynamic organization. Knockout or knockdown of H2BC1 or H2AZ.2 reduced cellular H2AK119ub levels, establishing H2BC1 and H2AZ.2 as critical determinants of H2AK119ub. Furthermore, genomic binding profiles of H2BC1 and H2AZ.2 overlapped significantly with H2AK119ub binding, with the most significant overlapping in the gene body and intergenic regions. Finally, assays in developing embryos reveal an interaction of H2AZ.2, H2BC1, and RING1A in vivo . Thus, this study revealed, for the first time, that the H2AK119ub nucleosome has a unique composition, and this composition is required for H2AK119ub and Polycomb gene silencing.

Molecular breeding of livestock for disease resistance.

Animal infectious diseases pose a significant threat to the global agriculture and biomedicine industries, leading to significant economic losses and public health risks. The emergence and spread of viral infections such as African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), and avian influenza virus (AIV) have highlighted the need for innovative approaches to develop resilient and disease-resistant animal populations. Gene editing technologies, such as CRISPR/Cas9, offer a promising avenue for generating animals with enhanced disease resistance. This review summarizes recent advances in molecular breeding strategies for generating disease-resistant animals, focusing on the development of disease-resistant livestock. We also highlight the potential applications of genome-wide CRISPR/Cas9 library screening and base editors in producing precise gene modified livestock for disease resistance in the future. Overall, gene editing technologies have the potential to revolutionize animal breeding and improve animal health and welfare.

Influence of season and conditions of surrogate sows on efficiency of somatic cell cloning production.

This study was focused on the effects of ovary acquisition season, embryo transfer season, and conditions of surrogate sows on cloning efficiency, with the objective of improving the production of cloned pigs. The statistical analysis documented that cloning efficiency was highest when ovary extraction and embryo transfer occurred in the spring, and lowest when such operations occurred in the autumn. This was evidenced by the higher number of recovered oocytes (3.2 ± 0.47 vs. 2.5 ± 0.51), rate of mature oocytes (57.4 ± 0.07% vs. 48.9 ± 0.06%), rate of developed cloned blastocysts (35.7 ± 0.12% vs. 34.4 ± 0.07%), pregnancy rate of surrogate sows (73.5% vs. 33.3%), delivery rate (67.6% vs. 16.7%), litter size (6.9 ± 2.3 vs. 2.3 ± 2.5), and the number of alive newborns (5.7 ± 2.2 vs. 1.3 ± 1.2). Cloning efficiency was little affected by the ovulatory status of the surrogate sow prior to embryo transfer. The length of pregnancy, the parity, and the length of labor of the surrogate sow significantly affected the efficiency of generating pigs cloned from somatic cells. Specifically, when length of pregnancy ranged from 111 to 117 days, surrogate sows with shorter gestation period had larger litter size (8.9 ± 2.8) and a higher number of stillbirths per litter (2.1 ± 2.0). Moreover, statistical analysis indicated that selecting sows with 2-4 parities as surrogates led to increased litter size (7.7 ± 3.0) and the number of alive newborns (6.4 ± 3.1). In comparison with naturally breeding sows, the surrogate sows spent more time giving birth and suffered higher rates of stillbirth. The data obtained in this study provide valuable insights for improving the production efficiency of somatic cell cloned pigs.

A HIT-trapping strategy for rapid generation of reversible and conditional alleles using a universal donor.

Targeted mutagenesis in model organisms is key for gene functional annotation and biomedical research. Despite technological advances in gene editing by the CRISPR-Cas9 systems, rapid and efficient introduction of site-directed mutations remains a challenge in large animal models. Here, we developed a robust and flexible insertional mutagenesis strategy, homology-independent targeted trapping (HIT-trapping), which is generic and can efficiently target-trap an endogenous gene of interest independent of homology arm and embryonic stem cells. Further optimization and equipping the HIT-trap donor with a site-specific DNA inversion mechanism enabled one-step generation of reversible and conditional alleles in a single experiment. As a proof of concept, we successfully created mutant alleles for 21 disease-related genes in primary porcine fibroblasts with an average knock-in frequency of 53.2%, a great improvement over previous approaches. The versatile HIT-trapping strategy presented here is expected to simplify the targeted generation of mutant alleles and facilitate large-scale mutagenesis in large mammals such as pigs.

FcRn is not the receptor mediating the transfer of serum IgG to colostrum in pigs.

In contrast to humans or rabbits, in which maternal IgG is transmitted to offspring prenatally via the placenta or the yolk sac, large domestic animals such as pigs, cows and sheep transmit IgG exclusively through colostrum feeding after delivery. The extremely high IgG content in colostrum is absorbed by newborns via the small intestine. Although it is widely accepted that the neonatal Fc receptor, FcRn, is the receptor mediating IgG transfer across both the placenta and small intestine, it remains unclear whether FcRn also mediates serum IgG transfer across the mammary barrier to colostrum/milk, especially in large domestic animals. In this study, using a FcRn knockout pig model generated with a CRISPR-Cas9-based approach, we clearly demonstrate that FcRn is not responsible for the IgG transfer from serum to colostrum in pigs, although like in other mammals, it is involved in IgG homeostasis and mediates IgG absorption in the small intestine of newborns.

A comprehensive analysis of the genomic organization, expression and phylogeny of immunoglobulin light chain genes in pigeon (Columba livia).

Previous studies on immunoglobulin light chain (IgL) genes in avian species are limited to Galloanseres, and few studies have investigated IgL genes in Neoaves, which includes most living birds. Based on published genome data, we demonstrate that the pigeon (Columba livia) IgL locus spans approximately 24 kb of DNA and contains twenty Vλ segments located upstream of a single pair of Jλ-Cλ. Among the identified Vλ gene segments, four segments are structurally intact and all four segments are able to recombine with Jλ. Moreover, the four functional Vλ segments are preferentially utilized in VλJλ recombination. Phylogenetic analysis suggests that the presence of the four functional Vλ segments in pigeon was likely generated by gene duplication that occurred after the divergence of pigeon and other birds. Our study provides insight into IgL gene evolution and evolutionary diversity of Ig genes in birds.

Identification of a Transcriptionally Forward α Gene and Two υ Genes within the Pigeon (Columba livia) IgH Gene Locus.

Compared with mammals, the bird Ig genetic system relies on gene conversion to create an Ab repertoire, with inversion of the IgA-encoding gene and very few cases of Ig subclass diversification. Although gene conversion has been studied intensively, class-switch recombination, a mechanism by which the IgH C region is exchanged, has rarely been investigated in birds. In this study, based on the published genome of pigeon (Columba livia) and high-throughput transcriptome sequencing of immune-related tissues, we identified a transcriptionally forward α gene and found that the pigeon IgH gene locus is arranged as µ-α-υ1-υ2. In this article, we show that both DNA deletion and inversion may result from IgA and IgY class switching, and similar junction patterns were observed for both types of class-switch recombination. We also identified two subclasses of υ genes in pigeon, which share low sequence identity. Phylogenetic analysis suggests that divergence of the two pigeon υ genes occurred during the early stage of bird evolution. The data obtained in this study provide new insight into class-switch recombination and Ig gene evolution in birds.

[Cashmere goat bacterial artificial chromosome recombination and cell transfection system].

The Cashmere goat is mainly used to produce cashmere, which is very popular for its delicate fiber, luscious softness and natural excellent warm property. Keratin associated protein (KAP) and bone morphogenetic protein (BMP) of the Cashmere goat play an important role in the proliferation and development of cashmere fiber follicle cells. Bacterial artificial chromosome containing kap6.3, kap8.1 and bmp4 genes were used to increase the production and quality of Cashmere. First, we constructed bacterial artificial chromosomes by homology recombination. Then Tol2 transposon was inserted into bacterial artificial chromosomes that were then transfected into Cashmere goat fibroblasts by Amaxa Nucleofector technology according to the manufacture's instructions. We successfully constructed the BAC-Tol2 vectors containing target genes. Each vector contained egfp report gene with UBC promoter, Neomycin resistant gene for cell screening and two loxp elements for resistance removing after transfected into cells. The bacterial artificial chromosome-Tol2 vectors showed a high efficiency of transfection that can reach 1% to 6% with a highest efficiency of 10%. We also obtained Cashmere goat fibroblasts integrated exogenous genes (kap6.3, kap8.1 and bmp4) preparing for the clone of Cashmere goat in the future. Our research demonstrates that the insertion of Tol2 transposons into bacterial artificial chromosomes improves the transfection efficiency and accuracy of bacterial artificial chromosome error-free recombination.

Molecular analysis of the immunoglobulin genes in goose.

Immunoglobulins play an important role in adaptive immune system as defense molecules against pathogens. However, our knowledge on avian immunoglobulin genes has been limited to a few species. In this study, we analyzed goose (Anser cygnoides orientalis) immunoglobulin genes. Three IgH classes including IgM, IgA, IgY and λ light chain were identified. The IgM and IgA heavy chain constant regions are characteristically similar to their counterparts described in other vertebrates. In addition to the classic Ig isotypes, we also detected a transcript that encoded a truncated form of IgY (IgY(ΔFc)) in goose. Similar to duck, the IgY(ΔFc) in goose was generated by using different transcriptional termination signal of the same υ gene. Limited variability and only one leader peptide were observed in VH and VL domains, which suggested that gene conversion was the primary mechanism involved in goose antibody diversity. Our study provides more insights into the immunoglobulin genes in goose that had not been fully explored before.

Transgenesis of Tol2-mediated seamlessly constructed BAC mammary gland expression vectors in Mus musculus.

Bacterial artificial chromosomes (BACs) are vectors that are capable of carrying gene fragments of up to 300 kb in size, and in theory, harbor cis-regulatory elements that are necessary for the expression of specific genes. Therefore, BACs can effectively alleviate or even eliminate the position effect induced by gene-integration, rendering these as ideal expression vectors of exogenous genes. However, the number of relevant studies involving BACs as vectors of exogenous genes are limited. In the present study, we converted the BAC regulatory region of the Mus musculus Wap gene into a mammary gland-specific expression vector. Using the galK-based positive-negative selection method, we seamlessly replaced the Wap gene in a BAC with Homo sapiens GPX3, MT2, and Luc genes while keeping the original mammary gland-specific regulatory sequence intact, without introducing any extra sequences (Loxp/Frt). To improve the efficiency of creating BAC transgenic mice, we used a Tol2 transposon system optimized for mammalian codons and eliminated 100 kb of sequence from the BAC 5' end (173 kb), which resulted in an 8.5% rate of successful gene transmission via pronuclear injection. The results of the present study indicate that seamlessly constructed BAC expression vectors can be used for the transmission of the GPX3 gene.

Higher methylation in genomic DNA indicates incomplete reprogramming in induced pluripotent stem cells.

Pluripotent stem cells can be created successfully through the inner cell mass (ICM), nuclear transfer, and defined-factor induction. Unfortunately, the epigenetic characteristics of the cells produced are poorly understood. In this article, we compared expression levels of enzymes involved in epigenetic modifications across six pluripotent stem cell lines. Six of the 11 genes evaluated here (Dnmt3a, Dnmt3b, Tet1, Ezh2, Mll1, and Lsd1) showed abnormally low levels of expression in the two germ-line chimeric induced pluripotent stem cell (iPSC) lines. We also conducted locus-specific analysis of DNA methylation at 9 loci. Although iPSCs did express Oct4, the Oct4 promoter region was shown to have a higher level of DNA methylation. The Xist and Line-1 repeating sequences differed relatively little in methylation level across the cell lines, but Peg3, Peg10, and H19 exhibited high degrees of variation in the pattern of DNA methylation. Meg3 in the Dlk1-Dio3 imprinting cluster was incompletely methylated in embryonic stem cells (ESCs) and nuclear transfer (nt) ESCs. However, in germ-line chimeric iPSCs, Meg3 was almost entirely methylated. ESC and ntESC lines showed twice as much Meg3 expression than in the iPSC lines. The genomic 5mC contents detected by reverse-phase high-performance liquid chromatography (HPLC) indicated that, despite their germ-line chimeric abilities, iPSCs remained incompletely reprogrammed, even though no direct evidence is shown here.

[Immunofluorescence patterns of histone H3 lysine 4 dimethylation on X chromosomes of different cloned cattle].

Using immunofluorescence staining, the patterns of histone H3 K4m2 of metaphase X chromosomes were measured in cloned cattle derived from FFB and FOV donor cells. The results demonstrated that the modification pattern of H3 K4m2 in the ear tissue of all clones was largely consistent with that of the donor cell line FFB and conventionally produced cattle, but different from that of the donor cell line FOV.

[Exploration of the hidden layers of genome].

In recent years, geneticists have been exploring the less visible parts of genome more thoroughly. They are coming to realize that these much more layers of genomic information, distinct from the protein-coding genes, connect in surprisingly deep and potent ways to growth and development in the high eukaryotes. This article reviews a number of startling observations about the extent of non-protein-coding RNA transcriptions, DNA methylation and covalent histone modifications in the complex organisms.