TRIB1 rs17321515 gene polymorphism increases the risk of coronary heart disease in general population and non-alcoholic fatty liver disease patients in Chinese Han population.

BACKGROUND: Present evidences suggested that TRIB1 rs17321515 polymorphism was tightly associated with the increased risk of NAFLD and CHD. CHD is one of the main complications of NAFLD, whether TRIB1 rs17321515 polymorphism could affect the risk of CHD in general population and NAFLD patients in Chinese Han population was remain unknown. The present study was designed to investigate the association between TRIB1 rs17321515 polymorphism and the risk of CHD in general population and NAFLD patients in Chinese Han population, and investigate the effect of TRIB1 rs17321515 polymorphism on serum lipid levels. PATIENTS AND METHODS: TRIB1 rs17321515 gene polymorphism was genotyped using the polymerase chain reaction (PCR) in healthy controls (n = 175), CHD patients (n = 155), NAFLD patients (n = 146), and NAFLD+CHD patients (n = 156). Serum lipid profiles were determined using biochemical methods. Statistical analyses were performed using SPSS 24.0 statistical software. RESULTS: The TRIB1 rs17321515 AA+GA genotypes were the significant risk factors for the CHD in general population (OR = 1.788; 95% CI: 1.104-2.897; P = 0.018) and in the NAFLD patients (OR = 1.760; 95% CI: 1.071-2.891; P = 0.026). After adjusted for age, gender, and body mass index, the risk for CHD in general population (OR = 1.857; 95% CI: 1.116-3.089; P = 0.017) and NAFLD patients was still significant (OR = 1.723; 95% CI: 1.033-2.873; P = 0.037). In addition, TRIB1 rs17321515 A carriers possess the higher lipid profiles in the included subjects. CONCLUSIONS: TRIB1 rs17321515 AA+GA genotypes were significant associated with the risk of CHD in general population and in NAFLD patients in Chinese Han population. The rs17321515 A allele increases the serum lipid profiles in included subjects.

BMI1 enables interspecies chimerism with human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) exhibit very limited contribution to interspecies chimeras. One explanation is that the conventional hPSCs are in a primed state and so unable  to form chimeras in pre-implantation embryos. Here, we show that the conventional hPSCs undergo rapid apoptosis when injected into mouse pre-implantation embryos. While, forced-expression of BMI1, a polycomb factor in hPSCs overcomes the apoptosis and enables hPSCs to integrate into mouse pre-implantation embryos and subsequently contribute to chimeras with both embryonic and extra-embryonic tissues. In addition, BMI1 also enables hPSCs to integrate into pre-implantation embryos of other species, such as rabbit and pig. Notably, BMI1 high expression and anti-apoptosis are also indicators for naïve hPSCs to form chimera in mouse embryos. Together, our findings reveal that the apoptosis is an initial barrier in interspecies chimerism using hPSCs and provide a rational to improve it.

Chromatin Accessibility Dynamics during Chemical Induction of Pluripotency.

Despite its exciting potential, chemical induction of pluripotency (CIP) efficiency remains low and the mechanisms are poorly understood. We report the development of an efficient two-step serum- and replating-free CIP protocol and the associated chromatin accessibility dynamics (CAD) by assay for transposase-accessible chromatin (ATAC)-seq. CIP reorganizes the somatic genome to an intermediate state that is resolved under 2iL condition by re-closing previously opened loci prior to pluripotency acquisition with gradual opening of loci enriched with motifs for the OCT/SOX/KLF families. Bromodeoxyuridine, a critical ingredient of CIP, is responsible for both closing and opening critical loci, at least in part by preventing the opening of loci enriched with motifs for the AP1 family and facilitating the opening of loci enriched with SOX/KLF/GATA motifs. These changes differ markedly from CAD observed during Yamanaka-factor-driven reprogramming. Our study provides insights into small-molecule-based reprogramming mechanisms and reorganization of nuclear architecture associated with cell-fate decisions.

XIST Derepression in Active X Chromosome Hinders Pig Somatic Cell Nuclear Transfer.

Pig cloning by somatic cell nuclear transfer (SCNT) remains extremely inefficient, and many cloned embryos undergo abnormal development. Here, by profiling transcriptome expression, we observed dysregulated chromosome-wide gene expression in every chromosome and identified a considerable number of genes that are aberrantly expressed in the abnormal cloned embryos. In particular, XIST, a long non-coding RNA gene, showed high ectopic expression in abnormal embryos. We also proved that nullification of the XIST gene in donor cells can normalize aberrant gene expression in cloned embryos and enhance long-term development capacity of the embryos. Furthermore, the increased quality of XIST-deficient embryos was associated with the global H3K9me3 reduction. Injection of H3K9me demethylase Kdm4A into NT embryos could improve the development of pre-implantation stage embryos. However, Kdm4A addition also induced XIST derepression in the active X chromosome and thus was not able to enhance the in vivo long-term developmental capacity of porcine NT embryos.

Cre-dependent Cas9-expressing pigs enable efficient in vivo genome editing.

Despite being time-consuming and costly, generating genome-edited pigs holds great promise for agricultural, biomedical, and pharmaceutical applications. To further facilitate genome editing in pigs, we report here establishment of a pig line with Cre-inducible Cas9 expression that allows a variety of ex vivo genome editing in fibroblast cells including single- and multigene modifications, chromosome rearrangements, and efficient in vivo genetic modifications. As a proof of principle, we were able to simultaneously inactivate five tumor suppressor genes (TP53, PTEN, APC, BRCA1, and BRCA2) and activate one oncogene (KRAS), achieved by delivering Cre recombinase and sgRNAs, which caused rapid lung tumor development. The efficient genome editing shown here demonstrates that these pigs can serve as a powerful tool for dissecting in vivo gene functions and biological processes in a temporal manner and for streamlining the production of genome-edited pigs for disease modeling.

Generation of Hoxc13 knockout pigs recapitulates human ectodermal dysplasia-9.

Atrichia and sparse hair phenotype cause distress to many patients. Ectodermal dysplasia-9 (ED-9) is a congenital condition characterized by hypotrichosis and nail dystrophy without other disorders, and Hoxc13 is a pathogenic gene for ED-9. However, mice carrying Hoxc13 mutation present several other serious disorders, such as skeletal defects, progressive weight loss and low viability. Mouse models cannot faithfully mimic human ED-9. In this study, we generated an ED-9 pig model via Hoxc13 gene knockout through single-stranded oligonucleotides (c.396C > A) combined with CRISPR/Cas9 and somatic cell nuclear transfer. Eight cloned piglets with three types of biallelic mutations (five piglets with Hoxc13c.396C > A/c.396C > A, two piglets with Hoxc13c.396C > A/c.396C > A + 1 and one piglet with Hoxc13Δ40/Δ40) were obtained. Hoxc13 was not expressed in pigs with all three mutation types, and the expression levels of Hoxc13-regulated genes, namely, Foxn1, Krt85 and Krt35, were decreased. The hair follicles displayed various abnormal phenotypes, such as reduced number of follicles and disarrayed hair follicle cable without normal hair all over the body. By contrast, the skin structure, skeleton phenotype, body weight gain and growth of Hoxc13 knockout pigs were apparently normal. The phenotypes of Hoxc13 mutation in pigs were similar to those in ED-9 patients. Therefore, Hoxc13 knockout pigs could be utilized as a model for ED-9 pathogenesis and as a hairless model for hair regeneration research. Moreover, the hairless pigs without other major abnormal phenotypes generated in this study could be effective models for other dermatological research because of the similarity between pig and human skins.

Generation of Knock-In Pigs Carrying Oct4-tdTomato Reporter through CRISPR/Cas9-Mediated Genome Engineering.

The porcine pluripotent cells that can generate germline chimeras have not been developed. The Oct4 promoter-based fluorescent reporter system, which can be used to monitor pluripotency, is an important tool to generate authentic porcine pluripotent cells. In this study, we established a porcine Oct4 reporter system, wherein the endogenous Oct4 promoter directly controls red fluorescent protein (RFP). 2A-tdTomato sequence was inserted to replace the stop codon of the porcine Oct4 gene by homogenous recombination (HR). Thus, the fluorescence can accurately show the activation of endogenous Oct4. Porcine fetal fibroblast (PFF) lines with knock-in (KI) of the tdTomato gene in the downstream of endogenous Oct4 promoter were achieved using the CRISPR/CAS9 system. Transgenic PFFs were used as donor cells for somatic cell nuclear transfer (SCNT). Strong RFP expression was detected in the blastocysts and genital ridges of SCNT fetuses but not in other tissues. Two viable transgenic piglets were also produced by SCNT. Reprogramming of fibroblasts from the fetuses and piglets by another round of SCNT resulted in tdTomato reactivation in reconstructed blastocysts. Result indicated that a KI porcine reporter system to monitor the pluripotent status of cells was successfully developed.

Cytoplasmic mislocalization of RNA splicing factors and aberrant neuronal gene splicing in TDP-43 transgenic pig brain.

BACKGROUND: TAR DNA-binding protein 43 (TDP-43) is a nuclear protein, but it is redistributed in the neuronal cytoplasm in both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Because small transgenic animal models often lack cytoplasmic TDP-43, how the cytoplasmic accumulation of TDP-43 contributes to these diseases remains unclear. The current study is aimed at studying the mechanism of cytoplasmic pathology of TDP-43. RESULTS: We established transgenic pigs expressing mutant TDP-43 (M337V). This pig model shows severe phenotypes and early death. We found that transgenic TDP-43 is also distributed in the cytoplasm of neuronal cells in the spinal cord and brain. Transgenic TDP-43 interacts with PSF, an RNA splicing factor that associates with NeuN to regulate neuronal RNA splicing. The interaction of TDP-43, PSF and NeuN causes PSF and NeuN mislocalize into the neuronal cytoplasm in transgenic pigs. Consistently, abnormal PSF-related neuronal RNA splicing is seen in TDP-43 transgenic pigs. The cytoplasmic localization of PSF and NeuN as well as abnormal PSF-related neuronal RNA splicing was also found in ALS patient brains. CONCLUSION: Our findings from a large mammalian model suggest that cytoplasmic mutant TDP-43 could reduce the nuclear function of RNA splicing factors, contributing to neuropathology.