Global chromosome rearrangement induced by CRISPR-Cas9 reshapes the genome and transcriptome of human cells.

Chromosome rearrangement plays important roles in development, carcinogenesis and evolution. However, its mechanism and subsequent effects are not fully understood. Large-scale chromosome rearrangement has been performed in the simple eukaryote, wine yeast, but the relative research in mammalian cells remains at the level of individual chromosome rearrangement due to technical limitations. In this study, we used CRISPR-Cas9 to target the highly repetitive human endogenous retrotransposons, LINE-1 and Alu, resulting in a large number of DNA double-strand breaks in the chromosomes. While this operation killed the majority of the cells, we eventually obtained live cell groups. Karyotype analysis and genome re-sequencing proved that we have achieved global chromosome rearrangement (GCR) in human cells. The copy number variations of the GCR genomes showed typical patterns observed in tumor genomes. The ATAC-seq and RNA-seq further revealed that the epigenetic and transcriptomic landscapes were deeply reshaped by GCR. Gene expressions related to p53 pathway, DNA repair, cell cycle and apoptosis were greatly altered to facilitate the cell survival. Our study provided a new application of CRISPR-Cas9 and a practical approach for GCR in complex mammalian genomes.

The composition dynamics of transposable elements in human blastocysts.

Transposable elements (TEs) are mobile DNA sequences that can replicate themselves and play significant roles in embryo development and chromosomal structure remodeling. In this study, we investigated the variation of TEs in blastocysts with different parental genetic backgrounds. We analyzed the proportions of 1137 TEs subfamilies from six classes at the DNA level using Bowtie2 and PopoolationTE2 in 196 blastocysts with abnormal parental chromosomal diseases. Our findings revealed that the parental karyotype was the dominant factor influencing TEs frequencies. Out of the 1116 subfamilies, different frequencies were observed in blastocysts with varying parental karyotypes. The development stage of blastocysts was the second most crucial factor influencing TEs proportions. A total of 614 subfamilies exhibited different proportions at distinct blastocyst stages. Notably, subfamily members belonging to the Alu family showed a high proportion at stage 6, while those from the LINE class exhibited a high proportion at stage 3 and a low proportion at stage 6. Moreover, the proportions of some TEs subfamilies also varied depending on blastocyst karyotype, inner cell mass status, and outer trophectoderm status. We found that 48 subfamilies displayed different proportions between balanced and unbalanced blastocysts. Additionally, 19 subfamilies demonstrated varying proportions among different inner cell mass scores, and 43 subfamilies exhibited different proportions among outer trophectoderm scores. This study suggests that the composition of TEs subfamilies may be influenced by various factors and undergoes dynamic modulation during embryo development.

A Study of Combined Genotype Effects of SHCBP1 on Wool Quality Traits in Chinese Merino.

SHCBP1 (Shc SH2-domain binding protein 1) is a member of the Src and collagen homolog (Shc) protein family and is closely associated with multiple signaling pathways that play important roles during hair follicle induction, morphogenesis, and cycling. The purpose of this study was to investigate SHCBP1 gene expression, polymorphisms, and the association between SHCBP1 and wool quality traits in Chinese Merino sheep. The SHCBP1 gene was shown, by qPCR, to be ubiquitously expressed in sheep tissues and differentially expressed in the adult skin of Chinese Merino and Suffolk sheep. Four SNPs (termed SHCBP1SNPs 1-4) were identified by Sanger sequencing and were located in exon 2, intron 9, intron 12, and exon 13 of the sheep SHCBP1 gene, respectively. SHCBP1SNPs 3 and 4 (rs411176240 and rs160910635) were significantly associated with wool crimp (P < 0.05). The combined polymorphism (SHCBP1SNP3-SHCBP1SNP4) was significantly associated with wool crimp (P < 0.05). Bioinformatics analysis showed that the SNPs associated with wool crimp (SHCBP1SNPs 3 and 4) might affect the pre-mRNA splicing by creating binding sites for serine-arginine-rich proteins and that SHCBP1SNP4 might alter the SHCBP1 mRNA and protein secondary structure. Our results suggest that SHCBP1 influences wool crimp and SHCBP1SNPs 3 and 4 might be useful markers for marker-assisted selection and sheep breeding.

The Characterization of Mitochondrial Genome of Spotted Pond Turtle (Geoclemys hamiltonii).

The spotted pond turtle Geoclemys hamiltonii (Gray, 1830) is widely distributed in the Indus, Ganges, and Brahmaputra river basins. In this study, the complete mitochondrial genome (mitogenome) of G. hamiltonii was sequenced using the next-generation sequencing (NGS) and Sanger sequencing, and the essential characteristics, gene arrangement, and phylogenetic relationship were analyzed. The results showed that the G. hamiltonii mitogenome was 16,505 bp in length (A: 33.6%, C: 27.1%, G: 13.4%, T: 25.8%) and consisted of 22 tRNAs, 13 protein-coding genes, two ribosomal RNA genes, and a non-coding control region (GenBank accession ON243873). The genome composition of G. hamiltonii presented a slight A + T bias (59.4%), and showed a positive AT skew (0.131) and a negative GC skew (- 0.338). All tRNAs had the typical clover structure, except trnS1 (GCT). The gene order of the G. hamiltonii mitogenome was the same as other Geoemydidae mitogenomes. A phylogenetic analysis based on the complete mitogenome indicated that the G. hamiltonii grouped independently of other species in the family Geoemydidae, supporting the species' placement in the monotypic genus Geoclemys. Our results describe a novel genome at the species level. As the first complete mitogenome of G. hamiltonii, it provided valuable molecular information for phylogenetic and conservation genetics analyses of G. hamiltonii.

The wide distribution and horizontal transfers of beta satellite DNA in eukaryotes.

Beta satellite DNA (satDNA), also known as Sau3A sequences, are repetitive DNA sequences reported in human and primate genomes. It is previously thought that beta satDNAs originated in old world monkeys and bursted in great apes. In this study, we searched 7821 genome assemblies of 3767 eukaryotic species and found that beta satDNAs are widely distributed across eukaryotes. The four major branches of eukaryotes, animals, fungi, plants and Harosa/SAR, all have multiple clades containing beta satDNAs. These results were also confirmed by searching whole genome sequencing data (SRA) and PCR assay. Beta satDNA sequences were found in all the primate clades, as well as in Dermoptera and Scandentia, indicating that the beta satDNAs in primates might originate in the common ancestor of Primatomorpha or Euarchonta. In contrast, the widely patchy distribution of beta satDNAs across eukaryotes presents a typical scenario of multiple horizontal transfers.

Functional Analysis of Sheep POU2F3 Isoforms.

POU domain class 2 transcription factor 3 (POU2F3) plays an important role in keratinocyte proliferation and differentiation. Our previous study identified four sheep POU2F3 transcript variants (POU2F3-1, POU2F3-2, POU2F3-3, and POU2F3-4), encoding three POU2F3 protein isoforms (POU2F3-1, POU2F3-2, and POU2F3-3). However, the functional differences among the three POU2F3 isoforms remain unknown. The objective of this study was to determine the tissue expression pattern of the four POU2F3 transcript variants in sheep and to investigate the functional differences in cell proliferation among the three POU2F3 isoforms. Quantitative RT-PCR analysis showed that the four POU2F3 transcripts were ubiquitously expressed in all tested adult sheep tissues, and POU2F3-1 exhibited higher expression level than the other three POU2F3 transcript variants in skin (P < 0.05). Cell proliferation assay showed that overexpression of any one of the three POU2F3 isoforms significantly inhibited the proliferation of sheep fetal fibroblasts and HaCaT cells at 48 and 72 h after transfection (P < 0.05). POU2F3-3 had less inhibitory effect on cell proliferation than POU2F3-1 and POU2F3-2 (P < 0.05), and POU2F3-1 and POU2F3-2 had similar inhibitory effects (P > 0.05). Dual luciferase reporter assays demonstrated that overexpression of any one of the three POU2F3 isoforms significantly inhibited the promoter activities of keratin 14 (KRT14) and matrix metalloproteinase 19 (MMP19) genes (P < 0.05). POU2F3-3 had less inhibitory effect on the promoter activities of KRT14 and MMP19 genes than POU2F3-1 and POU2F3-2 (P < 0.05), and POU2F3-1 and POU2F3-2 had similar inhibitory effects (P > 0.05). These results suggest three sheep POU2F3 isoforms have similar functional effects, but to a different extent.

The characterization of the mitochondrial genome of Graptemys ouachitensis.

Graptemys ouachitensis (CAGLE, 1953) belongs to the Graptemys genus, the Emydidae family, and the Testudines order. This study involved sequencing the complete mitochondrial genome (mitogenome) of G. ouachitensis using next-generation sequencing, and analyzing the essential characteristics, and phylogenetic relationship. The results revealed that the G. ouachitensis mitogenome was 16,674 bp in length (A: 34.1%, C: 26.0%, G: 13.0%, T: 26.9%) and included 22 tRNAs, 13 protein-coding genes, two ribosomal RNA genes, and a non-coding control region (GenBank accession: NC071766). The genome composition of G. ouachitensis presented a slight A + T bias (61.0%) and exhibited a positive AT skew (0.118) and a negative GC skew (-0.333). A phylogenetic analysis based on the complete mitogenome indicated that the G. ouachitensis was more closely associated with Malaclemys terrapin than the other eight known Emydidae species. Thus, our findings present a novel mitogenome at the species level. This study introduces the first complete mitogenome of G. ouachitensis, providing valuable molecular information for phylogenetic and conservation genetics analyses of G. ouachitensis.

Single-cell transcriptomic landscape of peripheral blood cells provides insights into adaptation of red-eared sliders (Trachemys scripta elegans).

Red-eared sliders (Trachemys scripta elegans), as one of the 100 most threatening aliens, have stronger immunity than the native species in response to environmental stress. Blood cells are an important component of immunity in the body. However, the blood cell researches of turtle are still in the traditional blood cell classification and morphological structure observation. Furthermore, turtle granulocytes cannot be accurately identified using traditional methods. Single-cell RNA sequencing techniques have been successfully implemented to study cells based on the mRNA expression patterns of each cell. The present study profiled the transcriptomes of peripheral blood cells in red-eared sliders to construct a single-cell transcriptional landscape of the different cell types and explored environmental adaptation mechanism from the perspective of hematology. All 14 transcriptionally distinct clusters (platelets, erythrocytes1, erythrocytes2, CSF1R monocytes, POF1B monocytes, neutrophils, GATA2high basophils, GATA2low basophils, CD4 T cells, CD7 T cells, B cells, ACKR4 cells, serotriflin cells, and ficolin cells) were identified in the peripheral blood cells of the red-eared sliders. In particular, a subtype of erythrocytes (erythrocytes1) that expressed immune signals was identified. Peripheral blood cells were grouped into three lineages: platelet, erythroid/lymphoid, and myeloid cell lineages. Furthermore, based on differentiation trajectory and up-regulated gene expression, ACKR4 cells were newly identified as lymphocytes, and serotriflin and ficolin cells as granulocytes. The single-cell transcriptional atlas of the peripheral blood cells in red-eared sliders provided in the present study will offer a comprehensive transcriptome reference for the exploration of physiological and pathological hematology in this species.

Comparison of the Single-Cell Immune Landscape of Testudines from Different Habitats.

Testudines, also known as living fossils, have evolved diversely and comprise many species that occupy a variety of ecological niches. However, the immune adaptation of testudines to the different ecological niches remains poorly understood. This study compared the composition, function, and differentiation trajectories of peripheral immune cells in testudines (Chelonia mydas, Trachemys scripta elegans, Chelonoidis carbonaria, and Pelodiscus sinensis) from different habitats using the single-cell RNA sequencing (scRNA-seq) technique. The results showed that T. scripta elegans, which inhabits freshwater and brackish environments, had the most complex composition of peripheral immune cells, with 11 distinct immune cell subsets identified in total. The sea turtle C. mydas, had the simplest composition of peripheral immune cells, with only 5 distinct immune cell clusters. Surprisingly, neither basophils were found in C. mydas nor T cells in C. carbonaria. Basophil subsets in peripheral blood were identified for the first time; two basophil subtypes (GATA2-high-basophils and GATA2-low-basophils) were observed in the peripheral blood of T. scripta elegans. In addition, ACKR4 cells, CD4 T cells, CD7 T cells, serotriflin cells, and ficolin cells were specifically identified in the peripheral blood of T. scripta elegans. Furthermore, LY6G6C cells, SPC24 cells, and NKT cells were specifically observed in C. carbonaria. Moreover, there were differences in the functional status and developmental trajectory of peripheral immune cells among the testudine species. The identification of specific features of peripheral immune cells in testudines from different habitats may enable elucidation of the adaptation mechanism of testudines to various ecological niches.

Interleukin 13 participates in terminal differentiation of esophageal squamous cell carcinoma cells.

Background: In China, esophageal squamous cell carcinoma (ESCC) accounts for more than 90% of all esophageal cancer cases. Interleukin 13 (IL-13) was widely reported to play a key role in tumor progression. Our previous study reported that IL-13 was a favorable predictive marker for the overall survival of esophageal squamous cell carcinoma (ESCC) patients, but how IL-13 contributes to ESCC progression remains unknown. This study aims to explore the role of IL-13 and its underlying downstream molecular mechanisms in ESCC progression. Methods: Tissue microarrays including 262 primary ESCC tumor tissues were collected and analyzed. The expression of IL-13 in ESCC tumor tissue was detected with immunohistochemistry staining (IHC). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to qualify the expressions of KRT13, KRT4 and 15-lipoxygenase-1 (15-LOX-1) in cultured ESCC cell lines with recombinant IL-13 treatment. Results: IL-13 was expressed in the esophageal epithelium cells and ESCC tumor cells. High IL-13 expression in ESCC tumor cells predicted a good prognosis for patients. Recombinant human IL-13 raised KRT13 and 15-LOX-1 mRNA levels, but lowered KRT4 mRNA level 15-LOX-1 in ESCC cells in vitro. Conclusions: In summary, our study suggests that IL-13 might improve the prognosis of ESCC by promoting the terminal differentiation of ESCC cells. This may offer potential new therapeutic target for early treatment of ESCC.

The Integration Preference of Sleeping Beauty at Non-TA Site Is Related to the Transposon End Sequences.

Recently, we proved that Sleeping Beauty (SB) transposon integrates into non-TA sites at a lower frequency. Here, we performed a further study on the non-TA integration of SB and showed that (1) SB can integrate into non-TA sites in HEK293T cells as well as in mouse cell lines; (2) Both the hyperactive transposase SB100X and the traditional SB11 catalyze integrations at non-TA sites; (3) The consensus sequence of the non-TA target sites only occurs at the opposite side of the sequenced junction between the transposon end and the genomic sequences, indicating that the integrations at non-TA sites are mainly aberrant integrations; and (4) The consensus sequence of the non-TA target sites is corresponding to the transposon end sequence. The consensus sequences changed following the changes of the transposon ends. This result indicated that the interaction between the SB transposon end and genomic DNA (gDNA) may be involved in the target site selection of the SB integrations at non-TA sites.

STAT3 Partly Inhibits Cell Proliferation via Direct Negative Regulation of FST Gene Expression.

Follistatin (FST) is a secretory glycoprotein and belongs to the TGF-ß superfamily. Previously, we found that two single nucleotide polymorphisms (SNPs) of sheep FST gene were significantly associated with wool quality traits in Chinese Merino sheep (Junken type), indicating that FST is involved in the regulation of hair follicle development and hair trait formation. The transcription regulation of human and mouse FST genes has been widely investigated, and many transcription factors have been identified to regulate FST gene. However, to date, the transcriptional regulation of sheep FST is largely unknown. In the present study, genome walking was used to close the genomic gap upstream of the sheep genomic FST gene and to obtain the FST gene promoter sequence. Transcription factor binding site analysis showed sheep FST promoter region contained a conserved putative binding site for signal transducer and activator of transcription 3 (STAT3), located at nucleotides -423 to -416 relative to the first nucleotide (A, +1) of the initiation codon (ATG) of sheep FST gene. The dual-luciferase reporter assay demonstrated that STAT3 inhibited the FST promoter activity and that the mutation of the putative STAT3 binding site attenuated the inhibitory effect of STAT3 on the FST promoter activity. Additionally, chromatin immunoprecipitation assay (ChIP) exhibited that STAT3 is directly bound to the FST promoter. Cell proliferation assay displayed that FST and STAT3 played opposite roles in cell proliferation. Overexpression of sheep FST significantly promoted the proliferation of sheep fetal fibroblasts (SFFs) and human keratinocyte (HaCaT) cells, and overexpression of sheep STAT3 displayed opposite results, which was accompanied by a significantly reduced expression of FST gene (P < 0.05). Taken together, STAT3 directly negatively regulates sheep FST gene and depresses cell proliferation. Our findings may contribute to understanding molecular mechanisms that underlie hair follicle development and morphogenesis.

Reduced Plasma Levels of α-Klotho and Their Correlation With Klotho Polymorphisms in Elderly Patients With Major Depressive Disorders.

Background: Recent literature suggests that α-Klotho, a widely recognized anti-aging protein, is involved in longevity as well as in many diseases, including Alzheimer's disease, and depression. Although the Klotho gene encodes α-Klotho, a single transmembrane protein with intracellular and extracellular domains, the relationship between Klotho gene polymorphism and circulating α-Klotho levels in patients with major depressive disorder (MDD) is not clear. Methods: A total of 144 MDD patients and 112 age-matched healthy controls were included in this study. The Klotho genetic polymorphisms (rs9536314, rs9527025, and rs9315202) and plasma α-Klotho levels were measured by PCR and ELISA, respectively. The severity of depressive symptoms was estimated using the Hamilton Depression Scale (HAMD). Results: We found a significantly lower level of plasma α-Klotho in the MDD patients than in controls. Among them, only elderly MDD patients (first episode) showed significantly lower α-Klotho levels than the age-matched controls, while elderly recurrent and young MDD patients showed no difference in plasma α-Klotho levels from age-matched controls. The young MDD group showed a significantly earlier onset age, higher plasma α-Klotho levels, and lower HAMD scores than those in the elderly MDD group. While the plasma α-Klotho levels were higher in rs9315202 T alleles carrier regardless age or sex, the rs9315202 T allele was negatively correlated with disease severity only in the elderly MDD patients. Conclusion: The results of our study showed that only elderly MDD patients showed a decrease in plasma α-Klotho levels along with an increase in disease severity as well as an association with the number of rs9315202 T alleles, and not young MDD patients compared to age-matched controls. Our data suggest that circulating α-Klotho levels combined with Klotho genetic polymorphisms are important in elderly MDD patients, particularly carriers of the Klotho gene rs9315202 T allele.

Could α-Klotho Unlock the Key Between Depression and Dementia in the Elderly: from Animal to Human Studies.

α-Klotho is known for its aging-related functions and is associated with neurodegenerative diseases, accelerated aging, premature morbidity, and mortality. Recent literature suggests that α-Klotho is also involved in the regulation of mental functions, such as cognition and psychosis. While most of studies of α-Klotho are focusing on its anti-aging functions and protective role in dementia, increasing evidence showed many shared symptoms between depression and dementia, while depression has been proposed as the preclinical stage of dementia such as Alzheimer's disease (AD). To see whether and how α-Klotho can be a key biological link between depression and dementia, in this review, we first gathered the evidence on biological distribution and function of α-Klotho in psychiatric functions from animal studies to human clinical investigations with a focus on the regulation of cognition and mood. Then, we discussed and highlighted the potential common underlying mechanisms of α-Klotho between psychiatric diseases and cognitive impairment. Finally, we hypothesized that α-Klotho might serve as a neurobiological link between depression and dementia through the regulation of oxidative stress and inflammation.

An Upstream Open Reading Frame Represses Translation of Chicken PPARγ Transcript Variant 1.

Peroxisome proliferator-activated receptor γ (PPARγ) is a master regulator of adipogenesis. The PPARγ gene produces various transcripts with different 5'-untranslated regions (5' UTRs) because of alternative promoter usage and splicing. The 5' UTR plays important roles in posttranscriptional gene regulation. However, to date, the regulatory role and underlying mechanism of 5' UTRs in the posttranscriptional regulation of PPARγ expression remain largely unclear. In this study, we investigated the effects of 5' UTRs on posttranscriptional regulation using reporter assays. Our results showed that the five PPARγ 5' UTRs exerted different effects on reporter gene activity. Bioinformatics analysis showed that chicken PPARγ transcript 1 (PPARγ1) possessed an upstream open reading frame (uORF) in its 5' UTR. Mutation analysis showed that a mutation in the uORF led to increased Renilla luciferase activity and PPARγ protein expression, but decreased Renilla luciferase and PPARγ1 mRNA expression. mRNA stability analysis using real-time RT-PCR showed that the uORF mutation did not interfere with mRNA stability, but promoter activity analysis of the cloned 5' UTR showed that the uORF mutation reduced promoter activity. Furthermore, in vitro transcription/translation assays demonstrated that the uORF mutation markedly increased the translation of PPARγ1 mRNA. Collectively, our results indicate that the uORF represses the translation of chicken PPARγ1 mRNA.

Structural Characterization and Association of Ovine Dickkopf-1 Gene with Wool Production and Quality Traits in Chinese Merino.

Dickkopf-1 (DKK1) is an inhibitor of canonical Wnt signaling pathway and regulates hair follicle morphogenesis and cycling. To investigate the potential involvement of DKK1 in wool production and quality traits, we characterized the genomic structure of ovine DKK1, performed polymorphism detection and association analysis of ovine DKK1 with wool production and quality traits in Chinese Merino. Our results showed that ovine DKK1 consists of four exons and three introns, which encodes a protein of 262 amino acids. The coding sequence of ovine DKK1 and its deduced amino acid sequence were highly conserved in mammals. Eleven single nucleotide polymorphisms (SNPs) were identified within the ovine DKK1 genomic region. Gene-wide association analysis showed that SNP5 was significantly associated with mean fiber diameter (MFD) in the B (selected for long wool fiber and high-quality wool), PW (selected for high reproductive capacity, high clean wool yield and high-quality wool) and U (selected for long wool fiber with good uniformity, high wool yield and lower fiber diameter) strains (p < 4.55 × 10-3 = 0.05/11). Single Nucleotide Polymorphisms wide association analysis showed that SNP8 was significantly associated with MFD in A strain and fleece weight in A (selected for large body size), PM (selected for large body size, high reproductive capacity and high meat yield) and SF (selected for mean fiber diameter less than 18 µm and wool fiber length between 5 and 9 cm) strains (p < 0.05), SNP9 was significantly associated with curvature in B and U strains (p < 0.05) and SNP10 was significantly associated with coefficient of variation of fiber diameter in A, PW and PM strains and standard deviation of fiber diameter in A and PM strains (p < 0.05). The haplotypes derived from these 11 identified SNPs were significantly associated with MFD (p < 0.05). In conclusion, our results suggest that DKK1 may be a major gene controlling wool production and quality traits, also the identified SNPs (SNPs5, 8, 9 and 10) might be used as potential molecular markers for improving sheep wool production and quality in sheep breeding.

Polymorphisms of FST gene and their association with wool quality traits in Chinese Merino sheep.

Follistatin (FST) is involved in hair follicle morphogenesis. However, its effects on hair traits are not clear. This study was designed to investigate the effects of FST gene single nucleotide polymorphisms (SNP) on wool quality traits in Chinese Merino sheep (Junken Type). We performed gene expression analysis, SNP detection, and association analysis of FST gene with sheep wool quality traits. The real-time RT-PCR analysis showed that FST gene was differentially expressed in adult skin between Chinese Merino sheep (Junken Type) and Suffolk sheep. Immunostaining showed that FST was localized in inner root sheath (IRS) and matrix of hair follicle (HF) in both SF and Suffolk sheep. Sequencing analysis identified a total of seven SNPs (termed SNPs 1-7) in the FST gene in Chinese Merino sheep (Junken Type). Association analysis showed that SNP2 (Chr 16. 25,633,662 G>A) was significantly associated with average wool fiber diameter, wool fineness SD, and wool crimp (P < 0.05). SNP4 (Chr 16. 25,633,569 C>T) was significantly associated with wool fineness SD and CV of fiber diameter (P < 0.05). Similarly, the haplotypes derived from these seven identified SNPs were also significantly associated with average wool fiber diameter, wool fineness SD, CV of fiber diameter, and wool crimp (P < 0.05). Our results suggest that FST influences wool quality traits and its SNPs 2 and 4 might be useful markers for marker-assisted selection and sheep breeding.