Maternal factor Trim75 contributes to zygotic genome activation program in mouse early embryos.

BACKGROUND: Zygotic genome activation (ZGA) is an important event in the early embryo development, and human embryo developmental arrest has been highly correlated with ZGA failure in clinical studies. Although a few studies have linked maternal factors to mammalian ZGA, more studies are needed to fully elucidate the maternal factors that are involved in ZGA. METHODS AND RESULTS: In this study, we utilized published single-cell RNA sequencing data from a Dux-mediated mouse embryonic stem cell to induce a 2-cell-like transition state and selected potential drivers for the transition according to an RNA velocity analysis. CONCLUSIONS: An overlap of potential candidate markers of 2-cell-like-cells identified in this research with markers generated by various data sets suggests that Trim75 is a potential driver of minor ZGA and may recruit EP300 and establish H3K27ac in the gene body of minor ZGA genes, thereby contributing to mammalian preimplantation embryo development.

Lactate regulates major zygotic genome activation by H3K18 lactylation in mammals.

Lactate is present at a high level in the microenvironment of mammalian preimplantation embryos in vivo and in vitro. However, its role in preimplantation development is unclear. Here, we report that lactate is highly enriched in the nuclei of early embryos when major zygotic genome activation (ZGA) occurs in humans and mice. The inhibition of its production and uptake results in developmental arrest at the 2-cell stage, major ZGA failure, and loss of lactate-derived H3K18lac, which could be rescued by the addition of Lac-CoA and recapitulated by overexpression of H3K18R mutation. By profiling the landscape of H3K18lac during mouse preimplantation development, we show that H3K18lac is enriched on the promoter regions of most major ZGA genes and correlates with their expressions. In humans, H3K18lac is also enriched in ZGA markers and temporally concomitant with their expressions. Taken together, we profile the landscapes of H3K18lac in mouse and human preimplantation embryos, and demonstrate the important role for H3K18lac in major ZGA, showing that a conserved metabolic mechanism underlies preimplantation development of mammalian embryos.

COX6C expression driven by copy amplification of 8q22.2 regulates cell proliferation via mediation of mitosis by ROS-AMPK signaling in lung adenocarcinoma.

Copy number variations (CNVs) play a vital role in regulating genes expression and tumorigenesis. We explored the copy number alterations in early-stage lung adenocarcinoma using high-throughput sequencing and nucleic acid flight mass spectrometry technology, and found that 8q22.1-22.2 is frequently amplified in lung adenocarcinoma tissues. COX6C localizes on the region and its expression is notably enhanced that driven by amplification in lung adenocarcinoma. Knockdown of COX6C significantly inhibits the cell proliferation, and induces S-G2/M cell cycle arrest, mitosis deficiency and apoptosis. Moreover, COX6C depletion causes a deficiency in mitochondrial fusion, and impairment of oxidative phosphorylation. Mechanistically, COX6C-induced mitochondrial deficiency stimulates ROS accumulation and activates AMPK pathway, then leading to abnormality in spindle formation and chromosome segregation, activating spindle assemble checkpoint, causing mitotic arrest, and ultimately inducing cell apoptosis. Collectively, we suggested that copy amplification-mediated COX6C upregulation might serves as a prospective biomarker for prognosis and targeting therapy in patients with lung adenocarcinoma.

Deep annotation of long noncoding RNAs by assembling RNA-seq and small RNA-seq data.

Long noncoding RNAs (lncRNAs) are increasingly being recognized as modulators in various biological processes. However, due to their low expression, their systematic characterization is difficult to determine. Here, we performed transcript annotation by a newly developed computational pipeline, termed RNA-seq and small RNA-seq combined strategy (RSCS), in a wide variety of cellular contexts. Thousands of high-confidence potential novel transcripts were identified by the RSCS, and the reliability of the transcriptome was verified by analysis of transcript structure, base composition, and sequence complexity. Evidenced by the length comparison, the frequency of the core promoter and the polyadenylation signal motifs, and the locations of transcription start and end sites, the transcripts appear to be full length. Furthermore, taking advantage of our strategy, we identified a large number of endogenous retrovirus-associated lncRNAs, and a novel endogenous retrovirus-lncRNA that was functionally involved in control of Yap1 expression and essential for early embryogenesis was identified. In summary, the RSCS can generate a more complete and precise transcriptome, and our findings greatly expanded the transcriptome annotation for the mammalian community.

Network characterization linc1393 in the maintenance of pluripotency provides the principles for lncRNA targets prediction.

Long non-coding RNAs (lncRNAs) have been implicated in diverse biological processes. However, the functional mechanisms have not yet been fully explored. Characterizing the interactions of lncRNAs with chromatin is central to determining their functions but, due to precise and efficient approaches lacking, our understanding of their functional mechanisms has progressed slowly. In this study, we demonstrate that a nuclear lncRNA linc1393 maintains mouse ESC pluripotency by recruiting SET1A near its binding sites, to establish H3K4me3 status and activate the expression of specific pluripotency-related genes. Moreover, we characterized the principles of lncRNA-chromatin interaction and transcriptional regulation. Accordingly, we developed a computational framework based on the XGBoost model, LncTargeter, to predict the targets of a given lncRNA, and validated its reliability in various cellular contexts. Together, these findings elucidate the roles and mechanisms of lncRNA on pluripotency maintenance, and provide a promising tool for predicting the regulatory networks of lncRNAs.

Partial erosion on under-methylated regions and chromatin reprogramming contribute to oncogene activation in IDH mutant gliomas.

BACKGROUND: IDH1/2 hotspot mutations are well known to drive oncogenic mutations in gliomas and are well-defined in the WHO 2021 classification of central nervous system tumors. Specifically, IDH mutations lead to aberrant hypermethylation of under-methylated regions (UMRs) in normal tissues through the disruption of TET enzymes. However, the chromatin reprogramming and transcriptional changes induced by IDH-related hypermethylation in gliomas remain unclear. RESULTS: Here, we have developed a precise computational framework based on Hidden Markov Model to identify altered methylation states of UMRs at single-base resolution. By applying this framework to whole-genome bisulfite sequencing data from 75 normal brain tissues and 15 IDH mutant glioma tissues, we identified two distinct types of hypermethylated UMRs in IDH mutant gliomas. We named them partially hypermethylated UMRs (phUMRs) and fully hypermethylated UMRs (fhUMRs), respectively. We found that the phUMRs and fhUMRs exhibit distinct genomic features and chromatin states. Genes related to fhUMRs were more likely to be repressed in IDH mutant gliomas. In contrast, genes related to phUMRs were prone to be up-regulated in IDH mutant gliomas. Such activation of phUMR genes is associated with the accumulation of active H3K4me3 and the loss of H3K27me3, as well as H3K36me3 accumulation in gene bodies to maintain gene expression stability. In summary, partial erosion on UMRs was accompanied by locus-specific changes in key chromatin marks, which may contribute to oncogene activation. CONCLUSIONS: Our study provides a computational strategy for precise decoding of methylation encroachment patterns in IDH mutant gliomas, revealing potential mechanistic insights into chromatin reprogramming that contribute to oncogenesis.

Inhibition of TGF-ß pathway improved the pluripotency of porcine pluripotent stem cells.

Porcine pluripotent stem cells had been derived from different culture systems. PeNK6 is a porcine pluripotent stem cell line that we established from an E5.5 embryo in a defined culture system. Signaling pathways related with pluripotency had been assessed in this cell line, and TGF-ß signaling pathway-related genes were found upregulated significantly. In this study, we elucidated the role of the TGF-ß signaling pathway in PeNK6 through adding small molecule inhibitors, SB431542 (KOSB) or A83-01 (KOA), into the original culture medium (KO) and analyzing the expression and activity of key factors involved in the TGF-ß signaling pathway. In KOSB/KOA medium, the morphology of PeNK6 became compact and the nuclear-to-cytoplasm ratio was increased. The expression of the core transcription factor SOX2 was significantly upregulated compared with cell lines in the control KO medium, and the differentiation potential became balanced among three germ layers rather than bias to neuroectoderm/endoderm as the original PeNK6 did. The results indicated that inhibition of TGF-ß has positive effects on the porcine pluripotency. Based on these results, we established a pluripotent cell line (PeWKSB) from E5.5 blastocyst by employing TGF-ß inhibitors, and the cell line showed improved pluripotency.

RRP15 deficiency induces ribosome stress to inhibit colorectal cancer proliferation and metastasis via LZTS2-mediated ß-catenin suppression.

Ribosome biogenesis (RiBi) plays a pivotal role in carcinogenesis by regulating protein translation and stress response. Here, we find that RRP15, a nucleolar protein critical for RiBi and checkpoint control, is frequently upregulated in primary CRCs and higher RRP15 expression positively correlated with TNM stage (P < 0.0001) and poor survival of CRC patients (P = 0.0011). Functionally, silencing RRP15 induces ribosome stress, cell cycle arrest, and apoptosis, resulting in suppression of cell proliferation and metastasis. Overexpression of RRP15 promotes cell proliferation and metastasis. Mechanistically, ribosome stress induced by RRP15 deficiency facilitates translation of TOP mRNA LZTS2 (Leucine zipper tumor suppressor 2), leading to the nuclear export and degradation of ß-catenin to suppress Wnt/ß-catenin signaling in CRC. In conclusion, ribosome stress induced by RRP15 deficiency inhibits CRC cell proliferation and metastasis via suppressing the Wnt/ß-catenin pathway, suggesting a potential new target in high-RiBi CRC patients.

Metabolic control of histone acetylation for precise and timely regulation of minor ZGA in early mammalian embryos.

Metabolism feeds into the regulation of epigenetics via metabolic enzymes and metabolites. However, metabolic features, and their impact on epigenetic remodeling during mammalian pre-implantation development, remain poorly understood. In this study, we established the metabolic landscape of mouse pre-implantation embryos from zygote to blastocyst, and quantified some absolute carbohydrate metabolites. We integrated these data with transcriptomic and proteomic data, and discovered the metabolic characteristics of the development process, including the activation of methionine cycle from 8-cell embryo to blastocyst, high glutaminolysis metabolism at blastocyst stage, enhanced TCA cycle activity from the 8-cell embryo stage, and active glycolysis in the blastocyst. We further demonstrated that oxidized nicotinamide adenine dinucleotide (NAD+) synthesis is indispensable for mouse pre-implantation development. Mechanistically, in part, NAD+ is required for the exit of minor zygotic gene activation (ZGA) by cooperating with SIRT1 to remove zygotic H3K27ac. In human, NAD+ supplement can promote the removal of zygotic H3K27ac and benefit pre-implantation development. Our findings demonstrate that precise and timely regulation of minor ZGA is controlled by metabolic dynamics, and enhance our understanding of the metabolism of mammalian early embryos.

Umbilical Cord Mesenchymal Stem Cell-Derived Small Extracellular Vesicles Deliver miR-21 to Promote Corneal Epithelial Wound Healing through PTEN/PI3K/Akt Pathway.

Objective: Rapid restoration of corneal epithelium integrity after injury is particularly important for preserving corneal transparency and vision. Mesenchymal stem cells (MSCs) can be taken into account as the promising regenerative therapeutics for improvement of wound healing processes based on the variety of the effective components. The extracellular vesicles form MSCs, especially exosomes, have been considered as important paracrine mediators though transferring microRNAs into recipient cell. This study investigated the mechanism of human umbilical cord MSC-derived small extracellular vesicles (HUMSC-sEVs) on corneal epithelial wound healing. Methods: HUMSC-sEVs were identified by transmission electron microscopy, nanoparticle tracking analysis, and Western blot. Corneal fluorescein staining and histological staining were evaluated in a corneal mechanical wound model. Changes in HCEC proliferation after HUMSC-sEVs or miR-21 mimic treatment were evaluated by CCK-8 and EdU assays, while migration was assessed by in vitro scratch wound assay. Full-length transcriptome sequencing was performed to identify the differentially expressed genes associated with HUMSC-sEVs treatment, followed by validation via real-time PCR and Western blot. Results: The sEVs derived from HUMSCs can significantly promote corneal epithelial cell proliferation, migration in vitro, and corneal epithelial wound healing in vivo. Similar effects were obtained after miR-21 transfection, while the beneficial effects of HUMSC-sEVs were partially negated by miR-21 knockdown. Results also show that the benefits are associated with decreased PTEN level and activated the PI3K/Akt signaling pathway in HCECs. Conclusion: HUMSC-sEVs could enhance the recovery of corneal epithelial wounds though restraining PTEN by transferring miR-21 and may represent a promising novel therapeutic agent for corneal wound repair.

Porcine Pluripotent Stem Cells Established from LCDM Medium with Characteristics Differ from Human and Mouse Extended Pluripotent Stem Cells.

Pluripotent stem cells (PSCs) have unlimited self-renewal and multifunctional development potential in vitro. Porcine PSCs are highly desirable due to the conserved characteristics between pigs and humans. Extended PSCs (EPSCs) are additionally capable of differentiating into embryonic (Em) and extraembryonic (E×Em) parts. Here, we employed the LCDM culture system (consisting of human LIF, CHIR99021, (S)-(+)-dimethindene maleate, and minocycline hydrochloride), which can establish EPSCs from humans and mice, to derive and maintain stable porcine PSCs (pLCDM) from in vivo blastocysts. Transcriptome analysis revealed the unique molecular characteristics of pLCDMs compared with early-stage embryos. Meanwhile, the parallels and differences in the transcriptome features among pLCDMs, human EPSCs, and mouse EPSCs were carefully analyzed and evaluated. Most noteworthy, the trophoblast lineage differentiation tendency of pLCDMs was clarified by inducing trophoblast-like cells and trophoblast stem cells (TSCs) in vitro. Further research found that 2 of the small molecules in LCDM culture system, (S)-(+)-dimethindene maleate (DiM) and minocycline hydrochloride (MiH), probably play a crucial role in promoting trophoblast lineage differentiation potential of pLCDMs.

Argonaute 2 is a key regulator of maternal mRNA degradation in mouse early embryos.

In mammalian early embryos, the transition from maternal to embryonic control of gene expression requires timely degradation of a subset of maternal mRNAs (MRD). Recently, zygotic genome activation (ZGA)-dependent MRD has been characterized in mouse 2-cell embryo. However, in early embryos, the dynamics of MRD is still poorly understood, and the maternal factor-mediated MRD before and along with ZGA has not been investigated. Argonaute 2 (Ago2) is highly expressed in mouse oocyte and early embryos. In this study, we showed that Ago2-dependent degradation involving RNA interference (RNAi) and RNA activation (RNAa) pathways contributes to the decay of over half of the maternal mRNAs in mouse early embryos. We demonstrated that AGO2 guided by endogenous small interfering RNAs (endosiRNAs), generated from double-stranded RNAs (dsRNAs) formed by maternal mRNAs with their complementary long noncoding RNAs (CMR-lncRNAs), could target maternal mRNAs and cooperate with P-bodies to promote MRD. In addition, we also showed that AGO2 may interact with small activating RNAs (saRNAs) to activate Yap1 and Tead4, triggering ZGA-dependent MRD. Thus, Ago2-dependent degradation is required for timely elimination of subgroups of maternal mRNAs and facilitates the transition between developmental states.

Lineage specification and pluripotency revealed by transcriptome analysis from oocyte to blastocyst in pig.

The inner cell mass (ICM) in blastocyst is the origin of all somatic and germ cells in mammals and pluripotent stem cells (PSCs) in vitro. As the conserved principles between pig and human, here we performed comprehensive single-cell RNA-seq for porcine early embryos from oocyte to early blastocyst (EB). We show the specification of the ICM and trophectoderm in morula and the molecular signature of the precursors. We demonstrate the existence of naïve pluripotency signature in morula and ICM of EB, and the specific pluripotent genes and the activity of signalling pathways highlight the characteristics of the naïve pluripotency. We observe the absence of dosage compensation with respect to X-chromosome (XC) in morula, and incomplete dosage compensation in the EB. However, the dynamics of dosage compensation may be independent of the expression of XIST induced XC inactivation. Our study describes molecular landmarks of embryogenesis in pig that will provide a better strategy for derivation of porcine PSCs and improve research in regenerative medicine.

Cellular reprogramming by single-cell fusion with mouse embryonic stem cells in pig.

Fusion of differentiated somatic cells with pluripotent stem cells can be used for cellular reprogramming, but the efficiency to obtain hybrid cells is extremely low. Here, we explored a novel cell fusion system, termed single-cell fusion, the efficiency was significantly improved verified by fusion of mouse embryonic stem cells (mESCs), comparing to traditional polyethylene glycol fusion. Then, we employed the optimized system to perform cell fusion of porcine embryonic fibroblasts (PEFs) and porcine pluripotent stem cells (pPSCs) with mESCs. The hybrid cells showed both red and green fluorescence and expressed species-specific genes of mouse and pig to evidence that the fusion was successful. The hybrid cells displayed characteristics similar with mESCs, including colony morphology, alkaline phosphatase positive and formation of embryoid body, and the expressions of core pluripotent factors OCT4, NANOG, and SOX2 of the pig were induced in the mESC/PEF hybrid cells. The results indicate PEFs and pPSCs could be reprogrammed by mESCs via the single-cell fusion. Taking advantage of the hybrid cells to investigate the signaling pathways depended on the pluripotency of pig, we suggest the transforming growth factor-ß signaling pathways may play important roles. In summary, the single-cell fusion is highly efficient, and we believe in the future it will be widely used in the application and fundamental research.

Long noncoding RNAs sustain high expression levels of exogenous octamer-binding protein 4 by sponging regulatory microRNAs during cellular reprogramming.

Long noncoding RNAs (lncRNAs) modulate gene expression as competing endogenous RNAs (ceRNAs) that sponge regulatory microRNAs (miRNAs). During cellular reprogramming, genes associated with pluripotency establishment need to be up-regulated, and developmental genes need to be silenced. However, how ceRNAs control cellular reprogramming still awaits full elucidation. Here, we used doxycycline-inducible expression of the four transcription factors octamer-binding protein 4 (OCT4), SRY-box 2 (SOX2), Krüppel-like factor 4 (KLF4), and proto-oncogene c-Myc (c-Myc) to generate induced pluripotent stem cells (iPSCs) from mouse embryonic fibroblasts (MEFs). Using RNA-Seq and bioinformatics approaches, we found that the expression levels of miRNAs from MEFs remain high from day 0 to 6 after the doxycycline induction. Many genes targeted by these miRNAs were up-regulated, and long intergenic noncoding RNAs (lincRNAs) and circular RNAs (circRNAs), which have complementary binding sites to these miRNAs, were highly expressed, indicating lincRNAs and circRNAs may function as ceRNAs. Intriguingly, knockdown of the linc/circRNAs that sponge the miRNAs, which target OCT4 down-regulated exogenous OCT4, decreased reprogramming efficiency, and resulted in low-grade iPSCs. Our results suggest that the ceRNA network plays an important role in cellular reprogramming.

The length of guide RNA and target DNA heteroduplex effects on CRISPR/Cas9 mediated genome editing efficiency in porcine cells.

The clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a versatile genome editing tool with high efficiency. A guide sequence of 20 nucleotides (nt) is commonly used in application of CRISPR/Cas9; however, the relationship between the length of the guide sequence and the efficiency of CRISPR/Cas9 in porcine cells is still not clear. To illustrate this issue, guide RNAs of different lengths targeting the EGFP gene were designed. Specifically, guide RNAs of 17 nt or longer were sufficient to direct the Cas9 protein to cleave target DNA sequences, while 15 nt or shorter guide RNAs had loss-of-function. Full-length guide RNAs complemented with mismatches also showed loss-of-function. When the shortened guide RNA and target DNA heteroduplex (gRNA:DNA heteroduplex) was blocked by mismatch, the CRISPR/Cas9 would be interfered with. These results suggested the length of the gRNA:DNA heteroduplex was a key factor for maintaining high efficiency of the CRISPR/Cas9 system rather than weak bonding between shortened guide RNA and Cas9 in porcine cells.

Endo-siRNAs repress expression of SINE1B during in vitro maturation of porcine oocyte.

Approximately 40% of mammalian genome is made of transposable elements (TEs), and during specific biological processes, such as gametogenesis, they may be activated by global demethylation, so strict silencing mechanism is indispensable for genomic stability. Here, we performed small RNA-seq on Dicer1 knockdown (KD) oocytes in pig, and observed short interspersed nuclear elements 1B (SINE1B) derived endogenous small interfering RNAs (endo-siRNAs), termed SINE1B-siRNAs, were significantly decreased and their biogenesis was dependent on Dicer1 and transcript of SINE1B. Furthermore, by injection of mimics and inhibitors of the SINE1B-siRNAs into germinal vesicle-stage (GV-stage) oocytes, we found the maturation rate was significantly decreased by SINE1B-siRNAs, indicating the SINE1B-siRNAs are indispensible for in vitro maturation (IVM) of porcine oocyte. To figure out the mechanism, we checked the expression pattern and DNA methylation status of SINE1B during IVM of porcine oocytes, and demonstrated the SINE1B-siRNAs could repress SINE1B expression induced by hypomethylation at a post-transcriptional level. Our results suggest that during gametogenesis when the erasure of DNA methylation occurs, endo-siRNAs act as a chronic response to limit retrotransposon activation.

Endo-siRNAs regulate early embryonic development by inhibiting transcription of long terminal repeat sequence in pig†.

Activity of some endogenous retroviruses (ERVs) has been proven to be important for development of early mammalian embryo. However, abnormal activation of ERVs can also cause genetic diseases due to their ability to retrotranspose, so the regulatory mechanism to limit transcription of ERVs needs to be clarified. Endogenous small interfering RNA (endo-siRNA) has been reported to protect cells against transposable elements (TEs). Here, we determined the role of ERVs long terminal repeat sequences (LTRs) derived endo-siRNAs (LTR-siRNAs) on inhibition of the activity of ERVs during early embryonic development in pig. Seven most highly expressed LTR-siRNAs were identified in porcine zygote by high-throughput small RNA sequencing. We verified that the biogenesis of the LTR-siRNAs was DICER-dependent and they were generated from double-stranded RNA (dsRNA) formed by sense and antisense transcripts of LTRs. And, the expression of sense and antisense of LTRs might be due to the loss of DNA methylation at some LTR loci. Furthermore, we showed that the LTR-siRNAs could regulate early embryonic development by repression of LTRs expression at a post-transcriptional level. So, we propose here, during early embryonic development when epigenetic reprogramming occurs, the endo-siRNA pathway acts as a sophisticated balance of regulatory mechanism for ERV activity.

Electrofusion of 2-Cell Embryos for Porcine Tetraploid Embryo Production.

The electrofusion of 2-cell embryos proves to be a simple and efficient way of generating mammalian tetraploid embryos. Many factors affect the fusion efficiency, such as fusion medium, electric field intensity, and fusion pulse length. In mice, production of tetraploid embryos by electrofusion has already been investigated; however, the investigation to produce porcine tetraploid embryos is seldom reported. In this chapter, we will describe oocytes in vitro maturation, in vitro fertilization, and the optimum conditions for electrofusion of 2-cell embryos to produce tetraploid embryos in pig.

Histone variant H3.3-mediated chromatin remodeling is essential for paternal genome activation in mouse preimplantation embryos.

Derepression of chromatin-mediated transcriptional repression of paternal and maternal genomes is considered the first major step that initiates zygotic gene expression after fertilization. The histone variant H3.3 is present in both male and female gametes and is thought to be important for remodeling the paternal and maternal genomes for activation during both fertilization and embryogenesis. However, the underlying mechanisms remain poorly understood. Using our H3.3B-HA-tagged mouse model, engineered to report H3.3 expression in live animals and to distinguish different sources of H3.3 protein in embryos, we show here that sperm-derived H3.3 (sH3.3) protein is removed from the sperm genome shortly after fertilization and extruded from the zygotes via the second polar bodies (PBII) during embryogenesis. We also found that the maternal H3.3 (mH3.3) protein is incorporated into the paternal genome as early as 2 h postfertilization and is detectable in the paternal genome until the morula stage. Knockdown of maternal H3.3 resulted in compromised embryonic development both of fertilized embryos and of androgenetic haploid embryos. Furthermore, we report that mH3.3 depletion in oocytes impairs both activation of the Oct4 pluripotency marker gene and global de novo transcription from the paternal genome important for early embryonic development. Our results suggest that H3.3-mediated paternal chromatin remodeling is essential for the development of preimplantation embryos and the activation of the paternal genome during embryogenesis.