R-loop-dependent promoter-proximal termination ensures genome stability.

The proper regulation of transcription is essential for maintaining genome integrity and executing other downstream cellular functions1,2. Here we identify a stable association between the genome-stability regulator sensor of single-stranded DNA (SOSS)3 and the transcription regulator Integrator-PP2A (INTAC)4-6. Through SSB1-mediated recognition of single-stranded DNA, SOSS-INTAC stimulates promoter-proximal termination of transcription and attenuates R-loops associated with paused RNA polymerase II to prevent R-loop-induced genome instability. SOSS-INTAC-dependent attenuation of R-loops is enhanced by the ability of SSB1 to form liquid-like condensates. Deletion of NABP2 (encoding SSB1) or introduction of cancer-associated mutations into its intrinsically disordered region leads to a pervasive accumulation of R-loops, highlighting a genome surveillance function of SOSS-INTAC that enables timely termination of transcription at promoters to constrain R-loop accumulation and ensure genome stability.

Unveiling Epigenetic Vulnerabilities in Triple-Negative Breast Cancer through 3D Organoid Drug Screening.

Triple-negative breast cancer (TNBC) poses a therapeutic challenge due to its aggressive nature and lack of targeted therapies. Epigenetic modifications contribute to TNBC tumorigenesis and drug resistance, offering potential therapeutic targets. Recent advancements in three-dimensional (3D) organoid cultures, enabling precise drug screening, hold immense promise for identifying novel compounds targeting TNBC. In this study, we established two patient-derived TNBC organoids and implemented a high-throughput drug screening system using these organoids and two TNBC cell lines. Screening a library of 169 epigenetic compounds, we found that organoid-based systems offer remarkable precision in drug response assessment compared to cell-based models. The top 30 compounds showing the highest drug sensitivity in the initial screening were further assessed in a secondary screen. Four compounds, panobinostat, pacritinib, TAK-901, and JIB-04, targeting histone deacetylase, JAK/STAT, histone demethylases, and aurora kinase pathways, respectively, exhibited potent anti-tumor activity in TNBC organoids, surpassing the effect of paclitaxel. Our study highlights the potential of these novel epigenetic drugs as effective therapeutic agents for TNBC and demonstrates the valuable role of patient-derived organoids in advancing drug discovery.

Loss of Tet hydroxymethylase activity causes mouse embryonic stem cell differentiation bias and developmental defects.

The TET family is well known for active DNA demethylation and plays important roles in regulating transcription, the epigenome and development. Nevertheless, previous studies using knockdown (KD) or knockout (KO) models to investigate the function of TET have faced challenges in distinguishing its enzymatic and nonenzymatic roles, as well as compensatory effects among TET family members, which has made the understanding of the enzymatic role of TET not accurate enough. To solve this problem, we successfully generated mice catalytically inactive for specific Tet members (Tetm/m). We observed that, compared with the reported KO mice, mutant mice exhibited distinct developmental defects, including growth retardation, sex imbalance, infertility, and perinatal lethality. Notably, Tetm/m mouse embryonic stem cells (mESCs) were successfully established but entered an impaired developmental program, demonstrating extended pluripotency and defects in ectodermal differentiation caused by abnormal DNA methylation. Intriguingly, Tet3, traditionally considered less critical for mESCs due to its lower expression level, had a significant impact on the global hydroxymethylation, gene expression, and differentiation potential of mESCs. Notably, there were common regulatory regions between Tet1 and Tet3 in pluripotency regulation. In summary, our study provides a more accurate reference for the functional mechanism of Tet hydroxymethylase activity in mouse development and ESC pluripotency regulation.

BMP4 preserves the developmental potential of mESCs through Ube2s- and Chmp4b-mediated chromosomal stability safeguarding.

Chemically defined medium is widely used for culturing mouse embryonic stem cells (mESCs), in which N2B27 works as a substitution for serum, and GSK3ß and MEK inhibitors (2i) help to promote ground-state pluripotency. However, recent studies suggested that MEKi might cause irreversible defects that compromise the developmental potential of mESCs. Here, we demonstrated the deficient bone morphogenetic protein (BMP) signal in the chemically defined condition is one of the main causes for the impaired pluripotency. Mechanistically, activating the BMP signal pathway by BMP4 could safeguard the chromosomal integrity and proliferation capacity of mESCs through regulating downstream targets Ube2s and Chmp4b. More importantly, BMP4 promotes a distinct in vivo developmental potential and a long-term pluripotency preservation. Besides, the pluripotent improvements driven by BMP4 are superior to those by attenuating MEK suppression. Taken together, our study shows appropriate activation of BMP signal is essential for regulating functional pluripotency and reveals that BMP4 should be applied in the serum-free culture system.

CRISPR/Cas9 mediated generation of a iPSC line EHTJUi005-A-1 with homozygous knockout of the SUV39H1 gene.

SUV39H1 is a histone methyltransferase involve numerous biological processes, including of aging, embryo development, tumor growth and mitosis via catalysis of dimethylation and trimethylation of lysine 9 of histone H3. Here we report a human induced pluripotent stem cell line (EHTJUi005-A-1) which is generated from a wildtype human iPSC previously established in our laboratory, and this iPSC has a homozygous knockout of 8 bp in Exon 2 of SUV39H1. This iPSC model provides a valuable resource to study epigenetic regulation in extensive biological processes as mentioned above.

An induced pluripotent stem cell line (EHTJUi003-A) generated from a neonate with c.1377delC mutation in the gene MYBPC3 causing hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is an autosomal dominant heart disease. An induced pluripotent stem cell line (EHTJUi003-A) was generated from umbilical cord blood mononuclear cells (UCBMCs) of a female neonate with heterozygous mutation of p.L460Wfs (c.1377delC) in the MYBPC3 gene. This iPSC model offers a very valuable resource to study the pathological mechanism of HCM in vitro.

An induced pluripotent stem cell line (EHTJUi004-A) generated from a neonate with c.4683_4684delCT:p.Leu1563fs mutation in the gene DSP causing Familial Arrhythmogenic Right Ventricular Dysplasia (ARVD).

Familial Arrhythmogenic Right Ventricular Dysplasia (ARVD) is a primary cardiomyopathy characterized by the abnormality of the right ventricular muscle. ARVD may be life-threatening due to the induction of paroxysmal refractory ventricular tachycardia or supraventricular arrhythmia. A human induced pluripotent stem cell line (EHTJUi004-A) was generated from human umbilical cord blood mononuclear cells (UCBMCs) of a female neonate with heterozygous mutation of p.Leu1563fs (c.4683_4684delCT) in the DSP gene. This iPS cell line resource provides an ideal in vitro model to study the pathological mechanism of ARVD.

Generation of a laminopathies-specific iPSC line EHTJUi005-A-3 with homozygous knockout of the LMNA gene by CRISPR/Cas9 technology.

LAMIN A/C, encoded by the LMNA gene, supports the normal structure of the cell nucleus and regulates the connection between the nucleus and the cytoskeleton as a component of the nucleus envelope. The loss of expression and function of the LMNA gene would lead to the occurrence of congenital muscular dystrophy and Emery-Dreifuss muscular dystrophy which are collectively named as laminopathies. Here, we report a human induced pluripotent stem cell (iPSC) line (EHTJUi005-A-3) generated from a wild iPSC (EHTJUi005-A) with homozygous knockout of the gene LMNA through CRISPR/Cas9. This iPSC line provides a useful research model for studying laminopathies disease.

Dux-Mediated Corrections of Aberrant H3K9ac during 2-Cell Genome Activation Optimize Efficiency of Somatic Cell Nuclear Transfer.

Differentiated somatic cells can be reprogrammed to totipotent embryos through somatic cell nuclear transfer (SCNT) with low efficiency. The histone deacetylase inhibitor trichostatin A (TSA) has been found to improve SCNT efficiency, but the underlying mechanism remains undetermined. Here, we examined genome-wide H3K9ac during SCNT embryo development and found that aberrant H3K9ac regions resulted in reduced 2-cell genome activation. TSA treatment largely corrects aberrant acetylation in SCNT embryos with an efficiency that is dictated by the native epigenetic environment. We further identified that the overexpression of Dux greatly improves SCNT efficiency by correcting the aberrant H3K9ac signal at its target sites, ensuring appropriate 2-cell genome activation. Intriguingly, the improvement in development mediated by TSA and Kdm4b is impeded by Dux knockout in SCNT embryos. Together, our study reveals that reprogramming of H3K9ac is important for optimal SCNT efficiency and identifies Dux as a crucial transcription factor in this process.

Nuclear Exosome Targeting Complex Core Factor Zcchc8 Regulates the Degradation of LINE1 RNA in Early Embryos and Embryonic Stem Cells.

The nuclear exosome targeting (NEXT) complex is responsible for specific nuclear RNA degradation in mammalian cells. However, its function in development remains unknown. Here, we find that the depletion of a central factor of the NEXT complex, Zcchc8, in mouse results in developmental defects, a shortened lifespan, and infertility. We find that Zcchc8-deficient embryonic stem cells (ESCs) exhibit proliferation abnormalities and reduced developmental potencies. Importantly, the transcripts of retrotransposon element LINE1 are found to be targeted by Zcchc8 and degraded by a Zcchc8-mediated mechanism. We further find that sustained expression of higher levels of LINE1 RNA is detected in maternal Zcchc8-depleted oocytes and embryos. Zcchc8-depleted oocytes show higher chromatin accessibility and developmental defects in both meiotic maturation and embryogenesis after fertilization. Collectively, our study defines Zcchc8-mediated RNA degradation as an important post-transcription regulation of LINE1 transcripts in early embryos and ESCs, which play vital roles in the pluripotency and early development.

Inhibition of Aberrant DNA Re-methylation Improves Post-implantation Development of Somatic Cell Nuclear Transfer Embryos.

Somatic cell nuclear transfer (SCNT) enables cloning of differentiated cells by reprogramming their nuclei to a totipotent state. However, successful full-term development of SCNT embryos is a low-efficiency process and arrested embryos frequently exhibit epigenetic abnormalities. Here, we generated genome-wide DNA methylation maps from mouse pre-implantation SCNT embryos. We identified widespread regions that were aberrantly re-methylated, leading to mis-expression of genes and retrotransposons important for zygotic genome activation. Inhibition of DNA methyltransferases (Dnmts) specifically rescued these re-methylation defects and improved the developmental capacity of cloned embryos. Moreover, combining inhibition of Dnmts with overexpression of histone demethylases led to stronger reductions in inappropriate DNA methylation and synergistic enhancement of full-term SCNT embryo development. These findings show that excessive DNA re-methylation is a potent barrier that limits full-term development of SCNT embryos and that removing multiple epigenetic barriers is a promising approach to achieve higher cloning efficiency.

Reduced Self-Diploidization and Improved Survival of Semi-cloned Mice Produced from Androgenetic Haploid Embryonic Stem Cells through Overexpression of Dnmt3b.

Androgenetic haploid embryonic stem cells (AG-haESCs) hold great promise for exploring gene functions and generating gene-edited semi-cloned (SC) mice. However, the high incidence of self-diploidization and low efficiency of SC mouse production are major obstacles preventing widespread use of these cells. Moreover, although SC mice generation could be greatly improved by knocking out the differentially methylated regions of two imprinted genes, 50% of the SC mice did not survive into adulthood. Here, we found that the genome-wide DNA methylation level in AG-haESCs is extremely low. Subsequently, downregulation of both de novo methyltransferase Dnmt3b and other methylation-related genes was determined to be responsible for DNA hypomethylation. We further demonstrated that ectopic expression of Dnmt3b in AG-haESCs could effectively improve DNA methylation level, and the high incidence of self-diploidization could be markedly rescued. More importantly, the developmental potential of SC embryos was improved, and most SC mice could survive into adulthood.

[Rosiglitazone inhibits human HepG2 cell proliferation via PI3K/PTEN/Akt signaling pathway].

OBJECTIVE: To investigate the effects of rosiglitazone (ROZ) on proliferation and cell cycle of human hepatocellular carcinoma cell line HepG2 and explore the underlying mechanisms by detecting the related proteins. METHODS: After treated with ROZ of different concentrations, HepG2 cells were tested for the changes in the cell proliferation by MTT assay and in the cell cycle by flow cytometry. Western blotting and RT-PCR were performed to measure the expressions of PTEN, pAkt, S phase kinase associated protein 2 (Skp2) and P27(kip1); at protein and mRNA levels, respectively. RESULTS ROZ significantly inhibited HepG2 cell proliferation in a concentration-dependent and time-dependent manner (P<0.05). The proportion of HepG2 cells in G0/G1 phase increased, and that in S phase decreased significantly (P<0.05). ROZ reduced the expressions of pAkt and Skp2, and raised the expressions of PTEN and P27(kip1); in HepG2 cells (P<0.05). RT-PCR revealed that ROZ increased the expressions of PTEN mRNA, decreased the expressions of Skp2 mRNA, and had no effect on P27(kip1); mRNA. CONCLUSION: Our study demonstrated that ROZ could inhibit HepG2 cell proliferation and block G0/G1 phase, the mechanisms may be related to the regulation on the expressions of Skp2 and P27(kip1); through the PI3K/PTEN/Akt signaling pathway.