INTRODUCTION The Saccharomyces cerevisiae 2.0 project (Sc2.0) aims to modify the yeast genome with a series of densely spaced designer changes. Both a synthetic yeast chromosome arm (synIXR) and the entirely synthetic chromosome (synIII) function with high fitness in yeast. For designer genome synthesis projects, precise engineering of the physical sequence to match the specified design is important for the systematic evaluation of underlying design principles. Yeast can maintain nuclear chromosomes as rings, occurring by chance at repeated sequences, although the cyclized format is unfavorable in meiosis given the possibility of dicentric chromosome formation from meiotic recombination. Here, we describe the de novo synthesis of synthetic yeast chromosome V (synV) in the “Build-A-Genome China” course, perfectly matching the designer sequence and bearing loxPsym sites, distinguishable watermarks, and all the other features of the synthetic genome. We generated a ring synV derivative with user-specified cyclization coordinates and characterized its performance in mitosis and meiosis. RATIONALE Systematic evaluation of underlying Sc2.0 design principles requires that the final assembled synthetic genome perfectly match the designed sequence. Given the size of yeast chromosomes, synthetic chromosome construction is performed iteratively, and new mutations and unpredictable events may occur during synthesis; even a very small number of unintentional nucleotide changes across the genome could have substantial effects on phenotype. Therefore, precisely matching the physical sequence to the designed sequence is crucial for verification of the design principles in genome synthesis. Ring chromosomes can extend those design principles to provide a model for genomic rearrangement, ring chromosome evolution, and human ring chromosome disorders. RESULTS We chemically synthesized, assembled, and incorporated designer chromosome synV (536,024 base pairs) of S. cerevisiae according to Sc2.0 principles, based on the complete nucleotide sequence of native yeast chromosome V (576,874 base pairs). This work was performed as part of the “Build-A-Genome China” course in Tianjin University. We corrected all mutations found—including duplications, substitutions, and indels—in the initial synV strain by using integrative cotransformation of the precise desired changes and by means of a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)–based method. Altogether, 3331 corrected base pairs were required to match to the designed sequence. We generated a strain that exactly matches all designer sequence changes that displays high fitness under a variety of culture conditions. All corrections were verified with whole-genome sequencing; RNA sequencing revealed only minor changes in gene expression—most notably, decreases in expression of genes relocated near synthetic telomeres as a result of design. We constructed a functional circular synV (ring_synV) derivative in yeast by precisely joining both chromosome ends (telomeres) at specified coordinates. The ring chromosome showed restoration of subtelomeric gene expression levels. The ring_synV strain exhibited fitness comparable with that of the linear synV strain, revealed no change in sporulation frequency, but notably reduced spore viability. In meiosis, heterozygous or homozygous diploid ring_wtV and ring_synV chromosomes behaved similarly, exhibiting substantially higher frequency of the formation of zero-spore tetrads, a type that was not seen in the rod chromosome diploids. Rod synV chromosomes went through meiosis with high spore viability, despite no effort having been made to preserve meiotic competency in the design of synV. CONCLUSION The perfect designer-matched synthetic chromosome V provides strategies to edit sequence variants and correct unpredictable events, such as off-target integration of extra copies of synthetic DNA elsewhere in the genome. We also constructed a ring synthetic chromosome derivative and evaluated its fitness and stability in yeast. Both synV and synVI can be circularized and can power yeast cell growth without affecting fitness when gene content is maintained. These fitness and stability phenotypes of the ring synthetic chromosome in yeast provide a model system with which to probe the mechanism of human ring chromosome disorders. Synthesis, cyclization, and characterization of synV . ( A ) Synthetic chromosome V (synV, 536,024 base pairs) was designed in silico from native chromosome V (wtV, 576,874 base pairs), with extensive genotype modification designed to be phenotypically neutral. ( B ) CRISPR/Cas9 strategy for multiplex repair. ( C ) Colonies of wtV, synV, and ring_synV strains.

N-α-acetyltransferase D (NatD) mediates N-α-terminal acetylation (Nt-acetylation) of histone H4 known to be involved in cell growth. Here we report that NatD promotes the migratory and invasive capabilities of lung cancer cells in vitro and in vivo. Depletion of NatD suppresses the epithelial-to-mesenchymal transition (EMT) of lung cancer cells by directly repressing the expression of transcription factor Slug, a key regulator of EMT. We found that Nt-acetylation of histone H4 antagonizes histone H4 serine 1 phosphorylation (H4S1ph), and that downregulation of Nt-acetylation of histone H4 facilitates CK2α binding to histone H4 in lung cancer cells, resulting in increased H4S1ph and epigenetic reprogramming to suppress Slug transcription to inhibit EMT. Importantly, NatD is commonly upregulated in primary human lung cancer tissues where its expression level correlates with Slug expression, enhanced invasiveness, and poor clinical outcomes. These findings indicate that NatD is a crucial epigenetic modulator of cell invasion during lung cancer progression.

Summary Programmed cell death contributes to host defense against pathogens. To investigate the relative importance of pyroptosis, necroptosis, and apoptosis during Salmonella infection, we infected mice and macrophages deficient for diverse combinations of caspases-1, -11, -12, and -8 and receptor interacting serine/threonine kinase 3 (RIPK3). Loss of pyroptosis, caspase-8-driven apoptosis, or necroptosis had minor impact on Salmonella control. However, combined deficiency of these cell death pathways caused loss of bacterial control in mice and their macrophages, demonstrating that host defense can employ varying components of several cell death pathways to limit intracellular infections. This flexible use of distinct cell death pathways involved extensive cross-talk between initiators and effectors of pyroptosis and apoptosis, where initiator caspases-1 and -8 also functioned as executioners when all known effectors of cell death were absent. These findings uncover a highly coordinated and flexible cell death system with in-built fail-safe processes that protect the host from intracellular infections.

Cell death plays an important role during pathogen infections. Here, we report that interferon-γ (IFNγ) sensitizes macrophages to Toll-like receptor (TLR)-induced death that requires macrophage-intrinsic death ligands and caspase-8 enzymatic activity, which trigger the mitochondrial apoptotic effectors, BAX and BAK. The pro-apoptotic caspase-8 substrate BID was dispensable for BAX and BAK activation. Instead, caspase-8 reduced pro-survival BCL-2 transcription and increased inducible nitric oxide synthase (iNOS), thus facilitating BAX and BAK signaling. IFNγ-primed, TLR-induced macrophage killing required iNOS, which licensed apoptotic caspase-8 activity and reduced the BAX and BAK inhibitors, A1 and MCL-1. The deletion of iNOS or caspase-8 limited SARS-CoV-2-induced disease in mice, while caspase-8 caused lethality independent of iNOS in a model of hemophagocytic lymphohistiocytosis. These findings reveal that iNOS selectively licenses programmed cell death, which may explain how nitric oxide impacts disease severity in SARS-CoV-2 infection and other iNOS-associated inflammatory conditions.

Background Aberrant changes in epigenetic mechanisms such as histone modifications play an important role in cancer progression. PRMT1 which triggers asymmetric dimethylation of histone H4 on arginine 3 (H4R3me2a) is upregulated in human colorectal cancer (CRC) and is essential for cell proliferation. However, how this dysregulated modification might contribute to malignant transitions of CRC remains poorly understood. Methods In this study, we integrated biochemical assays including protein interaction studies and chromatin immunoprecipitation (ChIP), cellular analysis including cell viability, proliferation, colony formation, and migration assays, clinical sample analysis, microarray experiments, and ChIP-Seq data to investigate the potential genomic recognition pattern of H4R3me2s in CRC cells and its effect on CRC progression. Results We show that PRMT1 and SMARCA4, an ATPase subunit of the SWI/SNF chromatin remodeling complex, act cooperatively to promote colorectal cancer (CRC) progression. We find that SMARCA4 is a novel effector molecule of PRMT1-mediated H4R3me2a. Mechanistically, we show that H4R3me2a directly recruited SMARCA4 to promote the proliferative, colony-formative, and migratory abilities of CRC cells by enhancing EGFR signaling. We found that EGFR and TNS4 were major direct downstream transcriptional targets of PRMT1 and SMARCA4 in colon cells, and acted in a PRMT1 methyltransferase activity-dependent manner to promote CRC cell proliferation. In vivo, knockdown or inhibition of PRMT1 profoundly attenuated the growth of CRC cells in the C57BL/6 J-ApcMin/+ CRC mice model. Importantly, elevated expression of PRMT1 or SMARCA4 in CRC patients were positively correlated with expression of EGFR and TNS4, and CRC patients had shorter overall survival. These findings reveal a critical interplay between epigenetic and transcriptional control during CRC progression, suggesting that SMARCA4 is a novel key epigenetic modulator of CRC. Our findings thus highlight PRMT1/SMARCA4 inhibition as a potential therapeutic intervention strategy for CRC. Conclusion PRMT1-mediated H4R3me2a recruits SMARCA4, which promotes colorectal cancer progression by enhancing EGFR signaling.

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Aurora kinase B (AURKB) triggers the phosphorylation of serine 10 on histone H3 (H3S10ph), which is important for chromosome condensation and cytokinesis during mitosis in mammals. However, how exactly AURKB controls cell cycle and contributes to tumorigenesis as an oncoprotein under pathological conditions remains largely unknown. Here, we report that AURKB promotes gastric cancer cell proliferation in vitro and in vivo. Silencing AURKB expression inhibits gastric cell proliferation and arrests the cell cycle in G2/M phase. We demonstrate that cyclin D1 (CCND1) is a direct downstream target of AURKB that plays a key role in gastric cancer cell proliferation. AURKB is able to activate the expression of CCND1 through mediating H3S10ph in the promoter of the CCND1 gene. Furthermore, we show that AZD1152, a specific inhibitor of AURKB, can suppress the expression of CCND1 in the gastric cancer cells and inhibit cell proliferation in vitro and in vivo. Importantly, we found that high AURKB and CCND1 expression levels are correlated with shorter overall survival of gastric cancer patients. This study demonstrates that AURKB promotes gastric tumorigenesis potentially through epigenetically activating CCND1 expression, suggesting AURKB as a promising therapeutic target in gastric cancer.

Heterochromatin protein 1γ (HP1γ) has been implicated in carcinogenesis of various cancer types. However, the role of HP1γ in prostate cancer (PCa) progression and the underlying molecular mechanisms remain largely unknown. We found that HP1γ is upregulated in PCa and elevated levels of HP1γ in PCa predict poor outcome. In addition, depletion of HP1γ in PCa cells not only repressed proliferation and induced apoptosis but also impaired tumorigenicity. We also found that c-Myc was capable of upregulating HP1γ by directly binding to the E-box element in the first intron of HP1γ gene, and the upregulated HP1γ, in turn, repressed the expression of miR-451a by enhancing H3K9 methylation at the promoter region of miR-451a. Furthermore, reduction of miR-451a significantly reversed HP1γ loss-induced PCa cell apoptosis, whereas miR-451a overexpression repressed cell survival by targeting and downregulating c-Myc. The association among c-Myc, HP1γ and miR-451a was further confirmed in human clinical samples. Therefore, we propose that an HP1γ/miR-451a/c-Myc regulatory circuitry exists in PCa cells and this circuit has a crucial role in PCa progression.