Genes to Cells最新文献

Chromatin condensation state is the key for retrieving genetic information. High-mobility group protein (HMG) proteins exhibit DNA-binding and bending activities, playing an important role in the regulation of chromatin structure. We have shown that nucleosomes tightly packaged into heterochromatin undergo considerable dynamic histone H2A-H2B maintenance via the direct interaction between HP1/Swi6 and facilitate chromatin transcription (FACT), which is composed of the Spt16/Pob3 heterodimer and Nhp6. In this study, we analyzed the role of Nhp6, an HMG box protein, in the FACT at heterochromatin. Pob3 mutant strains showed derepressed heterochromatin-dependent gene silencing, whereas Nhp6 mutant strains did not show significant defects in chromatin regulation or gene expression, suggesting that these two modules play different roles in chromatin regulation. We expressed a protein fusing Nhp6 to the C-terminus of Pob3, which mimics the multicellular FACT component Ssrp1. The chromatin-binding activity of FACT increased with the number of Nhp6 fused to Pob3, and the heterochromatin formation rate was promoted more strongly. Furthermore, we demonstrated that this promotion of heterochromatinization inhibited the heterochromatic variegation caused by epe1⁺ disruption. Heterochromatic variegation can be observed in a variety of regulatory steps; however, when it is caused by fluctuations in chromatin arrangement, it can be eliminated through the strong recruitment of the FACT complex.

DNA methyltransferases and Ten-Eleven Translocation (TET) proteins regulate the DNA methylation and demethylation cycles during mouse embryonic development. Although DNMT1 mainly plays a role in the maintenance of DNA methylation after DNA replication, it is also reported to possess de novo methyltransferase capacity. However, its physiological significance remains unclear. Here, we demonstrate that full-length DNMT1 (FL) and a mutant lacking the N-terminus necessary for its maintenance activity (602) confer the differentiation potential of mouse Dnmt1, Dnmt3a, and Dnmt3b (Dnmts-TKO) embryonic stem cells (ESCs). Both FL and 602 inhibit the spontaneous differentiation of Dnmts-TKO ESCs in the undifferentiated state. Dnmts-TKO ESCs showed loss of DNA methylation and de-repression of primitive endoderm-related genes, but these defects were partially restored in Dnmts-TKO + FL and Dnmts-TKO + 602 ESCs. Upon differentiation, Dnmts-TKO + FL ESCs show increased 5mC and 5hmC levels across chromosomes, including pericentromeric regions. In contrast, Dnmts-TKO + 602 ESCs didn't accumulate 5mC, and sister chromatids showed 5hmC asynchronously. Furthermore, in comparison with DNMT1_602, DNMT1_FL effectively promoted commitment to the epiblast-like cells and beyond, driving cell-autonomous mesendodermal and germline differentiation through embryoid body-based methods. With precise target selectivity achieved by its N-terminal region, DNMT1 may play a role in gene regulation leading to germline development.

WT 9-12 is one of the cell lines commonly used for autosomal dominant polycystic kidney disease (ADPKD) studies. Previous studies had described the PKD gene mutations and polycystin expression in WT 9-12. Nonetheless, the mutations occurring in other ADPKD-associated genes have not been investigated. This study aims to revisit these mutations and protein profile of WT 9-12. Whole genome sequencing verified the presence of truncation mutation at amino acid 2556 (Q2556X) in PKD1 gene of WT 9-12. Besides, those variations with high impacts included single nucleotide polymorphisms (rs8054182, rs117006360, and rs12925771) and insertions and deletions (InDels) (rs145602984 and rs55980345) in PKD1L2; InDel (rs1296698195) in PKD1L3; and copy number variations in GANAB. Protein profiles generated from the total proteins of WT 9-12 and HK-2 cells were compared using isobaric tags for relative and absolute quantitation (iTRAQ) analysis. Polycystin-1 was absent in WT 9-12. The gene ontology enrichment and reactome pathway analyses revealed that the upregulated and downregulated proteins of WT 9-12 relative to HK-2 cell line leaded to signaling pathways related to immune response and amino acid metabolism, respectively. The ADPKD-related mutations and signaling pathways associated with differentially expressed proteins in WT 9-12 may help researchers in cell line selection for their studies.

Most cervical cancers are caused by human papillomavirus (HPV) infection. In HeLa cells, the HPV18 viral genome is integrated at chromosome 8q24.21 and activates transcription of the proto-oncogene c-Myc. However, the mechanism of how the integrated HPV genome and its transcribed RNAs exhibit transcription activation function has not been fully elucidated. In this study, we found that HPV18 transcripts contain an enhancer RNA-like function to activate proximal genes including CCAT1-5L and c-Myc. We showed that the human genome-integrated HPV18 genes are activated by transcription coregulators including BRD4 and Mediator. The transcribed HPV18 RNAs form a liquid-like condensate at chromosome 8q24.21 locus, which in turn accumulates RNA polymerase II. Moreover, we focused on a relatively uncharacterized transcript from the upstream region of CCAT1, named URC. The URC RNA is transcribed as a chimera RNA with HPV18 and is composed of the 3′-untranslated region of the HPV18 transcript. We experimentally showed that the URC contributes to stabilization of HPV18 RNAs by supplying a polyadenylation site for the HPV18 transcript. Our findings suggest that integrated HPV18 at 8q24.21 locus produces HPV18-URC chimera RNA and promotes tumorigenesis through RNA-based condensate formation.

Calcineurin (CN) is a conserved Ca²⁺/calmodulin-dependent phosphoprotein phosphatase that plays a key role in Ca²⁺ signaling. Regulator of calcineurin 1 (RCAN1), also known as Down syndrome critical region gene 1 (DSCR1), interacts with calcineurin and inhibits calcineurin-dependent signaling in various organisms. Ppb1, the fission yeast calcineurin regulates Cl⁻-homeostasis, and Ppb1 deletion induces MgCl₂ hypersensitivity. Here, we characterize the conserved and novel roles of the fission yeast RCAN1 homolog rcn1⁺. Consistent with its role as an endogenous calcineurin inhibitor, Rcn1 overproduction reproduced the calcineurin-null phenotypes, including MgCl₂ hypersensitivity and inhibition of calcineurin signaling upon extracellular Ca²⁺ stimuli as evaluated by the nuclear translocation and transcriptional activation of the calcineurin substrate Prz1. Notably, overexpression of rcn1⁺ causes hypersensitivity to arsenite, whereas calcineurin deletion induces arsenite tolerance, showing a phenotypic discrepancy between Rcn1 overexpression and calcineurin deletion. Importantly, although Rcn1 deletion induces modest sensitivities to arsenite and MgCl₂ in wild-type cells, the arsenite tolerance, but not MgCl₂ sensitivity, associated with Ppb1 deletion was markedly suppressed by Rcn1 deletion. Collectively, our findings reveal a previously unrecognized functional collaboration between Rcn1 and calcineurin, wherein Rcn1 not only negatively regulates calcineurin in the Cl⁻ homeostasis, but also Rcn1 mediates calcineurin signaling to modulate arsenite cytotoxicity.

Quality-based protein production and degradation in the endoplasmic reticulum (ER) are essential for eukaryotic cell survival. During protein maturation in the ER, misfolded or unassembled proteins are destined for disposal through a process known as ER-associated degradation (ERAD). EDEM1 is an ERAD-accelerating factor whose gene expression is upregulated by the accumulation of aberrant proteins in the ER, known as ER stress. Although the role of EDEM1 in ERAD has been studied in detail, the turnover of EDEM1 by intracellular degradation machinery, including the proteasome and autophagy, is not well understood. To clarify EDEM1 regulation in the protein level, degradation mechanism of EDEM1 was examined. Our results indicate that both ERAD and autophagy degrade EDEM1 alike misfolded degradation substrates, although each degradation machinery targets EDEM1 in different folded states of proteins. We also found that ERAD factors, including the SEL1L/Hrd1 complex, YOD1, XTP3B, ERdj3, VIMP, BAG6, and JB12, but not OS9, are involved in EDEM1 degradation in a mannose-trimming-dependent and -independent manner. Our results suggest that the ERAD accelerating factor, EDEM1, is turned over by the ERAD itself, similar to ERAD clients.

Bacillus subtilis was engineered to produce circular subgenomes that are directly transmittable to another B. subtilis. The conjugational plasmid pLS20 integrated into the B. subtilis genome supported not only subgenome replication but also transmission to another B. subtilis species. The subgenome system developed in this study completes a streamlined platform from the synthesis to the transmission of giant DNA by B. subtilis.

In fission yeast, Schizosaccharomyces pombe, constitutive heterochromatin defined by methylation of histone H3 lysine 9 (H3K9me) and its binding protein Swi6/HP1 localizes at the telomere, centromere, and mating-type loci. These loci contain DNA sequences called dg and dh, and the RNA interference (RNAi)-dependent system establishes and maintains heterochromatin at dg/dh. Bi-directional transcription at dg/dh induced by RNA polymerase II is critical in RNAi-dependent heterochromatin formation because the transcribed RNAs provide substrates for siRNA synthesis and a platform for assembling RNAi factors. However, a regulator of dg/dh transcription during the establishment of heterochromatin is not known. Here, we found that a zinc-finger protein Moc3 localizes dh and activates dh-forward transcription in its zinc-finger-dependent manner when heterochromatin structure or heterochromatin-dependent silencing is compromised. However, Moc3 does not localize at normal heterochromatin and does not activate the dh-forward transcription. Notably, the loss of Moc3 caused a retarded heterochromatin establishment, showing that Moc3-dependent dh-forward transcription is critical for RNAi-dependent heterochromatin establishment. Therefore, Moc3 is a transcriptional activator that induces RNAi to establish heterochromatin.

Identifying key genes from a list of differentially expressed genes (DEGs) is a critical step in transcriptome analysis. However, current methods, including Gene Ontology analysis and manual annotation, essentially rely on existing knowledge, which is highly biased depending on the extent of the literature. As a result, understudied genes, some of which may be associated with important molecular mechanisms, are often ignored or remain obscure. To address this problem, we propose Clover, a data-driven scoring method to specifically highlight understudied genes. Clover aims to prioritize genes associated with important molecular mechanisms by integrating three metrics: the likelihood of appearing in the DEG list, tissue specificity, and number of publications. We applied Clover to Alzheimer's disease data and confirmed that it successfully detected known associated genes. Moreover, Clover effectively prioritized understudied but potentially druggable genes. Overall, our method offers a novel approach to gene characterization and has the potential to expand our understanding of gene functions. Clover is an open-source software written in Python3 and available on GitHub at https://github.com/G708/Clover.

Macropinocytosis (MPC) is a large-scale endocytosis pathway that involves actin-dependent membrane ruffle formation and subsequent ruffle closure to generate macropinosomes for the uptake of fluid-phase cargos. MPC is categorized into two types: constitutive and stimuli-induced. Constitutive MPC in macrophages relies on extracellular Ca²⁺ sensing by a calcium-sensing receptor. However, the link between stimuli-induced MPC and Ca²⁺ remains unclear. Here, we find that both intracellular and extracellular Ca²⁺ are required for epidermal growth factor (EGF)-induced MPC in A431 human epidermoid carcinoma cells. Through investigation of mammalian homologs of coelomocyte uptake defective (CUP) genes, we identify ATP2B4, encoding for a Ca²⁺ pump called the plasma membrane calcium ATPase 4 (PMCA4), as a Ca²⁺-related regulator of EGF-induced MPC. Knockout (KO) of ATP2B4, as well as depletion of extracellular/intracellular Ca²⁺, inhibited ruffle closure and macropinosome formation, without affecting ruffle formation. We demonstrate the importance of PMCA4 activity itself, independent of interactions with other proteins via its C-terminus known as a PDZ domain-binding motif. Additionally, we show that ATP2B4-KO reduces EGF-stimulated Ca²⁺ oscillation during MPC. Our findings suggest that EGF-induced MPC requires ATP2B4-dependent Ca²⁺ dynamics.