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The associated publication reports proteogenomic analysis of human pancreatic ductal adenocarcinoma (PDAC), where we provided significantly mutated genes (SMGs)/biomarkers, cellular pathways, and cell types as potential therapeutic targets to improve stratification of patients with PDAC. This protocol describes the detailed methods for whole exome sequencing (WES) and RNA sequencing analyses of PDAC samples, including tissue processing for proteogenomic analysis, DNA and RNA extraction, experimental procedures for WES and RNA sequencing, and WES and RNA sequencing data analyses.

The associated publication reports proteogenomic analysis of human pancreatic ductal adenocarcinoma (PDAC), where we provided significantly mutated genes (SMGs)/biomarkers, cellular pathways, and cell types as potential therapeutic targets to improve stratification of patients with PDAC. This protocol describes the detailed methods for mass spectrometry-based proteomic analysis of PDAC samples, including sample preparation, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, and data analyses (peptide/protein identification and quantitation).

The associated publication reports proteogenomic analysis of human pancreatic ductal adenocarcinoma (PDAC), where we provided significantly mutated genes (SMGs)/biomarkers, cellular pathways, and cell types as potential therapeutic targets to improve stratification of patients with PDAC. This protocol describes the detailed methods for cell-based assays for potential prognostic biomarkers in PDAC, including cell culture, viral transduction, and cell-based assays.

The associated publication reports proteogenomic analysis of human pancreatic ductal adenocarcinoma (PDAC), where we provided significantly mutated genes (SMGs)/biomarkers, cellular pathways, and cell types as potential therapeutic targets to improve stratification of patients with PDAC. This protocol describes the detailed methods for assays for orthotopic PDAC mouse models, including mouse tumour tissue processing, ultrasound imaging, Masson-trichrome staining, and IHC analysis for immune cell markers.

The associated publication reports proteogenomic analysis of human pancreatic ductal adenocarcinoma (PDAC), where we provided significantly mutated genes (SMGs)/biomarkers, cellular pathways, and cell types as potential therapeutic targets to improve stratification of patients with PDAC. This protocol describes the detailed methods for bioinformatics analysis of PDAC subtypes, including tumour purity estimation, subtype prediction for tumour samples in previous cohorts, pathway enrichment analysis, kinase activity analysis, and pan-omics analysis.

An article on the Japanese military ‘comfort women’ of J. Mark Ramseyer, Mitsubishi professor of Japanese legal studies at Harvard Law School, made a fuss at the beginning of 2021. Ramseyer’s attack on the Japanese military ‘comfort women’ has been clearly established as a case to be withdrawn for academic infidelity by the global academic community within a short period of a month or so. Ramseyer’s writing, which was one of a series of articles on his racial-class hate theory that emerged to the fore in the late 2010s, is the declaration of his own identity as a the Japanese military ‘comfort women’ denalist under the influence of the Japanese military ‘comfort women’ denialism in Japan waging the ‘history war’. Ramseyer’s claim that forced mobilization and sexual slavery are simply untrue is the same as the conventional argument of the Japanese military ‘comfort women’ denialism in Japan since the late 1990s, and is only a flimsy slang or hope that can be used only within Japan. Ramseyer’s failure is a proof of that fact. Nevertheless, it is unlikely that the Japanese military ‘comfort women’ denialism even leaded by the Japanese government will soon end. Rather, amid the bizarre silence of the Japanese media, it is even questioned whether the entire Japanese society is becoming an ‘island of obstinacy and confirmation bias.’ In order to properly solve the the Japanese military ‘comfort women’ issue, and to establish its proper place in the history, the Korean society, together with the international community including Japanese citizens, must make more intense efforts.

We report a proteogenomic analysis of pancreatic ductal adenocarcinoma (PDAC). Mutation-phosphorylation correlations identified signaling pathways associated with somatic mutations in significantly mutated genes. Messenger RNA-protein abundance correlations revealed potential prognostic biomarkers correlated with patient survival. Integrated clustering of mRNA, protein and phosphorylation data identified six PDAC subtypes. Cellular pathways represented by mRNA and protein signatures, defining the subtypes and compositions of cell types in the subtypes, characterized them as classical progenitor (TS1), squamous (TS2-4), immunogenic progenitor (IS1) and exocrine-like (IS2) subtypes. Compared with the mRNA data, protein and phosphorylation data further classified the squamous subtypes into activated stroma-enriched (TS2), invasive (TS3) and invasive-proliferative (TS4) squamous subtypes. Orthotopic mouse PDAC models revealed a higher number of pro-tumorigenic immune cells in TS4, inhibiting T cell proliferation. Our proteogenomic analysis provides significantly mutated genes/biomarkers, cellular pathways and cell types as potential therapeutic targets to improve stratification of patients with PDAC.

Summary Post-meiotic spermatids become spermatozoa through developmental stages during spermiogenesis. Isolation of spermatid fractions is required to examine the change of protein expression during spermiogenesis. Here, we present a simple method to isolate spermatid fractions from mouse testes using unit gravity sedimentation in a BSA density gradient. Isolation of spermatid fractions can be used to analyze changes of transcript or protein during spermiogenesis. For complete details on the use and execution of this protocol, please refer to Kim et al. (2020)., Graphical Abstract, Highlights • A protocol for isolation of post-meiotic spermatids from mouse testes • Preparation of single-cell suspension through serial digestion of testes • Analysis of cell fractions from unit gravity sedimentation in BSA density gradient, Post-meiotic spermatids become spermatozoa through developmental stages during spermiogenesis. Isolation of spermatid fractions is required to examine the change of protein expression during spermiogenesis. Here, we present a simple method to isolate spermatid fractions from mouse testes using unit gravity sedimentation in a BSA density gradient. Isolation of spermatid fractions can be used to analyze changes of transcript or protein during spermiogenesis.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with poor prognosis, and the situation has not improved despite extensive clinical and scientific research. Here, we report proteogenomic analysis of PDAC. Mutation-phosphorylation correlations identified signaling pathways associated with somatic mutations in significantly mutated genes. mRNA-protein abundance correlations revealed oncogene and tumor suppressor candidates correlating with patient survival. Integrated clustering of mRNA, protein, and phosphorylation data identified six PDAC subtypes (Sub1-6), which were indistinguishable using mRNA data alone. mRNA and protein signatures defining Sub1-6 revealed that Sub1, Sub2-4, and Sub5-6 were precursor, invasive, and immunogenic tumors, respectively. In the Sub2-4 group, proliferation was highest for Sub4; Sub6 in the Sub5-6 group had an increased pancreatic secretion capacity. Orthotopic mouse PDAC models revealed higher numbers of pro-tumorigenic immune cells in Sub4 tumors, inhibiting T cell proliferation. Our proteogenomic analysis provides therapeutic targets and improves understanding of cancer biology and patient stratification in PDAC.

Chang Rok Kim, Taichi Noda, Hyunkyung Kim, Gibeom Kim, Seongwan Park, Yongwoo Na, Seiya Oura, Keisuke Shimada, Injin Bang, Jun-Yeong Ahn, Yong Ryoul Kim, Se Kyu Oh, Hee-Jung Choi, Jong-Seo Kim, Inkyung Jung, Ho Lee, Yuki Okada, Masahito Ikawa, Sung Hee Baek, PHF7 Modulates BRDT Stability and Histone-to-Protamine Exchange during Spermiogenesis, Cell Reports, Volume 32, Issue 4, 2020, 107950, ISSN 2211-1247, https://doi.org/10.1016/j.celrep.2020.107950., Spermatogenesis is a complex process of sperm generation, including mitosis, meiosis, and spermiogenesis. During spermiogenesis, histones in post-meiotic spermatids are removed from chromatin and replaced by protamines. Although histone-to-protamine exchange is important for sperm nuclear condensation, the underlying regulatory mechanism is still poorly understood. Here, we identify PHD finger protein 7 (PHF7) as an E3 ubiquitin ligase for histone H3K14 in post-meiotic spermatids. Generation of Phf7-deficient mice and Phf7 C160A knockin mice with impaired E3 ubiquitin ligase activity reveals defects in histone-to-protamine exchange caused by dysregulation of histone removal factor Bromodomain, testis-specific (BRDT) in early condensing spermatids. Surprisingly, E3 ubiquitin ligase activity of PHF7 on histone ubiquitination leads to stabilization of BRDT by attenuating ubiquitination of BRDT. Collectively, our findings identify PHF7 as a critical factor for sperm chromatin condensation and contribute to mechanistic understanding of fundamental phenomenon of histone-to-protamine exchange and potential for drug development for the male reproduction system. Histone-to-protamine exchange is essential for functional sperm. Kim et al. demonstrate that PHF7 is an E3 ubiquitin ligase for histone H3, and that PHF7-mediated histone ubiquitination regulates BRDT stability during histone removal. Mice with impaired Phf7 show dysfunctional sperm caused by defects in histone ubiquitination and histone-to-protamine exchange.

Janus tyrosine kinase 2 (JAK2)−signal transducer and activator of transcription 3 (STAT3) signaling pathway is essential for modulating cellular development, differentiation, and homeostasis. Thus, dysregulation of JAK2−STAT3 signaling pathway is frequently associated with human malignancies. Here, we provide evidence that lysine-specific demethylase 3A (KDM3A) functions as an essential epigenetic enzyme for the activation of JAK2−STAT3 signaling pathway. KDM3A is tyrosine-phosphorylated by JAK2 in the nucleus and functions as a STAT3-dependent transcriptional coactivator. JAK2−KDM3A signaling cascade induced by IL-6 leads to alteration of histone H3K9 methylation as a predominant epigenetic event, thereby providing the functional and mechanistic link between activation of JAK2−STAT3 signaling pathway and its epigenetic control. Together, our findings demonstrate that inhibition of KDM3A phosphorylation could be a potent therapeutic strategy to control oncogenic effect of JAK2−STAT3 signaling pathway.

The circadian clock is a self-sustaining oscillator that controls daily rhythms. For the proper circadian gene expression, dynamic changes in chromatin structure are important. Although chromatin modifiers have been shown to play a role in circadian gene expression, the in vivo role of circadian signal-modulated chromatin modifiers at an organism level remains to be elucidated. Here, we provide evidence that the lysine-specific demethylase 1 (LSD1) is phosphorylated by protein kinase Cα (PKCα) in a circadian manner and the phosphorylated LSD1 forms a complex with CLOCK:BMAL1 to facilitate E-box-mediated transcriptional activation. Knockin mice bearing phosphorylation-defective Lsd1(SA/SA) alleles exhibited altered circadian rhythms in locomotor behavior with attenuation of rhythmic expression of core clock genes and impaired phase resetting of circadian clock. These data demonstrate that LSD1 is a key component of the molecular circadian oscillator, which plays a pivotal role in rhythmicity and phase resetting of the circadian clock.

Summary: Unc-51-like-kinase 1 (ULK1) is a target of both the mechanistic target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK), whose role is to facilitate the initiation of autophagy in response to starvation. Upon glucose starvation, dissociation of mTOR from ULK1 and phosphorylation by AMPK leads to the activation of ULK1 activity. Here, we provide evidence that ULK1 is the attachment of O-linked N-acetylglucosamine (O-GlcNAcylated) on the threonine 754 site by O-linked N-acetylglucosamine transferase (OGT) upon glucose starvation. ULK1 O-GlcNAcylation occurs after dephosphorylation of adjacent mTOR-dependent phosphorylation on the serine 757 site by protein phosphatase 1 (PP1) and phosphorylation by AMPK. ULK1 O-GlcNAcylation is crucial for binding and phosphorylation of ATG14L, allowing the activation of lipid kinase VPS34 and leading to the production of phosphatidylinositol-(3)-phosphate (PI(3)P), which is required for phagophore formation and initiation of autophagy. Our findings provide insights into the crosstalk between dephosphorylation and O-GlcNAcylation during autophagy and specify a molecular framework for potential therapeutic intervention in autophagy-related diseases. : Autophagy initiation is regulated by ULK1, a serine/threonine kinase. Pyo et al. demonstrate that ULK1 is O-GlcNAcylated on threonine 754 by OGT upon glucose starvation. O-GlcNAcylation of ULK1 is crucial for activating VPS34 via ATG14L. Our findings provide new insights into the crosstalk between dephosphorylation and O-GlcNAcylation during the autophagy process. Keywords: autophagy initiation, O-GlcNACylation, ULK1, AMPK, mTORC1, PP1, phagophore formation

The actions of transcription factors, chromatin modifiers and noncoding RNAs are crucial for the programming of cell states. Although the importance of various epigenetic machineries for controlling pluripotency of embryonic stem (ES) cells has been previously studied, how chromatin modifiers cooperate with specific transcription factors still remains largely elusive. Here, we find that Pontin chromatin remodelling factor plays an essential role as a coactivator for Oct4 for maintenance of pluripotency in mouse ES cells. Genome-wide analyses reveal that Pontin and Oct4 share a substantial set of target genes involved in ES cell maintenance. Intriguingly, we find that the Oct4-dependent coactivator function of Pontin extends to the transcription of large intergenic noncoding RNAs (lincRNAs) and in particular linc1253, a lineage programme repressing lincRNA, is a Pontin-dependent Oct4 target lincRNA. Together, our findings demonstrate that the Oct4-Pontin module plays critical roles in the regulation of genes involved in ES cell fate determination., Long non-coding RNAs or lincRNAs identified in embryonic stem (ES) cells have been shown to regulate ES cell states; however, how these lincRNAs are regulated remains unclear. Here the authors show that the transcriptional coactivator Pontin regulates the expression of lincRNAs involved in ES cell maintenance in an Oct4-dependent manner.