PCDHGC3 hypermethylation as a potential biomarker of intestinal neuroendocrine carcinomas.

Neuroendocrine neoplasms (NENs) encompass tumors arising from neuroendocrine cells in various organs, including the gastrointestinal tract, pancreas, adrenal gland, and paraganglia. Despite advancements, accurately predicting the aggressiveness of gastroenteropancreatic (GEP) NENs based solely on pathological data remains challenging, thereby limiting optimal clinical management. Our previous research unveiled a crucial link between hypermethylation of the protocadherin PCDHGC3 gene and neuroendocrine tumors originating from the paraganglia and adrenal medulla. This epigenetic alteration was associated with increased metastatic potential and succinate dehydrogenase complex (SDH) dysfunction. Expanding upon this discovery, the current study explored PCDHGC3 gene methylation within the context of GEP-NENs in a cohort comprising 34 cases. We uncovered promoter hypermethylation of PCDHGC3 in 29% of GEP-NENs, with a significantly higher prevalence in gastrointestinal (GI) neuroendocrine carcinomas (NECs) compared with both pancreatic (Pan) NECs and neuroendocrine tumors (NETs) of GI and Pan origin. Importantly, these findings were validated in one of the largest multi-center GEP-NEN cohorts. Mechanistic analysis revealed that PCDHGC3 hypermethylation was not associated with SDH mutations or protein loss, indicating an SDH-independent epigenetic mechanism. Clinically, PCDHGC3 hypermethylation emerged as a significant prognostic factor, correlating with reduced overall survival rates in both patient cohorts. Significantly, whereas PCDHGC3 hypermethylation exhibited a strong correlation with TP53 somatic mutations, a hallmark of NEC, its predictive value surpassed that of TP53 mutations, with an area under the curve (AUC) of 0.95 (95% CI 0.83-1.0) for discriminating GI-NECs from GI-NETs, highlighting its superior predictive performance. In conclusion, our findings position PCDHGC3 methylation status as a promising molecular biomarker for effectively stratifying patients with GI-NENs. This discovery has the potential to advance patient care by enabling more precise risk assessments and tailored treatment strategies. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

An integrative epigenomic approach identifies ELF3 as an oncogenic regulator in ASCL1-positive neuroendocrine carcinoma.

Neuroendocrine carcinoma (NEC) is a highly aggressive subtype of the neuroendocrine tumor with an extremely poor prognosis. We have previously conducted a comprehensive genomic analysis of over 100 cases of NEC of the gastrointestinal system (GIS-NEC) and unraveled its unique and organ-specific genomic drivers. However, the epigenomic features of GIS-NEC remain unexplored. In this study, we have described the epigenomic landscape of GIS-NEC and small cell lung carcinoma (SCLC) by integrating motif enrichment analysis from the assay of transposase-accessible chromatin sequencing (ATAC-seq) and enhancer profiling from a novel cleavage under targets and tagmentation (CUT&Tag) assay for H3K27ac and identified ELF3 as one of the super-enhancer-related transcriptional factors in NEC. By combining CUT&Tag and knockdown RNA sequencing for ELF3, we uncovered the transcriptional network regulated by ELF3 and defined its distinctive gene signature, including AURKA, CDC25B, CLDN4, ITGB6, and YWAHB. Furthermore, a loss-of-function assay revealed that ELF3 depletion led to poor cell viability. Finally, using gene expression of clinical samples, we successfully divided GIS-NEC patients into two subgroups according to the ELF3 signature and demonstrated that tumor-promoting pathways were activated in the ELF3 signature-high group. Our findings highlight the transcriptional regulation of ELF3 as an oncogenic transcription factor and its tumor-promoting properties in NEC.

Comprehensive Genomic Profiling of Neuroendocrine Carcinomas of the Gastrointestinal System.

The neuroendocrine carcinoma of the gastrointestinal system (GIS-NEC) is a rare but highly malignant neoplasm. We analyzed 115 cases using whole-genome/exome sequencing, transcriptome sequencing, DNA methylation assays, and/or ATAC-seq and found GIS-NECs to be genetically distinct from neuroendocrine tumors (GIS-NET) in the same location. Clear genomic differences were also evident between pancreatic NECs (Panc-NEC) and nonpancreatic GIS-NECs (Nonpanc-NEC). Panc-NECs could be classified into two subgroups (i.e., "ductal-type" and "acinar-type") based on genomic features. Alterations in TP53 and RB1 proved common in GIS-NECs, and most Nonpanc-NECs with intact RB1 demonstrated mutually exclusive amplification of CCNE1 or MYC. Alterations of the Notch gene family were characteristic of Nonpanc-NECs. Transcription factors for neuroendocrine differentiation, especially the SOX2 gene, appeared overexpressed in most GIS-NECs due to hypermethylation of the promoter region. This first comprehensive study of genomic alterations in GIS-NECs uncovered several key biological processes underlying genesis of this very lethal form of cancer. SIGNIFICANCE: GIS-NECs are genetically distinct from GIS-NETs. GIS-NECs arising in different organs show similar histopathologic features and share some genomic features, but considerable differences exist between Panc-NECs and Nonpanc-NECs. In addition, Panc-NECs could be classified into two subgroups (i.e., "ductal-type" and "acinar-type") based on genomic and epigenomic features. This article is highlighted in the In This Issue feature, p. 587.

Reconstruction of proto-vertebrate, proto-cyclostome and proto-gnathostome genomes provides new insights into early vertebrate evolution.

Ancient polyploidization events have had a lasting impact on vertebrate genome structure, organization and function. Some key questions regarding the number of ancient polyploidization events and their timing in relation to the cyclostome-gnathostome divergence have remained contentious. Here we generate de novo long-read-based chromosome-scale genome assemblies for the Japanese lamprey and elephant shark. Using these and other representative genomes and developing algorithms for the probabilistic macrosynteny model, we reconstruct high-resolution proto-vertebrate, proto-cyclostome and proto-gnathostome genomes. Our reconstructions resolve key questions regarding the early evolutionary history of vertebrates. First, cyclostomes diverged from the lineage leading to gnathostomes after a shared tetraploidization (1R) but before a gnathostome-specific tetraploidization (2R). Second, the cyclostome lineage experienced an additional hexaploidization. Third, 2R in the gnathostome lineage was an allotetraploidization event, and biased gene loss from one of the subgenomes shaped the gnathostome genome by giving rise to remarkably conserved microchromosomes. Thus, our reconstructions reveal the major evolutionary events and offer new insights into the origin and evolution of vertebrate genomes.

CTCF looping is established during gastrulation in medaka embryos.

Chromatin looping plays an important role in genome regulation. However, because ChIP-seq and loop-resolution Hi-C (DNA-DNA proximity ligation) are extremely challenging in mammalian early embryos, the developmental stage at which cohesin-mediated loops form remains unknown. Here, we study early development in medaka (the Japanese killifish, Oryzias latipes) at 12 time points before, during, and after gastrulation (the onset of cell differentiation) and characterize transcription, protein binding, and genome architecture. We find that gastrulation is associated with drastic changes in genome architecture, including the formation of the first loops between sites bound by the insulator protein CTCF and a large increase in the size of contact domains. In contrast, the binding of the CTCF is fixed throughout embryogenesis. Loops form long after genome-wide transcriptional activation, and long after domain formation seen in mouse embryos. These results suggest that, although loops may play a role in differentiation, they are not required for zygotic transcription. When we repeated our experiments in zebrafish, loops did not emerge until gastrulation, that is, well after zygotic genome activation. We observe that loop positions are highly conserved in synteny blocks of medaka and zebrafish, indicating that the 3D genome architecture has been maintained for >110-200 million years of evolution.

Genomes as documents of evolutionary history: a probabilistic macrosynteny model for the reconstruction of ancestral genomes.

MOTIVATION: It has been argued that whole-genome duplication (WGD) exerted a profound influence on the course of evolution. For the purpose of fully understanding the impact of WGD, several formal algorithms have been developed for reconstructing pre-WGD gene order in yeast and plant. However, to the best of our knowledge, those algorithms have never been successfully applied to WGD events in teleost and vertebrate, impeded by extensive gene shuffling and gene losses. RESULTS: Here, we present a probabilistic model of macrosynteny (i.e. conserved linkage or chromosome-scale distribution of orthologs), develop a variational Bayes algorithm for inferring the structure of pre-WGD genomes, and study estimation accuracy by simulation. Then, by applying the method to the teleost WGD, we demonstrate effectiveness of the algorithm in a situation where gene-order reconstruction algorithms perform relatively poorly due to a high rate of rearrangement and extensive gene losses. Our high-resolution reconstruction reveals previously overlooked small-scale rearrangements, necessitating a revision to previous views on genome structure evolution in teleost and vertebrate. CONCLUSIONS: We have reconstructed the structure of a pre-WGD genome by employing a variational Bayes approach that was originally developed for inferring topics from millions of text documents. Interestingly, comparison of the macrosynteny and topic model algorithms suggests that macrosynteny can be regarded as documents on ancestral genome structure. From this perspective, the present study would seem to provide a textbook example of the prevalent metaphor that genomes are documents of evolutionary history. AVAILABILITY AND IMPLEMENTATION: The analysis data are available for download at http://www.gen.tcd.ie/molevol/supp_data/MacrosyntenyTGD.zip , and the software written in Java is available upon request. CONTACT: yoichiro.nakatani@tcd.ie or aoife.mclysaght@tcd.ie. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Associations between nucleosome phasing, sequence asymmetry, and tissue-specific expression in a set of inbred Medaka species.

BACKGROUND: Transcription start sites (TSSs) with pronounced and phased nucleosome arrays downstream and nucleosome-depleted regions upstream of TSSs are observed in various species. RESULTS: We have characterized sequence variation and expression properties of this set of TSSs (which we call "Nucleocyclic TSSs") using germline and somatic cells of three medaka (Oryzias latipes) inbred isolates from different locations. We found nucleocyclic TSSs in medaka to be associated with higher gene expression and characterized by a clear boundary in sequence composition with potentially-nucleosome-destabilizing A/T-enrichment upstream (p < 10(-60)) and nucleosome- accommodating C/G-enrichment downstream (p < 10(-40)) that was highly conserved from an ancestor. A substantial genetic distance between the strains facilitated the in-depth analysis of patterns of fixed mutations, revealing a localization-specific equilibrium between the rates of distinct mutation categories that would serve to maintain the conserved sequence anisotropy around TSSs. Downstream of nucleocyclic TSSs, C to T, T to C, and other mutation rates on the sense strand increased around first nucleosome dyads and decreased around first linkers, which contrasted with genomewide mutational patterns around nucleosomes (p < 5 %). C to T rates are higher than G to A rates around nucleosome associated with germline nucleocyclic TSS sites (p < 5 %), potentially due to the asymmetric effect of transcription-coupled repair. CONCLUSIONS: Our results demonstrate an atypical evolutionary process surrounding nucleocyclic TSSs.

Coordinated changes in DNA methylation in antigen-specific memory CD4 T cells.

Memory CD4(+) T cells are central regulators of both humoral and cellular immune responses. T cell differentiation results in specific changes in chromatin structure and DNA methylation of cytokine genes. Although the methylation status of a limited number of gene loci in T cells has been examined, the genome-wide DNA methylation status of memory CD4(+) T cells remains unexplored. To further elucidate the molecular signature of memory T cells, we conducted methylome and transcriptome analyses of memory CD4(+) T cells generated using T cells from TCR-transgenic mice. The resulting genome-wide DNA methylation profile revealed 1144 differentially methylated regions (DMRs) across the murine genome during the process of T cell differentiation, 552 of which were associated with gene loci. Interestingly, the majority of these DMRs were located in introns. These DMRs included genes such as CXCR6, Tbox21, Chsy1, and Cish, which are associated with cytokine production, homing to bone marrow, and immune responses. Methylation changes in memory T cells exposed to specific Ag appeared to regulate enhancer activity rather than promoter activity of immunologically relevant genes. In addition, methylation profiles differed between memory T cell subsets, demonstrating a link between T cell methylation status and T cell differentiation. By comparing DMRs between naive and Ag-specific memory T cells, this study provides new insights into the functional status of memory T cells.

Genome-wide genetic variations are highly correlated with proximal DNA methylation patterns.

5-methyl-cytosines at CpG sites frequently mutate into thymines, accounting for a large proportion of spontaneous point mutations. The repair system would leave substantial numbers of errors in neighboring regions if the synthesis of erased gaps around deaminated 5-methyl-cytosines is error-prone. Indeed, we identified an unexpected genome-wide role of the CpG methylation state as a major determinant of proximal natural genetic variation. Specifically, 507 Mbp (∼18%) of the human genome was within 10 bp of a CpG site; in these regions, the single nucleotide polymorphism (SNP) rate significantly increased by ∼50% (P < 10(-566) by a two-proportion z-test) if the neighboring CpG sites are methylated. To reconfirm this finding in another vertebrate, we compared six single-base resolution methylomes in two inbred medaka (Oryzias latipes) strains with sufficient genetic divergence (3.4%). We found that the SNP rate also increased by ∼50% (P < 10(-2170)), and the substitution rates in all dinucleotides increased simultaneously (P < 10(-441)) around methylated CpG sites. In the hypomethylated regions, the "CGCG" motif was significantly enriched (P < 10(-680)) and evolutionarily conserved (P = ∼ 0.203%), and slow CpG deamination rather than fast CpG gain was seen, indicating a possible role of CGCG as a candidate cis-element for the hypomethylation state. In regions that were hypermethylated in germline-like tissues but were hypomethylated in somatic liver cells, the SNP rate was significantly smaller than that in hypomethylated regions in both tissues, suggesting a positive selective pressure during DNA methylation reprogramming. This is the first report of findings showing that the CpG methylation state is significantly correlated with the characteristics of evolutionary change in neighboring DNA.

Genome-wide profiling of DNA methylation in human cancer cells.

Global changes in DNA methylation correlate with altered gene expression and genomic instability in cancer. We have developed a methylation-specific digital sequencing (MSDS) method that can assess DNA methylation on a genomic scale. MSDS is a simple, low-cost method that combines the use of methylation-sensitive restriction enzymes with second generation sequencing technology. DNA methylation in two colon cancer cell lines, HT29 and HCT116, was measured using MSDS. When methylation levels were compared between the two cell lines, many differentially methylated regions (DMRs) were identified in CpG island shore regions (located within 2kb of a CpG island), gene body regions and intergenic regions. The number of DMRs in the vicinity of gene transcription start sites correlated with the level of expression of TACC1, CLDN1, and PLEKHC1 (FERMT2) genes, which have been linked to carcinogenesis. The MSDS method has the potential to provide novel insight into the functional complexity of the human genome.

High-resolution analysis of the 5'-end transcriptome using a next generation DNA sequencer.

Massively parallel, tag-based sequencing systems, such as the SOLiD system, hold the promise of revolutionizing the study of whole genome gene expression due to the number of data points that can be generated in a simple and cost-effective manner. We describe the development of a 5'-end transcriptome workflow for the SOLiD system and demonstrate the advantages in sensitivity and dynamic range offered by this tag-based application over traditional approaches for the study of whole genome gene expression. 5'-end transcriptome analysis was used to study whole genome gene expression within a colon cancer cell line, HT-29, treated with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5Aza). More than 20 million 25-base 5'-end tags were obtained from untreated and 5Aza-treated cells and matched to sequences within the human genome. Seventy three percent of the mapped unique tags were associated with RefSeq cDNA sequences, corresponding to approximately 14,000 different protein-coding genes in this single cell type. The level of expression of these genes ranged from 0.02 to 4,704 transcripts per cell. The sensitivity of a single sequence run of the SOLiD platform was 100-1,000 fold greater than that observed from 5'end SAGE data generated from the analysis of 70,000 tags obtained by Sanger sequencing. The high-resolution 5'end gene expression profiling presented in this study will not only provide novel insight into the transcriptional machinery but should also serve as a basis for a better understanding of cell biology.

Chromatin-associated periodicity in genetic variation downstream of transcriptional start sites.

Might DNA sequence variation reflect germline genetic activity and underlying chromatin structure? We investigated this question using medaka (Japanese killifish, Oryzias latipes), by comparing the genomic sequences of two strains (Hd-rR and HNI) and by mapping approximately 37.3 million nucleosome cores from Hd-rR blastulae and 11,654 representative transcription start sites from six embryonic stages. We observed a distinctive approximately 200-base pair (bp) periodic pattern of genetic variation downstream of transcription start sites; the rate of insertions and deletions longer than 1 bp peaked at positions of approximately +200, +400, and +600 bp, whereas the point mutation rate showed corresponding valleys. This approximately 200-bp periodicity was correlated with the chromatin structure, with nucleosome occupancy minimized at positions 0, +200, +400, and +600 bp. These data exemplify the potential for genetic activity (transcription) and chromatin structure to contribute to molding the DNA sequence on an evolutionary time scale.

UTGB/medaka: genomic resource database for medaka biology.

Medaka (Oryzias latipes) is a small egg-laying freshwater teleost native to East Asia that has become an excellent model system for developmental genetics and evolutionary biology. The draft medaka genome sequence (700 Mb) was reported in June 2007, and its substantial genomic resources have been opened to the public through the University of Tokyo Genome Browser Medaka (UTGB/medaka) database. This database provides basic genomic information, such as predicted genes, expressed sequence tags (ESTs), guanine/cytosine (GC) content, repeats and comparative genomics, as well as unique data resources including (i) 2473 genetic markers and experimentally confirmed PCR primers that amplify these markers, (ii) 142,414 bacterial artificial chromosome (BAC) and 217,344 fosmid end sequences that amount to 15.0- and 11.1-fold clone coverage of the entire genome, respectively, and were used for draft genome assembly, (iii) 16,519,460 single nucleotide polymorphisms (SNPs), and 2 859 905 insertions/deletions detected between two medaka inbred strain genomes and (iv) 841 235 5'-end serial analyses of gene-expression (SAGE) tags that identified 344 266 transcription start sites on the genome. UTGB/medaka is available at: http://medaka.utgenome.org/.

Reconstruction of the vertebrate ancestral genome reveals dynamic genome reorganization in early vertebrates.

Although several vertebrate genomes have been sequenced, little is known about the genome evolution of early vertebrates and how large-scale genomic changes such as the two rounds of whole-genome duplications (2R WGD) affected evolutionary complexity and novelty in vertebrates. Reconstructing the ancestral vertebrate genome is highly nontrivial because of the difficulty in identifying traces originating from the 2R WGD. To resolve this problem, we developed a novel method capable of pinning down remains of the 2R WGD in the human and medaka fish genomes using invertebrate tunicate and sea urchin genes to define ohnologs, i.e., paralogs produced by the 2R WGD. We validated the reconstruction using the chicken genome, which was not considered in the reconstruction step, and observed that many ancestral proto-chromosomes were retained in the chicken genome and had one-to-one correspondence to chicken microchromosomes, thereby confirming the reconstructed ancestral genomes. Our reconstruction revealed a contrast between the slow karyotype evolution after the second WGD and the rapid, lineage-specific genome reorganizations that occurred in the ancestral lineages of major taxonomic groups such as teleost fishes, amphibians, reptiles, and marsupials.

The medaka draft genome and insights into vertebrate genome evolution.

Teleosts comprise more than half of all vertebrate species and have adapted to a variety of marine and freshwater habitats. Their genome evolution and diversification are important subjects for the understanding of vertebrate evolution. Although draft genome sequences of two pufferfishes have been published, analysis of more fish genomes is desirable. Here we report a high-quality draft genome sequence of a small egg-laying freshwater teleost, medaka (Oryzias latipes). Medaka is native to East Asia and an excellent model system for a wide range of biology, including ecotoxicology, carcinogenesis, sex determination and developmental genetics. In the assembled medaka genome (700 megabases), which is less than half of the zebrafish genome, we predicted 20,141 genes, including approximately 2,900 new genes, using 5'-end serial analysis of gene expression tag information. We found single nucleotide polymorphisms (SNPs) at an average rate of 3.42% between the two inbred strains derived from two regional populations; this is the highest SNP rate seen in any vertebrate species. Analyses based on the dense SNP information show a strict genetic separation of 4 million years (Myr) between the two populations, and suggest that differential selective pressures acted on specific gene categories. Four-way comparisons with the human, pufferfish (Tetraodon), zebrafish and medaka genomes revealed that eight major interchromosomal rearrangements took place in a remarkably short period of approximately 50 Myr after the whole-genome duplication event in the teleost ancestor and afterwards, intriguingly, the medaka genome preserved its ancestral karyotype for more than 300 Myr.

Diversity of Ca2+-induced morphology revealed by morphological phenotyping of Ca2+-sensitive mutants of Saccharomyces cerevisiae.

Yeast cell morphology can be treated as a quantitative trait using the image processing software CalMorph. In the present study, we investigated Ca(2+)-induced morphological changes in Ca(2+)-sensitive (cls) mutants of Saccharomyces cerevisiae, based on the discovery that the characteristic Ca(2+)-induced morphological changes in the Ca(2+)-sensitive mutant zds1 reflect changes in the Ca(2+) signaling-mediated cell cycle control pathway. By applying hierarchical cluster analysis to the quantitative morphological data of 58 cls mutants, 31 of these mutants were classified into seven classes based on morphological similarities. The patterns of morphological change induced by Ca(2+) in one class differed from those of another class. Based on the results obtained using versatile methods for phenotypic analysis, we conclude that a high concentration of Ca(2+) exerts a wide variety of effects on yeast and that there are multiple Ca(2+)-regulatory pathways that are distinct from the Zds1p-related pathway.

High-dimensional and large-scale phenotyping of yeast mutants.

One of the most powerful techniques for attributing functions to genes in uni- and multicellular organisms is comprehensive analysis of mutant traits. In this study, systematic and quantitative analyses of mutant traits are achieved in the budding yeast Saccharomyces cerevisiae by investigating morphological phenotypes. Analysis of fluorescent microscopic images of triple-stained cells makes it possible to treat morphological variations as quantitative traits. Deletion of nearly half of the yeast genes not essential for growth affects these morphological traits. Similar morphological phenotypes are caused by deletions of functionally related genes, enabling a functional assignment of a locus to a specific cellular pathway. The high-dimensional phenotypic analysis of defined yeast mutant strains provides another step toward attributing gene function to all of the genes in the yeast genome.

Data mining tools for the Saccharomyces cerevisiae morphological database.

For comprehensive understanding of precise morphological changes resulting from loss-of-function mutagenesis, a large collection of 1,899,247 cell images was assembled from 91,71 micrographs of 4782 budding yeast disruptants of non-lethal genes. All the cell images were processed computationally to measure approximately 500 morphological parameters in individual mutants. We have recently made this morphological quantitative data available to the public through the Saccharomyces cerevisiae Morphological Database (SCMD). Inspecting the significance of morphological discrepancies between the wild type and the mutants is expected to provide clues to uncover genes that are relevant to the biological processes producing a particular morphology. To facilitate such intensive data mining, a suite of new software tools for visualizing parameter value distributions was developed to present mutants with significant changes in easily understandable forms. In addition, for a given group of mutants associated with a particular function, the system automatically identifies a combination of multiple morphological parameters that discriminates a mutant group from others significantly, thereby characterizing the function effectively. These data mining functions are available through the World Wide Web at http://scmd.gi.k.u-tokyo.ac.jp/.