Network perturbation by recurrent regulatory variants in cancer.

Cancer driving genes have been identified as recurrently affected by variants that alter protein-coding sequences. However, a majority of cancer variants arise in noncoding regions, and some of them are thought to play a critical role through transcriptional perturbation. Here we identified putative transcriptional driver genes based on combinatorial variant recurrence in cis-regulatory regions. The identified genes showed high connectivity in the cancer type-specific transcription regulatory network, with high outdegree and many downstream genes, highlighting their causative role during tumorigenesis. In the protein interactome, the identified transcriptional drivers were not as highly connected as coding driver genes but appeared to form a network module centered on the coding drivers. The coding and regulatory variants associated via these interactions between the coding and transcriptional drivers showed exclusive and complementary occurrence patterns across tumor samples. Transcriptional cancer drivers may act through an extensive perturbation of the regulatory network and by altering protein network modules through interactions with coding driver genes.

Intersection of genetics and epigenetics in monozygotic twin genomes.

As a final function of various epigenetic mechanisms, chromatin regulation is a transcription control process that especially demonstrates active interaction with genetic elements. Thus, chromatin structure has become a principal focus in recent genomics researches that strive to characterize regulatory functions of DNA variants related to diseases or other traits. Although researchers have been focusing on DNA methylation when studying monozygotic (MZ) twins, a great model in epigenetics research, interactions between genetics and epigenetics in chromatin level are expected to be an imperative research trend in the future. In this review, we discuss how the genome, epigenome, and transcriptome of MZ twins can be studied in an integrative manner from this perspective.

Global transcription network incorporating distal regulator binding reveals selective cooperation of cancer drivers and risk genes.

Global network modeling of distal regulatory interactions is essential in understanding the overall architecture of gene expression programs. Here, we developed a Bayesian probabilistic model and computational method for global causal network construction with breast cancer as a model. Whereas physical regulator binding was well supported by gene expression causality in general, distal elements in intragenic regions or loci distant from the target gene exhibited particularly strong functional effects. Modeling the action of long-range enhancers was critical in recovering true biological interactions with increased coverage and specificity overall and unraveling regulatory complexity underlying tumor subclasses and drug responses in particular. Transcriptional cancer drivers and risk genes were discovered based on the network analysis of somatic and genetic cancer-related DNA variants. Notably, we observed that the risk genes were functionally downstream of the cancer drivers and were selectively susceptible to network perturbation by tumorigenic changes in their upstream drivers. Furthermore, cancer risk alleles tended to increase the susceptibility of the transcription of their associated genes. These findings suggest that transcriptional cancer drivers selectively induce a combinatorial misregulation of downstream risk genes, and that genetic risk factors, mostly residing in distal regulatory regions, increase transcriptional susceptibility to upstream cancer-driving somatic changes.

Genome-wide reorganization of histone H2AX toward particular fragile sites on cell activation.

γH2AX formation by phosphorylation of the histone variant H2AX is the key process in the repair of DNA lesions including those arising at fragile sites under replication stress. Here we demonstrate that H2AX is dynamically reorganized to preoccupy γH2AX hotspots on increased replication stress by activated cell proliferation and that H2AX is enriched in aphidicolin-induced replisome stalling sites in cycling cells. Interestingly, H2AX enrichment was particularly found in genomic regions that replicate in early S phase. High transcription activity, a hallmark of early replicating fragile sites, was a determinant of H2AX localization. Subtelomeric H2AX enrichment was also attributable to early replication and high gene density. In contrast, late replicating and infrequently transcribed regions, including common fragile sites and heterochromatin, lacked H2AX enrichment. In particular, heterochromatin was inaccessible to H2AX incorporation, maybe partly explaining the cause of mutation accumulation in cancer heterochromatin. Meanwhile, H2AX in actively dividing cells was intimately colocalized with INO80. INO80 silencing reduced H2AX levels, particularly at the INO80-enriched sites. Our findings suggest that active DNA replication is accompanied with the specific localization of H2AX and INO80 for efficient damage repair or replication-fork stabilization in actively transcribed regions.

Regulation of the boundaries of accessible chromatin.

Regulatory regions maintain nucleosome-depleted, open chromatin status but simultaneously require the presence of nucleosomes for specific histone modifications. It remains unclear how these can be achieved for proper regulatory function. Here we demonstrate that nucleosomes positioned within accessible chromatin regions near the boundaries provide platforms for histone modifications while preventing the occlusion of regulatory elements. These boundary nucleosomes were particularly enriched for active or poised regulatory marks in human, such as histone acetylations, H3K4 methylations, H3K9me3, H3K79me2, and H4K20me1. Additionally, we found that based on a genome-wide profiling of ~100 recombinant yeast strains, the location of open chromatin borders tends to vary mostly within 150 bp upon genetic perturbation whereas this positional variation increases in proportion to the sequence preferences of the underlying DNA for nucleosome formation. More than 40% of the local boundary shifts were associated with genetic variation in cis- or trans-acting factors. A sizeable fraction of the identified genetic factors was also associated with nearby gene expression, which was correlated with the distance between the transcription start site (tss) and the boundary that faces the tss. Taken together, the variation in the width of accessible chromatin regions may arise in conjunction with the modulation of the boundary nucleosomes by post-translational modifications or by chromatin regulators and in association with the activity of nearby gene transcription.

Genetic landscape of open chromatin in yeast.

Chromatin regulation underlies a variety of DNA metabolism processes, including transcription, recombination, repair, and replication. To perform a quantitative genetic analysis of chromatin accessibility, we obtained open chromatin profiles across 96 genetically different yeast strains by FAIRE (formaldehyde-assisted isolation of regulatory elements) assay followed by sequencing. While 5∼10% of open chromatin region (OCRs) were significantly affected by variations in their underlying DNA sequences, subtelomeric areas as well as gene-rich and gene-poor regions displayed high levels of sequence-independent variation. We performed quantitative trait loci (QTL) mapping using the FAIRE signal for each OCR as a quantitative trait. While individual OCRs were associated with a handful of specific genetic markers, gene expression levels were associated with many regulatory loci. We found multi-target trans-loci responsible for a very large number of OCRs, which seemed to reflect the widespread influence of certain chromatin regulators. Such regulatory hotspots were enriched for known regulatory functions, such as recombinational DNA repair, telomere replication, and general transcription control. The OCRs associated with these multi-target trans-loci coincided with recombination hotspots, telomeres, and gene-rich regions according to the function of the associated regulators. Our findings provide a global quantitative picture of the genetic architecture of chromatin regulation.

Antigen Coverage Presented by MHC Class I Has a Negative Correlation with SARS-CoV-2-Induced Mortality.

The COVID-19 pandemic has caused a health crisis worldwide; therefore, it is necessary to understand the factors related to its prognosis. In this study, we hypothesized that SARS-CoV-2-derived antigens presented by MHC class I may correlate with mortality in COVID-19 because they induce adaptive immune responses. Antigen coverage at the national level was inferred using country-specific HLA allele frequencies and relative predictions of binding antigens. We performed regression analysis between antigen coverage and the death rate due to COVID-19 across countries and found a negative correlation, although it was statistically significant only in HLA-B. This negative correlation was corroborated in multiple regression analysis with known risk factors, such as the prevalence of underlying disease. Furthermore, we analyzed antigen coverage in accordance with SARS-CoV-2 domains and identified a significant negative correlation when it was derived from the spike domain, which is reported to be favorable for COVID-19 prognosis. Taken together, the results indicate that the antigen coverage of SARS-CoV-2 specifically presented by HLA-B may act as a favorable factor when explaining COVID-19-induced mortality.

Recurrence-associated gene signature in patients with stage I non-small-cell lung cancer.

Recurrent gene mutations and fusions in cancer patients are likely to be associated with cancer progression or recurrence by Vogelstein et al. (Science (80-) 340, 1546-1558 (2013)). In this study, we investigated gene mutations and fusions that recurrently occurred in early-stage cancer patients with stage I non-small-cell cancer (NSCLC). Targeted exome sequencing was performed to profile the variants and confirmed their fidelity at the gene and pathway levels through comparison with data for stage I lung cancer patients, which was obtained from The Cancer Genome Atlas (TCGA). Next, we identified prognostic gene mutations (ATR, ERBB3, KDR, and MUC6), fusions (GOPC-ROS1 and NTRK1-SH2D2A), and VEGF signaling pathway associated with cancer recurrence. To infer the functional implication of the recurrent variants in early-stage cancers, the extent of their selection pattern was investigated, and they were shown to be under positive selection, implying a selective advantage for cancer progression. Specifically, high selection scores were observed in the variants with significantly high risks for recurrence. Taken together, the results of this study enabled us to identify recurrent gene mutations and fusions in a stage I NSCLC cohort and to demonstrate positive selection, which had implications regarding cancer recurrence.

DNA Methylation and gene expression patterns are widely altered in fetal growth restriction and associated with FGR development.

Fetal growth restriction (FGR) is the failure of the fetus toachieve its genetically determined growth potential, which increasesrisks for a variety of genetic diseases, such as type 2 diabetes mellitus, coronary artery disease, and stroke, during the lifetime. The dysregulation of DNA methylationis known to interact with environmental fluctuations, affect gene expressions comprehensively, and be fatal to fetus development in specific cases. Therefore, we set out to find out epigenetic and transcriptomic alterations associated with FGR development. We found a set of differentially expressed genes associated with differentially methylated regions in placentae and cord blood samples. Using dimensional reduction analysis, the expression and methylation variables of the epigenetically altered genes classified the FGR samples from the controls. These genes were also enriched in the biological pathways such as metabolism and developmental processes related to FGR. Furthermore, three genes of INS, MEG3, and ZFP36L2 are implicated in epigenetic imprinting, which has been associated with FGR. These results strongly suggest that DNA methylation is highly dysregulated during FGR development, and abnormal DNA methylation patterns are likely to alter gene expression.

Longitudinal change of genetic variations in cetuximab-treated metastatic colorectal cancer.

Recurrent gene mutations and copy number alterations in cancer patients are presumably associated with resistance to targeted therapy. In the present study, we assessed the gene mutations and copy number alterations that recurrently occurred in cetuximab-treated patients with metastatic colorectal cancer (mCRC). Targeted next-generation sequencing was performed in the tumor samples obtained pre- and postcetuximab treatment to assess the variations that occurred during cetuximab treatment. Moreover, we identified the emergent gene mutations (CDK6, EPHA3, ERCC2, MYC, PCMTD1, PIK3CA, PRIM2, RICTOR, and ZNRF3) and copy number alterations (ARAF, BCL2, BRCA2, EGFR, MYC, and SMAD4) that were recurrently observed only in postprogression samples and not in pretreatment or posttreatment samples from patients revealing clinical response. Furthermore, to identify the feasible candidate variations implicated in treatment resistance, we examined the variants with clonal expansion during treatment and discovered PCBP1 as a variant associated with posttreatment progression. Various recurrent mutations were enriched in the TGF-beta signaling pathway. Collectively, we identified recurrent variations in mCRC samples exhibiting post-cetuximab progression. Additionally, future studies are required to evaluate the therapeutic potential of these variations.

How to interpret and integrate multi-omics data at systems level.

Current parallel sequencing technologies generate biological sequence data explosively and enable omics studies that analyze collective biological features. The more omics data that is accumulated, the more they show the regulatory complexity of biological phenotypes. This high order regulatory complexity needs systems-level approaches, including network analysis, to understand it. There are a series of layers in the omics field that are closely connected to each other as described in 'central dogma.' We, therefore, have to not only interpret each single omics layer but also to integrate multi-omics layers systematically to get a full picture of the regulatory landscape of the biological phenotype. Especially, individual omics data has their own adequate biological network to apply systematic analysis appropriately. A full regulatory landscape can only be obtained when multi-omics data are incorporated within adequate networks. In this review, we discuss how to interpret and integrate multi-omics data systematically using recent studies. We also propose an analysis framework for systematic multi-omics interpretation by centering on the transcriptional core regulator, which can be incorporated in all omics networks.

Accelerated Evolution of the Regulatory Sequences of Brain Development in the Human Genome.

Genetic modifications in noncoding regulatory regions are likely critical to human evolution. Human-accelerated noncoding elements are highly conserved noncoding regions among vertebrates but have large differences across humans, which implies human-specific regulatory potential. In this study, we found that human-accelerated noncoding elements were frequently coupled with DNase I hypersensitive sites (DHSs), together with monomethylated and trimethylated histone H3 lysine 4, which are active regulatory markers. This coupling was particularly pronounced in fetal brains relative to adult brains, non-brain fetal tissues, and embryonic stem cells. However, fetal brain DHSs were also specifically enriched in deeply conserved sequences, implying coexistence of universal maintenance and human-specific fitness in human brain development. We assessed whether this coexisting pattern was a general one by quantitatively measuring evolutionary rates of DHSs. As a result, fetal brain DHSs showed a mixed but distinct signature of regional conservation and outlier point acceleration as compared to other DHSs. This finding suggests that brain developmental sequences are selectively constrained in general, whereas specific nucleotides are under positive selection or constraint relaxation simultaneously. Hence, we hypothesize that human- or primate-specific changes to universally conserved regulatory codes of brain development may drive the accelerated, and most likely adaptive, evolution of the regulatory network of the human brain.

Predicting clinical benefit of immunotherapy by antigenic or functional mutations affecting tumour immunogenicity.

Neoantigen burden is regarded as a fundamental determinant of response to immunotherapy. However, its predictive value remains in question because some tumours with high neoantigen load show resistance. Here, we investigate our patient cohort together with a public cohort by our algorithms for the modelling of peptide-MHC binding and inter-cohort genomic prediction of therapeutic resistance. We first attempt to predict MHC-binding peptides at high accuracy with convolutional neural networks. Our prediction outperforms previous methods in > 70% of test cases. We then develop a classifier that can predict resistance from functional mutations. The predictive genes are involved in immune response and EGFR signalling, whereas their mutation patterns reflect positive selection. When integrated with our neoantigen profiling, these anti-immunogenic mutations reveal higher predictive power than known resistance factors. Our results suggest that the clinical benefit of immunotherapy can be determined by neoantigens that induce immunity and functional mutations that facilitate immune evasion.

Chromatin structure-based prediction of recurrent noncoding mutations in cancer.

Recurrence is a hallmark of cancer-driving mutations. Recurrent mutations can arise at the same site or affect the same gene at different sites. Here we identified a set of mutations arising in individual samples and altering different cis-regulatory elements that converge on a common gene via chromatin interactions. The mutations and genes identified in this fashion showed strong relevance to cancer, in contrast to noncoding mutations with site-specific recurrence only. We developed a prediction method that identifies potentially recurrent mutations on the basis of the features shared by mutations whose recurrence is observed in a given cohort. Our method was capable of accurately predicting recurrent mutations at the level of target genes but not mutations recurring at the same site. We experimentally validated predicted mutations in distal regulatory regions of the TERT gene. In conclusion, we propose a novel approach to discovering potential cancer-driving mutations in noncoding regions.

Genetic Architecture of Transcription and Chromatin Regulation.

DNA microarray and next-generation sequencing provide data that can be used for the genetic analysis of multiple quantitative traits such as gene expression levels, transcription factor binding profiles, and epigenetic signatures. In particular, chromatin opening is tightly coupled with gene transcription. To understand how these two processes are genetically regulated and associated with each other, we examined the changes of chromatin accessibility and gene expression in response to genetic variation by means of quantitative trait loci mapping. Regulatory patterns commonly observed in yeast and human across different technical platforms and experimental designs suggest a higher genetic complexity of transcription regulation in contrast to a more robust genetic architecture of chromatin regulation.

Genetic factors underlying discordance in chromatin accessibility between monozygotic twins.

BACKGROUND: Open chromatin is implicated in regulatory processes; thus, variations in chromatin structure may contribute to variations in gene expression and other phenotypes. In this work, we perform targeted deep sequencing for open chromatin, and array-based genotyping across the genomes of 72 monozygotic twins to identify genetic factors regulating co-twin discordance in chromatin accessibility. RESULTS: We show that somatic mutations cause chromatin discordance mainly via the disruption of transcription factor binding sites. Structural changes in DNA due to C:G to A:T transversions are under purifying selection due to a strong impact on chromatin accessibility. We show that CpGs whose methylation is specifically regulated during cellular differentiation appear to be protected from high mutation rates of 5'-methylcytosines, suggesting that the spectrum of CpG variations may be shaped fully at the developmental level but not through natural selection. Based on the association mapping of within-pair chromatin differences, we search for cases in which twin siblings with a particular genotype had chromatin discordance at the relevant locus. We identify 1,325 chromatin sites that are differentially accessible, depending on the genotype of a nearby locus, suggesting that epigenetic differences can control regulatory variations via interactions with genetic factors. Poised promoters present high levels of chromatin discordance in association with either somatic mutations or genetic-epigenetic interactions. CONCLUSION: Our observations illustrate how somatic mutations and genetic polymorphisms may contribute to regulatory, and ultimately phenotypic, discordance.

Collagen triple helix repeat containing 1 (CTHRC1) acts via ERK-dependent induction of MMP9 to promote invasion of colorectal cancer cells.

Collagen triple helix repeat-containing 1 (CTHRC1) is known to be aberrantly upregulated in most human solid tumors, although the functional roles of CTHRC1 in colorectal cancer remain unclear. In this study, we investigated the occurrence of CTHRC1 upregulation and its role in vivo and in vitro. The expression profile and clinical importance of CTHRC1 were examined by reverse transcription-polymerase chain reaction and immunohistochemical analyses in normal and tumor patient samples. CTHRC1 was detectable in normal tissues, but also was highly expressed in tumor specimens. CTHRC1 upregulation was significantly associated with demethylation of the CTHRC1 promoter in colon cancer cell lines and tumor tissues. Clinicopathologic analyses showed that nodal status and expression of CTHRC1 (95% CI 0.999-3.984, p=0.05) were significant prognostic factors for disease-free survival. Promoter CpG methylation and hypermethylation status were measured by bisulfite sequencing and pyrosequencing analysis. Furthermore, we showed that overexpression of CTHRC1 in the SW480 and HT-29 cell lines increased invasiveness, an effect mediated by extracellular signal-regulated kinase (ERK)-dependent upregulation of matrix metalloproteinase 9 (MMP9). Consistent with this, we found that knockdown of CTHRC1 attenuated ERK activation and cancer cell invasivity. These results demonstrate that CTHRC1 expression is elevated in human colon cancer cell lines and clinical specimens, and promotes cancer cell invasivity through ERK-dependent induction of MMP9 expression. Our results further suggest that high levels of CTHRC1 expression are associated with poor clinical outcomes.

Epigenetic regulation of microRNA-10b and targeting of oncogenic MAPRE1 in gastric cancer.

MicroRNAs act as negative regulators of gene expression, and the altered expression of microRNAs by epigenetic mechanisms is strongly implicated in carcinogenesis. Here we report that the microRNA-10b gene (miR-10b) was silenced in gastric cancer cells by promoter methylation. In this study, using a methylation array and bisulfate pyrosequencing analysis, we found that miR-10b promoter CpGs were heavily methylated in gastric cancers. Clinicopathologic data showed that miR-10b methylation increased with patient age and occurred significantly more frequently in intestinal-type (28/44, 64%) than in diffuse-type (22/56, 39%) gastric cancers (P = 0.016). In addition, miR-10b methylation was also associated with an increase in expression of the oncogene that encodes microtubule-associated protein, RP/EB family, member 1 (MAPRE1; P = 0.004), which was identified as a potential miR-10b target. After 5-aza-2'-deoxycytidine treatment of gastric cancer cells, miR-10b methylation was significantly decreased, and expression of miR-10b and HOXD4, which is 1 kb downstream of miR-10b, was greatly restored. Moreover, decreased MAPRE1 expression coincided with increased miR-10b expression, suggesting that miR-10b targets MAPRE1 transcription. We also found that transfection with precursor miR-10b into gastric cancer cells dramatically decreased MAPRE1 mRNA and protein, resulting in a significant decrease in colony formation and cell growth rates. Thus, we show a tumor-suppressive role for miR-10b in gastric carcinogenesis. miR-10b methylation may be a useful molecular biomarker for assessing the risk of gastric cancer development, and modulation of miR-10b may represent a therapeutic approach for treating gastric cancer.