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Simple Summary: Cancer is a disease caused by mutations in DNA differentiating cancer cells from normal cells. Precision medicine could exploit these genetic differences to identify and create therapeutic opportunities that selectively kill cancer cells while sparing normal cells. In some cancers, the same mutations recur, making them desirable biomarkers for genetics-based therapeutic strategies. This is the case for the gene ARID1A which is lost in about 50% of ovarian clear cell carcinomas. We used genetic screening to identify targets for killing an ovarian clear cell carcinoma cell line lacking the ARID1A gene. We identified a cancer stress response regulator called KEAP1 as a potential target for ARID1A-deficient ovarian clear cell carcinomas. Using different approaches and cell lines, we observed a relationship where ARID1A-deficient cells required KEAP1 for fitness. Together these data suggest that KEAP1 could be pursued as a means to selectively kill certain types of ARID1A-deficient cancer. ARID1A is the core DNA-binding subunit of the BAF chromatin remodeling complex and is mutated in about 8% of all cancers. The frequency of ARID1A loss varies between cancer subtypes, with clear cell ovarian carcinoma (CCOC) presenting the highest incidence at > 50% of cases. Despite a growing understanding of the consequences of ARID1A loss in cancer, there remains limited targeted therapeutic options for ARID1A-deficient cancers. Using a genome-wide CRISPR screening approach, we identify KEAP1 as a genetic dependency of ARID1A in CCOC. Depletion or chemical perturbation of KEAP1 results in selective growth inhibition of ARID1A-KO cell lines and edited primary endometrial epithelial cells. While we confirm that KEAP1-NRF2 signalling is dysregulated in ARID1A-KO cells, we suggest that this synthetic lethality is not due to aberrant NRF2 signalling. Rather, we find that KEAP1 perturbation exacerbates genome instability phenotypes associated with ARID1A deficiency. Together, our findings identify a potentially novel synthetic lethal interaction of ARID1A-deficient cells. [ABSTRACT FROM AUTHOR]

Most human breast cancers have diversified genomically and biologically by the time they become clinically evident (1-3). Early events involved in their genesis and the cellular context in which these events occur have thus been difficult to characterize. Here we present the first formal evidence of the shared and independent ability of basal cells and luminal progenitors, isolated from normal human mammary tissue and transduced with a single oncogene ([KRAS.sup.GI2D]), to produce serially transplantable, polyclonal, invasive ductal carcinomas within 8 weeks of being introduced either subrenally or subcutaneously into immunodeficient mice (4). DNA barcoding (5,6) of the initial cells revealed a dramatic change in the numbers and sizes of clones generated from them within 2 weeks, and the first appearance of many 'new' clones in tumours passaged into secondary recipients. Both primary and secondary tumours were phenotypically heterogeneous and primary tumours were categorized transcriptionally as 'normal-like'. This system challenges previous concepts that carcinogenesis in normal human epithelia is necessarily a slow process requiring the acquisition of multiple driver mutations. It also presents the first description of initial events that accompany the genesis and evolution of malignant human mammary cell populations, thereby contributing new understanding of the rapidity with which heterogeneity in their properties can develop., To investigate the susceptibility of different normal human mammary cell types to transformation under the influence of known oncogenes, we isolated [CD49f.sup.+][EpCAM.sup.low] basal cells (BCs), [CD49f.sup.+][EpCAM.sup.+] luminal progenitors (LPs), [CD49f.sup.-][EpCAM.sup.+] [...]

Genome sequences of chicken (low pathogenic avian influenza [LPAI] and highly pathogenic avian influenza [HPAI]) and human isolates from a 2004 outbreak of H7N3 avian influenza in Canada showed a [...]

We present Epiclomal, a probabilistic clustering method arising from a hierarchical mixture model to simultaneously cluster sparse single-cell DNA methylation data and impute missing values. Using synthetic and published single-cell CpG datasets, we show that Epiclomal outperforms non-probabilistic methods and can handle the inherent missing data characteristic that dominates single-cell CpG genome sequences. Using newly generated single-cell 5mCpG sequencing data, we show that Epiclomal discovers sub-clonal methylation patterns in aneuploid tumour genomes, thus defining epiclones that can match or transcend copy number-determined clonal lineages and opening up an important form of clonal analysis in cancer. Epiclomal is written in R and Python and is available at https://github.com/shahcompbio/Epiclomal. Author summary: DNA methylation is an epigenetic mark that occurs when methyl groups are attached to the DNA molecule, thereby playing decisive roles in numerous biological processes. Advances in technology have allowed the generation of high-throughput DNA methylation sequencing data from single cells. One of the goals is to group cells according to their DNA methylation profiles; however, a major challenge arises due to a large amount of missing data per cell. To address this problem, we developed a novel statistical model and framework: Epiclomal. Our approach uses a hierarchical mixture model to borrow statistical strength across cells and neighboring loci to accurately define cell groups (clusters). We compare our approach to different methods on both synthetic and published datasets. We show that Epiclomal is more robust than other approaches, producing more accurate clusters of cells in the majority of experimental scenarios. We also apply Epiclomal to newly generated single-cell DNA methylation data from breast cancer xenografts. Our results show that methylation-based clusters can mirror or in some instances transcend the clusters defined by single-cell copy number analysis. This illustrates the importance of single-cell DNA methylation analysis in understanding cellular heterogeneity in cancer. [ABSTRACT FROM AUTHOR]

Summary Nucleosome position, density, and post-translational modification are widely accepted components of mechanisms regulating DNA transcription but still incompletely understood. We present a modified native ChIP-seq method combined with an analytical framework that allows MNase accessibility to be integrated with histone modification profiles. Application of this methodology to the primitive (CD34+) subset of normal human cord blood cells enabled genomic regions enriched in one versus two nucleosomes marked by histone 3 lysine 4 trimethylation (H3K4me3) and/or histone 3 lysine 27 trimethylation (H3K27me3) to be associated with their transcriptional and DNA methylation states. From this analysis, we defined four classes of promoter-specific profiles and demonstrated that a majority of bivalent marked promoters are heterogeneously marked at a single-cell level in this primitive cell type. Interestingly, extension of this approach to human embryonic stem cells revealed an altered relationship between chromatin modification state and nucleosome content at promoters, suggesting developmental stage-specific organization of histone methylation states. [ABSTRACT FROM AUTHOR]

Summary The normal adult human mammary gland is a continuous bilayered epithelial system. Bipotent and myoepithelial progenitors are prominent and unique components of the outer (basal) layer. The inner (luminal) layer includes both luminal-restricted progenitors and a phenotypically separable fraction that lacks progenitor activity. We now report an epigenomic comparison of these three subsets with one another, with their associated stromal cells, and with three immortalized, non-tumorigenic human mammary cell lines. Each genome-wide analysis contains profiles for six histone marks, methylated DNA, and RNA transcripts. Analysis of these datasets shows that each cell type has unique features, primarily within genomic regulatory regions, and that the cell lines group together. Analyses of the promoter and enhancer profiles place the luminal progenitors in between the basal cells and the non-progenitor luminal subset. Integrative analysis reveals networks of subset-specific transcription factors. [ABSTRACT FROM AUTHOR]

Endotoxin is recognized as one of the virulence factors of the Bordetella avium bird pathogen, and characterization of its structure and corresponding genomic features are important for an understanding of its role in pathogenicity and for an improved general knowledge of Bordetella spp virulence factors. The structure of the biologically active part of B. avium LPS, lipid A, is described and compared to those of another bird pathogen, opportunistic in humans, Bordetella hinzii, and to that of Bordetella trematum, a human pathogen. Sequence analyses showed that the three strains have homologues of acyl-chain modifying enzymes PagL, PagP and LpxO, of the 1-phosphatase LpxE, in addition to LgmA, LgmB and LgmC, which are required for the glucosamine modification. MALDI mass spectrometry identified a high amount of glucosamine substituting the phosphate groups of B. avium lipid A; this modification was absent from B. hinzii and B. trematum. The acylation patterns of the three lipid As were similar, but they differed from those of Bordetella pertussis and Bordetella parapertussis. They were also found to be close to the lipid A structure of Bordetella bronchiseptica, a mammalian pathogen, only differing from the latter by the degree of hydroxylation of the branched fatty acid. [ABSTRACT FROM AUTHOR]

Prenatal adversity or stress can have long-term consequences on developmental trajectories and health outcomes. Although the biological mechanisms underlying these effects are poorly understood, epigenetic modifications, such as DNA methylation, have the potential to link early-life environments to alterations in physiological systems, with long-term functional implications. We investigated the consequences of two prenatal insults, prenatal alcohol exposure (PAE) and food-related stress, on DNA methylation profiles of the rat brain during early development. As these insults can have sex-specific effects on biological outcomes, we analyzed epigenome-wide DNA methylation patterns in prefrontal cortex, a key brain region involved in cognition, executive function, and behavior, of both males and females. We found sex-dependent and sex-concordant influences of these insults on epigenetic patterns. These alterations occurred in genes and pathways related to brain development and immune function, suggesting that PAE and food-related stress may reprogram neurobiological/physiological systems partly through central epigenetic changes, and may do so in a sex-dependent manner. Such epigenetic changes may reflect the sex-specific effects of prenatal insults on long-term functional and health outcomes and have important implications for understanding possible mechanisms underlying fetal alcohol spectrum disorder and other neurodevelopmental disorders. [ABSTRACT FROM AUTHOR]

Background: Profiling the entire genome at base pair resolution in a single test offers novel insights into disease by means of dissection of genetic contributors to phenotypic features. Methods: We performed genome sequencing for a patient who presented with atypical hereditary sensory and autonomic neuropathy, severe epileptic encephalopathy, global developmental delay, and growth hormone deficiency. Results: Assessment of the variants detected by mapped sequencing reads followed by Sanger confirmation revealed that the proband is a compound heterozygote for rare variants within RETREG1 (FAM134B), a gene associated with a recessive form of hereditary sensory and autonomic neuropathy, but not with epileptic encephalopathy or global developmental delay. Further analysis of the data also revealed a heterozygous missense variant in DNM1L, a gene previously implicated in an autosomal dominant encephalopathy, epilepsy, and global developmental delay and confirmed by Sanger sequencing to be a de novo variant not present in parental genomes. Conclusions: Our findings emphasize the importance of genome‐wide sequencing in patients with a well‐characterized genetic disease with atypical presentation. This approach reduces the potential for misdiagnoses. [ABSTRACT FROM AUTHOR]

Although it is known that breast cancers are a heterogeneous collection of diseases, the extensive heterogeneity within individual tumours has yet to be characterized in terms of phenotype and functional activity. Recent studies demonstrate that there is a phenotypically distinct rare subset of tumour-initiating cells (TICs) in primary human breast tumours that are characterized by their unique ability to produce tumours in xenografted immunodeficient mice. Further investigation into the genetic evolution of these cells and their predominance over time will help to ascertain how their frequency and growth potential, growth rate, treatment response and metastatic ability are related. To enable the clonal progeny of individual TICs (and normal human mammary stem cells) to be monitored in vivo (and in vitro) under different conditions, we are developing a lentiviral-based strategy for "marking" these with integrated noncoding variable 'barcode' sequences. Sequencing results from a first set of 230 individual clones within a larger barcode lentiviral library has demonstrated a diversity of the library in which <1% of the clones lack the barcode sequence. To validate a non-invasive technique for monitoring in vivo tumour growth, highly immunodeficient mice, NOD/SCID/IL-2R c-null, were transplanted orthotopically or heterotopically with transduced malignant breast cell lines that had been selected for stable expression of luciferase. As few as 625 transplanted cells could be detected at both sites 24 hours post-transplant. Also, although transplants of fewer than 250 cells could be imaged and monitored for tumour growth, the amplitude of the signal detected varied between individual host mice. Future experiments will aim to apply our cellular barcoding strategy to track the clonal evolution of human breast TICs and their relative contributions to individual tumour landscapes while concurrently monitoring their in vivo growth activity, metastatic properties, and responsiveness to anti-cancer therapies. [ABSTRACT FROM AUTHOR]

The Mammalian Gene Collection (MGC) consortium (http://mgc.nci.nih.gov) seeks to establish publicly available collections of full-ORF cDNAs for several organisms of significance to biomedical research, including human. To date over 15,200 human cDNA clones containing full-length open reading frames (ORFs) have been identified via systematic expressed sequence tag (EST) analysis of a diverse set of cDNA libraries; however, further systematic EST analysis is no longer an efficient method for identifying new cDNAs. As part of our involvement in the MGC program, we have developed a scalable method for targeted recovery of cDNA clones to facilitate recovery of genes absent from the MGC collection. First, cDNA is synthesized from various RNAs, followed by polymerase chain reaction (PCR) amplification of transcripts in 96-well plates using gene-specific primer pairs flanking the ORFs. Amplicons are cloned into a sequencing vector, and full-length sequences are obtained. Sequences are processed and assembled using Phred and Phrap, and analyzed using Consed and a number of bioinformatics methods we have developed. Sequences are compared with the Reference Sequence (RefSeq) database, and validation of sequence discrepancies is attempted using other sequence databases including dbEST and dbSNP. Clones with identical sequence to RefSeq or containing only validated changes will become part of the MGC human gene collection. Clones containing novel splice variants or polymorphisms have also been identified. Our approach to clone recovery, applied at large scale, has the potential to recover many and possibly most of the genes absent from the MGC collection. [ABSTRACT FROM AUTHOR]

Li-Fraumeni syndrome (LFS) is a hereditary cancer predisposition syndrome associated with a highly penetrant cancer spectrum characterized by germline TP53 mutations. We characterized the first LFS zebrafish hotspot mutants, tp53 R217H and R242H (human R248H and R273H), and found these mutants exhibit partial-to-no activation of p53 target genes, have defective cell-cycle checkpoints, and display partial-to-full resistance to apoptosis, although the R217H mutation has hypomorphic characteristics. Spontaneous tumor development histologically resembling human sarcomas was observed as early as 6 months. tp53 R242H mutants had a higher lifetime tumor incidence compared to tp53 null and R217H mutants, suggesting it is a more aggressive mutation. We observed mutation-specific tumor phenotypes across tp53 mutants with associated diverse transcriptomic and DNA methylome profiles in tp53 mutant larvae, impacting metabolism, cell signalling, and biomacromolecule synthesis and degradation. These tp53 zebrafish mutants demonstrate fidelity to their human counterparts and provide new insights into underlying tumorigenesis mechanisms and kinetics that suggest metabolic rewiring and cellular signalling changes occur prior to tumor initiation, which will guide targeted therapeutics for LFS., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jason Berman reports financial support was provided by Canadian Institutes of Health Research. Jason Berman reports a relationship with Oxford Immune Algorithmics that includes: board membership. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. J.N.B. is a member of the Scientific Advisory Board of Oxford Immune Algorithmics., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The somatic missense point mutation c.402C>G (p.C134W) in the FOXL2 transcription factor is pathognomonic for adult-type granulosa cell tumors (AGCT) and a diagnostic marker for this tumor type. However, the molecular consequences of this mutation and its contribution to the mechanisms of AGCT pathogenesis remain unclear. To explore these mechanisms, we engineered V5-FOXL2 WT - and V5-FOXL2 C134W -inducible isogenic cell lines and performed chromatin immunoprecipitation sequencing and transcriptome profiling. FOXL2 C134W associated with the majority of the FOXL2 wild-type DNA elements as well as a large collection of unique elements genome wide. This model enabled confirmation of altered DNA-binding specificity for FOXL2 C134W and identification of unique targets of FOXL2 C134W including SLC35F2 , whose expression increased sensitivity to YM155. Our results suggest FOXL2 C134W drives AGCT by altering the binding affinity of FOXL2-containing complexes to engage an oncogenic transcriptional program. SIGNIFICANCE: A mechanistic understanding of FOXL2 C134W -induced regulatory state alterations drives discovery of a rationally designed therapeutic strategy., (©2020 American Association for Cancer Research.)

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

The yeast Sup35 protein is a subunit of the translation termination factor, and its conversion to the [PSI + ] prion state leads to more translational read-through. Although extensive studies have been done on [PSI + ], changes at the proteomic level have not been performed exhaustively. We therefore used a SILAC-based quantitative mass spectrometry approach and identified 4187 proteins from both [psi - ] and [PSI + ] strains. Surprisingly, there was very little difference between the two proteomes under standard growth conditions. We found however that several [PSI + ] strains harbored an additional chromosome, such as chromosome I. Albeit, we found no evidence to support that [PSI + ] induces chromosomal instability (CIN). Instead we hypothesized that the selective pressure applied during the establishment of [PSI + ]-containing strains could lead to a supernumerary chromosome due to the presence of the ade1-14 selective marker for translational read-through. We therefore verified that there was no prevalence of disomy among newly generated [PSI + ] strains in absence of strong selection pressure. We also noticed that low amounts of adenine in media could lead to higher levels of mitochondrial DNA in [PSI + ] in ade1-14 cells. Our study has important significance for the establishment and manipulation of yeast strains with the Sup35 prion.

Rhodobacter capsulatus is a model organism for studying a novel type of horizontal gene transfer mediated by a phage-like gene transfer agent (RcGTA). Here we report the draft genome sequences of six R. capsulatus strains that exhibit different RcGTA properties, including RcGTA overproducers, RcGTA nonproducers, and/or RcGTA nonreceivers.

Follicular lymphoma (FL) and the GCB subtype of diffuse large B-cell lymphoma (DLBCL) derive from germinal center B cells. Targeted resequencing studies have revealed mutations in various genes encoding proteins in the NF-kappaB pathway that contribute to the activated B-cell (ABC) DLBCL subtype, but thus far few GCB-specific mutations have been identified. Here we report recurrent somatic mutations affecting the polycomb-group oncogene EZH2, which encodes a histone methyltransferase responsible for trimethylating Lys27 of histone H3 (H3K27). After the recent discovery of mutations in KDM6A (UTX), which encodes the histone H3K27me3 demethylase UTX, in several cancer types, EZH2 is the second histone methyltransferase gene found to be mutated in cancer. These mutations, which result in the replacement of a single tyrosine in the SET domain of the EZH2 protein (Tyr641), occur in 21.7% of GCB DLBCLs and 7.2% of FLs and are absent from ABC DLBCLs. Our data are consistent with the notion that EZH2 proteins with mutant Tyr641 have reduced enzymatic activity in vitro.

Since its start, the Mammalian Gene Collection (MGC) has sought to provide at least one full-protein-coding sequence cDNA clone for every human and mouse gene with a RefSeq transcript, and at least 6200 rat genes. The MGC cloning effort initially relied on random expressed sequence tag screening of cDNA libraries. Here, we summarize our recent progress using directed RT-PCR cloning and DNA synthesis. The MGC now contains clones with the entire protein-coding sequence for 92% of human and 89% of mouse genes with curated RefSeq (NM-accession) transcripts, and for 97% of human and 96% of mouse genes with curated RefSeq transcripts that have one or more PubMed publications, in addition to clones for more than 6300 rat genes. These high-quality MGC clones and their sequences are accessible without restriction to researchers worldwide.

To facilitate discovery of novel human embryonic stem cell (ESC) transcripts, we generated 2.5 million LongSAGE tags from 9 human ESC lines. Analysis of this data revealed that ESCs express proportionately more RNA binding proteins compared with terminally differentiated cells, and identified novel ESC transcripts, at least one of which may represent a marker of the pluripotent state.