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Mutational processes acting on cancer genomes can be traced by investigating mutational signatures. Because high sequencing costs limit current studies to small numbers of good-quality samples, we propose a robust, cost- and time-effective method, called mutREAD, to detect mutational signatures from small quantities of DNA, including degraded samples. We show that mutREAD recapitulates mutational signatures identified by whole genome sequencing, and will ultimately allow the study of mutational signatures in larger cohorts and, by compatibility with formalin-fixed paraffin-embedded samples, in clinical settings. Sequencing tumour genomes can reveal information about the processes that drive the formation of cancer. Here, the authors describe a method that can detect these mutational signatures from small amounts of DNA and degraded samples.

Long non-coding RNAs are being increasingly recognised as important molecules involved in regulating a diverse array of biological functions. For example, many long non-coding RNAs have been associated with tumourigenesis and in this context their molecular functions often involves impacting on chromatin and transcriptional control processes. One important cellular control system that is often deregulated in cancer cells is the ERK MAP kinase pathway. Here we have investigated whether ERK pathway signaling in response to EGF stimulation, leads to changes in the production of long non-coding RNAs. We identify several different classes of EGF pathway-regulated lncRNAs. We focus on one of the inducible lincRNAs, EGF inducible long intergenic non-coding RNA 1 (EINCR1). EINCR1 is predominantly nuclear and shows delayed activation kinetics compared to other immediate-early EGF-inducible genes. In humans it is expressed in a tissue-specific manner and is mainly confined to the heart but it exhibits little evolutionary conservation. Importantly, in several cancers EINCR1 shows elevated expression levels which correlate with poor survival in lung adenocarcinoma patients. In the context of lung adenocarcinomas, EINCR1 expression is anti-correlated with the expression of several protein coding EGF-regulated genes. A potential functional connection is demonstrated as EINCR1 overexpression is shown to reduce the expression of EGF-regulated protein coding genes including FOS and FOSB.

BackgroundWhile p53 mutations occur early in Barrett’s oesophagus (BE) progression to oesophageal adenocarcinoma (EAC), their role in gastric cardia stem cells remains unclear.ObjectiveThis study investigates the impact of p53 mutation on the fate and function of cardia progenitor cells in BE to EAC progression, particularly under the duress of chronic injury.DesignWe used a BE mouse model (L2-IL1β) harbouring a Trp53 mutation (R172H) to study the effects of p53 on Cck2r+ cardia progenitor cells. We employed lineage tracing, pathological analysis, organoid cultures, single-cell RNA sequencing (scRNA-seq) and computational analyses to investigate changes in progenitor cell behaviour, differentiation patterns and tumour progression. Additionally, we performed orthotopic transplantation of sorted metaplastic and mutant progenitor cells to assess their tumourigenic potential in vivo.ResultsThe p53 mutation acts as a switch to expand progenitor cells and inhibit their differentiation towards metaplasia, but only amidst chronic injury. In L2-IL1β mice, p53 mutation increased progenitors expansion and lineage-tracing with a shift from metaplasia to dysplasia. scRNA-seq revealed dysplastic cells arise directly from mutant progenitors rather than progressing through metaplasia. In vitro, p53 mutation enhanced BE progenitors’ organoid-forming efficiency, growth, DNA damage resistance and progression to aneuploidy. Sorted metaplastic cells grew poorly with no progression to dysplasia, while mutant progenitors gave rise to dysplasia in orthotopic transplantation. Computational analyses indicated that p53 mutation inhibited stem cell differentiation through Notch activation.Conclusionsp53 mutation contributes to BE progression by increasing expansion and fitness of undifferentiated cardia progenitors and preventing their differentiation towards metaplasia.

Background: A non-endoscopic capsule-sponge device allows sampling the entire length of the esophagus. Here, we compared microbiomes of the oral cavity, esophagus, and gastric corpus collected by oral swab, capsule-sponge device, and endoscopic biopsy, respectively, in patients representing three distinct risk profiles for esophageal squamous cell carcinoma (ESCC). Methods: The study enrolled 11 patients with esophageal squamous intraepithelial neoplasia, 21 patients after curative treatment for head and neck squamous cell cancer (HNSCC) (HNSCC survivors), and 40 patients with functional dyspeptic (FD) symptoms. Microbial genomic DNA was analyzed using 16S rRNA gene amplicon sequencing. Results: The Shannon index of the capsule-sponge sample microbiota was significantly higher in FD group than in patients after treatment for HNSCC, and the Chao index of gastric samples differed between HNSCC survivors and FD patients. Analysis of the β-diversity of FD patients, HNSCC, and esophageal squamous intraepithelial neoplasia showed that different genera formed at each location. The abundance of 205, 116, and 9 genera differed between FD patients and HNSCC survivors in the gastric, capsule-sponge, and oral samples, respectively; 33 genera differed between the FD group and patients with esophageal squamous intraepithelial neoplasia in capsule-sponge samples. Conclusions: The bacterial communities of the upper digestive tract were clustered according to the anatomic site. Despite substantial differences in gastric and esophageal microbiota samples between FD patients and HNSCC survivors, the microbial members and diversity showed small differences between FD patients and those with esophageal squamous intraepithelial neoplasia. It remains unclear whether gastric and esophageal dysbiosis is associated with or is a consequence of treatment for HNSCC.

It has recently become apparent that cells encode a large number of novel non-protein-coding genes called long non-coding RNAs (lncRNAs). Whilst the biological function of many lncRNAs remains unknown, recent evidence has suggested that lncRNAs may be important regulators of cellular growth, differentiation and may play a significant role in cancer. Epidermal growth factor (EGF) - an activator of the ERK1/2 signalling cascade - is an important spatio-temporal regulator of transcription and, ultimately, of cellular growth and movement. EGF stimulation triggers a wave-like expression of immediate-early genes (IE genes), followed by delayed-early genes (DE genes) and secondary-response genes (SR genes). Over the years, considerable effort has been made to unravel the regulatory loops downstream of EGF signalling. This study investigated whether lncRNAs are sensitive to EGF signalling and whether they play a role in the transcriptional programme associated with EGF signalling. In order to identify lncRNAs regulated by EGF signalling, I sequenced nuclear RNA in the presence or absence of EGF stimulation. RNA-seq data showed that 173 lncRNAs are upregulated by EGF, of which 89 were intergenic lncRNAs (lincRNAs). The time-dependent expression profile of EGF-upregulated lincRNAs followed the well-established expression pattern of IE genes. Finally, investigation of the expression of lincRNAs in primary breast and lung cancer cells showed that EGF-upregulated lincRNAs were differentially expressed in cancer. The EGF-dependent induction profile and cancer enrichment were particularly strong for one of the transcripts - EGF-induced lncRNA 1 (EIN1) - and I selected it for further studies.Firstly, using bioinformatics and biochemical approaches, I confirmed the non-coding status of the EIN1 transcript. Secondly, I confirmed that EIN1 transcription is ERK1/2-dependent and is independent of protein synthesis. Investigation of EIN1 expression in normal tissues showed its high enrichment in the human cardiovascular system. At the cellular level, the EIN1 transcript was predominantly found in the nucleus. Functionally, the depletion of endogenous EIN1 transcripts (using the newly developed CRISPRi approach) led to changes in the EGF-dependent transcription programme. EIN1 downregulation resulted in the addition of normally EGF-independent genes into the EGF-dependent expression programme.Collectively, these results show that EGF (via the ERK1/2 pathway) can regulate transcription of lincRNAs. The EIN1 example suggests that lincRNAs may play a crucial role in the modulation of the EGF-dependent expression programme by limiting of the scope of the programme.

Gastric epithelial cells maintain homeostasis through dynamic self-renewal mechanisms involving stem and progenitor cells; however, identifying them has been challenging. This study aims to identify stem cells of healthy gastric epithelium and cell type-specific regulators defining gastric epithelial homeostasis via single-nucleus multiome analysis. Ten unique gastric samples were collected from 8-12 week old wildtype mice. Isolated nuclei were subjected to simultaneous profiling of gene expression and chromatin accessibility. After quality control, 31,598 cells were analyzed with Seurat and Signac using weighted-nearest neighbors analysis for joint RNA and ATAC clustering. Furthermore, SCENIC+, MultiVelo, EpiCHAOS and Cell plasticity score were used to uncover gene regulatory networks, cell state dynamics and lineage trajectories. Our analyses were validated by the identification of known regulators of stem-cell differentiation into mature cell types. More importantly, it revealed previously uncharacterized regulatory networks comprising novel transcription factor combinations that define cell identities, including , , and as candidate regulators of parietal, foveolar, chief and neck cells, respectively. Further, our data support the identity of isthmus cells as stem-like cells of healthy gastric epithelium, as evidenced by epigenetic plasticity that simultaneously contains open chromatin states of all differentiated cell types in the absence of transcriptional reprogramming. Consistent with Waddington's epigenetic landscape hypothesis, gastric epithelial homeostasis is controlled by orchestrated epigenetic and transcriptional programs. Contrary to the prevailing hypothesis, stem cells can be defined not by a separate epigenetic state but by epigenetic superposition of differentiated cell states. Future work is needed to define the universality of these results.

Background: The Human Cell Atlas is a large international collaborative effort to map all cell types of the human body. Single cell RNA sequencing can generate high quality data for the delivery of such an atlas. However, delays between fresh sample collection and processing may lead to poor data and difficulties in experimental design. Despite this, there has not yet been a systematic assessment of the effect of cold storage time on the quality of scRNAseq. Results: This study assessed the effect of cold storage on fresh healthy spleen, oesophagus and lung from ≥5 donors over 72 hours. We collected 240,000 high quality single cell transcriptomes with detailed cell type annotations and whole genome sequences of donors, enabling future eQTL studies. Our data provide a valuable resource for the study of these three organs and will allow cross-organ comparison of cell types. We see little effect of cold ischaemic time on cell viability, yield, total number of reads per cell and other quality control metrics in any of the tissues within the first 24 hours. However, we observed higher percentage of mitochondrial reads, indicative of cellular stress, and increased contamination by background ambient RNA reads in the 72h samples in spleen, which is cell type specific. Conclusions: In conclusion, we present robust protocols for tissue preservation for up to 24 hours prior to scRNAseq analysis. This greatly facilitates the logistics of sample collection for Human Cell Atlas or clinical studies since it increases the time frames for sample processing. Footnotes * https://prod.data.humancellatlas.org/explore/projects/c4077b3c-5c98-4d26-a614-246d12c2e5d7 * https://www.tissuestabilitycellatlas.org

High levels of histone acetylation are associated with the regulatory elements of active genes, suggesting a link between acetylation and gene activation. However, several studies have shown that histone acetylation dynamics rather than hyperacetylation per se are important determinants in gene activation, particularly at inducible genes. We revisited this model, in the context of EGF-inducible gene expression and found that rather than a simple unifying model, there are two broad classes of genes; one in which high lysine acetylation activity is required for efficient gene activation, and a second group where the opposite occurs and high acetylation activity is inhibitory. We examined the latter class in more detail using EGR2 as a model gene and found that lysine acetylation levels are critical for several activation parameters, including the timing of expression onset, and overall amplitudes of the transcriptional response. In contrast, DUSP1 responds in the canonical manner and its transcriptional activity is promoted by acetylation. Single cell approaches demonstrate heterogenous DUSP1 activation kinetics and that acetylation levels influence allele activation frequencies. Our data therefore point to a complex interplay between acetylation dynamics and target gene induction, which cannot simply be explained by a unified response to acetylation activity. Instead, acetylation level thresholds are an important determinant of transcriptional induction dynamics that are sensed in a gene-specific manner.