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The function of histone modifications in initiating and regulating the chromosomal events of the meiotic prophase remains poorly understood. In Saccharomyces cerevisiae , we examined the genome‐wide localization of histone H3 lysine 4 trimethylation (H3K4me3) along meiosis and its relationship to gene expression and position of the programmed double‐strand breaks (DSBs) that initiate interhomologue recombination, essential to yield viable haploid gametes. We find that the level of H3K4me3 is constitutively higher close to DSB sites, independently of local gene expression levels. Without Set1, the H3K4 methylase, 84% of the DSB sites exhibit a severely reduced DSB frequency, the reduction being quantitatively correlated with the local level of H3K4me3 in wild‐type cells. Further, we show that this differential histone mark is already established in vegetative cells, being higher in DSB‐prone regions than in regions with no or little DSB. Taken together, our results demonstrate that H3K4me3 is a prominent and preexisting mark of active meiotic recombination initiation sites. Novel perspectives to dissect the various layers of the controls of meiotic DSB formation are discussed.

piRNAs keep Nanos in check Small RNAs of the piRNA (Piwi-associated RNA) class have various functions in the germline — repressing transposable elements, maintaining germline stem cells and promoting genome stability. Rouget et al . have now uncovered a function for piRNAs outside the germline, in the fruit fly embryo. Specifically, piRNAs that are complementary to a sequence in the 3′-untranslated region of an mRNA for the embryonic posterior morphogen Nanos facilitate adenylation of the mRNA and its subsequent decay. Without piRNAs, Nanos accumulates and developmental defects result. Piwi-associated RNAs (piRNAs) are small RNAs with several functions in the germline, such as repressing transposable elements and helping to maintain germline stem cells. Now, a function for piRNAs has been discovered outside the germline, in the fruitfly embryo. Specifically, piRNAs are required for the decay of the messenger RNA encoding the posterior morphogen Nanos. When piRNA-induced regulation is impaired, this mRNA is stabilized and developmental defects ensue. Piwi-associated RNAs (piRNAs), a specific class of 24- to 30-nucleotide-long RNAs produced by the Piwi-type of Argonaute proteins, have a specific germline function in repressing transposable elements. This repression is thought to involve heterochromatin formation and transcriptional and post-transcriptional silencing 1 , 2 , 3 , 4 , 5 , 6 . The piRNA pathway has other essential functions in germline stem cell maintenance 7 and in maintaining germline DNA integrity 8 , 9 , 10 . Here we uncover an unexpected function of the piRNA pathway in the decay of maternal messenger RNAs and in translational repression in the early embryo. A subset of maternal mRNAs is degraded in the embryo at the maternal-to-zygotic transition. In Drosophila , maternal mRNA degradation depends on the RNA-binding protein Smaug and the deadenylase CCR4 11 , 12 , 13 , as well as the zygotic expression of a microRNA cluster 14 . Using mRNA encoding the embryonic posterior morphogen Nanos (Nos) as a paradigm to study maternal mRNA decay, we found that CCR4-mediated deadenylation of nos depends on components of the piRNA pathway including piRNAs complementary to a specific region in the nos 3′ untranslated region. Reduced deadenylation when piRNA-induced regulation is impaired correlates with nos mRNA stabilization and translational derepression in the embryo, resulting in head development defects. Aubergine, one of the Argonaute proteins in the piRNA pathway, is present in a complex with Smaug, CCR4, nos mRNA and piRNAs that target the nos 3′ untranslated region, in the bulk of the embryo. We propose that piRNAs and their associated proteins act together with Smaug to recruit the CCR4 deadenylation complex to specific mRNAs, thus promoting their decay. Because the piRNAs involved in this regulation are produced from transposable elements, this identifies a direct developmental function for transposable elements in the regulation of gene expression.

Acute myeloid leukemias (AML) are characterized by mutations of tumor suppressor and oncogenes, involving distinct genes in adults and children. While certain mutations have been associated with the increased risk of AML relapse, the genomic landscape of primary chemotherapy-resistant AML is not well defined. As part of the TARGET initiative, we performed whole-genome DNA and transcriptome RNA and miRNA sequencing analysis of pediatric AML with failure of induction chemotherapy. We identified at least three genetic groups of patients with induction failure, including those with NUP98 rearrangements, somatic mutations of WT1 in the absence of apparent NUP98 mutations, and additional recurrent variants including those in KMT2C and MLLT10. Comparison of specimens before and after chemotherapy revealed distinct and invariant gene expression programs. While exhibiting overt therapy resistance, these leukemias nonetheless showed diverse forms of clonal evolution upon chemotherapy exposure. This included selection for mutant alleles of FRMD8, DHX32, PIK3R1, SHANK3, MKLN1, as well as persistence of WT1 and TP53 mutant clones, and elimination of FLT3, PTPN11, and NRAS mutant clones. These findings delineate genetic mechanisms of primary chemotherapy resistance in pediatric AML, which should inform improved approaches for its diagnosis and therapy.

Canonical animal microRNAs (miRNAs) are generated by sequential cleavage of precursor substrates by the Drosha and Dicer RNase III enzymes. Several variant pathways exploit other RNA metabolic activities to generate functional miRNAs. However, all of these pathways culminate in Dicer cleavage, suggesting that this is a unifying feature of miRNA biogenesis. Here, we show that maturation of miR-451, a functional miRNA that is perfectly conserved among vertebrates, is independent of Dicer. Instead, structure-function and knockdown studies indicate that Drosha generates a short pre-mir-451 hairpin that is directly cleaved by Ago2 and followed by resection of its 3′ terminus. We provide stringent evidence for this model by showing that Dicer knockout cells can generate mature miR-451 but not other miRNAs, whereas Ago2 knockout cells reconstituted with wild-type Ago2, but not Slicer-deficient Ago2, can process miR-451. Finally, we show that the mir-451 backbone is amenable to reprogramming, permitting vector-driven expression of diverse functional miRNAs in the absence of Dicer. Beyond the demonstration of an alternative strategy to direct gene silencing, these observations open the way for transgenic rescue of Dicer conditional knockouts.

Senescence is a state of stable cell cycle exit with important implications for development and disease. Here, we demonstrate that the chromatin remodeling enzyme ATRX is required for therapy-induced senescence. ATRX accumulates in nuclear foci and is required for therapy-induced senescence in multiple types of transformed cells exposed to either DNA damaging agents or CDK4 inhibitors. Mobilization into foci depends on the ability of ATRX to interact with H3K9me3 histone and HP1. Foci form soon after cells exit the cell cycle, before other hallmarks of senescence appear. Eliminating ATRX in senescent cells destabilizes the senescence-associated heterochromatic foci. Additionally, ATRX binds to and suppresses expression from the HRAS locus; repression of HRAS is sufficient to promote the transition of quiescent cells into senescence and preventing repression blocks progression into senescence. Thus ATRX is a critical regulator of therapy-induced senescence and acts in multiple ways to drive cells into this state. Therapy induced senescence (TIS) is a growth suppressive program activated by cytostatic agents in some cancer cells. Here the authors show that the chromatin remodeling enzyme ATRX is a regulator of TIS and drives cells into this state via multiple mechanisms.

Ductal carcinoma in situ (DCIS) is considered a non-invasive precursor to breast cancer, and although associated with an increased risk of developing invasive disease, many women with DCIS will never progress beyond their in situ diagnosis. The path from normal duct to invasive ductal carcinoma (IDC) is not well understood, and efforts to do so are hampered by the substantial heterogeneity that exists between patients, and even within patients. Here we show gene expression analysis from > 2,000 individually micro-dissected ductal lesions representing 145 patients. Combining all samples into one continuous trajectory we show there is a progressive loss in basal layer integrity heading towards IDC, coupled with two epithelial to mesenchymal transitions, one early and a second coinciding with the convergence of DCIS and IDC expression profiles. We identify early processes and potential biomarkers, including CAMK2N1 , MNX1 , ADCY5 , HOXC11 and ANKRD22 , whose reduced expression is associated with the progression of DCIS to invasive breast cancer. Progression from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) remains poorly understood. Here, the authors analyse over 2700 micro-dissected samples using transcriptomics to identify genes that characterise different stages of DCIS to IDC progression, and identify IDC-associated markers within early-stage lesions.

To test the performance of a new sequencing platform, develop an updated somatic calling pipeline and establish a reference for future benchmarking experiments, we performed whole-genome sequencing of 3 common cancer cell lines (COLO-829, HCC-1143 and HCC-1187) along with their matched normal cell lines to great sequencing depths (up to 278x coverage) on both Illumina HiSeqX and NovaSeq sequencing instruments. Somatic calling was generally consistent between the two platforms despite minor differences at the read level. We designed and implemented a novel pipeline for the analysis of tumor-normal samples, using multiple variant callers. We show that coupled with a high-confidence filtering strategy, the use of combination of tools improves the accuracy of somatic variant calling. We also demonstrate the utility of the dataset by creating an artificial purity ladder to evaluate the somatic pipeline and benchmark methods for estimating purity and ploidy from tumor-normal pairs. The data and results of the pipeline are made accessible to the cancer genomics community.

Heterogeneity is a hallmark of cancer. The advent of single-cell technologies has helped uncover heterogeneity in a high-throughput manner in different cancers across varied contexts. Here we apply single-cell sequencing technologies to reveal inherent heterogeneity in assumptively monoclonal pancreatic cancer (PDAC) cell lines and patient-derived organoids (PDOs). Our findings reveal a high degree of both genomic and transcriptomic polyclonality in monolayer PDAC cell lines, custodial variation induced by growing apparently identical cell lines in different laboratories, and transcriptomic shifts in transitioning from 2D to 3D spheroid growth models. Our findings also call into question the validity of widely available immortalized, non-transformed pancreatic lines as contemporaneous “control” lines in experiments. We confirm these findings using a variety of independent assays, including but not limited to whole exome sequencing, single-cell copy number variation sequencing (scCNVseq), single-nuclei assay for transposase-accessible chromatin with sequencing, fluorescence in-situ hybridization, and single-cell RNA sequencing (scRNAseq). We map scRNA expression data to unique genomic clones identified by orthogonally-gathered scCNVseq data of these same PDAC cell lines. Further, while PDOs are known to reflect the cognate in vivo biology of the parental tumor, we identify transcriptomic shifts during ex vivo passage that might hamper their predictive abilities over time. The impact of these findings on rigor and reproducibility of experimental data generated using established preclinical PDAC models between and across laboratories is uncertain, but a matter of concern. It is unclear if the molecular profiles of pancreatic ductal adenocarcinoma (PDAC) preclinical models remain stable during propagation. Here, the authors characterise clonal evolution throughout propagation in PDAC cell lines and a patient-derived organoid using single-cell genomics, transcriptomics and epigenomics.

While the genomes of normal tissues undergo dynamic changes over time, little is understood about the temporal-spatial dynamics of genomes in premalignant tissues that progress to cancer compared to those that remain cancer-free. Here we use whole genome sequencing to contrast genomic alterations in 427 longitudinal samples from 40 patients with stable Barrett’s esophagus compared to 40 Barrett’s patients who progressed to esophageal adenocarcinoma (ESAD). We show the same somatic mutational processes are active in Barrett’s tissue regardless of outcome, with high levels of mutation, ESAD gene and focal chromosomal alterations, and similar mutational signatures. The critical distinction between stable Barrett’s versus those who progress to cancer is acquisition and expansion of TP53 −/− cell populations having complex structural variants and high-level amplifications, which are detectable up to six years prior to a cancer diagnosis. These findings reveal the timing of common somatic genome dynamics in stable Barrett’s esophagus and define key genomic features specific to progression to esophageal adenocarcinoma, both of which are critical for cancer prevention and early detection strategies. Barrett’s esophagus is a pre-malignant condition that can progress to esophageal cancer. Here, the authors carry out whole genome sequencing of samples from patients who did or did not progress to cancer and find that mutations in many genes occur regardless of progression status, but also find features associated with progressive disease.

The role of the immune system in regulating tissue stem cells remains poorly understood, as does the relationship between immune-mediated tissue damage and regeneration. Graft vs. host disease (GVHD) occurring after allogeneic bone marrow transplantation (allo-BMT) involves immune-mediated damage to the intestinal epithelium and its stem cell compartment. To assess impacts of T-cell-driven injury on distinct epithelial constituents, we have performed single cell RNA sequencing on intestinal crypts following experimental BMT. Intestinal stem cells (ISCs) from GVHD mice have exhibited global transcriptomic changes associated with a substantial Interferon-γ response and upregulation of STAT1. To determine its role in crypt function, STAT1 has been deleted within murine intestinal epithelium. Following allo-BMT, STAT1 deficiency has resulted in reduced epithelial proliferation and impaired ISC recovery. Similarly, epithelial Interferon-γ receptor deletion has also attenuated proliferation and ISC recovery post-transplant. Investigating the mechanistic basis underlying this epithelial response, ISC STAT1 expression in GVHD has been found to correlate with upregulation of ISC c-Myc. Furthermore, activated T cells have stimulated Interferon-γ-dependent epithelial regeneration in co-cultured organoids, and Interferon-γ has directly induced STAT1-dependent c-Myc expression and ISC proliferation. These findings illustrate immunologic regulation of a core tissue stem cell program after damage and support a role for Interferon-γ as a direct contributor to epithelial regeneration. The role of the immune system in tissue regeneration remains poorly understood. Here, in bone marrow transplantation and organoid culture models, T-cell-derived Interferon-γ is shown to promote STAT1-dependent intestinal stem cell c-MYC expression and epithelial regeneration.