Linking CRISPR-Cas9 double-strand break profiles to gene editing precision with BreakTag.

Cas9 can cleave DNA in both blunt and staggered configurations, resulting in distinct editing outcomes, but what dictates the type of Cas9 incisions is largely unknown. In this study, we developed BreakTag, a versatile method for profiling Cas9-induced DNA double-strand breaks (DSBs) and identifying the determinants of Cas9 incisions. Overall, we assessed cleavage by SpCas9 at more than 150,000 endogenous on-target and off-target sites targeted by approximately 3,500 single guide RNAs. We found that approximately 35% of SpCas9 DSBs are staggered, and the type of incision is influenced by DNA:gRNA complementarity and the use of engineered Cas9 variants. A machine learning model shows that Cas9 incision is dependent on the protospacer sequence and that human genetic variation impacts the configuration of Cas9 cuts and the DSB repair outcome. Matched datasets of Cas9 and engineered variant incisions with repair outcomes show that Cas9-mediated staggered breaks are linked with precise, templated and predictable single-nucleotide insertions, demonstrating that a scission-based gRNA design can be used to correct clinically relevant pathogenic single-nucleotide deletions.

Adipokines and risk of rheumatoid arthritis: A two-sample multivariable Mendelian randomisation study.

Adiponectin, leptin, and resistin are thought to be involved in the pathogenesis of rheumatoid arthritis (RA). However, the causal relationship between these adipokines and the risk for RA is unclear. We performed a range of two-sample Mendelian randomisation (MR) analyses to assess the causal effect of circulating adiponectin, leptin, and resistin on RA risk in European and East Asian individuals. Different sets of adiponectin-, leptin-, and resistin-related genetic variants were used as instruments for genetically determined adipokine levels. As body mass index (BMI) is a risk factor for RA and affects adipokine levels, multivariable MR was used to calculate the causal effect of each adipokine on RA risk taking BMI into account. Several MR analyses revealed no evidence of a causal relationship between circulating adiponectin, leptin, or resistin levels and RA risk in either Europeans or East Asians. Similarly, multivariable MR did not provide evidence of any causal effect of adiponectin, leptin, or resistin on RA risk when taking BMI into account. This MR study shows for the first time that genetically determined levels of adiponectin, leptin, or resistin do not have a direct causal effect on the risk of developing RA after adjustment for BMI.

rrvgo: a Bioconductor package for interpreting lists of Gene Ontology terms.

Gene Ontology (GO) annotation is often used to guide the biological interpretation of high-throughput omics experiments, e.g. by analysing lists of differentially regulated genes for enriched GO terms. Due to the hierarchical nature of GOs, the resulting lists of enriched terms are usually redundant and difficult to summarise and interpret. To facilitate the interpretation of large lists of GO terms, I developed rrvgo, a Bioconductor package that aims at simplifying the redundancy of GO lists by grouping similar terms based on their semantic similarity. rrvgo also provides different visualization options to guide the interpretation of the summarized GO terms. Considering that several software tools have been developed for this purpose, rrvgo is unique at combining powerful visualizations in a programmatic interface coupled with up-to-date GO gene annotation provided by the Bioconductor project.

E3 ubiquitin ligase Atrogin-1 mediates adaptive resistance to KIT-targeted inhibition in gastrointestinal stromal tumor.

KIT/PDGFRA oncogenic tyrosine kinase signaling is the central oncogenic event in most gastrointestinal stromal tumors (GIST), which are human malignant mesenchymal neoplasms that often feature myogenic differentiation. Although targeted inhibition of KIT/PDGFRA provides substantial clinical benefit, GIST cells adapt to KIT/PDGFRA driver suppression and eventually develop resistance. The specific molecular events leading to adaptive resistance in GIST remain unclear. By using clinically representative in vitro and in vivo GIST models and GIST patients' samples, we found that the E3 ubiquitin ligase Atrogin-1 (FBXO32)-the main effector of muscular atrophy in cachexia-resulted in the most critical gene derepressed in response to KIT inhibition, regardless the type of KIT primary or secondary mutation. Atrogin-1 in GISTs is transcriptionally controlled by the KIT-FOXO3a axis, thus indicating overlap with Atrogin-1 regulation mechanisms in nonneoplastic muscle cells. Further, Atrogin-1 overexpression was a GIST-cell-specific pro-survival mechanism that enabled the adaptation to KIT-targeted inhibition by apoptosis evasion through cell quiescence. Buttressed on these findings, we established in vitro and in vivo the preclinical proof-of-concept for co-targeting KIT and the ubiquitin pathway to maximize the therapeutic response to first-line imatinib treatment.

DNA Methylation Profiling of Human Hepatocarcinogenesis.

BACKGROUND AND AIMS: Mutations in TERT (telomerase reverse transcriptase) promoter are established gatekeepers in early hepatocarcinogenesis, but little is known about other molecular alterations driving this process. Epigenetic deregulation is a critical event in early malignancies. Thus, we aimed to (1) analyze DNA methylation changes during the transition from preneoplastic lesions to early HCC (eHCC) and identify candidate epigenetic gatekeepers, and to (2) assess the prognostic potential of methylation changes in cirrhotic tissue. APPROACH AND RESULTS: Methylome profiling was performed using Illumina HumanMethylation450 (485,000 cytosine-phosphateguanine, 96% of known cytosine-phosphateguanine islands), with data available for a total of 390 samples: 16 healthy liver, 139 cirrhotic tissue, 8 dysplastic nodules, and 227 HCC samples, including 40 eHCC below 2cm. A phylo-epigenetic tree derived from the Euclidean distances between differentially DNA-methylated sites (n = 421,997) revealed a gradient of methylation changes spanning healthy liver, cirrhotic tissue, dysplastic nodules, and HCC with closest proximity of dysplasia to HCC. Focusing on promoter regions, we identified epigenetic gatekeeper candidates with an increasing proportion of hypermethylated samples (beta value > 0.5) from cirrhotic tissue (<1%), to dysplastic nodules (≥25%), to eHCC (≥50%), and confirmed inverse correlation between DNA methylation and gene expression for TSPYL5 (testis-specific Y-encoded-like protein 5), KCNA3 (potassium voltage-gated channel, shaker-related subfamily, member 3), LDHB (lactate dehydrogenase B), and SPINT2 (serine peptidase inhibitor, Kunitz type 2) (all P < 0.001). Unsupervised clustering of genome-wide methylation profiles of cirrhotic tissue identified two clusters, M1 and M2, with 42% and 58% of patients, respectively, which correlates with survival (P < 0.05), independent of etiology. CONCLUSIONS: Genome-wide DNA-methylation profiles accurately discriminate the different histological stages of human hepatocarcinogenesis. We report on epigenetic gatekeepers in the transition between dysplastic nodules and eHCC. DNA-methylation changes in cirrhotic tissue correlate with clinical outcomes.

Genome-wide detection of DNA double-strand breaks by in-suspension BLISS.

sBLISS (in-suspension breaks labeling in situ and sequencing) is a versatile and widely applicable method for identification of endogenous and induced DNA double-strand breaks (DSBs) in any cell type that can be brought into suspension. sBLISS provides genome-wide profiles of the most consequential DNA lesion implicated in a variety of pathological, but also physiological, processes. In sBLISS, after in situ labeling, DSB ends are linearly amplified, followed by next-generation sequencing and DSB landscape analysis. Here, we present a step-by-step experimental protocol for sBLISS, as well as a basic computational analysis. The main advantages of sBLISS are (i) the suspension setup, which renders the protocol user-friendly and easily scalable; (ii) the possibility of adapting it to a high-throughput or single-cell workflow; and (iii) its flexibility and its applicability to virtually every cell type, including patient-derived cells, organoids, and isolated nuclei. The wet-lab protocol can be completed in 1.5 weeks and is suitable for researchers with intermediate expertise in molecular biology and genomics. For the computational analyses, basic-to-intermediate bioinformatics expertise is required.

Signalling codes for the maintenance and lineage commitment of embryonic gastric epithelial progenitors.

The identity of embryonic gastric epithelial progenitors is unknown. We used single-cell RNA-sequencing, genetic lineage tracing and organoid assays to assess whether Axin2- and Lgr5-expressing cells are gastric progenitors in the developing mouse stomach. We show that Axin2+ cells represent a transient population of embryonic epithelial cells in the forestomach. Lgr5+ cells generate both glandular corpus and squamous forestomach organoids ex vivo Only Lgr5+ progenitors give rise to zymogenic cells in culture. Modulating the activity of the WNT, BMP and Notch pathways in vivo and ex vivo, we found that WNTs are essential for the maintenance of Lgr5+ epithelial cells. Notch prevents differentiation of the embryonic epithelial cells along all secretory lineages and hence ensures their maintenance. Whereas WNTs promote differentiation of the embryonic progenitors along the zymogenic cell lineage, BMPs enhance their differentiation along the parietal lineage. In contrast, WNTs and BMPs are required to suppress differentiation of embryonic gastric epithelium along the pit cell lineage. Thus, coordinated action of the WNT, BMP and Notch pathways controls cell fate determination in the embryonic gastric epithelium.

The RNA fold interactome of evolutionary conserved RNA structures in S. cerevisiae.

RNA-binding proteins play key roles in regulation of gene expression via recognition of structural features in RNA molecules. Here we apply a quantitative RNA pull-down approach to 186 evolutionary conserved RNA structures and report 162 interacting proteins. Unlike global RNA interactome capture, we associate individual RNA structures within messenger RNA with their interacting proteins. Of our binders 69% are known RNA-binding proteins, whereas some are previously unrelated to RNA binding and do not harbor canonical RNA-binding domains. While current knowledge about RNA-binding proteins relates to their functions at 5' or 3'-UTRs, we report a significant number of them binding to RNA folds in the coding regions of mRNAs. Using an in vivo reporter screen and pulsed SILAC, we characterize a subset of mRNA-RBP pairs and thus connect structural RNA features to functionality. Ultimately, we here present a generic, scalable approach to interrogate the increasing number of RNA structural motifs.

High-throughput identification of synthetic riboswitches by barcode-free amplicon-sequencing in human cells.

Synthetic riboswitches mediating ligand-dependent RNA cleavage or splicing-modulation represent elegant tools to control gene expression in various applications, including next-generation gene therapy. However, due to the limited understanding of context-dependent structure-function relationships, the identification of functional riboswitches requires large-scale-screening of aptamer-effector-domain designs, which is hampered by the lack of suitable cellular high-throughput methods. Here we describe a fast and broadly applicable method to functionally screen complex riboswitch libraries (~1.8 × 104 constructs) by cDNA-amplicon-sequencing in transiently transfected and stimulated human cells. The self-barcoding nature of each construct enables quantification of differential mRNA levels without additional pre-selection or cDNA-manipulation steps. We apply this method to engineer tetracycline- and guanine-responsive ON- and OFF-switches based on hammerhead, hepatitis-delta-virus and Twister ribozymes as well as U1-snRNP polyadenylation-dependent RNA devices. In summary, our method enables fast and efficient high-throughput riboswitch identification, thereby overcoming a major hurdle in the development cascade for therapeutically applicable gene switches.

Spatial Chromosome Folding and Active Transcription Drive DNA Fragility and Formation of Oncogenic MLL Translocations.

How spatial chromosome organization influences genome integrity is still poorly understood. Here, we show that DNA double-strand breaks (DSBs) mediated by topoisomerase 2 (TOP2) activities are enriched at chromatin loop anchors with high transcriptional activity. Recurrent DSBs occur at CCCTC-binding factor (CTCF) and cohesin-bound sites at the bases of chromatin loops, and their frequency positively correlates with transcriptional output and directionality. The physiological relevance of this preferential positioning is indicated by the finding that genes recurrently translocating to drive leukemias are highly transcribed and are enriched at loop anchors. These genes accumulate DSBs at recurrent hotspots that give rise to chromosomal fusions relying on the activity of both TOP2 isoforms and on transcriptional elongation. We propose that transcription and 3D chromosome folding jointly pose a threat to genomic stability and are key contributors to the occurrence of genome rearrangements that drive cancer.

Quantifying post-transcriptional regulation in the development of Drosophila melanogaster.

Even though proteins are produced from mRNA, the correlation between mRNA levels and protein abundances is moderate in most studies, occasionally attributed to complex post-transcriptional regulation. To address this, we generate a paired transcriptome/proteome time course dataset with 14 time points during Drosophila embryogenesis. Despite a limited mRNA-protein correlation (ρ = 0.54), mathematical models describing protein translation and degradation explain 84% of protein time-courses based on the measured mRNA dynamics without assuming complex post transcriptional regulation, and allow for classification of most proteins into four distinct regulatory scenarios. By performing an in-depth characterization of the putatively post-transcriptionally regulated genes, we postulate that the RNA-binding protein Hrb98DE is involved in post-transcriptional control of sugar metabolism in early embryogenesis and partially validate this hypothesis using Hrb98DE knockdown. In summary, we present a systems biology framework for the identification of post-transcriptional gene regulation from large-scale, time-resolved transcriptome and proteome data.

Role for the transcriptional activator ZRF1 in early metastatic events in breast cancer progression and endocrine resistance.

Breast cancer is one of the most common malignancies among women which is often treated with hormone therapy and chemotherapy. Despite the improvements in detection and treatment of breast cancer, the vast majority of breast cancer patients are diagnosed with metastatic disease either at the beginning of the disease or later during treatment. Still, the molecular mechanisms causing a therapy resistant metastatic breast cancer are still elusive. In the present study we addressed the function of the transcriptional activator ZRF1 during breast cancer progression. We provide evidence that ZRF1 plays an essential role for the early metastatic events in vitro and acts like a tumor suppressor protein during the progression of breast invasive ductal carcinoma into a more advanced stage. Hence, depletion of ZRF1 results in the acquisition of metastatic behavior by facilitating the initiation of the metastatic cascade, notably for cell adhesion, migration and invasion. Furthermore absence of ZRF1 provokes endocrine resistance via misregulation of cell death and cell survival related pathways. Taken together, we have identified ZRF1 as an important regulator of breast cancer progression that holds the potential to be explored for new treatment strategies in the future.

PM20D1 is a quantitative trait locus associated with Alzheimer's disease.

The chances to develop Alzheimer's disease (AD) result from a combination of genetic and non-genetic risk factors 1 , the latter likely being mediated by epigenetic mechanisms 2 . In the past, genome-wide association studies (GWAS) have identified an important number of risk loci associated with AD pathology 3 , but a causal relationship remains difficult to establish. In contrast, locus-specific or epigenome-wide association studies (EWAS) have revealed site-specific epigenetic alterations, which provide mechanistic insights for a particular risk gene but often lack the statistical power of GWAS 4 . Here, combining both approaches, we report a previously unidentified association of the peptidase M20-domain-containing protein 1 (PM20D1) with AD. We find that PM20D1 is a methylation and expression quantitative trait locus coupled to an AD-risk associated haplotype, which displays enhancer-like characteristics and contacts the PM20D1 promoter via a haplotype-dependent, CCCTC-binding-factor-mediated chromatin loop. Furthermore, PM20D1 is increased following AD-related neurotoxic insults at symptomatic stages in the APP/PS1 mouse model of AD and in human patients with AD who are carriers of the non-risk haplotype. In line, genetically increasing or decreasing the expression of PM20D1 reduces and aggravates AD-related pathologies, respectively. These findings suggest that in a particular genetic background, PM20D1 contributes to neuroprotection against AD.

Liver X Receptor-Binding DNA Motif Associated With Atherosclerosis-Specific DNA Methylation Profiles of Alu Elements and Neighboring CpG Islands.

BACKGROUND: The signals that determine atherosclerosis-specific DNA methylation profiles are only partially known. We previously identified a 29-bp DNA motif (differential methylation motif [DMM]) proximal to CpG islands (CGIs) that undergo demethylation in advanced human atheromas. Those data hinted that the DMM docks modifiers of DNA methylation and transcription. METHODS AND RESULTS: We sought to functionally characterize the DMM. We showed that the DMM overlaps with the RNA polymerase III-binding B box of Alu short interspersed nuclear elements and contains a DR2 nuclear receptor response element. Pointing to a possible functional role for an Alu DMM, CGIs proximal (<100 bp) to near-intact DMM-harboring Alu are significantly less methylated relative to CGIs proximal to degenerate DMM-harboring Alu or to DMM-devoid mammalian-wide interspersed repeat short interspersed nuclear elements in human arteries. As for DMM-binding factors, LXRB (liver X receptor ß) binds the DMM in a DR2-dependent fashion, and LXR (liver X receptor) agonists induce significant hypermethylation of the bulk of Alu in THP-1 cells. Furthermore, we describe 3 intergenic long noncoding RNAs that harbor a DMM, are under transcriptional control by LXR agonists, and are differentially expressed between normal and atherosclerotic human aortas. Notably, CGIs adjacent to those long noncoding RNAs tend to be hypomethylated in symptomatic relative to stable human atheromas. CONCLUSIONS: Collectively, the data suggest that a DMM is associated with 2 distinct methylation states: relatively low methylation of in cis CGIs and Alu element hypermethylation. Based on the known atheroprotective role of LXRs, we propose that LXR agonist-induced Alu hypermethylation, a landmark of atherosclerosis, is a compensatory rather than proatherogenic response.

Distinct populations of embryonic epithelial progenitors generate Lgr5+ intestinal stem cells.

The adult intestinal stem cells (ISCs) are transcriptionally heterogeneous. As the mechanisms governing their developmental specification are still poorly understood, whether this heterogeneity reflects an early determination of distinct cellular sub-types with potentially distinct physiological functions remains an open question. We investigate the cellular heterogeneity within the mouse embryonic midgut epithelium at the molecular and functional levels. Cell fate mapping analysis revealed that multiple early embryonic epithelial progenitors give rise to Lgr5+ ISCs. The origin of the molecularly distinct early precursors along the anterior-posterior axis defines the transcriptional signature of embryonic Lgr5+ ISC progenitors. We further show that the early epithelial progenitors have different capacity to generate Lgr5+ ISC progenitors and Axin2+ early precursors display the highest potential.

DNA Methylomes Reveal Biological Networks Involved in Human Eye Development, Functions and Associated Disorders.

This work provides a comprehensive CpG methylation landscape of the different layers of the human eye that unveils the gene networks associated with their biological functions and how these are disrupted in common visual disorders. Herein, we firstly determined the role of CpG methylation in the regulation of ocular tissue-specification and described hypermethylation of retinal transcription factors (i.e., PAX6, RAX, SIX6) in a tissue-dependent manner. Second, we have characterized the DNA methylome of visual disorders linked to internal and external environmental factors. Main conclusions allow certifying that crucial pathways related to Wnt-MAPK signaling pathways or neuroinflammation are epigenetically controlled in the fibrotic disorders involved in retinal detachment, but results also reinforced the contribution of neurovascularization (ETS1, HES5, PRDM16) in diabetic retinopathy. Finally, we had studied the methylome in the most frequent intraocular tumors in adults and children (uveal melanoma and retinoblastoma, respectively). We observed that hypermethylation of tumor suppressor genes is a frequent event in ocular tumors, but also unmethylation is associated with tumorogenesis. Interestingly, unmethylation of the proto-oncogen RAB31 was a predictor of metastasis risk in uveal melanoma. Loss of methylation of the oncogenic mir-17-92 cluster was detected in primary tissues but also in blood from patients.

ZBTB48 is both a vertebrate telomere-binding protein and a transcriptional activator.

Telomeres constitute the ends of linear chromosomes and together with the shelterin complex form a structure essential for genome maintenance and stability. In addition to the constitutive binding of the shelterin complex, other direct, yet more transient interactions are mediated by the CST complex and HOT1/HMBOX1, while subtelomeric variant repeats are recognized by NR2C/F transcription factors. Recently, the Kruppel-like zinc finger protein ZBTB48/HKR3/TZAP has been described as a novel telomere-associated factor in the vertebrate lineage. Here, we show that ZBTB48 binds directly both to telomeric and to subtelomeric variant repeat sequences. ZBTB48 is found at telomeres of human cancer cells regardless of the mode of telomere maintenance and it acts as a negative regulator of telomere length. In addition to its telomeric function, we demonstrate through a combination of RNAseq, ChIPseq and expression proteomics experiments that ZBTB48 acts as a transcriptional activator on a small set of target genes, including mitochondrial fission process 1 (MTFP1). This discovery places ZBTB48 at the interface of telomere length regulation, transcriptional control and mitochondrial metabolism.

The developmental proteome of Drosophila melanogaster.

Drosophila melanogaster is a widely used genetic model organism in developmental biology. While this model organism has been intensively studied at the RNA level, a comprehensive proteomic study covering the complete life cycle is still missing. Here, we apply label-free quantitative proteomics to explore proteome remodeling across Drosophila's life cycle, resulting in 7952 proteins, and provide a high temporal-resolved embryogenesis proteome of 5458 proteins. Our proteome data enabled us to monitor isoform-specific expression of 34 genes during development, to identify the pseudogene Cyp9f3Ψ as a protein-coding gene, and to obtain evidence of 268 small proteins. Moreover, the comparison with available transcriptomic data uncovered examples of poor correlation between mRNA and protein, underscoring the importance of proteomics to study developmental progression. Data integration of our embryogenesis proteome with tissue-specific data revealed spatial and temporal information for further functional studies of yet uncharacterized proteins. Overall, our high resolution proteomes provide a powerful resource and can be explored in detail in our interactive web interface.

Dynamic changes in chromatin states during specification and differentiation of adult intestinal stem cells.

Epigenetic mechanisms, including chromatin structure, chromatin dynamics and histone modifications play an important role for maintenance and differentiation of pluripotent embryonic stem cells. However, little is known about the molecular mechanisms of adult stem cell specification and differentiation. Here, we used intestinal stem cells (ISCs) as a model system to reveal the epigenetic changes coordinating gene expression programs during these processes. We found that two distinct epigenetic mechanisms participate in establishing the transcriptional program promoting ISC specification from embryonic progenitors. A large number of adult ISC signature genes are targets of repressive DNA methylation in embryonic intestinal epithelial progenitors. On the other hand, genes essential for embryonic development acquire H3K27me3 and are silenced during ISC specification. We also show that the repression of ISC signature genes as well as the activation of enterocyte specific genes is accompanied by a global loss of H2A.Z during ISCs differentiation. Our results reveal that, already during ISC specification, an extensive remodeling of chromatin both at promoters and distal regulatory elements organizes transcriptional landscapes operating in differentiated enterocytes, thus explaining similar chromatin modification patterns in the adult gut epithelium.

Id2 controls specification of Lgr5+ intestinal stem cell progenitors during gut development.

The adult intestinal stem cells (ISCs), their hierarchies, mechanisms of maintenance and differentiation have been extensively studied. However, when and how ISCs are established during embryogenesis remains unknown. We show here that the transcription regulator Id2 controls the specification of embryonic Lgr5+ progenitors in the developing murine small intestine. Cell fate mapping analysis revealed that Lgr5+ progenitors emerge at E13.5 in wild-type embryos and differ from the rest on the intestinal epithelium by a characteristic ISC signature. In the absence of Id2, the intestinal epithelium differentiates into Lgr5+ cells already at E9.5. Furthermore, the size of the Lgr5+ cell pool is significantly increased. We show that Id2 restricts the activity of the Wnt signalling pathway at early stages and prevents precocious differentiation of the embryonic intestinal epithelium. Id2-deficient embryonic epithelial cells cultured ex vivo strongly activate Wnt target genes as well as markers of neoplastic transformation and form fast growing undifferentiated spheroids. Furthermore, adult ISCs from Id2-deficient mice display a distinct transcriptional signature, supporting an essential role for Id2 in the correct specification of ISCs.