The agouti-related protein (Agrp) is a powerful orexigenic peptide, but little is known about its transcriptional regulation. The objective of this study was to determine molecular mechanisms for the activation of hypothalamic Agrp and identify compounds that stimulate appetite.

Timely and accurate assembly of the mitotic spindle is critical for the faithful segregation of chromosomes, and centrosome separation is a key step in this process. The timing of centrosome separation varies dramatically between cell types; however, the mechanisms responsible for these differences and its significance are unclear. Here, we show that activation of epidermal growth factor receptor (EGFR) signaling determines the timing of centrosome separation. Premature separation of centrosomes decreases the requirement for the major mitotic kinesin Eg5 for spindle assembly, accelerates mitosis, and decreases the rate of chromosome missegregation. Importantly, EGF stimulation impacts upon centrosome separation and mitotic progression to different degrees in different cell lines. Cells with high EGFR levels fail to arrest in mitosis upon Eg5 inhibition. This has important implications for cancer therapy because cells with high centrosomal response to EGF are more susceptible to combinatorial inhibition of EGFR and Eg5.

A remarkable observation emerging from recent cancer genome analyses is the identification of chromothripsis as a one-off genomic catastrophe, resulting in massive somatic DNA structural rearrangements (SRs). Largely due to lack of suitable model systems, the mechanistic basis of chromothripsis has remained elusive. We developed an integrative method termed "complex alterations after selection and transformation (CAST)," enabling efficient in vitro generation of complex DNA rearrangements including chromothripsis, using cell perturbations coupled with a strong selection barrier followed by massively parallel sequencing. We employed this methodology to characterize catastrophic SR formation processes, their temporal sequence, and their impact on gene expression and cell division. Our in vitro system uncovered a propensity of chromothripsis to occur in cells with damaged telomeres, and in particular in hyperploid cells. Analysis of primary medulloblastoma cancer genomes verified the link between hyperploidy and chromothripsis in vivo. CAST provides the foundation for mechanistic dissection of complex DNA rearrangement processes.

Epigenetic mechanisms have emerged as links between prenatal environmental exposure and disease risk later in life. Here, we studied epigenetic changes associated with maternal smoking at base pair resolution by mapping DNA methylation, histone modifications, and transcription in expectant mothers and their newborn children. We found extensive global differential methylation and carefully evaluated these changes to separate environment associated from genotype-related DNA methylation changes. Differential methylation is enriched in enhancer elements and targets in particular "commuting" enhancers having multiple, regulatory interactions with distal genes. Longitudinal whole-genome bisulfite sequencing revealed that DNA methylation changes associated with maternal smoking persist over years of life. Particularly in children prenatal environmental exposure leads to chromatin transitions into a hyperactive state. Combined DNA methylation, histone modification, and gene expression analyses indicate that differential methylation in enhancer regions is more often functionally translated than methylation changes in promoters or non-regulatory elements. Finally, we show that epigenetic deregulation of a commuting enhancer targeting c-Jun N-terminal kinase 2 (JNK2) is linked to impaired lung function in early childhood.

Double-strand breaks (DSBs) are the most toxic type of DNA lesions. Cells repair these lesions using either end protection- or end resection-coupled mechanisms. To study DSB repair choice, we present the Color Assay Tracing-Repair (CAT-R) to simultaneously quantify DSB repair via end protection and end resection pathways. CAT-R introduces DSBs using CRISPR/Cas9 in a tandem fluorescent reporter, whose repair distinguishes small insertions/deletions from large deletions. We demonstrate CAT-R applications in chemical and genetic screens. First, we evaluate 21 compounds currently in clinical trials which target the DNA damage response. Second, we examine how 417 factors involved in DNA damage response influence the choice between end protection and end resection. Finally, we show that impairing nucleotide excision repair favors error-free repair, providing an alternative way for improving CRISPR/Cas9-based knock-ins. CAT-R is a high-throughput, versatile assay to assess DSB repair choice, which facilitates comprehensive studies of DNA repair and drug efficiency testing.

Synthetic lethality describes a genetic interaction between two perturbations, leading to cell death, whereas neither event alone has a significant effect on cell viability. This concept can be exploited to specifically target tumor cells. CRISPR viability screens have been widely employed to identify cancer vulnerabilities. However, an approach to systematically infer genetic interactions from viability screens is missing.

The occurrence and formation of genomic structural variants (SVs) is known to be influenced by the 3D chromatin architecture, but the extent and magnitude have been challenging to study. Here, we apply Hi-C to study chromatin organization before and after induction of chromothripsis in human cells. We use Hi-C to manually assemble the derivative chromosomes following the occurrence of massive complex rearrangements, which allows us to study the sources of SV formation and their consequences on gene regulation. We observe an action-reaction interplay whereby the 3D chromatin architecture directly impacts the location and formation of SVs. In turn, the SVs reshape the chromatin organization to alter the local topologies, replication timing, and gene regulation in cis We show that SVs have a strong tendency to occur between similar chromatin compartments and replication timing regions. Moreover, we find that SVs frequently occur at 3D loop anchors, that SVs can cause a switch in chromatin compartments and replication timing, and that this is a major source of SV-mediated effects on nearby gene expression changes. Finally, we provide evidence for a general mechanistic bias of the 3D chromatin on SV occurrence using data from more than 2700 patient-derived cancer genomes.

Multiple myeloma is a hematologic malignancy of monoclonal plasma cells that accumulate in the bone marrow. Despite their clinical and pathophysiologic relevance, the roles of bone marrow-infiltrating T cells in treatment-naïve patients are incompletely understood. We investigated whether clonally expanded T cells (i) were detectable in multiple myeloma bone marrow, (ii) showed characteristic immune phenotypes, and (iii) whether dominant clones recognized antigens selectively presented on multiple myeloma cells. Single-cell index sorting and T-cell receptor (TCR) αβ sequencing of bone marrow T cells from 13 treatment-naïve patients showed dominant clonal expansion within CD8+ cytolytic effector compartments, and only a minority of expanded T-cell clones expressed the classic immune-checkpoint molecules PD-1, CTLA-4, or TIM-3. To identify their molecular targets, TCRs of 68 dominant bone marrow clones from five selected patients were reexpressed and incubated with multiple myeloma and non-multiple myeloma cells from corresponding patients. Only 1 of 68 TCRs recognized antigen presented on multiple myeloma cells. This TCR was HLA-C-restricted, self-peptide-specific and could be activated by multiple myeloma cells of multiple patients. The remaining dominant T-cell clones did not recognize multiple myeloma cells and were, in part, specific for antigens associated with chronic viral infections. In conclusion, we showed that dominant bone marrow T-cell clones in treatment-naïve patients rarely recognize antigens presented on multiple myeloma cells and exhibit low expression of classic immune-checkpoint molecules. Our data provide experimental context for experiences from clinical immune-checkpoint inhibition trials and will inform future T cell-dependent therapeutic strategies.

As a consequence of the accumulation of insertion events over evolutionary time, mobile elements now comprise nearly half of the human genome. The Alu, L1, and SVA mobile element families are still duplicating, generating variation between individual genomes. Mobile element insertions (MEI) have been identified as causes for genetic diseases, including hemophilia, neurofibromatosis, and various cancers. Here we present a comprehensive map of 7,380 MEI polymorphisms from the 1000 Genomes Project whole-genome sequencing data of 185 samples in three major populations detected with two detection methods. This catalog enables us to systematically study mutation rates, population segregation, genomic distribution, and functional properties of MEI polymorphisms and to compare MEI to SNP variation from the same individuals. Population allele frequencies of MEI and SNPs are described, broadly, by the same neutral ancestral processes despite vastly different mutation mechanisms and rates, except in coding regions where MEI are virtually absent, presumably due to strong negative selection. A direct comparison of MEI and SNP diversity levels suggests a differential mobile element insertion rate among populations.

Chromosome instability (CIN) is associated with poor survival and therapeutic outcome in a number of malignancies. Despite this correlation, CIN can also lead to growth disadvantages. Here, we show that simultaneous overexpression of the mitotic checkpoint protein Mad2 with Kras(G12D) or Her2 in mammary glands of adult mice results in mitotic checkpoint overactivation and a delay in tumor onset. Time-lapse imaging of organotypic cultures and pathologic analysis prior to tumor establishment reveals error-prone mitosis, mitotic arrest, and cell death. Nonetheless, Mad2 expression persists and increases karyotype complexity in Kras tumors. Faced with the selective pressure of oncogene withdrawal, Mad2-positive tumors have a higher frequency of developing persistent subclones that avoid remission and continue to grow.

To ensure genomic integrity, living organisms have evolved diverse molecular processes for sensing and repairing damaged DNA. If improperly repaired, DNA damage can give rise to different types of mutations, an important class of which are genomic structural variants (SVs). In spite of their importance for phenotypic variation and genome evolution, potential contributors to SV formation in Saccharomyces cerevisiae (budding yeast), a highly tractable model organism, are not fully recognized. Here, we developed and applied a genome-wide assay to identify yeast gene knockout mutants associated with de novo deletion formation, in particular single-strand annealing (SSA)-mediated deletion formation, in a systematic manner. In addition to genes previously linked to genome instability, our approach implicates novel genes involved in chromatin remodeling and meiosis in affecting the rate of SSA-mediated deletion formation in the presence or absence of stress conditions induced by DNA-damaging agents. We closely examined two candidate genes, the chromatin remodeling gene IOC4 and the meiosis-related gene MSH4, which when knocked-out resulted in gene expression alterations affecting genes involved in cell division and chromosome organization, as well as DNA repair and recombination, respectively. Our high-throughput approach facilitates the systematic identification of processes linked to the formation of a major class of genetic variation.

Neuroblastoma is the most common extra-cranial solid tumor of early childhood. Standard therapies are not effective in case of poor prognosis and chemotherapy resistance. To improve drug therapy, it is imperative to discover new targets that play a substantial role in tumorigenesis of neuroblastoma. The mitotic machinery is an attractive target for therapeutic interventions and inhibitors can be developed to target mitotic entry, spindle apparatus, spindle activation checkpoint, and mitotic exit. We present an elaborate analysis pipeline to determine cancer specific therapeutic targets by first performing a focused gene expression analysis to select genes followed by a gene knockdown screening assay of live cells. We interrogated gene expression studies of neuroblastoma tumors and selected 240 genes relevant for tumorigenesis and cell cycle. With these genes we performed time-lapse screening of gene knockdowns in neuroblastoma cells. We classified cellular phenotypes and used the temporal context of the perturbation effect to determine the sequence of events, particularly the mitotic entry preceding cell death. Based upon this phenotype kinetics from the gene knockdown screening, we inferred dynamic gene functions in mitosis and cell proliferation. We identified six genes (DLGAP5, DSCC1, SMO, SNRPD1, SSBP1, and UBE2C) with a vital role in mitosis and these are promising therapeutic targets for neuroblastoma. Images and movies of every time point of all screened genes are available at https://ichip.bioquant.uni-heidelberg.de.

Detailed knowledge of the molecular biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is crucial for understanding of viral replication, host responses, and disease progression. Here, we report gene expression profiles of three SARS-CoV- and SARS-CoV-2-infected human cell lines. SARS-CoV-2 elicited an approximately two-fold higher stimulation of the innate immune response compared to SARS-CoV in the human epithelial cell line Calu-3, including induction of miRNA-155. Single-cell RNA sequencing of infected cells showed that genes induced by virus infections were broadly upregulated, whereas interferon beta/lambda genes, a pro-inflammatory cytokines such as IL-6, were expressed only in small subsets of infected cells. Temporal analysis suggested that transcriptional activities of interferon regulatory factors precede those of nuclear factor κB. Lastly, we identified heat shock protein 90 (HSP90) as a protein relevant for the infection. Inhibition of the HSP90 activity resulted in a reduction of viral replication and pro-inflammatory cytokine expression in primary human airway epithelial cells.

Chromosome loss that results in monosomy is detrimental to viability, yet it is frequently observed in cancers. How cancers survive with monosomy is unknown. Using p53-deficient monosomic cell lines, we find that chromosome loss impairs proliferation and genomic stability. Transcriptome and proteome analysis demonstrates reduced expression of genes encoded on the monosomes, which is partially compensated in some cases. Monosomy also induces global changes in gene expression. Pathway enrichment analysis reveals that genes involved in ribosome biogenesis and translation are downregulated in all monosomic cells analyzed. Consistently, monosomies display defects in protein synthesis and ribosome assembly. We further show that monosomies are incompatible with p53 expression, likely due to defects in ribosome biogenesis. Accordingly, impaired ribosome biogenesis and p53 inactivation are associated with monosomy in cancer. Our systematic study of monosomy in human cells explains why monosomy is so detrimental and reveals the importance of p53 for monosomy occurrence in cancer.

The genome engineering capability of the CRISPR/Cas system depends on the DNA repair machinery to generate the final outcome. Several genes can have an impact on mutations created, but their exact function and contribution to the result of the repair are not completely characterised. This lack of knowledge has limited the ability to comprehend and regulate the editing outcomes. Here, we measure how the absence of 21 repair genes changes the mutation outcomes of Cas9-generated cuts at 2,812 synthetic target sequences in mouse embryonic stem cells. Absence of key non-homologous end joining genes Lig4, Xrcc4, and Xlf abolished small insertions and deletions, while disabling key microhomology-mediated repair genes Nbn and Polq reduced frequency of longer deletions. Complex alleles of combined insertion and deletions were preferentially generated in the absence of Xrcc6. We further discover finer structure in the outcome frequency changes for single nucleotide insertions and deletions between large microhomologies that are differentially modulated by the knockouts. We use the knowledge of the reproducible variation across repair milieus to build predictive models of Cas9 editing results that outperform the current standards. This work improves our understanding of DNA repair gene function, and provides avenues for more precise modulation of CRISPR/Cas9-generated mutations.

Investigating genomic structural variants at basepair resolution is crucial for understanding their formation mechanisms. We identify and analyse 8,943 deletion breakpoints in 1,092 samples from the 1000 Genomes Project. We find breakpoints have more nearby SNPs and indels than the genomic average, likely a consequence of relaxed selection. By investigating the correlation of breakpoints with DNA methylation, Hi-C interactions, and histone marks and the substitution patterns of nucleotides near them, we find that breakpoints with the signature of non-allelic homologous recombination (NAHR) are associated with open chromatin. We hypothesize that some NAHR deletions occur without DNA replication and cell division, in embryonic and germline cells. In contrast, breakpoints associated with non-homologous (NH) mechanisms often have sequence microinsertions, templated from later replicating genomic sites, spaced at two characteristic distances from the breakpoint. These microinsertions are consistent with template-switching events and suggest a particular spatiotemporal configuration for DNA during the events.

TCF3-HLF-positive acute lymphoblastic leukemia (ALL) is currently incurable. Using an integrated approach, we uncovered distinct mutation, gene expression and drug response profiles in TCF3-HLF-positive and treatment-responsive TCF3-PBX1-positive ALL. We identified recurrent intragenic deletions of PAX5 or VPREB1 in constellation with the fusion of TCF3 and HLF. Moreover somatic mutations in the non-translocated allele of TCF3 and a reduction of PAX5 gene dosage in TCF3-HLF ALL suggest cooperation within a restricted genetic context. The enrichment for stem cell and myeloid features in the TCF3-HLF signature may reflect reprogramming by TCF3-HLF of a lymphoid-committed cell of origin toward a hybrid, drug-resistant hematopoietic state. Drug response profiling of matched patient-derived xenografts revealed a distinct profile for TCF3-HLF ALL with resistance to conventional chemotherapeutics but sensitivity to glucocorticoids, anthracyclines and agents in clinical development. Striking on-target sensitivity was achieved with the BCL2-specific inhibitor venetoclax (ABT-199). This integrated approach thus provides alternative treatment options for this deadly disease.

Genomic structural variants (SVs) are abundant in humans, differing from other forms of variation in extent, origin and functional impact. Despite progress in SV characterization, the nucleotide resolution architecture of most SVs remains unknown. We constructed a map of unbalanced SVs (that is, copy number variants) based on whole genome DNA sequencing data from 185 human genomes, integrating evidence from complementary SV discovery approaches with extensive experimental validations. Our map encompassed 22,025 deletions and 6,000 additional SVs, including insertions and tandem duplications. Most SVs (53%) were mapped to nucleotide resolution, which facilitated analysing their origin and functional impact. We examined numerous whole and partial gene deletions with a genotyping approach and observed a depletion of gene disruptions amongst high frequency deletions. Furthermore, we observed differences in the size spectra of SVs originating from distinct formation mechanisms, and constructed a map of SV hotspots formed by common mechanisms. Our analytical framework and SV map serves as a resource for sequencing-based association studies.

Copy-number variations (CNVs) are widespread in the human genome, but comprehensive assignments of integer locus copy-numbers (i.e., copy-number genotypes) that, for example, enable discrimination of homozygous from heterozygous CNVs, have remained challenging. Here we present CopySeq, a novel computational approach with an underlying statistical framework that analyzes the depth-of-coverage of high-throughput DNA sequencing reads, and can incorporate paired-end and breakpoint junction analysis based CNV-analysis approaches, to infer locus copy-number genotypes. We benchmarked CopySeq by genotyping 500 chromosome 1 CNV regions in 150 personal genomes sequenced at low-coverage. The assessed copy-number genotypes were highly concordant with our performed qPCR experiments (Pearson correlation coefficient 0.94), and with the published results of two microarray platforms (95-99% concordance). We further demonstrated the utility of CopySeq for analyzing gene regions enriched for segmental duplications by comprehensively inferring copy-number genotypes in the CNV-enriched >800 olfactory receptor (OR) human gene and pseudogene loci. CopySeq revealed that OR loci display an extensive range of locus copy-numbers across individuals, with zero to two copies in some OR loci, and two to nine copies in others. Among genetic variants affecting OR loci we identified deleterious variants including CNVs and SNPs affecting ~15% and ~20% of the human OR gene repertoire, respectively, implying that genetic variants with a possible impact on smell perception are widespread. Finally, we found that for several OR loci the reference genome appears to represent a minor-frequency variant, implying a necessary revision of the OR repertoire for future functional studies. CopySeq can ascertain genomic structural variation in specific gene families as well as at a genome-wide scale, where it may enable the quantitative evaluation of CNVs in genome-wide association studies involving high-throughput sequencing.

Sequencing of cell-free DNA in the blood of cancer patients (liquid biopsy) provides attractive opportunities for early diagnosis, assessment of treatment response, and minimally invasive disease monitoring. To unlock liquid biopsy analysis for pediatric tumors with few genetic aberrations, we introduce an integrated genetic/epigenetic analysis method and demonstrate its utility on 241 deep whole-genome sequencing profiles of 95 patients with Ewing sarcoma and 31 patients with other pediatric sarcomas. Our method achieves sensitive detection and classification of circulating tumor DNA in peripheral blood independent of any genetic alterations. Moreover, we benchmark different metrics for cell-free DNA fragmentation analysis, and we introduce the LIQUORICE algorithm for detecting circulating tumor DNA based on cancer-specific chromatin signatures. Finally, we combine several fragmentation-based metrics into an integrated machine learning classifier for liquid biopsy analysis that exploits widespread epigenetic deregulation and is tailored to cancers with low mutation rates. Clinical associations highlight the potential value of cfDNA fragmentation patterns as prognostic biomarkers in Ewing sarcoma. In summary, our study provides a comprehensive analysis of circulating tumor DNA beyond recurrent genetic aberrations, and it renders the benefits of liquid biopsy more readily accessible for childhood cancers.