Search

Auditory synaptopathy/neuropathy (AS/AN) is a distinct type of sensorineural hearing loss in which the cochlear sensitivity to sound (i.e. active cochlear amplification by outer hair cells) is preserved whereas sound encoding by inner hair cells and/or auditory nerve fibers is disrupted owing to genetic or environmental factors. Autosomal-dominant auditory neuropathy type 2 (AUNA2) was linked either to chromosomal bands 12q24 or 13q34 in a large German family in 2017. By whole-genome sequencing, we now detected a 5500 bp deletion in ATP11A on chromosome 13q34 segregating with the phenotype in this family. ATP11A encodes a P-type ATPase that translocates phospholipids from the exoplasmic to the cytoplasmic leaflet of the plasma membrane. The deletion affects both isoforms of ATP11A and activates a cryptic splice site leading to the formation of an alternative last exon. ATP11A carrying the altered C-terminus loses its flippase activity for phosphatidylserine. Atp11a is expressed in fibers and synaptic contacts of the auditory nerve and in the cochlear nucleus in mice, and conditional Atp11a knockout mice show a progressive reduction of the spiral ganglion neuron compound action potential, recapitulating the human phenotype of AN. By combining whole-genome sequencing, immunohistochemistry, in vitro functional assays and generation of a mouse model, we could thus identify a partial deletion of ATP11A as the genetic cause of AUNA2.

Supplementary Figure from PTEN Loss Contributes to Erlotinib Resistance in EGFR-Mutant Lung Cancer by Activation of Akt and EGFR

Clinical resistance to epidermal growth factor receptor (EGFR) inhibition in lung cancer has been linked to the emergence of the EGFR T790M resistance mutation or amplification of MET. Additional mechanisms contributing to EGFR inhibitor resistance remain elusive. By applying combined analyses of gene expression, copy number, and biochemical analyses of EGFR inhibitor responsiveness, we identified homozygous loss of PTEN to segregate EGFR-dependent and EGFR-independent cells. We show that in EGFR-dependent cells, PTEN loss partially uncouples mutant EGFR from downstream signaling and activates EGFR, thereby contributing to erlotinib resistance. The clinical relevance of our findings is supported by the observation of PTEN loss in 1 out of 24 primary EGFR-mutant non–small cell lung cancer (NSCLC) tumors. These results suggest a novel resistance mechanism in EGFR-mutant NSCLC involving PTEN loss. [Cancer Res 2009;69(8):3256–61]

Supplementary Materials & Methods and Figure Legend from PTEN Loss Contributes to Erlotinib Resistance in EGFR-Mutant Lung Cancer by Activation of Akt and EGFR

Supplementary Table from PTEN Loss Contributes to Erlotinib Resistance in EGFR-Mutant Lung Cancer by Activation of Akt and EGFR

Promoting brown adipose tissue (BAT) activity has been recognized as innovative therapeutic approach to improve obesity and metabolic disease. Whilst the molecular circuitry underlying thermogenic activation of BAT is well understood, the processes underlying rheostatic regulation of BAT to maintain homeostasis and avoid excessive energy dissipation remain ill-defined. Increasing cyclic AMP (cAMP) biosynthesis is key for BAT activation. Here, we demonstrate that ADCY3, an adenylyl cyclase whose expression is induced during cold exposure and regulates cAMP homeostasis in thermogenic fat, is dispensable for BAT function in lean mice, but becomes critical during obesity. Furthermore, by combining RNA-seq with epigenomic H3K4me3 profiling, we detected a novel, cold-inducible promoter that generates a 5’ truncated Adcy3-at mRNA isoform, Adcy3-at. Mice lacking only Adcy3-at, but not full-length Adcy3, displayed increased energy expenditure already under lean conditions and were protected against obesity and ensuing metabolic imbalances. Subcellularly, translated ADCY3-AT proteins are retained in the endoplasmic reticulum (ER), did not translocate to the cell membrane, and lacked enzymatic activity. By interacting with ADCY3, ADCY3-AT retained ADCY3 in the ER and, thereby, reduced the plasma membrane pool of ADCYs available for G-protein mediated cAMP synthesis. Thereby, ADCY3-AT acts as a signaling rheostat in BAT, limiting adverse consequences of uncurbed cAMP activity after long-term BAT activation. Adcy3-at induction was driven by a cold-induced, truncated isoform of the transcriptional cofactor PPARGC1A (PPARG Coactivator 1 Alpha, PPARGC1A-AT). Expression of Ppargc1a-at and Adcy3-at are evolutionary conserved, indicating that transcriptional rewiring by commissioning of alternative promoters is key for thermogenic fat function.

Most manipulations that extend lifespan also increase resistance to various stress factors and environmental cues in a range of animals from yeast to mammals. However, the underlying molecular mechanisms regulating stress resistance during aging are still largely unknown. Here we identify Krüppel-like factor 1 (KLF-1) as a mediator of a cytoprotective response that dictates longevity induced by reduced mitochondrial function. A redox-regulated KLF-1 activation and transfer to the nucleus coincides with the peak of somatic mitochondrial biogenesis that occurs around a transition from larval stage L3 to D1. We further show that KLF-1 activates genes involved in the xenobiotic detoxification programme and identified cytochrome P450 oxidases, the KLF-1 main effectors, as longevity-assurance factors of mitochondrial mutants. Collectively, these findings underline the importance of the xenobiotic detoxification in the mitohormetic, longevity assurance pathway and identify KLF-1 as a central factor in orchestrating this response., Cytochrome P450 oxidases (CYPs) are enzymes that participate in the xenobiotic detoxification and their expression is enhanced in long-lived model organisms. Here the authors show that KLF-1 promotes cyp expression and ensures lifespan extension in C. elegans mitomutants by activating mitohormesis.

AATF is a central regulator of the cellular outcome upon p53 activation, a finding that has primarily been attributed to its function as a transcription factor. Recent data showed that AATF is essential for ribosome biogenesis and plays a role in rRNA maturation. AATF has been implicated to fulfil this role through direct interaction with rRNA and was identified in several RNA-interactome capture experiments. Here, we provide a first comprehensive analysis of the RNA bound by AATF using CLIP-sequencing. Interestingly, this approach shows predominant binding of the 45S pre-ribosomal RNA precursor molecules. Furthermore, AATF binds to mRNAs encoding for ribosome biogenesis factors as well as snoRNAs. These findings are complemented by an in-depth analysis of the protein interactome of AATF containing a large set of proteins known to play a role in rRNA maturation with an emphasis on the protein-RNA-complexes known to be required for the generation of the small ribosomal subunit (SSU). In line with this finding, the binding sites of AATF within the 45S rRNA precursor localize in close proximity to the SSU cleavage sites. Consequently, our multilayer analysis of the protein-RNA interactome of AATF reveals this protein to be an important hub for protein and RNA interactions involved in ribosome biogenesis.

Up to 40% of esophageal carcinomas have a biallelic intact TP53 gene. It is largely unclear how these carcinoma cells prevent apoptosis, what is the kind of pathway alterations, or whether therapeutically relevant alterations exist in this subgroup. We evaluated The Cancer Genome Atlas (TCGA) data to compare TP53-mutated with TP53–wild-type tumors regarding copy number variations, gene mutations, and expression patterns of protein-coding genes and miRNAs. Additionally, we analyzed up to 428 esophageal adenocarcinomas (EACs) in total using an ultra-deep parallel sequencing panel, immunohistochemistry, as well as fluorescence in situ hybridization. In the TCGA cohort, 17.3% has a biallelic intact TP53 gene. This group has a smaller average total size of somatic copy number variations. Some protein coding genes and miRNAs were differentially expressed between the TP53-wild-type and TP53-mutated group to emphasize mdm2, CCND2, TP73, or miRNA 150, 488, or 4662a. In addition, 50% of the TP53–wild-type tumors carry somatic mutations in at least one of the genes involved in the TP53 pathway. Our patient cohort revealed 41.3% TP53–wild-type tumors; 5.6% were MDM2 amplified. In accordance with the TCGA data, we did not find a prognostic relevance of TP53 in our tumor cohort as well. The mutation status of TP53 defines an important subtype in esophageal carcinoma. Our comprehensive molecular analysis revealed important and potentially therapeutically relevant genomic alterations in this subgroup.

Regulation of the cellular metabolism is now recognized as a crucial mechanism for the homeostasis of innate and adaptive immune cells upon diverse extracellular stimuli. Macrophages, for instance, increase glycolysis upon stimulation with pathogen-associated molecular patterns (PAMPs). Conceivably, pathogens also counteract these metabolic changes for their own survival in the host. However, despite this dynamic interplay in host-pathogen interactions, the role of immunometabolism in the context of intracellular bacterial infections is still unclear. Here, employing unbiased metabolomic and transcriptomic approaches, we investigated the role of metabolic adaptations of macrophages upon Salmonella enterica serovar Typhimurium (S. Typhimurium) infections. Importantly, our results suggested that S. Typhimurium abrogates glycolysis and its modulators such as insulin-signaling to impair macrophage defense. Mechanistically, glycolytic enzyme aldolase A is critical for v-ATPase assembly and the acidification of phagosomes upon S. Typhimurium infection, and impairment in the glycolytic machinery eventually leads to decreased bacterial clearance and antigen presentation in macrophages. Collectively, our results highlight a vital molecular link between metabolic adaptation and phagosome maturation in macrophages, which is targeted by S. Typhimurium to evade cell-autonomous defense.

Among autoimmune rheumatic diseases, systemic sclerosis (SSc) is especially challenging due to its clinical diversity, reflected by the extent of skin and internal organ involvement 1 . Besides the two major subsets, limited and diffuse SSc (lcSSc, dcSSc), a substantial number of SSc cases exhibit overlap features of another rheumatic disease, summarized as SSc-overlap syndromes. A few of these are already well characterized by their specific clinical course and circulating antibodies (e.g., mixed connective tissue disease, polymyositis-scleroderma syndrome).

Obesity and type 2 diabetes mellitus are global emergencies and long noncoding RNAs (lncRNAs) are regulatory transcripts with elusive functions in metabolism. Here we show that a high fraction of lncRNAs, but not protein-coding mRNAs, are repressed during diet-induced obesity (DIO) and refeeding, whilst nutrient deprivation induced lncRNAs in mouse liver. Similarly, lncRNAs are lost in diabetic humans. LncRNA promoter analyses, global cistrome and gain-of-function analyses confirm that increased MAFG signaling during DIO curbs lncRNA expression. Silencing Mafg in mouse hepatocytes and obese mice elicits a fasting-like gene expression profile, improves glucose metabolism, de-represses lncRNAs and impairs mammalian target of rapamycin (mTOR) activation. We find that obesity-repressed LincIRS2 is controlled by MAFG and observe that genetic and RNAi-mediated LincIRS2 loss causes elevated blood glucose, insulin resistance and aberrant glucose output in lean mice. Taken together, we identify a MAFG-lncRNA axis controlling hepatic glucose metabolism in health and metabolic disease., Despite widespread transcription of LncRNA in mammalian systems, their contribution to metabolic homeostasis at the cellular and tissue level remains elusive. Here Pradas-Juni et al. describe a transcription factor–LncRNA pathway that couples hepatocyte nutrient sensing to regulation of glucose metabolism in mice.

Regulation of the cellular metabolism is now recognized as a crucial mechanism for the homeostasis of innate and adaptive immune cells upon diverse extracellular stimuli. Macrophages, for instance, increase glycolysis upon stimulation with pathogen-associated molecular patterns (PAMPs). Conceivably, pathogens also counteract these metabolic changes for their own survival in the host. However, despite this dynamic interplay in host-pathogen interactions, the role of immunometabolism in the context of intracellular bacterial infections is still unclear. Here, employing unbiased metabolomic and transcriptomic approaches, we investigated the role of metabolic adaptations of macrophages upon Salmonella enterica serovar Typhimurium (S. Typhimurium) infections. Importantly, our results suggested that S. Typhimurium abrogates glycolysis and its modulators such as insulin-signaling to impair macrophage defense. Mechanistically, glycolytic enzyme aldolase A is critical for v-ATPase assembly and the acidification of phagosomes upon S. Typhimurium infection, and impairment in the glycolytic machinery eventually leads to decreased bacterial clearance and antigen presentation in macrophages. Collectively, our results highlight a vital molecular link between metabolic adaptation and phagosome maturation in macrophages, which is targeted by S. Typhimurium to evade cell-autonomous defense.

While Next-Generation Sequencing (NGS) can now be considered an established analysis technology for research applications across the life sciences, the analysis workflows still require substantial bioinformatics expertise. Typical challenges include the appropriate selection of analytical software tools, the speedup of the overall procedure using HPC parallelization and acceleration technology, the development of automation strategies, data storage solutions and finally the development of methods for full exploitation of the analysis results across multiple experimental conditions. Recently, NGS has begun to expand into clinical environments, where it facilitates diagnostics enabling personalized therapeutic approaches, but is also accompanied by new technological, legal and ethical challenges. There are probably as many overall concepts for the analysis of the data as there are academic research institutions. Among these concepts are, for instance, complex IT architectures developed in-house, ready-to-use technologies installed on-site as well as comprehensive Everything as a Service (XaaS) solutions. In this mini-review, we summarize the key points to consider in the setup of the analysis architectures, mostly for scientific rather than diagnostic purposes, and provide an overview of the current state of the art and challenges of the field.

Next-Generation Sequencing (NGS) has turned from a new and experimental technology into a standard procedure for cancer genome studies and clinical investigation. While a multitude of software packages for cancer genome data analysis have been made available, these need to be combined into efficient analytical workflows that cover multiple aspects relevant to a clinical environment and that deliver handy results within a reasonable time frame. Here, we introduce QuickNGS Cancer as a new suite of bioinformatics pipelines which is focused on cancer genomics and significantly reduces the analytical hurdles that still limit a broader applicability of NGS technology, particularly to clinically driven research. QuickNGS Cancer allows a highly efficient analysis of a broad variety of NGS data types, specifically considering cancer-specific issues, such as biases introduced by tumor impurity and aneuploidy or the assessment of genomic variations regarding their biomedical relevance. It delivers highly reproducible analysis results ready for interpretation within only a few days after sequencing, as shown by a re-analysis of 140 tumor/normal pairs from The Cancer Genome Atlas (TCGA) in which QuickNGS Cancer detected a significant number of mutations in key cancer genes missed by a well-established mutation calling pipeline. Finally, QuickNGS Cancer obtained several unexpected mutations in leukemias which could be confirmed by Sanger sequencing. This article is protected by copyright. All rights reserved

Background Recent cancer genome studies on many human cancer types have relied on multiple molecular high-throughput technologies. Given the vast amount of data that has been generated, there are surprisingly few databases which facilitate access to these data and make them available for flexible analysis queries in the broad research community. If used in their entirety and provided at a high structural level, these data can be directed into constantly increasing databases which bear an enormous potential to serve as a basis for machine learning technologies with the goal to support research and healthcare with predictions of clinically relevant traits. Results We have developed the Cancer Systems Biology Database (CancerSysDB), a resource for highly flexible queries and analysis of cancer-related data across multiple data types and multiple studies. The CancerSysDB can be adopted by any center for the organization of their locally acquired data and its integration with publicly available data from multiple studies. A publicly available main instance of the CancerSysDB can be used to obtain highly flexible queries across multiple data types as shown by highly relevant use cases. In addition, we demonstrate how the CancerSysDB can be used for predictive cancer classification based on whole-exome data from 9091 patients in The Cancer Genome Atlas (TCGA) research network. Conclusions Our database bears the potential to be used for large-scale integrative queries and predictive analytics of clinically relevant traits. Electronic supplementary material The online version of this article (10.1186/s12859-018-2157-7) contains supplementary material, which is available to authorized users.

Activation of Agouti-related peptide (AgRP) neurons potently promotes feeding, and chronically altering their activity also affects peripheral glucose homeostasis. We demonstrate that acute activation of AgRP neurons causes insulin resistance through impairment of insulin-stimulated glucose uptake into brown adipose tissue (BAT). AgRP neuron activation acutely reprograms gene expression in BAT toward a myogenic signature, including increased expression of myostatin. Interference with myostatin activity improves insulin sensitivity that was impaired by AgRP neurons activation. Optogenetic circuitry mapping reveals that feeding and insulin sensitivity are controlled by both distinct and overlapping projections. Stimulation of AgRP → LHA projections impairs insulin sensitivity and promotes feeding while activation of AgRP → anterior bed nucleus of the stria terminalis (aBNST)vl projections, distinct from AgRP → aBNSTdm projections controlling feeding, mediate the effect of AgRP neuron activation on BAT-myostatin expression and insulin sensitivity. Collectively, our results suggest that AgRP neurons in mice induce not only eating, but also insulin resistance by stimulating expression of muscle-related genes in BAT, revealing a mechanism by which these neurons rapidly coordinate hunger states with glucose homeostasis.

Activation of brown adipose tissue (BAT) controls energy homeostasis in rodents and humans and has emerged as an innovative strategy for the treatment of obesity and type 2 diabetes mellitus. Here we show that ageing- and obesity-associated dysfunction of brown fat coincides with global microRNA downregulation due to reduced expression of the microRNA-processing node Dicer1. Consequently, heterozygosity of Dicer1 in BAT aggravated diet-induced-obesity (DIO)-evoked deterioration of glucose metabolism. Analyses of differential microRNA expression during preadipocyte commitment and mouse models of progeria, longevity and DIO identified miR-328 as a regulator of BAT differentiation. Reducing miR-328 blocked preadipocyte commitment, whereas miR-328 overexpression instigated BAT differentiation and impaired muscle progenitor commitment-partly through silencing of the β-secretase Bace1. Loss of Bace1 enhanced brown preadipocyte specification in vitro and was overexpressed in BAT of obese and progeroid mice. In vivo Bace1 inhibition delayed DIO-induced weight gain and improved glucose tolerance and insulin sensitivity. These experiments reveal Dicer1-miR-328-Bace1 signalling as a determinant of BAT function, and highlight the potential of Bace1 inhibition as a therapeutic approach to improve not only neurodegenerative diseases but also ageing- and obesity-associated impairments of BAT function.

Aging-associated diseases and their comorbidities affect the life of a constantly growing proportion of the population in developed countries. At the center of these comorbidities are changes of kidney structure and function as age-related chronic kidney disease predisposes to the development of cardiovascular diseases such as stroke, myocardial infarction or heart failure. To detect molecular mechanisms involved in kidney aging, we analyzed gene expression profiles of kidneys from adult and aged wild-type mice by transcriptomic, proteomic and targeted lipidomic methodologies. Interestingly, transcriptome and proteome analyses revealed differential expression of genes primarily involved in lipid metabolism and immune response. Additional lipidomic analyses uncovered significant age-related differences in the total amount of phosphatidylethanolamines, phosphatidylcholines and sphingomyelins as well as in subspecies of phosphatidylserines and ceramides with age. By integration of these datasets we identified Aldh1a1, a key enzyme in vitamin A metabolism specifically expressed in the medullary ascending limb, as one of the most prominent upregulated proteins in old kidneys. Moreover, ceramidase Asah1 was highly expressed in aged kidneys, consistent with a decrease in ceramide C16. In summary, our data suggest that changes in lipid metabolism are involved in the process of kidney aging and in the development of chronic kidney disease.

Cancer is a highly heterogeneous disease, both intra- and inter-individually consisting of complex phenotypes and systems biology. Although genomic data has contributed greatly towards the identification of cancer-specific mutations and the progress of precision medicine, genomic alterations are only one of several important biological drivers of cancer. Furthermore, single-layer omics represent only a small piece of the cancer biology puzzle and provide only partial clues to connecting genotype with phenotype. A more integrated approach is urgently needed to unravel the underpinnings of molecular signatures and the phenotypic manifestation of cancer hallmarks. We have developed IndivuType, a knowledge and discovery platform that combines genomics, transcriptomics, and proteomics datasets to identify putative therapeutic targets in a colorectal cancer (CRC) cohort of 500 patients. We identified hundreds of proteins dysregulated in CRC that are dependent on the genetic background of the patient. Further, we characterized these proteins using clinical parameters from the IndivuType database to prioritize targets for precision medicine approaches. This project highlights the utility of moving beyond solely genomics approaches to better understand the molecular mechanisms underpinning cancer. Citation Format: Margarita Krawczyk, Jonathan Woodsmith, Marcel Nutsoa, Thomas Corwin, Silvia von der Heyde, Vincent Piras, Mark Whittaker, Alex Hergovich, Peter Frommolt, Hartmut Juhl. Proteogenomic characterization of colorectal cancer using the IndivuType multiomics database [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6587.

e16063 Background: Cancer is a highly heterogeneous disease, both intra- and inter-individually consisting of complex phenotypes and systems biology. Although genomic data has contributed greatly towards the identification of cancer-specific mutations and the progress of precision medicine, genomic alterations are only one of several important biological drivers of cancer. Furthermore, single-layer omics represent only a small piece of the cancer biology puzzle and provide only partial clues to connecting genotype with clinically relevant phenotypic data. A more integrated approach is urgently needed to unravel the underpinnings of molecular signatures and the phenotypic manifestation of cancer hallmarks. Methods: Here we characterize a colorectal cancer (CRC) cohort of 500 patients across multiple distinct omic data types. Across this CRC cohort, we defined clinically relevant whole genome sequencing based metrics such as micro-satellite-instability (MSI) status, and furthermore investigate gene expression at the transcript level using RNA-Seq, as well as at the proteomic level using tandem mass spectrometry. We further characterized a subgroup of 100 of these patients through 16s rRNA sequencing to identify associated microbiome profiles. Results: We combined these analyses with comprehensive clinical data to observe the impact of ascertained molecular signatures on the CRC patient cohort. Here, we report how patient survival correlates both with specific molecular events across individual omic data types, as well as with combined multi-omic analyses. Conclusions: This project highlights the utility of integrating multiple distinct data types to obtain a more comprehensive overview of the molecular mechanisms underpinning colo-rectal cancer. Furthermore, through combining identified aberrant molecular mechanisms with clinical reports, multi-omic data can be prioritized through their impact on patient cohort survival.

Increasing brown adipose tissue (BAT) thermogenesis in mice and humans improves metabolic health and understanding BAT function is of interest for novel approaches to counteract obesity. The role of long noncoding RNAs (lncRNAs) in these processes remains elusive. We observed maternally expressed, imprinted lncRNA H19 increased upon cold-activation and decreased in obesity in BAT. Inverse correlations of H19 with BMI were also observed in humans. H19 overexpression promoted, while silencing of H19 impaired adipogenesis, oxidative metabolism and mitochondrial respiration in brown but not white adipocytes. In vivo, H19 overexpression protected against DIO, improved insulin sensitivity and mitochondrial biogenesis, whereas fat H19 loss sensitized towards HFD weight gains. Strikingly, paternally expressed genes (PEG) were largely absent from BAT and we demonstrated that H19 recruits PEG-inactivating H19-MBD1 complexes and acts as BAT-selective PEG gatekeeper. This has implications for our understanding how monoallelic gene expression affects metabolism in rodents and, potentially, humans., Brown adipose tissue (BAT) thermogenesis counteracts obesity and promotes metabolic health. The role of long non-coding RNAs (lncRNAs) in the regulation of this process is not well understood. Here the authors identify a maternally expressed lncRNA, H19, that increases BAT oxidative metabolism and energy expenditure.

T-cell prolymphocytic leukemia (T-PLL) is a rare and poor-prognostic mature T-cell malignancy. Here we integrated large-scale profiling data of alterations in gene expression, allelic copy number (CN), and nucleotide sequences in 111 well-characterized patients. Besides prominent signatures of T-cell activation and prevalent clonal variants, we also identify novel hot-spots for CN variability, fusion molecules, alternative transcripts, and progression-associated dynamics. The overall lesional spectrum of T-PLL is mainly annotated to axes of DNA damage responses, T-cell receptor/cytokine signaling, and histone modulation. We formulate a multi-dimensional model of T-PLL pathogenesis centered around a unique combination of TCL1 overexpression with damaging ATM aberrations as initiating core lesions. The effects imposed by TCL1 cooperate with compromised ATM toward a leukemogenic phenotype of impaired DNA damage processing. Dysfunctional ATM appears inefficient in alleviating elevated redox burdens and telomere attrition and in evoking a p53-dependent apoptotic response to genotoxic insults. As non-genotoxic strategies, synergistic combinations of p53 reactivators and deacetylase inhibitors reinstate such cell death execution., T-cell prolymphocytic leukemia (T-PLL) is a rare malignancy with a poor prognosis. Here, the authors investigate the genomic landscape, gene expression profiles and functional mechanisms in 111 patients, highlighting TCL1 overexpression and ATM aberrations as core lesions which co-operate to impair DNA damage processing.

Genome maintenance defects cause complex disease phenotypes characterized by developmental failure, cancer susceptibility and premature ageing. It remains poorly understood how DNA damage responses function during organismal development and maintain tissue functionality when DNA damage accumulates with ageing. Here we show that the FOXO transcription factor DAF-16 is activated in response to DNA damage during development, whereas the DNA damage responsiveness of DAF-16 declines with ageing. We find that in contrast to its established role in mediating starvation arrest, DAF-16 alleviates DNA-damage-induced developmental arrest and even in the absence of DNA repair promotes developmental growth and enhances somatic tissue functionality. We demonstrate that the GATA transcription factor EGL-27 co-regulates DAF-16 target genes in response to DNA damage and together with DAF-16 promotes developmental growth. We propose that EGL-27/GATA activity specifies DAF-16-mediated DNA damage responses to enable developmental progression and to prolong tissue functioning when DNA damage persists.

In the arcuate nucleus of the hypothalamus (ARH) satiety signaling (anorexigenic) pro-opiomelanocortin (POMC)-expressing and hunger signaling (orexigenic) agouti-related peptide (AgRP)-expressing neurons are key components of the neuronal circuits that control food intake and energy homeostasis. Here, we assessed whether the catecholamine noradrenalin directly modulates the activity of these neurons in mice. Perforated patch clamp recordings showed that noradrenalin changes the activity of these functionally antagonistic neurons in opposite ways, increasing the activity of the orexigenic NPY/AgRP neurons and decreasing the activity of the anorexigenic POMC neurons. Cell type-specific transcriptomics and pharmacological experiments revealed that the opposing effect on these neurons is mediated by the activation of excitatory α1A - and β- adrenergic receptors in NPY/AgRP neurons, while POMC neurons are inhibited via α2A – adrenergic receptors. Thus, the coordinated differential modulation of the key hypothalamic neurons in control of energy homeostasis assigns noradrenalin an important role to promote feeding. DOI: http://dx.doi.org/10.7554/eLife.25770.001

A fundamental principle in malignant tranformation is the ability of cancer cells to escape the naturally occurring cell-intrinsic responses to DNA damage. Tumors progress despite the accumulation of DNA lesions. However, the underlying mechanisms of this tolerance to genotoxic stress are still poorly characterized. Here, we show that replication stress occurs in Kras-driven murine lung adenocarcinomas, as well as in proliferating murine embryonic and adult tissues. We identify the transcriptional regulator AATF/CHE-1 as a key molecule to sustain proliferative tissues and tumor progression in parts by inhibiting p53-driven apoptosis in vivo. In an autochthonous Kras-driven lung adenocarcinoma model, deletion of Aatf delayed lung cancer formation predominantly in a p53-dependent manner. Moreover, targeting Aatf in existing tumors through a dual recombinase strategy caused a halt in tumor progression. Taken together, these data suggest that AATF may serve as a drug target to treat KRAS-driven malignancies.

Poised enhancers marked by H3K27me3 in pluripotent stem cells have been implicated in the establishment of somatic expression programs during embryonic stem cell (ESC) differentiation. However, the functional relevance and mechanism of action of poised enhancers remain unknown. Using CRISPR/Cas9 technology to engineer precise genetic deletions, we demonstrate that poised enhancers are necessary for the induction of major anterior neural regulators. Interestingly, circularized chromosome conformation capture sequencing (4C-seq) shows that poised enhancers already establish physical interactions with their target genes in ESCs in a polycomb repressive complex 2 (PRC2)-dependent manner. Loss of PRC2 does not activate poised enhancers or induce their putative target genes in undifferentiated ESCs; however, loss of PRC2 in differentiating ESCs severely and specifically compromises the induction of major anterior neural genes representing poised enhancer targets. Overall, our work illuminates an unexpected function for polycomb proteins in facilitating neural induction by endowing major anterior neural loci with a permissive regulatory topology, Work in the Rada-Iglesias laboratory is supported by CMMC intramural funding, the DFG (research grants RA 2547/1-1, RA 2547/2-1, and TE 1007/3-1), a UoC Advanced Researcher Group Grant, and a CECAD grant

CLUH is a cytosolic mRNA-binding protein that specifically binds a subset of mRNAs encoding mitochondrial proteins and may regulate their localized translation., Mitochondrial function requires coordination of two genomes for protein biogenesis, efficient quality control mechanisms, and appropriate distribution of the organelles within the cell. How these mechanisms are integrated is currently not understood. Loss of the Clu1/CluA homologue (CLUH) gene led to clustering of the mitochondrial network by an unknown mechanism. We find that CLUH is coregulated both with genes encoding mitochondrial proteins and with genes involved in ribosomal biogenesis and translation. Our functional analysis identifies CLUH as a cytosolic messenger ribonucleic acid (RNA; mRNA)–binding protein. RNA immunoprecipitation experiments followed by next-generation sequencing demonstrated that CLUH specifically binds a subset of mRNAs encoding mitochondrial proteins. CLUH depletion decreased the levels of proteins translated by target transcripts and caused mitochondrial clustering. A fraction of CLUH colocalizes with tyrosinated tubulin and can be detected close to mitochondria, suggesting a role in regulating transport or translation of target transcripts close to mitochondria. Our data unravel a novel mechanism linking mitochondrial biogenesis and distribution.

Background Although the pattern of lateral organ formation from apical meristems establishes species-specific plant architecture, the positional information that confers cell fate to cells as they transit to the meristem flanks where they differentiate, remains largely unknown. We have combined fluorescence-activated cell sorting and RNA-seq to characterise the cell-type-specific transcriptome at the earliest developmental time-point of lateral organ formation using DORNRÖSCHEN-LIKE::GFP to mark founder-cell populations at the periphery of the inflorescence meristem (IM) in apetala1 cauliflower double mutants, which overproliferate IMs. Results Within the lateral organ founder-cell population at the inflorescence meristem, floral primordium identity genes are upregulated and stem-cell identity markers are downregulated. Additional differentially expressed transcripts are involved in polarity generation and boundary formation, and in epigenetic and post-translational changes. However, only subtle transcriptional reprogramming within the global auxin network was observed. Conclusions The transcriptional network of differentially expressed genes supports the hypothesis that lateral organ founder-cell specification involves the creation of polarity from the centre to the periphery of the IM and the establishment of a boundary from surrounding cells, consistent with bract initiation. However, contrary to the established paradigm that sites of auxin response maxima pre-pattern lateral organ initiation in the IM, auxin response might play a minor role in the earliest stages of lateral floral initiation. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3189-x) contains supplementary material, which is available to authorized users.

The pathological consequences of structural variants disrupting 3D genome organization can be difficult to elucidate in vivo due to differences in gene dosage sensitivity between mice and humans. This is illustrated by branchiooculofacial syndrome (BOFS), a rare congenital disorder caused by heterozygous mutations within TFAP2A, a neural crest regulator for which humans, but not mice, are haploinsufficient. Here, we present a BOFS patient carrying a heterozygous inversion with one breakpoint located within a topologically associating domain (TAD) containing enhancers essential for TFAP2A expression in human neural crest cells (hNCCs). Using patient-specific hiPSCs, we show that, although the inversion shuffles the TFAP2A hNCC enhancers with novel genes within the same TAD, this does not result in enhancer adoption. Instead, the inversion disconnects one TFAP2A allele from its cognate enhancers, leading to monoallelic and haploinsufficient TFAP2A expression in patient hNCCs. Our work illustrates the power of hiPSC differentiation to unveil long-range pathomechanisms., The Tomo Šarić laboratory participates in the EBiSC project, which received support from the Innovative Medicines Initiative Joint Undertaking under grant agreement no. 115582, resources of which are composed of financial contributions from the European Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies’ in-kind contributions. K.K.’s work was supported by the Deutsche Forschungsgemeinschaft (DFG) under grant agreement KO 3588/2-1. Work in the Rada-Iglesias laboratory was supported by CMMC intramural funding and grants from Fritz Thyssen Stiftung and Else Kröner Fresenius Stiftung.

Autophagy is a highly conserved intracellular degradative pathway that is crucial for cellular homeostasis. During autophagy, the core autophagy protein ATG12 plays, together with ATG5 and ATG16, an essential role in the expansion of the autophagosomal membrane. In this study we analyzed gene replacement mutants of atg12 in Dictyostelium discoideum AX2 wild-type and ATG16&oline, cells. RNAseq analysis revealed a strong enrichment of, firstly, autophagy genes among the up-regulated genes and, secondly, genes implicated in cell motility and phagocytosis among the down-regulated genes in the generated ATG12&oline, ATG16&oline, and ATG12&oline, /16&oline, cells. The mutant strains showed similar defects in fruiting body formation, autolysosome maturation, and cellular viability, implying that ATG12 and ATG16 act as a functional unit in canonical autophagy. In contrast, ablation of ATG16 or of ATG12 and ATG16 resulted in slightly more severe defects in axenic growth, macropinocytosis, and protein homeostasis than ablation of only ATG12, suggesting that ATG16 fulfils an additional function in these processes. Phagocytosis of yeast, spore viability, and maximal cell density were much more affected in ATG12&oline, cells, indicating that both proteins also have cellular functions independent of each other. In summary, we show that ATG12 and ATG16 fulfil autophagy-independent functions in addition to their role in canonical autophagy.

Autosomal recessive primary microcephaly (MCPH) is characterized by reduced head circumference, reduction in the size of the cerebral cortex with otherwise grossly normal brain structure and variable intellectual disability. MCPH is caused by mutations of 11 different genes which code for proteins implicated in cell division and cell cycle regulation. We studied a consanguineous eight-generation family from Pakistan with ten microcephalic children using homozygosity mapping and found a new MCPH locus at HSA 7q21.11-q21.3. Sanger sequencing of the most relevant candidate genes in this region revealed a homozygous single nucleotide substitution c.589GA in CDK6, which encodes cyclin-dependent kinase 6. The mutation changes a highly conserved alanine at position 197 into threonine (p.Ala197Thr). Post hoc whole-exome sequencing corroborated this mutation's identification as the causal variant. CDK6 is an important protein for the control of the cell cycle and differentiation of various cell types. We show here for the first time that CDK6 associates with the centrosome during mitosis; however, this was not observed in patient fibroblasts. Moreover, the mutant primary fibroblasts exhibited supernumerary centrosomes, disorganized microtubules and mitotic spindles, an increased centrosome nucleus distance, reduced cell proliferation and impaired cell motility and polarity. Upon ectopic expression of the mutant protein and knockdown of CDK6 through shRNA, we noted similar effects. We propose that the identified CDK6 mutation leads to reduced cell proliferation and impairs the correct functioning of the centrosome in microtubule organization and its positioning near the nucleus which are key determinants during neurogenesis.

Cellular heterogeneity within embryonic and adult tissues is involved in multiple biological and pathological processes. Here, we present a simple epigenomic strategy that allows the functional dissection of cellular heterogeneity. By integrating H3K27me3 chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) data, we demonstrate that the presence of broad H3K27me3 domains at transcriptionally active genes reflects the heterogeneous expression of major cell identity regulators. Using dorsoventral patterning of the spinal neural tube as a model, the proposed approach successfully identifies the majority of previously known dorsoventral patterning transcription factors with high sensitivity and precision. Moreover, poorly characterized patterning regulators can be similarly predicted, as shown for ZNF488, which confers p1/p2 neural progenitor identity. Finally, we show that, as our strategy is based on universal chromatin features, it can be used to functionally dissect cellular heterogeneity within various organisms and tissues, thus illustrating its potential applicability to a broad range of biological and pathological contexts.