The genome and population genomics of allopolyploid Coffea arabica reveal the diversification history of modern coffee cultivars.

Coffea arabica, an allotetraploid hybrid of Coffea eugenioides and Coffea canephora, is the source of approximately 60% of coffee products worldwide, and its cultivated accessions have undergone several population bottlenecks. We present chromosome-level assemblies of a di-haploid C. arabica accession and modern representatives of its diploid progenitors, C. eugenioides and C. canephora. The three species exhibit largely conserved genome structures between diploid parents and descendant subgenomes, with no obvious global subgenome dominance. We find evidence for a founding polyploidy event 350,000-610,000 years ago, followed by several pre-domestication bottlenecks, resulting in narrow genetic variation. A split between wild accessions and cultivar progenitors occurred ~30.5 thousand years ago, followed by a period of migration between the two populations. Analysis of modern varieties, including lines historically introgressed with C. canephora, highlights their breeding histories and loci that may contribute to pathogen resistance, laying the groundwork for future genomics-based breeding of C. arabica.

AMPK promotes induction of the tumor suppressor FLCN through activation of TFEB independently of mTOR.

AMPK is a central regulator of energy homeostasis. AMPK not only elicits acute metabolic responses but also promotes metabolic reprogramming and adaptations in the long-term through regulation of specific transcription factors and coactivators. We performed a whole-genome transcriptome profiling in wild-type (WT) and AMPK-deficient mouse embryonic fibroblasts (MEFs) and primary hepatocytes that had been treated with 2 distinct classes of small-molecule AMPK activators. We identified unique compound-dependent gene expression signatures and several AMPK-regulated genes, including folliculin (Flcn), which encodes the tumor suppressor FLCN. Bioinformatics analysis highlighted the lysosomal pathway and the associated transcription factor EB (TFEB) as a key transcriptional mediator responsible for AMPK responses. AMPK-induced Flcn expression was abolished in MEFs lacking TFEB and transcription factor E3, 2 transcription factors with partially redundant function; additionally, the promoter activity of Flcn was profoundly reduced when its putative TFEB-binding site was mutated. The AMPK-TFEB-FLCN axis is conserved across species; swimming exercise in WT zebrafish induced Flcn expression in muscle, which was significantly reduced in AMPK-deficient zebrafish. Mechanistically, we have found that AMPK promotes dephosphorylation and nuclear localization of TFEB independently of mammalian target of rapamycin activity. Collectively, we identified the novel AMPK-TFEB-FLCN axis, which may function as a key cascade for cellular and metabolic adaptations.-Collodet, C., Foretz, M., Deak, M., Bultot, L., Metairon, S., Viollet, B., Lefebvre, G., Raymond, F., Parisi, A., Civiletto, G., Gut, P., Descombes, P., Sakamoto, K. AMPK promotes induction of the tumor suppressor FLCN through activation of TFEB independently of mTOR.

MitoRS, a method for high throughput, sensitive, and accurate detection of mitochondrial DNA heteroplasmy.

BACKGROUND: Mitochondrial dysfunction is linked to numerous pathological states, in particular related to metabolism, brain health and ageing. Nuclear encoded gene polymorphisms implicated in mitochondrial functions can be analyzed in the context of classical genome wide association studies. By contrast, mitochondrial DNA (mtDNA) variants are more challenging to identify and analyze for several reasons. First, contrary to the diploid nuclear genome, each cell carries several hundred copies of the circular mitochondrial genome. Mutations can therefore be present in only a subset of the mtDNA molecules, resulting in a heterogeneous pool of mtDNA, a situation referred to as heteroplasmy. Consequently, detection and quantification of variants requires extremely accurate tools, especially when this proportion is small. Additionally, the mitochondrial genome has pseudogenized into numerous copies within the nuclear genome over the course of evolution. These nuclear pseudogenes, named NUMTs, must be distinguished from genuine mtDNA sequences and excluded from the analysis. RESULTS: Here we describe a novel method, named MitoRS, in which the entire mitochondrial genome is amplified in a single reaction using rolling circle amplification. This approach is easier to setup and of higher throughput when compared to classical PCR amplification. Sequencing libraries are generated at high throughput exploiting a tagmentation-based method. Fine-tuned parameters are finally applied in the analysis to allow detection of variants even of low frequency heteroplasmy. The method was thoroughly benchmarked in a set of experiments designed to demonstrate its robustness, accuracy and sensitivity. The MitoRS method requires 5 ng total DNA as starting material. More than 96 samples can be processed in less than a day of laboratory work and sequenced in a single lane of an Illumina HiSeq flow cell. The lower limit for accurate quantification of single nucleotide variants has been measured at 1% frequency. CONCLUSIONS: The MitoRS method enables the robust, accurate, and sensitive analysis of a large number of samples. Because it is cost effective and simple to setup, we anticipate this method will promote the analysis of mtDNA variants in large cohorts, and may help assessing the impact of mtDNA heteroplasmy on metabolic health, brain function, cancer progression, or ageing.

Network Analysis of Metabolite GWAS Hits: Implication of CPS1 and the Urea Cycle in Weight Maintenance.

BACKGROUND AND SCOPE: Weight loss success is dependent on the ability to refrain from regaining the lost weight in time. This feature was shown to be largely variable among individuals, and these differences, with their underlying molecular processes, are diverse and not completely elucidated. Altered plasma metabolites concentration could partly explain weight loss maintenance mechanisms. In the present work, a systems biology approach has been applied to investigate the potential mechanisms involved in weight loss maintenance within the Diogenes weight-loss intervention study. METHODS AND RESULTS: A genome wide association study identified SNPs associated with plasma glycine levels within the CPS1 (Carbamoyl-Phosphate Synthase 1) gene (rs10206976, p-value = 4.709e-11 and rs12613336, p-value = 1.368e-08). Furthermore, gene expression in the adipose tissue showed that CPS1 expression levels were associated with successful weight maintenance and with several SNPs within CPS1 (cis-eQTL). In order to contextualize these results, a gene-metabolite interaction network of CPS1 and glycine has been built and analyzed, showing functional enrichment in genes involved in lipid metabolism and one carbon pool by folate pathways. CONCLUSIONS: CPS1 is the rate-limiting enzyme for the urea cycle, catalyzing carbamoyl phosphate from ammonia and bicarbonate in the mitochondria. Glycine and CPS1 are connected through the one-carbon pool by the folate pathway and the urea cycle. Furthermore, glycine could be linked to metabolic health and insulin sensitivity through the betaine osmolyte. These considerations, and the results from the present study, highlight a possible role of CPS1 and related pathways in weight loss maintenance, suggesting that it might be partly genetically determined in humans.

Medium-chain fatty acids inhibit mitochondrial metabolism in astrocytes promoting astrocyte-neuron lactate and ketone body shuttle systems.

Medium-chain triglycerides have been used as part of a ketogenic diet effective in reducing epileptic episodes. The health benefits of the derived medium-chain fatty acids (MCFAs) are thought to result from the stimulation of liver ketogenesis providing fuel for the brain. We tested whether MCFAs have direct effects on energy metabolism in induced pluripotent stem cell-derived human astrocytes and neurons. Using single-cell imaging, we observed an acute pronounced reduction of the mitochondrial electrical potential and a concomitant drop of the NAD(P)H signal in astrocytes, but not in neurons. Despite the observed effects on mitochondrial function, MCFAs did not lower intracellular ATP levels or activate the energy sensor AMP-activated protein kinase. ATP concentrations in astrocytes were unaltered, even when blocking the respiratory chain, suggesting compensation through accelerated glycolysis. The MCFA decanoic acid (300 µM) promoted glycolysis and augmented lactate formation by 49.6%. The shorter fatty acid octanoic acid (300 µM) did not affect glycolysis but increased the rates of astrocyte ketogenesis 2.17-fold compared with that of control cells. MCFAs may have brain health benefits through the modulation of astrocyte metabolism leading to activation of shuttle systems that provide fuel to neighboring neurons in the form of lactate and ketone bodies.-Thevenet, J., De Marchi, U., Santo Domingo, J., Christinat, N., Bultot, L., Lefebvre, G., Sakamoto, K., Descombes, P., Masoodi, M., Wiederkehr, A. Medium-chain fatty acids inhibit mitochondrial metabolism in astrocytes promoting astrocyte-neuron lactate and ketone body shuttle systems.

Perinatal exposure to bisphenol a increases adult mammary gland progesterone response and cell number.

Bisphenol A [BPA, 2,2,-bis (hydroxyphenyl) propane] is one of the highest-volume chemicals produced worldwide. It is detected in body fluids of more than 90% of the human population. Originally synthesized as an estrogenic compound, it is currently utilized to manufacture food and beverage containers resulting in uptake with food and drinks. There is concern that exposure to low doses of BPA, defined as less than or equal to 5 mg/kg body weight /d, may have developmental effects on various hormone-responsive organs including the mammary gland. Here, we asked whether perinatal exposure to a range of low doses of BPA is sufficient to alter mammary gland hormone response later on in life, with a possible impact on breast cancer risk. To mimic human exposure, we added BPA to the drinking water of C57/Bl6 breeding pairs. Analysis of the mammary glands of their daughters at puberty showed that estrogen-dependent transcriptional events were perturbed and the number of terminal end buds, estrogen-induced proliferative structures, was altered in a dose-dependent fashion. Importantly, adult females showed an increase in mammary epithelial cell numbers comparable to that seen in females exposed to diethylbestrol, a compound exposure to which was previously linked to increased breast cancer risk. Molecularly, the mRNAs encoding Wnt-4 and receptor activator of nuclear factor κB ligand, two key mediators of hormone function implicated in control of mammary stem cell proliferation and carcinogenesis, showed increased induction by progesterone in the mammary tissue of exposed mice. Thus, perinatal exposure to environmentally relevant doses of BPA alters long-term hormone response that may increase the propensity to develop breast cancer.

Analysis of HIV-1 expression level and sense of transcription by high-throughput sequencing of the infected cell.

Next-generation sequencing offers an unprecedented opportunity to jointly analyze cellular and viral transcriptional activity without prerequisite knowledge of the nature of the transcripts. SupT1 cells were infected with a vesicular stomatitis virus G envelope protein (VSV-G)-pseudotyped HIV vector. At 24 h postinfection, both cellular and viral transcriptomes were analyzed by serial analysis of gene expression followed by high-throughput sequencing (SAGE-Seq). Read mapping resulted in 33 to 44 million tags aligning with the human transcriptome and 0.23 to 0.25 million tags aligning with the genome of the HIV-1 vector. Thus, at peak infection, 1 transcript in 143 is of viral origin (0.7%), including a small component of antisense viral transcription. Of the detected cellular transcripts, 826 (2.3%) were differentially expressed between mock- and HIV-infected samples. The approach also assessed whether HIV-1 infection modulates the expression of repetitive elements or endogenous retroviruses. We observed very active transcription of these elements, with 1 transcript in 237 being of such origin, corresponding on average to 123,123 reads in mock-infected samples (0.40%) and 129,149 reads in HIV-1-infected samples (0.45%) mapping to the genomic Repbase repository. This analysis highlights key details in the generation and interpretation of high-throughput data in the setting of HIV-1 cellular infection.

Generation of a genomic tiling array of the human major histocompatibility complex (MHC) and its application for DNA methylation analysis.

BACKGROUND: The major histocompatibility complex (MHC) is essential for human immunity and is highly associated with common diseases, including cancer. While the genetics of the MHC has been studied intensively for many decades, very little is known about the epigenetics of this most polymorphic and disease-associated region of the genome. METHODS: To facilitate comprehensive epigenetic analyses of this region, we have generated a genomic tiling array of 2 Kb resolution covering the entire 4 Mb MHC region. The array has been designed to be compatible with chromatin immunoprecipitation (ChIP), methylated DNA immunoprecipitation (MeDIP), array comparative genomic hybridization (aCGH) and expression profiling, including of non-coding RNAs. The array comprises 7832 features, consisting of two replicates of both forward and reverse strands of MHC amplicons and appropriate controls. RESULTS: Using MeDIP, we demonstrate the application of the MHC array for DNA methylation profiling and the identification of tissue-specific differentially methylated regions (tDMRs). Based on the analysis of two tissues and two cell types, we identified 90 tDMRs within the MHC and describe their characterisation. CONCLUSION: A tiling array covering the MHC region was developed and validated. Its successful application for DNA methylation profiling indicates that this array represents a useful tool for molecular analyses of the MHC in the context of medical genomics.

Genome-wide response of the human Hep3B hepatoma cell to proinflammatory cytokines, from transcription to translation.

Given the unknown timing of the onset of an acute systemic inflammation in humans, the fine tuning of cascades and pathways involved in the associated hepatocyte response cannot be appraised in vivo. Therefore, the authors used a genome-wide and kinetic analysis in the human Hep3B hepatoma cell line challenged with a conditioned medium from bacterial lipopolysaccharide-stimulated macrophages. A complete coverage of the liver transcriptome disclosed 648 mRNAs whose change in abundance allowed for their clustering in mRNA subsets with an early, intermediate, or late regulation. The contribution of transcription, stability, or translation was appraised with genome-wide studies of the changes in nuclear primary transcripts, mRNA decay, or polysome-associated mRNAs. A predominance of mRNAs with decreased stability and the fact that translation alone controls a significant number of acute phase-associated proteins are prominent findings. Transcription and stability act independently or, more rarely, cooperate or even counteract in a gene-by-gene manner, which results in a unidirectional change in mRNA abundance. Waves of mRNAs for groups of functionally related proteins are up- or downregulated in an ordered fashion. This includes an early regulation of transcription-associated proteins, an intermediate repression of detoxication and metabolism proteins, and finally an enhanced translation and transport of a number of membranous or secreted proteins along with an enhanced protein degradation. In conclusion, this study provides a comprehensive and simultaneous overview of events in the human hepatocyte during the inflammatory acute phase.

Global gene repression in hepatocellular carcinoma and fetal liver, and suppression of dudulin-2 mRNA as a possible marker for the cirrhosis-to-tumor transition.

BACKGROUND/AIMS: Whether the transcriptional reprogramming process induced by hepatocellular carcinoma recapitulates that of the developing liver is at present unclear. METHODS: With a complete coverage of the liver transcriptome by microarray using adult livers as controls, we searched for similarities and differences in mRNA abundances between hepatocellular carcinoma nodules and fetal livers taken before (early) or after (late) the 22-24th week of gestation. Changes in some mRNA levels were studied in further liver samples by quantitative RT-PCR. RESULTS: Altered gene expression in hepatocellular carcinoma mostly results in down-regulated mRNAs which largely overlap with those repressed in the late fetal liver. Different frequencies of transcription factor binding sites in the down-regulated genes vs control genes as well as changes in abundance of mRNAs for relevant transcription factors point to a transcriptional repression. The down-regulated mRNAs code for proteins involved in (i) transcription and translation, (ii) specific functions of the differentiated hepatocyte or (iii) activation of proliferation and apoptosis. CONCLUSIONS: Apoptosis limitation is likely to predominate over active proliferation during liver development and hepatocellular carcinoma. Repression of the apoptosis-associated dudulin-2 mRNA points to a potential marker for the transition from a carcinoma-free to carcinoma-associated cirrhosis.

Altered gene expression in acute systemic inflammation detected by complete coverage of the human liver transcriptome.

The goal of the current study was to provide complete coverage of the liver transcriptome with human probes corresponding to every gene expressed in embryonic, adult, and/or cancerous liver. We developed dedicated tools, namely, the Liverpool nylon array of complementary DNA (cDNA) probes for approximately 10,000 nonredundant genes and the LiverTools database. Inflammation-induced transcriptome changes were studied in liver tissue samples from patients with an acute systemic inflammation and from control subjects. One hundred and fifty-four messenger RNAs (mRNA) correlated statistically with the extent of inflammation. Of these, 134 mRNA samples were not associated previously with an acute-phase (AP) response. The hepatocyte origin and proinflammatory cytokine responsiveness of these mRNAs were confirmed by quantitative reverse-transcription polymerase chain reaction (Q-RT-PCR) in cytokine-challenged hepatoma cells. The corresponding gene promoters were enriched in potential binding sites for inflammation-driven transcription factors in the liver. Some of the corresponding proteins may provide novel blood markers of clinical relevance. The mRNAs whose level is most correlated with the AP extent (P <.05) were enriched in intracellular signaling molecules, transcription factors, glycosylation enzymes, and up-regulated plasma proteins. In conclusion, the hepatocyte responded to the AP extent by fine tuning some mRNA levels, controlling most, if not all, intracellular events from early signaling to the final secretion of proteins involved in innate immunity. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).