DNA methylation by CcrM contributes to genome maintenance in the Agrobacterium tumefaciens plant pathogen.

The cell cycle-regulated DNA methyltransferase CcrM is conserved in most Alphaproteobacteria, but its role in bacteria with complex or multicentric genomes remains unexplored. Here, we compare the methylome, the transcriptome and the phenotypes of wild-type and CcrM-depleted Agrobacterium tumefaciens cells with a dicentric chromosome with two essential replication origins. We find that DNA methylation has a pleiotropic impact on motility, biofilm formation and viability. Remarkably, CcrM promotes the expression of the repABCCh2 operon, encoding proteins required for replication initiation/partitioning at ori2, and represses gcrA, encoding a conserved global cell cycle regulator. Imaging ori1 and ori2 in live cells, we show that replication from ori2 is often delayed in cells with a hypo-methylated genome, while ori2 over-initiates in cells with a hyper-methylated genome. Further analyses show that GcrA promotes the expression of the RepCCh2 initiator, most likely through the repression of a RepECh2 anti-sense RNA. Altogether, we propose that replication at ori1 leads to a transient hemi-methylation and activation of the gcrA promoter, allowing repCCh2 activation by GcrA and contributing to initiation at ori2. This study then uncovers a novel and original connection between CcrM-dependent DNA methylation, a conserved epigenetic regulator and genome maintenance in an Alphaproteobacterial pathogen.

Robust landscapes of ribosome dwell times and aminoacyl-tRNAs in response to nutrient stress in liver.

Translation depends on messenger RNA (mRNA)-specific initiation, elongation, and termination rates. While translation elongation is well studied in bacteria and yeast, less is known in higher eukaryotes. Here we combined ribosome and transfer RNA (tRNA) profiling to investigate the relations between translation elongation rates, (aminoacyl-) tRNA levels, and codon usage in mammals. We modeled codon-specific ribosome dwell times from ribosome profiling, considering codon pair interactions between ribosome sites. In mouse liver, the model revealed site- and codon-specific dwell times that differed from those in yeast, as well as pairs of adjacent codons in the P and A site that markedly slow down or speed up elongation. While translation efficiencies vary across diurnal time and feeding regimen, codon dwell times were highly stable and conserved in human. Measured tRNA levels correlated with codon usage and several tRNAs showed reduced aminoacylation, which was conserved in fasted mice. Finally, we uncovered that the longest codon dwell times could be explained by aminoacylation levels or high codon usage relative to tRNA abundance.

Transcription factor activity rhythms and tissue-specific chromatin interactions explain circadian gene expression across organs.

Temporal control of physiology requires the interplay between gene networks involved in daily timekeeping and tissue function across different organs. How the circadian clock interweaves with tissue-specific transcriptional programs is poorly understood. Here, we dissected temporal and tissue-specific regulation at multiple gene regulatory layers by examining mouse tissues with an intact or disrupted clock over time. Integrated analysis uncovered two distinct regulatory modes underlying tissue-specific rhythms: tissue-specific oscillations in transcription factor (TF) activity, which were linked to feeding-fasting cycles in liver and sodium homeostasis in kidney; and colocalized binding of clock and tissue-specific transcription factors at distal enhancers. Chromosome conformation capture (4C-seq) in liver and kidney identified liver-specific chromatin loops that recruited clock-bound enhancers to promoters to regulate liver-specific transcriptional rhythms. Furthermore, this looping was remarkably promoter-specific on the scale of less than 10 kilobases (kb). Enhancers can contact a rhythmic promoter while looping out nearby nonrhythmic alternative promoters, confining rhythmic enhancer activity to specific promoters. These findings suggest that chromatin folding enables the clock to regulate rhythmic transcription of specific promoters to output temporal transcriptional programs tailored to different tissues.

Genomic history of the Italian population recapitulates key evolutionary dynamics of both Continental and Southern Europeans.

BACKGROUND: The cline of human genetic diversity observable across Europe is recapitulated at a micro-geographic scale by variation within the Italian population. Besides resulting from extensive gene flow, this might be ascribable also to local adaptations to diverse ecological contexts evolved by people who anciently spread along the Italian Peninsula. Dissecting the evolutionary history of the ancestors of present-day Italians may thus improve the understanding of demographic and biological processes that contributed to shape the gene pool of European populations. However, previous SNP array-based studies failed to investigate the full spectrum of Italian variation, generally neglecting low-frequency genetic variants and examining a limited set of small effect size alleles, which may represent important determinants of population structure and complex adaptive traits. To overcome these issues, we analyzed 38 high-coverage whole-genome sequences representative of population clusters at the opposite ends of the cline of Italian variation, along with a large panel of modern and ancient Euro-Mediterranean genomes. RESULTS: We provided evidence for the early divergence of Italian groups dating back to the Late Glacial and for Neolithic and distinct Bronze Age migrations having further differentiated their gene pools. We inferred adaptive evolution at insulin-related loci in people from Italian regions with a temperate climate, while possible adaptations to pathogens and ultraviolet radiation were observed in Mediterranean Italians. Some of these adaptive events may also have secondarily modulated population disease or longevity predisposition. CONCLUSIONS: We disentangled the contribution of multiple migratory and adaptive events in shaping the heterogeneous Italian genomic background, which exemplify population dynamics and gene-environment interactions that played significant roles also in the formation of the Continental and Southern European genomic landscapes.

Apolipoprotein E4 Expression Causes Gain of Toxic Function in Isogenic Human Induced Pluripotent Stem Cell-Derived Endothelial Cells.

OBJECTIVE: The ApoE (apolipoprotein) allele epsilon 4 is a major genetic risk factor for Alzheimer disease, cardiovascular disorders, and stroke, indicating that it significantly impacts cerebral and vascular systems. However, very little is known about how APOE genotype affects brain endothelial cells, which form a network of tight junctions to regulate communication between the brain and circulating blood factors. Approach and Results: Here, we present a novel model of endothelial dysfunction using isogenic human induced pluripotent stem cell-derived cells harboring different alleles of the APOE gene, specifically ApoE 3/3, 3/4, and 4/4. We show for the first time that ApoE4 expression by endothelial cells is sufficient to cause a toxic gain of cellular dysfunction. Using RNAseq, we found significant effects of ApoE4 on signaling pathways involved in blood coagulation and barrier function. These changes were associated with altered cell function, including increased binding of platelets to ECs with the 3/4 or 4/4 genotype. ApoE4-positive cells exhibited a proinflammatory state and prothrombotic state, evidenced by higher secretion of Aß (amyloid-ß) 40 and 42, increased release of cytokines, and overexpression of the platelet-binding protein VWF (vonWillebrand factor). Immunohistochemistry of human brain Alzheimer disease brains also showed increased VWF expression with the ApoE4/4 genotype. Finally, pharmacological inhibition of inflammation in ECs by celastrol rescued overexpression of VWF in cells expressing ApoE4. CONCLUSIONS: These cells provide novel insight into ApoE4-mediated endothelial dysfunction and provide a new platform to test potential therapies for vascular disorders.

Circadian and feeding rhythms differentially affect rhythmic mRNA transcription and translation in mouse liver.

Diurnal oscillations of gene expression are a hallmark of rhythmic physiology across most living organisms. Such oscillations are controlled by the interplay between the circadian clock and feeding rhythms. Although rhythmic mRNA accumulation has been extensively studied, comparatively less is known about their transcription and translation. Here, we quantified simultaneously temporal transcription, accumulation, and translation of mouse liver mRNAs under physiological light-dark conditions and ad libitum or night-restricted feeding in WT and brain and muscle Arnt-like 1 (Bmal1)-deficient animals. We found that rhythmic transcription predominantly drives rhythmic mRNA accumulation and translation for a majority of genes. Comparison of wild-type and Bmal1 KO mice shows that circadian clock and feeding rhythms have broad impact on rhythmic gene expression, Bmal1 deletion affecting surprisingly both transcriptional and posttranscriptional levels. Translation efficiency is differentially regulated during the diurnal cycle for genes with 5'-Terminal Oligo Pyrimidine tract (5'-TOP) sequences and for genes involved in mitochondrial activity, many harboring a Translation Initiator of Short 5'-UTR (TISU) motif. The increased translation efficiency of 5'-TOP and TISU genes is mainly driven by feeding rhythms but Bmal1 deletion also affects amplitude and phase of translation, including TISU genes. Together this study emphasizes the complex interconnections between circadian and feeding rhythms at several steps ultimately determining rhythmic gene expression and translation.

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.

De novo DNA methylation of endogenous retroviruses is shaped by KRAB-ZFPs/KAP1 and ESET.

Endogenous retroviruses (ERVs) undergo de novo DNA methylation during the first few days of mammalian embryogenesis, although the factors that control the targeting of this process are largely unknown. We asked whether KAP1 (KRAB-associated protein 1) is involved in this mechanism because of its previously defined role in maintaining the silencing of ERVs through the histone methyltransferase ESET and histone H3 lysine 9 trimethylation. Here, we demonstrate that introduced ERV sequences are sufficient to direct rapid de novo methylation of a flanked promoter in embryonic stem (ES) cells. This mechanism requires the presence of an ERV sequence-recognizing KRAB zinc-finger protein (ZFP) and both KAP1 and ESET. Furthermore, this process can also take place on a strong cellular promoter and leads to methylation signatures that are subsequently maintained in vivo throughout embryogenesis. Finally, we show that methylation of ERVs residing in the genome is affected by knockout of KAP1 in early embryos. KRAB-ZFPs, KAP1 and ESET are thus likely to be responsible for the early embryonic instatement of stable epigenetic marks at ERV-containing loci.

KAP1 controls endogenous retroviruses in embryonic stem cells.

More than forty per cent of the mammalian genome is derived from retroelements, of which about one-quarter are endogenous retroviruses (ERVs). Some are still active, notably in mice the highly polymorphic early transposon (ETn)/MusD and intracisternal A-type particles (IAP). ERVs are transcriptionally silenced during early embryogenesis by histone and DNA methylation (and reviewed in ref. 7), although the initiators of this process, which is essential to protect genome integrity, remain largely unknown. KAP1 (KRAB-associated protein 1, also known as tripartite motif-containing protein 28, TRIM28) represses genes by recruiting the histone methyltransferase SETDB1, heterochromatin protein 1 (HP1) and the NuRD histone deacetylase complex, but few of its physiological targets are known. Two lines of evidence suggest that KAP1-mediated repression could contribute to the control of ERVs: first, KAP1 can trigger permanent gene silencing during early embryogenesis, and second, a KAP1 complex silences the retrovirus murine leukaemia virus in embryonic cells. Consistent with this hypothesis, here we show that KAP1 deletion leads to a marked upregulation of a range of ERVs, in particular IAP elements, in mouse embryonic stem (ES) cells and in early embryos. We further demonstrate that KAP1 acts synergistically with DNA methylation to silence IAP elements, and that it is enriched at the 5' untranslated region (5'UTR) of IAP genomes, where KAP1 deletion leads to the loss of histone 3 lysine 9 trimethylation (H3K9me3), a hallmark of KAP1-mediated repression. Correspondingly, IAP 5'UTR sequences can impose in cis KAP1-dependent repression on a heterologous promoter in ES cells. Our results establish that KAP1 controls endogenous retroelements during early embryonic development.

Metabolome-associated psychological comorbidities improvement in irritable bowel syndrome patients receiving a probiotic.

Our recent randomized, placebo-controlled study in Irritable Bowel Syndrome (IBS) patients with diarrhea or alternating bowel habits showed that the probiotic Bifidobacterium longum (BL) NCC3001 improves depression scores and decreases brain emotional reactivity. However, the involved metabolic pathways remain unclear. This analysis aimed to investigate the biochemical pathways underlying the beneficial effects of BL NCC3001 using metabolomic profiling. Patients received probiotic (1x 1010CFU, n=16) or placebo (n=19) daily for 6 weeks. Anxiety and depression were measured using the Hospital Anxiety and Depression Scale. Brain activity in response to negative emotional stimuli was assessed by functional Magnetic Resonance Imaging. Probiotic fecal abundance was quantified by qPCR. Quantitative measurement of specific panels of plasma host-microbial metabolites was performed by mass spectrometry-based metabolomics. Probiotic abundance in feces was associated with improvements in anxiety and depression scores, and a decrease in amygdala activation. The probiotic treatment increased the levels of butyric acid, tryptophan, N-acetyl tryptophan, glycine-conjugated bile acids, and free fatty acids. Butyric acid concentration correlated with lower anxiety and depression scores, and decreased amygdala activation. Furthermore, butyric acid concentration correlated with the probiotic abundance in feces. In patients with non-constipation IBS, improvements in psychological comorbidities and brain emotional reactivity were associated with an increased abundance of BL NCC3001 in feces and specific plasma metabolites, mainly butyric acid. These findings suggest the importance of a probiotic to thrive in the gut and highlight butyric acid as a potential biochemical marker linking microbial metabolism with beneficial effects on the gut-brain axis.