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Actinobacteria produce numerous antibiotics and other specialized metabolites that have important applications in medicine and agriculture.sup.1. Diffusible hormones frequently control the production of such metabolites by binding TetR family transcriptional repressors (TFTRs), but the molecular basis for this remains unclear.sup.2. The production of methylenomycin antibiotics in Streptomyces coelicolor A3(2) is initiated by the binding of 2-alkyl-4-hydroxymethylfuran-3-carboxylic acid (AHFCA) hormones to the TFTR MmfR.sup.3. Here we report the X-ray crystal structure of an MmfR-AHFCA complex, establishing the structural basis for hormone recognition. We also elucidate the mechanism for DNA release upon hormone binding through the single-particle cryo-electron microscopy structure of an MmfR-operator complex. DNA binding and release assays with MmfR mutants and synthetic AHFCA analogues define the role of individual amino acid residues and hormone functional groups in ligand recognition and DNA release. These findings will facilitate the exploitation of actinobacterial hormones and their associated TFTRs in synthetic biology and in the discovery of new antibiotics. X-ray crystallography and cryo-electron microscopy structures of the transcriptional repressor of the methylomycin gene cluster, MmfR, reveal the molecular basis for regulation of antibiotic biosynthesis by AHFCA hormones in Actinobacteria., Author(s): Shanshan Zhou [sup.1] , Hussain Bhukya [sup.4] , Nicolas Malet [sup.1] , Peter J. Harrison [sup.1] , Dean Rea [sup.2] , Matthew J. Belousoff [sup.5] , Hariprasad Venugopal [sup.6] [...]

Long noncoding RNA activated by transforming growth factor-beta (lnc-ATB) is abnormally expressed in a number of tumor types. The aim of this study was to investigate the expression of lnc-ATB and miR-141-3p, and to determine whether lnc-ATB can regulate epithelial-mesenchymal transition (EMT) by miR-141-3p in breast cancer. Here, we found that lnc-ATB was highly expressed, whereas there was low expression of miR-141-3p in breast cancer tissues and cells. Knockdown of lnc-ATB in two breast cancer cell lines (MDA-MB-231 and BT549) significantly increased miR-141-3p expression. Down-regulation of lnc-ATB resulted in a morphological change of breast cancer cells from spindle-like to a round shape, and in a remarkable inhibition of cell migration and invasion, which were reversed by miR-141-3p inhibitor. Furthermore, we demonstrated that lnc-ATB knockdown decreased ZEB1, ZEB2, N-cadherin, and vimentin expression, and promoted E-cadherin expression, while miR-141-3p inhibitor could reverse those effects. Moreover, we proved that miR-141-3p directly bound to the 3= untranslated region (UTR) of ZEB1 and ZEB2 and negatively regulated ZEB1 and ZEB2 expression. Taken together, our results show that knockdown of lnc-ATB significantly inhibits the EMT process of breast cancer cells by increasing the expression of miR-141-3p, indicating that lnc-ATB might serve as a novel therapeutic target for breast cancer. Key words: breast cancer, lncRNA-ATB, miR-141-3p, epithelial-mesenchymal transition. Le long ARN non codant active par le facteur de croissance transformant-beta (lnc-ATB) est exprime de maniere anormale dans plusieurs types de tumeurs. L'objectif de cette etude etait d'examiner l'expression de lnc-ATB et de miR-141-3p dans le cancer du sein et de determiner si lnc-ATB peut reguler la transition epithelio-mesenchymateuse (TEM) par miR-141-3p. Les auteurs ont trouve ici que lnc-ATB etait hautement exprime dans les tissus et les cellules de cancer du sein alors que mi-R-141-3p etait faiblement exprime. Le knockdown de lnc-ATB dans deux lignees cellulaires de cancer du sein (MDA-MB-231 et BT549) augmentait significativement l'expression de miR-141-3p. La regulation a la baisse de lnc-ATB donnait lieu a un changement morphologique des cellules de cancer du sein, d'une forme en fuseau a une forme arrondie, et a une inhibition remarquable de la migration et de l'invasion cellulaire, qui etaient renverses par un inhibiteur de miR-141-3p. Par ailleurs, les auteurs ont demontre que le knockdown de lnc-ATB diminuait l'expression de ZEB1 et ZEB2, de la N-cadherine et de la vimentine, et favorisait l'expression de E-cadherine, alors que l'inhibiteur de miR-141-3p pouvait renverser ces effets. Qui plus est, ils ont prouve que miR-141-3p se liait directement a la region 3= non traduite de ZEB1 et ZEB2 et regulait negativement l'expression de ZEB1 et ZEB2. Dans l'ensemble, leurs resultats montrent que le knockdown de lnc-ATB inhibe significativement le processus de TEM des cellules de cancer du sein en augmentant l'expression de miR-141-3p, indiquant que lnc-ATB pourrait servir de nouvelle cible therapeutique dans le cancer du sein. [Traduit par la Redaction] Mots-cles : cancer du sein, lnc-ARN-ATB, miR-141-3p, transition epithelio-mesenchymateuse., Introduction Breast cancer is one of the most common malignant tumors in females (DeSantis et al. 2014). The improvement in early diagnosis and treatment has significantly improved prognosis (Zhang et [...]

Normal hematopoiesis is sustained through a carefully orchestrated balance between hematopoietic stem cell (HSC) self-renewal and differentiation. The functional importance of this axis is underscored by the severity of disease phenotypes initiated by abnormal HSC function, including myelodysplastic syndromes and hematopoietic malignancies. Major advances in the understanding of transcriptional regulation of primitive hematopoietic cells have been achieved; however, the post-transcriptional regulatory layer that may impinge on their behavior remains underexplored by comparison. Key players at this level include RNA-binding proteins (RBPs), which execute precise and highly coordinated control of gene expression through modulation of RNA properties that include its splicing, polyadenylation, localization, degradation, or translation. With the recent identification of RBPs having essential roles in regulating proliferation and cell fate decisions in other systems, there has been an increasing appreciation of the importance of post-transcriptional control at the stem cell level. Here we discuss our current understanding of RBP-driven post-transcriptional regulation in HSCs, its implications for normal, perturbed, and malignant hematopoiesis, and the most recent technological innovations aimed at RBP-RNA network characterization at the systems level. Emerging evidence highlights RBP-driven control as an underappreciated feature of primitive hematopoiesis, the greater understanding of which has important clinical implications.Key words: hematopoietic stem cells, leukemia stem cells, post-transcriptional regulation, RNA-binding proteins, RNA regulons.L'hematopoiese normale est soutenue par un equilibre soigneusement orchestre entre l'autorenouvelement des cellules souches hematopoietiques (CSH) et leur differenciation. L'importance fonctionnelle de cet axe est mise en evidence par la severite des phenotypes des maladies initiees par une fonction anormale des CSH, dont les syndromes myelodysplasiques et les cancers hematopoietiques. Des percees importantes dans la comprehension de la regulation transcriptionnelle des cellules hematopoietiques primitives ont ete realisees; toutefois, en comparaison, les composantes de la regulation post-transcriptionnelle qui pourraient affecter leur comportement demeurent peu explorees. Les joueurs cles de ce niveau de regulation comprennent les proteines de liaison de l'ARN qui executent un controle hautement coordonne de l'expression genique par la modulation des proprietes de l'ARN dont l'epissage, la polyadenylation, la localisation, la degradation ou la traduction. Avec l'identification recente de proteines de liaison de l'ARN qui jouent un role essentiel dans la regulation de la proliferation et des decisions cellulaires dans d'autres systemes, l'importance du controle post-transcriptionnel a l'echelle des cellules souches est de plus en plus reconnue. Les auteurs discutent ici de la comprehension actuelle de la regulation post-transcriptionnelle assuree par les proteines de liaison de l'ARN dans les CSH, de son implication dans l'hematopoiese normale, perturbee et maligne, de meme que des innovations technologiques les plus recentes visant a caracteriser le reseau ARN-proteines de liaison de l'ARN a l'echelle des systemes. De nouvelles donnees soulignent que le controle exerce par les proteines de liaison de l'ARN constitue une caracteristique negligee de l'hematopoiese primitive, dont une meilleure connaisance aurait d'importantes retombees cliniques. [Traduit par la Redaction]Mots-cles : cellules souches hematopoietiques, cellules souches leucemiques, regulation post-transcriptionnelle, proteines de liaison de l'ARN, regulons d'ARN., IntroductionThe machinery underlying stem cells' capacity to orchestrate their self-maintenance and differentiation can be represented as integrated circuits that are genetically programmed. As demonstrated by the capacity to reprogram somatic [...]

Mutations that attenuate homologous recombination (HR)-mediated repair promote tumorigenesis and sensitize cells to chemotherapeutics that cause replication fork collapse, a phenotype known as 'BRCAness'.sup.1. BRCAness tumours arise from loss-of-function mutations in 22 genes.sup.1. Of these genes, all but one (CDK12) function directly in the HR repair pathway.sup.1. CDK12 phosphorylates serine 2 of the RNA polymerase II C-terminal domain heptapeptide repeat.sup.2-7, a modification that regulates transcription elongation, splicing, and cleavage and polyadenylation.sup.8,9. Genome-wide expression studies suggest that depletion of CDK12 abrogates the expression of several HR genes relatively specifically, thereby blunting HR repair.sup.3-7,10,11. This observation suggests that the mutational status of CDK12 may predict sensitivity to targeted treatments against BRCAness, such as PARP1 inhibitors, and that CDK12 inhibitors may induce sensitization of HR-competent tumours to these treatments.sup.6,7,10,11. Despite growing clinical interest, the mechanism by which CDK12 regulates HR genes remains unknown. Here we show that CDK12 globally suppresses intronic polyadenylation events in mouse embryonic stem cells, enabling the production of full-length gene products. Many HR genes harbour more intronic polyadenylation sites than other expressed genes, and these sites are particularly sensitive to loss of CDK12. The cumulative effect of these sites accounts for the enhanced sensitivity of HR gene expression to CDK12 loss, and we find that this mechanism is conserved in human tumours that contain loss-of-function CDK12 mutations. This work clarifies the function of CDK12 and underscores its potential both as a chemotherapeutic target and as a tumour biomarker.The kinase CDK12 suppresses usage of intronic polyadenylation sites and thereby promotes the expression of genes involved in homologous recombination DNA repair., Author(s): Sara J. Dubbury [sup.1] [sup.2] , Paul L. Boutz [sup.1] [sup.3] , Phillip A. Sharp [sup.1] [sup.2] Author Affiliations:(1) Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, [...]

Epigenomics regulates gene expression and is as important as genomics in precision personal health, as it is heavily influenced by environment and lifestyle. We profiled whole-genome DNA methylation and the corresponding transcriptome of peripheral blood mononuclear cells collected from a human volunteer over a period of 36 months, generating 28 methylome and 57 transcriptome datasets. We found that DNA methylomic changes are associated with infrequent glucose level alteration, whereas the transcriptome underwent dynamic changes during events such as viral infections. Most DNA meta-methylome changes occurred 80-90 days before clinically detectable glucose elevation. Analysis of the deep personal methylome dataset revealed an unprecedented number of allelic differentially methylated regions that remain stable longitudinally and are preferentially associated with allele-specific gene regulation. Our results revealed that changes in different types of 'omics' data associate with different physiological aspects of this individual: DNA methylation with chronic conditions and transcriptome with acute events. Personal 'omics' analysis of a single individual over a period of 3 years reveals that different types of omics data associate with episodes of acute and chronic disease., Author(s): Rui Chen [sup.1] [sup.3] , Lin Xia [sup.2] , Kailing Tu [sup.2] , Meixue Duan [sup.2] , Kimberly Kukurba [sup.1] , Jennifer Li-Pook-Than [sup.1] , Dan Xie [sup.1] [sup.2] [...]

Histone H3 lysine 9 methylation (H3K9me) mediates heterochromatic gene silencing and is important for genome stability and the regulation of gene expression.sup.1-4. The establishment and epigenetic maintenance of heterochromatin involve the recruitment of H3K9 methyltransferases to specific sites on DNA, followed by the recognition of pre-existing H3K9me by the methyltransferase and methylation of proximal histone H3.sup.5-11. This positive feedback loop must be tightly regulated to prevent deleterious epigenetic gene silencing. Extrinsic anti-silencing mechanisms involving histone demethylation or boundary elements help to limit the spread of inappropriate H3K9me.sup.12-15. However, how H3K9 methyltransferase activity is locally restricted or prevented from initiating random H3K9me--which would lead to aberrant gene silencing and epigenetic instability--is not fully understood. Here we reveal an autoinhibited conformation in the conserved H3K9 methyltransferase Clr4 (also known as Suv39h) of the fission yeast Schizosaccharomyces pombe that has a critical role in preventing aberrant heterochromatin formation. Biochemical and X-ray crystallographic data show that an internal loop in Clr4 inhibits the catalytic activity of this enzyme by blocking the histone H3K9 substrate-binding pocket, and that automethylation of specific lysines in this loop promotes a conformational switch that enhances the H3K9me activity of Clr4. Mutations that are predicted to disrupt this regulation lead to aberrant H3K9me, loss of heterochromatin domains and inhibition of growth, demonstrating the importance of the intrinsic inhibition and auto-activation of Clr4 in regulating the deposition of H3K9me and in preventing epigenetic instability. Conservation of the Clr4 autoregulatory loop in other H3K9 methyltransferases and the automethylation of a corresponding lysine in the human SUV39H2 homologue.sup.16 suggest that the mechanism described here is broadly conserved.An autoinhibitory conformation of the histone H3K9 methyltransferase Clr4 of Schizosaccharomyces pombe helps to prevent aberrant heterochromatin formation and maintains epigenetic stability., Author(s): Nahid Iglesias [sup.1] [sup.2] , Mark A. Currie [sup.1] [sup.2] , Gloria Jih [sup.1] [sup.2] , Joao A. Paulo [sup.1] , Nertila Siuti [sup.1] [sup.2] , Marian Kalocsay [sup.1] [...]

Author(s): Gloria Jih [1, 2]; Nahid Iglesias [1, 2]; Mark A. Currie [1, 2]; Natarajan V. Bhanu [3]; Joao A. Paulo [1]; Steven P. Gygi [1]; Benjamin A. Garcia [3]; [...]

Autophagy is a highly conserved self-digestion process, which is essential for maintaining homeostasis and viability in response to nutrient starvation (1-4). Although the components of autophagy in the cytoplasm have been well studied (5,6), the molecular basis for the transcriptional and epigenetic regulation of autophagy is poorly understood. Here we identify co-activator-associated arginine methyltransferase 1 (CARM1) as a crucial component of autophagy in mammals. Notably, CARM1 stability is regulated by the SKP2-containing SCF (SKP1-cullin1-F-box protein) E3 ubiquitin ligase in the nucleus, but not in the cytoplasm, under nutrient-rich conditions. Furthermore, we show that nutrient starvation results in AMP-activated protein kinase (AMPK)-dependent phosphorylation of FOXO3a in the nucleus, which in turn transcriptionally represses SKP2. This repression leads to increased levels of CARM1 protein and subsequent increases in histone H3 Arg17 dimethylation. Genome-wide analyses reveal that CARM1 exerts transcriptional co-activator function on autophagy-related and lysosomal genes through transcription factor EB (TFEB). Our findings demonstrate that CARMl-dependent histone arginine methylation is a crucial nuclear event in autophagy, and identify a new signalling axis of AMPK-SKP2-CARM1 in the regulation of autophagy induction after nutrient starvation., To explore the importance of nuclear events in autophagy, we proposed that specific histone marks are involved in the epigenetic and transcriptional regulation of autophagy in the nucleus leading to [...]

The mTOR pathway orchestrates cellular homeostasis. The rapamycin-sensitive mTOR complex (mTORC1) in the kidney has been widely studied; however, mTORC2 function in renal tubules is poorly characterized. Here, we generated mice lacking mTORC2 in the distal tubule ([Rictor.sup.fl/fl] Ksp-Cre mice), which were viable and had no obvious phenotype, except for a 2.5-fold increase in plasma aldosterone. Challenged with a low-[Na.sup.+] diet, these mice adequately reduced [Na.sup.+] excretion; however, [Rictor.sup.fl/fl] Ksp-Cre mice rapidly developed hyperkalemia on a high-[K.sup.+] diet, despite a 10-fold increase in serum aldosterone levels, implying that mTORC2 regulates kaliuresis. Phosphorylation of serum- and glucocorticoid-inducible kinase 1 (SGK1) and PKC-α was absent in [Rictor.sup.fl/fl] Ksp-Cre mice, indicating a functional block in [K.sup.+] secretion activation via ROMK channels. Indeed, patch-clamp experiments on split-open tubular segments from the transition zone of the late connecting tubule and early cortical collecting duct demonstrated that [Ba.sup.2+]-sensitive apical [K.sup.+] currents were barely detectable in the majority of [Rictor.sup.fl/fl] Ksp-Cre mice. Conversely, epithelial sodium channel (ENaC) activity was largely preserved, suggesting that the reduced ability to maintain [K.sup.+] homeostasis is the result of impaired apical [K.sup.+] conductance and not a reduced electrical driving force for [K.sup.+] secretion. Thus, these data unravel a vital and nonredundant role of mTORC2 for distal tubular [K.sup.+] handling., Introduction Since their discovery in 2002, the mTOR complexes have attracted a tremendous interest. The mTOR complexes belong to the AGC family of kinases and control a diverse range of [...]

How tissue regeneration programs are triggered by injury has received limited research attention. Here we investigate the existence of enhancer regulatory elements that are activated in regenerating tissue. Transcriptomic analyses reveal that leptin b (lepb) is highly induced in regenerating hearts and fins of zebrafish. Epigenetic profiling identified a short DNA sequence element upstream and distal to lepb that acquires open chromatin marks during regeneration and enables injury-dependent expression from minimal promoters. This element could activate expression in injured neonatal mouse tissues and was divisible into tissue-specific modules sufficient for expression in regenerating zebrafish fins or hearts. Simple enhancer-effector transgenes employing lepb-linked sequences upstream of pro- or anti-regenerative factors controlled the efficacy of regeneration in zebrafish. Our findings provide evidence for 'tissue regeneration enhancer elements' (TREEs) that trigger gene expression in injury sites and can be engineered to modulate the regenerative potential of vertebrate organs., The capacity for complex tissue regeneration is unevenly distributed among vertebrate tissues and species. Salamanders and zebrafish possess remarkable potential to regenerate tissues like amputated appendages, resected heart muscle, and [...]

Tissue morphogenesis arises from coordinated changes in cell shape driven by actomyosin contractions. Patterns of gene expression regionalize cell behaviours by controlling actomyosin contractility. Here we report two modes of control over Rho1 and myosin II (MyoII) activation in the Drosophila endoderm. First, Rho1-MyoII are induced in a spatially restricted primordium via localized transcription of the G-protein-coupled receptor ligand Fog. Second, a tissue-scale wave of Rho1-MyoII activation and cell invagination progresses anteriorly away from the primordium. The wave does not require sustained gene transcription, and is not governed by regulated Fog delivery. Instead, MyoII inhibition blocks Rho1 activation and propagation, revealing a mechanical feedback driven by MyoII. We find that MyoII activation and invagination in each row of cells drives adhesion to the vitelline membrane mediated by integrins, apical spreading, MyoII activation and invagination in the next row. Endoderm morphogenesis thus emerges from local transcriptional initiation and a mechanically driven cycle of cell deformation. Tissue shape changes in the posterior endoderm of the early Drosophila embryo are driven by actomyosin contractions emerging from a transcriptional induction followed by a mechanically-driven propagation of RhoI-myosin II activation., Author(s): Anaïs Bailles [sup.1] [sup.2] , Claudio Collinet [sup.1] [sup.2] , Jean-Marc Philippe [sup.1] [sup.2] , Pierre-François Lenne [sup.1] [sup.2] , Edwin Munro [sup.3] , Thomas Lecuit [sup.1] [sup.2] [sup.4] [...]

Wing polyphenism is an evolutionarily successful feature found in a wide range of insects (1). Long-winged morphs can fly, which allows them to escape adverse habitats and track changing resources, whereas short-winged morphs are flightless, but usually possess higher fecundity than the winged morphs (1-3). Studies on aphids, crickets and planthoppers have revealed that alternative wing morphs develop in response to various environmental cues (1,2,4-8), and that the response to these cues may be mediated by developmental hormones, although research in this area has yielded equivocal and conflicting results about exactly which hormones are involved (4,8-10). As it stands, the molecular mechanism underlying wing morph determination in insects has remained elusive. Here we show that two insulin receptors in the migratory brown planthopper Nilaparvata lugens, InR1 and InR2, have opposing roles in controlling long wing versus short wing development by regulating the activity of the forkhead transcription factor Foxo. InR1, acting via the phosphatidylinositol-3-OH kinase (PI(3)K)-protein kinase B (Akt) signalling cascade, leads to the long-winged morph if active and the short-winged morph if inactive. InR2, by contrast, functions as a negative regulator of the InR1-PI(3)K-Akt pathway: suppression of InR2 results in development of the long-winged morph. The brain-secreted ligand Ilp3 triggers development of long-winged morphs. Our findings provide the first evidence of a molecular basis for the regulation of wing polyphenism in insects, and they are also the first demonstration--to our knowledge--of binary control over alternative developmental outcomes, and thus deepen our understanding of the development and evolution of phenotypic plasticity., The migratory brown planthopper (BPH) N. lugens (order Hemiptera), can develop into short-winged or long-winged morphs in response to environmental cues (Fig. 1a and Supplementary Notes 1,2). The long-winged BPHs [...]

The structure of messenger RNA is important for post-transcriptional regulation, mainly because it affects binding of trans-acting factors (1). However, little is known about the in vivo structure of full-length [...]

Visualizing the physical basis for molecular behaviour inside living cells is a great challenge for biology. RNAs are central to biological regulation, and the ability of RNA to adopt specific structures intimately controls every step of the gene expression program (1). However, our understanding of physiological RNA structures is limited; current in vivo RNA structure profiles include only two of the four nucleotides that make up RNA (2,3). Here we present a novel biochemical approach, in vivo click selective 2'-hydroxyl acylation and profiling experiment (icSHAPE), which enables the first global view, to our knowledge, of RNA secondary structures in living cells for all four bases. icSHAPE of the mouse embryonic stem cell transcriptome versus purified RNA folded in vitro shows that the structural dynamics of RNA in the cellular environment distinguish different classes of RNAs and regulatory elements. Structural signatures at translational start sites and ribosome pause sites are conserved from in vitro conditions, suggesting that these RNA elements are programmed by sequence. In contrast, focal structural rearrangements in vivo reveal precise interfaces of RNA with RNA-binding proteins or RNAmodification sites that are consistent with atomic-resolution structural data. Such dynamic structural footprints enable accurate prediction of RNA-protein interactions and [N.sup.6]-methyladenosine ([m.sup.6]A) modification genome wide. These results open the door for structural genomics of RNA in living cells and reveal key physiological structures controlling gene expression., SHAPE accurately identifies flexible (single-stranded) bases in RNA for all four nucleotides. However, current methods are potentially limited by high background rates (>70% of RNA molecules have no modification due [...]

Tumour oncogenes include transcription factors that co-opt the general transcriptional machinery to sustain the oncogenic state (1), but direct pharmacological inhibition of transcription factors has so far proven difficult (2). However, the transcriptional machinery contains various enzymatic cofactors that can be targeted for the development of new therapeutic candidates (3), including cyclin-dependent kinases (CDKs) (4). Here we present the discovery and characterization of a covalent CDK7 inhibitor, THZ1, which has the unprecedented ability to target a remote cysteine residue located outside of the canonical kinase domain, providing an unanticipated means of achieving selectivity for CDK7. Cancer cell-line profiling indicates that a subset of cancer cell lines, including human T-cell acute lymphoblastic leukaemia (T-ALL), have exceptional sensitivity to THZ1. Genome-wide analysis in Jurkat T-ALL cells shows that THZ1 disproportionally affects transcription of RUNX1 and suggests that sensitivity to THZ1 may be due to vulnerability conferred by the RUNX1 super-enhancer and the key role of RUNX1 in the core transcriptional regulatory circuitry of these tumour cells. Pharmacological modulation of CDK7 kinase activity may thus provide an approach to identify and treat tumour types that are dependent on transcription for maintenance of the oncogenic state., In an effort to discover new inhibitors of kinases that regulate gene transcription, we performed cell-based screening and kinase selectivity profiling of a library of known and novel ATP-site-directed kinase [...]