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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] [...]

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 [...]

A unique resource of longitudinal childhood height data available in Finnish population cohorts was used to study the largely unknown genetic underpinnings of height growth. The partially correlated variants at LIN28B which influence the entire period of postnatal growth implied a critical role for LIN28B in the regulation of human growth.

A module for implementing robust feedback control in synthetic cellular networks has been reported. Its design is first proved mathematically to be universal for all networks, and then implemented in living cells. A module for robust perfect adaptation., Author(s): Noah Olsman, Johan Paulsson Author Affiliations: A universal control system for synthetic gene networks Anyone who has lived without central heating and cooling has had to learn the right [...]

Whether DNA segments that amplify gene activity represent a previously unrecognized form of gene regulation is still under debate.Whether DNA segments that amplify gene activity represent a previously unrecognized form of gene regulation is still under debate., Author(s): Heidi LedfordAuthor Affiliations:Cancer researchers seek to harness mysterious DNA 'super-enhancers' Double helix illustration [see PDF for image]Experimental cancer treatments that harness souped-up segments of DNA called super-enhancers to activate [...]

An approach developed for mapping cis-regulatory variation based on interindividual variation in allelic expression (AE) is used to identify putative causal DNA variants that regulate long-range chromatin interactions. Studies on the regulation of asthma and autoimmune disease reveal that the genetic variants associated with them affect the expression of the ZPBP2/GSDMB/ORMDL3 gene locus in the chromosome 17q12-q21 region through allele-specific chromatin remodeling.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2008.10.037 Byline: Chong Pyo Choe, Susan J. Brown Keywords: Segmentation; Pair-rule; Segment polarity; Tribolium Abstract: In Drosophila, primary pair-rule genes establish the parasegmental boundaries and indirectly control the periodic expression of the segment polarity genes engrailed (en) and wingless (wg) via regulation of secondary pair-rule genes. Although orthologs of some Drosophila pair-rule genes are not required for proper segmentation in Tribolium, segmental expression of Tc-en and Tc-wg is conserved. To understand how these segment polarity genes are regulated, we examined the results of expressing one or two pair-rule genes in the absence of the other known pair-rule genes. Expression of one or both of the secondary pair-rule genes, Tc-sloppy-paired (Tc-slp) and Tc-paired (Tc-prd), activated Tc-wg in the absence of the primary pair-rule genes, Tc-even-skipped (Tc-eve), Tc-runt (Tc-run) and Tc-odd-skipped (Tc-odd). Tc-eve alone failed to activate Tc-wg or Tc-en, but in combination with Tc-run or Tc-prd activated Tc-en. These results, interpreted within the pair-rule gene expression patterns, suggest separate models for the genetic regulation of the juxtaposed expression of Tc-wg and Tc-en at odd- and even-numbered parasegmental boundaries, respectively. Conserved interactions between eve and prd at the anterior boundary of odd-numbered parasegments may reflect an ancestral segmentation mechanism that functioned in every segment prior to the evolution of pair-rule segmentation. Author Affiliation: Division of Biology, Kansas State University, Manhattan, KS 66506, USA Article History: Received 16 August 2008; Revised 18 October 2008; Accepted 28 October 2008

Coordination of animal behavior with reproductive status is often achieved through elaboration of hormones by the gonad. In the nematode Caenorhabditis elegans, adult males explore their environment to locate mates. Mate searching is regulated by presence of mates, nutritional status, and a signal from the gonad. Here we show that the gonadal signal acts via the nuclear receptor DAF-12, a protein known to regulate several C. elegans life-history traits. DAF-12 has both activational and organizational functions to stimulate exploratory behavior and acts downstream of the gonadal signal, outside of the gonad. DAF-12 acts upstream of sensory input from mating partners and physiological signals indicating nutritional status. Mate searching was rescued in germ-line ablated animals, but not if both germ line and somatic gonad were ablated, by a precursor of the DAF-12 ligand, dafachronic acid (DA). The results are interpreted to suggest that the germ line produces a DA precursor that is converted to DA outside of the germ line, possibly in the somatic gonad. As it does in other pathways in which it functions, in regulation of male mate searching behavior DAF-12 acts at a choice point between alternatives favoring reproduction (mate searching) vs. survival (remaining on food).

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2007.12.011 Byline: Takayoshi Inoue (a)(b), Yukiko U. Inoue (a), Junko Asami (a), Hitomi Izumi (a), Shun Nakamura (a), Robb Krumlauf (b) Keywords: Cdh6 (cadherin-6); Mouse; Gene regulation; BAC; Transposon; Homologous recombination; Forebrain; Hindbrain; Compartment; Neural crest cells Abstract: Classic cadherins are cell adhesion molecules whose expression patterns are dynamically modulated in association with their diverse functions during morphogenesis. The large size and complexity of cadherin loci have made it a challenge to investigate the organization of cis-regulatory modules that control their spatiotemporal patterns of expression. Towards this end, we utilized bacterial artificial chromosomes (BACs) containing the Cdh6 gene, a mouse type II classic cadherin, to systematically identify cis-regulatory modules that govern its expression. By inserting a lacZ reporter gene into the Cdh6 BAC and generating a series of modified variants via homologous recombination or transposon insertions that have been examined in transgenic mice, we identified an array of genomic regions that contribute to specific regulation of the gene. These regions span [approximately equal to]350 kb of the locus between 161-kb upstream and 186-kb downstream of the Cdh6 transcription start site. Distinct modules independently regulate compartmental expression (i.e. forebrain, hindbrain rhombomeres, and spinal cord) and/or cell lineage-specific expression patterns (i.e. neural crest subpopulations such as Schwann cells) of Cdh6 at the early developmental stages. With respect to regulation of expression in neural crest cells, we have found that distinct regions contribute to different aspects of expression and have identified a short 79-bp region that is implicated in regulating expression in cells once they have emigrated from the neural tube. These results build a picture of the complex organization of Cdh6 cis-regulatory modules and highlight the diverse inputs that contribute to its dynamic expression during early mouse embryonic development. Author Affiliation: (a) Department of Biochemistry and Cellular Biology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Ogawahigashi 4-1-1, Kodaira, Tokyo 187-8502, Japan (b) Stowers Institute for Medical Research, 1000 East, 50th Street, Kansas City, MO 64110, USA Article History: Received 29 June 2007; Revised 5 November 2007; Accepted 9 December 2007

Internal bases in mRNA can be subjected to modifications that influence the fate of mRNA in cells. One of the most prevalent modified bases is found at the 5 end of mRNA, at the first encoded nucleotide adjacent to the 7-methylguanosine cap. Here we show that this nucleotide, N[sup.6],2-O-dimethyladenosine (m[sup.6]A[sub.m]), is a reversible modification that influences cellular mRNA fate. Using a transcriptome-wide map of m[sup.6]A[sub.m] we find that m[sup.6]A[sub.m]-initiated transcripts are markedly more stable than mRNAs that begin with other nucleotides. We show that the enhanced stability of m[sup.6]A[sub.m]-initiated transcripts is due to resistance to the mRNA-decapping enzyme DCP2. Moreover, we find that m[sup.6]A[sub.m] is selectively demethylated by fat mass and obesity-associated protein (FTO). FTO preferentially demethylates m[sup.6]A[sub.m] rather than N[sup.6]-methyladenosine (m[sup.6]A), and reduces the stability of m[sup.6]A[sub.m] mRNAs. Together, these findings show that the methylation status of m[sup.6]A[sub.m] in the 5 cap is a dynamic and reversible epitranscriptomic modification that determines mRNA stability., Author(s): Jan Mauer [1]; Xiaobing Luo [2]; Alexandre Blanjoie [3]; Xinfu Jiao [2]; Anya V. Grozhik [1]; Deepak P. Patil [1]; Bastian Linder [1]; Brian F. Pickering [1]; Jean-Jacques Vasseur [...]

Genetic variation in functionally integrated skeletal traits can be maintained over 10 million years despite bottlenecks and stringent selection. Here, we describe an analysis of the genetic architecture of the canid axial skeleton using populations of the Portuguese Water Dog Canis familiaris) and silver fox (Vulpes vulpes). Twenty-one skeletal metrics taken from radiographs of the forelimbs and hind limbs of the fox and dog were used to construct separate anatomical principal component (PC) matrices of the two species. In both species, 15 of the 21 PCs exhibited significant heritability, ranging from 25% to 70%. The second PC, in both species, represents a trade-off in which limb-bone width is inversely correlated with limb-bone length. PC2 accounts for approximately 15% of the observed skeletal variation, ~30% of the variation in shape. Many of the other significant PCs affect very small amounts of variation (e.g., 0.2-2%) along trade-off axes that partition function between the forelimbs and hind limbs. These PCs represent shape axes in which an increase in size of an element of the forelimb is associated with a decrease in size of an element of the hind limb and vice versa. In most cases, these trade-offs are heritable in both species and genetic loci have been identified in the Portuguese Water Dog for many of these. These PCs, present in both the dog and the fox, include ones that affect lengths of the forelimb versus the hind limb, length of the forefoot versus that of the hind foot, muscle moment (i.e., lever) arms of the forelimb versus hind limb, and cortical thickness of the bones of the forelimb versus hind limb. These inverse relationships suggest that genetic regulation of the axial skeleton results, in part, from the action of genes that influence suites of functionally integrated traits. Their presence in both dogs and foxes suggests that the genes controlling the regulation of these PCs of the forelimb versus hind limb may be found in other tetrapod taxa.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2007.03.020 Byline: Federico Gonzalez, Denis Duboule, Francois Spitz Keywords: Hox genes; Global and shared enhancers; Regulatory landscapes; Limb morphogenesis; Evolution of gene regulation Abstract: In tetrapods, posterior Hoxd genes (from groups 10 to 13) are necessary to properly pattern the developing autopods, including the number and identities of digits. Their coordinated expression is achieved by sharing a global control region (GCR), which was isolated and localized 200 kb 5' (centromeric) of the gene cluster. However, in transgenic assays, the GCR was unable to fully recapitulate all aspects of the endogenous Hoxd expression patterns during distal limb development. In this paper, we further analyze the regulatory potential of this locus and report the characterization of Prox, a second enhancer element that contributes to the transcriptional activity of posterior Hoxd genes in developing distal limb buds. We show that the GCR and Prox elements complement each other and work in combination to correctly establish the late phase of Hoxd genes expression. Based on DNA sequence conservation and transgenic assays, we discuss the functions of these regulatory regions as well as a potential evolutionary scheme accounting for their emergence along with the evolution of tetrapod limbs. Author Affiliation: National Center for Competence in Research 'Frontiers in Genetics', Department of Zoology and Animal Biology, University of Geneva, Sciences III, Quai Ernest Ansermet 30, 1211 Geneva 4, Switzerland Article History: Received 11 January 2007; Revised 27 February 2007; Accepted 19 March 2007

Competence-stimulating peptide (CSP) and ComD of the streptococcal species are a pheromone and its receptor, respectively, involved in the regulation of competence for natural genetic transformation. We show here that these molecules have undergone positive selection. This study is the first report of positive selection due to competition among bacterial populations.

A study on regulation of notochord examines the role of gene in skeleton development of mice.

Two Bacillus subtilis transcriptional factors, TnrA and GlnR, regulate gene expression in response to changes in nitrogen availability. These two proteins have similar amino acid sequences in their DNA binding domains and bind to DNA sites (GlnR/TnrA sites) that have the same consensus sequence. Expression of the mrA gene was found to be activated by TnrA and repressed by GlnR. Mutational analysis demonstrated that a GlnR/ TnrA site which lies immediately upstream of the -35 region of the tnrA promoter is required for regulation of tnrA expression by both GlnR and TnrA. Expression of the glnRA operon, which contains two GlnR/TnrA binding sites (glnRAo1 and glnRAo2) in its promoter region, is repressed by both GlnR and TnrA. The glnRAo2 site, which overlaps the -35 region of the glnRA promoter, was shown to be required for regulation by both GlnR and TnrA, while the glnRAol site which lies upstream of the -35 promoter region is only involved in GlnR-mediated regulation. Examination of TnrA binding to mrA and glnRA promoter DNA in gel mobility shift experiments showed that TnrA bound with an equilibrium dissociation binding constant of 55 nM to the GlnR/TnrA site in the tnrA promoter region, while the affinities of TnrA for the two GlnR/TnrA sites in the glnRA promoter region were greater than 3 [micro]M. These results demonstrate that GlnR and TnrA cross-regulate each other's expression and that there are differences in their DNA-binding specificities.

The identification, isolation and characterization of the nitroreductase genes, nprA and nprB, involved in 2,4-dinitrophenol reduction in the phototrophic bacterium Rhodobacter capsulatus B10 are described. The induction of the nprA gene by paraquat and by an oxidized redox status in the cell indicates that the SoxRS system can also be implicated in controlling nprA gene expression.

The role of the aristaless-related homeobox gene Alx4 in antero-postefior (AP-) patterning of the developing vertebrate limb has remained somewhat elusive. Polydactyly of Alx4 mutant mice is known to be accompanied by ectopic anterior expression of genes like Shh, Fgf4 and 5'Hoxd. We reported previously that polydactyly in Alx4 mutant mice requires SHH signaling, but we now show that in early [Alx4.sup.-/-] limb buds the anterior ectopic expression of Fgf4 and Hoxdl3, and therefore disruption of AP-patterning, occurs independently of SHH signaling. To better understand how Alx4 functions in the pathways that regulate AP-patterning, we also studied genomic regulatory sequences that are capable of directing expression of a reporter gene in a pattern corresponding to endogenous Alx4 expression in anterior limb bud mesenchyme. We observed, as expected for authentic Alx4 expression, expansion of reporter construct expression in a [Shh.sup.-/-] background. Total lack of reporter expression in a [Gli3.sup.-/-] background confirms the existence of Gli3-dependent and--independent Alx4 expression in the limb bud. Apparently, these two modules of Alx4 expression are linked to dissimilar functions. Keywords: Limb development; Alx4; Gli3; Antero-posterior patterning; Gene regulation; Sonic hedgehog; FGF4; Polydactyly

The ceplR genes encode an N-acyl homoserine lactone (AHL)-dependent quorum-sensing system consisting of an AHL synthase that directs the synthesis of N-octanoyI-L-homoserine lactone (OHL) and N-hexanoyI-L-homoserine lactone and a transcriptional regulator. The virulence of cepIR mutants was examined in two animal models. Rats were infected with agar beads containing Burkholderia cenocepacia K56-2, K56-12 (cepl: :[Tp.sup.r]) or K56-R2 (cepR'' Tn5-OT182). At 10 days post-infection, the extent of lung histopathological changes was significantly lower in lungs infected with K56-12 or K56-R2 compared to the parent strain. Intranasal infections were performed in [Cftr.sup.(-/-)] mice and their wild-type siblings. K56-2 was more virulent in both groups of mice. K56-12 was the least virulent strain and was not invasive in the [Cftr.sup.(-/-)] mice. OHL was readily detected in lung homogenates from [Cftr.sup.(-/-)] mice infected with K56-2 but was only detected at levels slightly above background in a few mice infected with K56-12. Lung homogenates from mice infected with K56-2 had significantly higher levels of the inflammatory mediators murine macrophage inflammatory protein-2, KC/N51, interleukin-1 [beta] and interleukin-6 than those from K56-12-infected animals. These studies indicate that a functional CepIR quorum-sensing system contributes to the severity of B. cenocepacia infections. A zinc metalloprotease gene (zmpA) was shown to be regulated by CepR and may be one of the factors that accounts for the difference in virulence between the cepl mutant and the parent strain.

The Bacillus subtilis PurR mediates adenine repression and guanosine induction of purA. PRPP inhibits binding of PurR to DNA in vitro. Mutations in the PRPP binding motif of PurR caused strong repression regardless of purine exclusions or additions, establishing the role of PRPP as regulator of PurR.

Streptococcus pneumoniae normally resides in the human nasopharynx in a nondisease state. In response to unknown triggers this organism can descend to the lower respiratory tract and/or invade the bloodstream. Regulation and activation of virulence genes play essential roles in this process of disease development. Characterization of S. pneumoniae regulatory networks has been a recent area of interest, but despite inroads little is known about regulation of virulence genes in this pathogen. A putative transcriptional regulator in S. pneumoniae, mgrA, which exhibits homology to the virulence gene activator mga of group A streptococcus, was previously identified as a regulator that is required for development of pneumonia in a murine model. In this study we confirmed that mgrA plays a role in both nasopharyngeal carriage and pneumonia. Transcriptional profiling by microarray technology was used to show that mgrA acts as a repressor of the previously characterized rlrA pathogenicity islet. This is manifested phenotypically by a decrease in adherence to epithelial cells in tissue culture since the rlrA pathogenicity islet contains genes mediating adherence.

Genomic studies with bacteria have identified redox-responsive genes without known roles in counteracting oxidative damage. Previous transcriptional profiling showed that expression of one such gene, yggX, was activated by superoxide stress in Escherichia coli. Here we show that this activation could be mimicked by artificial expression of the regulatory protein SoxS. Northern analysis confirmed the transcriptional activation of yggX by oxidative stress or SoxS expression but not in response to the related MarA or Rob proteins. Northern analysis showed that mltC, which codes for a peptidoglycan hydrolase and is positioned immediately downstream of yggX, was also regulated by oxidative stress or ectopic expression of SoxS. Purified SoxS protein bound to the predicted yggX promoter region, between positions 223 and 163 upstream from the yggX translational start site. Within this region, a 20-bp sequence was found to be necessary for oxidative stress-mediated activation of yggX transcription. A yggX deletion strain was hypersensitive to the redox-cycling agent paraquat, and a plasmid expressing YggX complemented the sensitivity of the deletion strain. Under exposure to paraquat, the yggX deletion strain showed a deficiency in aconitase activity compared to the isogenic wild-type strain, while expression of YggX from a multicopy plasmid increased the aconitase levels above those of the wild-type strain. These results demonstrate the direct regulation of the yggX gene by the redox-sensing SoxRS system and provide further evidence for the involvement of yggX in protection of iron-sulfur proteins against oxidative damage.

CTP synthase is encoded by the pyrG gene and catalyzes the conversion of UTP to CTP. A Lactococcus lactis pyrG mutant with a cytidine requirement was constructed, in which [beta]-galactosidase activity in a pyrG-lacLM transcriptional fusion was used to monitor gene expression of pyrG. A 10-fold decrease in the CTP pool induced by cytidine limitation was found to immediately increase expression of the L. lactis pyrG gene. The final level of expression of pyrG is 37-fold higher than the uninduced level. CTP limitation has pronounced effects on central cellular metabolism, and both RNA and protein syntheses are inhibited. Expression of pyrG responds only to the cellular level of CTP, since expression of pyrG has no correlation to alterations in UTP, GTP, and ATP pool sizes. In the untranslated pyrG leader sequence a potential terminator structure can be identified, and this structure is required for regulation of the pyrG gene. It is possible to fold the pyrG leader in an alternative structure that would prevent the formation of the terminator. We suggest a model for pyrG regulation in L. lactis, and probably in other gram-positive bacteria as well, in which pyrG expression is directly dependent on the CTP concentration through an attenuator mechanism. At normal CTP concentrations a terminator is preferentially formed in the pyrG leader, thereby reducing expression of CTP synthase. At low CTP concentrations the RNA polymerase pauses at a stretch of C residues in the pyrG leader, thereby allowing an antiterminator to form and transcription to proceed. This model therefore does not include any trans-acting protein for sensing the CTP concentration as previously proposed for Bacillus subtilis.

Streptomyces linear plasmids and linear chromosomes can replicate also in a circular form when their telomeres are deleted. The 17-kb linear plasmid pSLA2 has been a useful model in studies of such replicons. Here we report that the minimal origin initiating replication of pSLA2-derived plasmids as circular molecules cannot propagate these plasmids in a linear mode unless they also contain a novel plasmid-encoded locus, here interferes with the establishment of pSLA2 in circular form in Streptomyces lividans transformants. The additional presence of an adjacent divergently transcribed locus, rorA (rlrA override), which strongly resembles the kor (kil override) transcription control genes identified previously on Streptomyces plasmids, reversed the detrimental effects of rlrA on plasmid establishment and additionally stabilized circular plasmid inheritance by spores during the S. lividans life cycle. While the effects of the rlrA/rorA locus of pSLA2 were seen also on linear plasmids derived from the unrelated SLP2 replicon, they did not extend to plasmids whose replication was initiated at a cloned chromosomal origin. Our results establish the existence of, and provide the initial description of, a novel plasmid-borne regulatory system that differentially affects the propagation of linear and circular plasmids in Streptomyces.

Regulation of transcription of the tryptophanase operon requires that translation of its leader peptide coding region, tnaC, be coupled with its transcription. We show in vitro that a transcription pause site exists at the end of the tnaC coding region and that translation of tnaC releases the paused transcription complex, coupling transcription with translation.

The PhoP/PhoQ two-component system controls the extracellular magnesium deprivation response in Salmonella enterica. In addition, several virulence-associated genes that are mainly required for intramacrophage survival during the infection process are under the control of its transcriptional regulation. Despite shared [Mg.sup.2+] modulation of the expression of the PhoP-activated genes, no consensus sequence common to all of them could be detected in their promoter regions. We have investigated the transcriptional regulation and the interaction of the response regulator PhoP with the promoter regions of the PhoP-activated loci phoPQ, mgtA, slyB, pmrD, pcgL, phoN, pagC, and mgtCB. A direct repeat of the heptanucleotide sequence (G/T)GTTTA(A/T) was identified as the conserved motif recognized by PhoP to directly control the gene expression of the first five loci, among which the first four are ancestral to enterobacteria. On the other hand, no direct interaction of the response regulator with the promoter of phoN, pagC, or mgtCB was apparent by either in vitro or in vivo assays. These loci are Salmonella specific and were probably acquired by horizontal DNA transfer. Besides, sequence analysis of pag promoters revealed the presence of a conserved PhoP box in 6 out of the 12 genes analyzed. Our results strongly suggest that the expression of a set of [Mg.sup.2+]-controlled genes is driven by PhoP via unknown intermediate regulatory mechanisms that could also involve ancillary factors.

We characterized the sac operon, a global regulator for virulence gene expression in Staphylococcus aureus. A Tn917 sae mutant was obtained by screening a Tn917 library of the agr mutant ISP479Mu for clones with altered hemolytic activity. Sequence analysis of the sac operon revealed two additional open reading frames (ORFs) (ORF3 and ORF4) upstream of the two-component regulatory genes saeR and saeS. Four overlapping sae-specific transcripts (T1 to T4) were detected by Northern blot analysis, and the transcriptional initiation points were mapped by primer extension analysis. The T1, T2, and T3 mRNAs are probably terminated at the same stem-loop sequence downstream of saeS. The T1 message (3.1 kb) initiates upstream of ORF4, T2 (2.4 kb) initiates upstream of ORF3, and T3 (2.0 kb) initiates in front of saeR. T4 (0.7 kb) represents a monocistronic mRNA encompassing ORF4 only. sae-specific transcripts were detectable in all of the 40 different clinical S. aureus isolates investigated. Transcript levels were at maximum during the post-exponential growth phase. The sae mutant showed a significantly reduced rate of invasion of human endothelial cells, consistent with diminished transcription and expression of fnbA. The expression of type 5 capsular polysaccharide is activated in the sae mutant of strain Newman, as shown by immunofluorescence and promoter-reporter fusion experiments. In summary, the sae operon constitutes a four-component regulator system which acts on virulence gene expression in S. aureus.

The regulation of expression of the genes encoding the cellulases and hemicellulases of Clostridium cellulovorans was studied at the mRNA level with cells grown under various culture conditions. A basic pattern of gene expression and of relative expression levels was obtained from cells grown in media containing poly-, di- or monomeric sugars. The cellulase (cbpA and engE) and hemicellulase (xynA) genes were coordinately expressed in medium containing cellobiose or cellulose. Growth in the presence of cellulose, xylan, and pectin gave rise to abundant expression of most genes (cbpA-exgS, engH, hbpA, manA, engM, engE, xynA, and/or pelA) studied. Moderate expression of cbpA, engH, manA, engE, and xynA was observed when cellobiose or fructose was used as the carbon source. Low levels of mRNA from cbpA, manA, engE, and xynA were observed with cells grown in lactose, mannose, and locust bean gum, and very little or no expression of cbpA, engH, manA, engE, and xynA was detected in glucose-, galactose-, maltose-, and sucrose-grown cells. The cbpA-exgS and engE genes were most frequently expressed under all conditions studied, whereas expression of xynA and pelA was more specifically induced at higher levels in xylan- or pectin-containing medium, respectively. Expression of the genes (cbpA, hbpA, manA, engM, and engE) was nat observed in the presence of most soluble di- or monosaccharides such as glucose. These results support the hypotheses that there is coordinate expression of some cellulases and hemicellulases, that a catabolite repression type of mechanism regulates cellulase expression in rapidly growing cells, and that the presence of hemicelluloses has an effect on cellulose utilization by the cell.

The multicellular developmental cycle of Myxococcus xanthus requires large-scale changes in gene transcription, and recent findings indicate that NtrC-like activators play a prominent role in regulating these changes. In this study, we made insertions in 28 uncharacterized ntrC-like activator (nla) genes and found that eight of these insertions cause developmental defects. Hence, these results are consistent with the idea that M. xanthus uses a series of different NtrC-like activators during fruiting body development. Four of the eight developmental mutants we identified have motility defects. The nlal, nla19, and nla23 mutants show S-motility defects, while the nla24 mutant shows defects in both S-motility and A-motility. During development, aggregation of the nlal, nla19, and nla23 mutants is delayed slightly and the nla24 mutant shows no signs of aggregation or sporulation. The nla4, nla6, nla18, and nla28 mutants have no appreciable loss in motility, but they fail to aggregate and to sporulate normally. The nla18 mutant belongs to a special class of developmental mutants whose defects can be rescued when they are codeveloped with wild-type cells, suggesting that nla18 fails to produce a cell-cell signal required for development. The three remaining activator mutants, nla4, nla6, and nla28, appear to have complex developmental phenotypes that include deficiencies in cell-cell developmental signals.

Regulation of the [sigma] factor RpoS occurs at the levels of transcription, translation, and protein stability activity, and it determines whether Escherichia coli turns on or off the stationary-phase response. To better understand the regulation of RpoS, we conducted genetic screens and found that mutations in the pst locus cause accumulation of RpoS during exponential growth. The pst locus encodes for the components of the high-affinity transport system for inorganic phosphate ([P.sub.i]), which is involved in sensing [P.sub.i] levels in the environment. When the Pst transporter is compromised (either by mutation or by [P.sub.i] starvation), the two-component system PhoBR activates the transcription of the Pho regulon, a subset of genes that encode proteins for transporting and metabolizing alternative phosphate sources. Our data show that strains carrying mutations which constitutively activate the Pho regulon have increased rpoS translation during exponential growth. This upregulation of rpoS translation is Hfq dependent, suggesting the involvement of a small regulatory RNA (sRNA). The transcription of this yet-to-be-identified sRNA is regulated by the PhoBR two-component system.