A Novel Mechanism of Transposon-Mediated Gene Activation

Transposable Insertion Sequences (IS elements) have been shown to provide various benefits to their hosts via gene activation or inactivation under stress conditions by appropriately inserting into specific chromosomal sites. Activation is usually due to derepression or introduction of a complete or partial promoter located within the element. Here we define a novel mechanism of gene activation by the transposon IS5 in Escherichia coli. The glycerol utilization operon, glpFK, that is silent in the absence of the cAMP-Crp complex, is activated by IS5 when inserted upstream of its promoter. High-level expression is nearly constitutive, only mildly dependent on glycerol, glucose, GlpR, and Crp, and allows growth at a rate similar to or more rapid than that of wild-type cells. Expression is from the glpFK promoter and dependent on (1) the DNA phase, (2) integration host factor (IHF), and (3) a short region at the 3′ end of IS5 harboring a permanent bend and an IHF binding site. The lacZYA operon is also subject to such activation in the absence of Crp. Thus, we have defined a novel mechanism of gene activation involving transposon insertion that may be generally applicable to many organisms

Duplication of 7q34 is specific to juvenile pilocytic astrocytomas and a hallmark of cerebellar and optic pathway tumours

BACKGROUND: Juvenile pilocytic astrocytomas (JPA), a subgroup of low-grade astrocytomas (LGA), are common, heterogeneous and poorly understood subset of brain tumours in children. Chromosomal 7q34 duplication leading to fusion genes formed between KIAA1549 and BRAF and subsequent constitutive activation of BRAF was recently identified in a proportion of LGA, and may be involved in their pathogenesis. Our aim was to investigate additional chromosomal unbalances in LGA and whether incidence of 7q34 duplication is associated with tumour type or location. METHODS AND RESULTS: Using Illumina-Human-Hap300-Duo and 610-Quad high-resolution-SNP-based arrays and quantitative PCR on genes of interest, we investigated 84 paediatric LGA. We demonstrate that 7q34 duplication is specific to sporadic JPA (35 of 53-66%) and does not occur in other LGA subtypes (0 of 27) or NFI-associated-JPA (0 of 4). We also establish that it is site specific as it occurs in the majority of cerebellar JPA (24 of 30-80%) followed by brainstem, hypothalamic/optic pathway JPA (10 of 16-62.5%) and is rare in hemispheric JPA (1 of 7-14%). The MAP-kinase pathway, assessed through ERK phosphorylation, was active in all tumours regardless of 7q34 duplication. Gain of function studies performed on hTERT-immortalised astrocytes show that overexpression of wild-type BRAF does not increase cell proliferation or baseline MAPK signalling even if it sensitises cells to EGFR stimulation. CONCLUSIONS AND INTERPRETATION: Our results suggest that variants of JPA might arise from a unique site-restricted progenitor cell where 7q34 duplication, a hallmark of this tumour-type in association to MAPK-kinase pathway activation, potentially plays a site-specific role in their pathogenesis. Importantly, gain of function abnormalities in components of MAP-Kinase signalling are potentially present in all JPA making this tumour amenable to therapeutic targeting of this pathway. British Journal of Cancer (2009) 101, 722-733. doi: 10.1038/sj.bjc.6605179 www.bjcancer.com Published online 14 July 2009 (C) 2009 Cancer Research U

Transcriptional control in the prereplicative phase of T4 development

Control of transcription is crucial for correct gene expression and orderly development. For many years, bacteriophage T4 has provided a simple model system to investigate mechanisms that regulate this process. Development of T4 requires the transcription of early, middle and late RNAs. Because T4 does not encode its own RNA polymerase, it must redirect the polymerase of its host, E. coli, to the correct class of genes at the correct time. T4 accomplishes this through the action of phage-encoded factors. Here I review recent studies investigating the transcription of T4 prereplicative genes, which are expressed as early and middle transcripts. Early RNAs are generated immediately after infection from T4 promoters that contain excellent recognition sequences for host polymerase. Consequently, the early promoters compete extremely well with host promoters for the available polymerase. T4 early promoter activity is further enhanced by the action of the T4 Alt protein, a component of the phage head that is injected into E. coli along with the phage DNA. Alt modifies Arg265 on one of the two α subunits of RNA polymerase. Although work with host promoters predicts that this modification should decrease promoter activity, transcription from some T4 early promoters increases when RNA polymerase is modified by Alt. Transcription of T4 middle genes begins about 1 minute after infection and proceeds by two pathways: 1) extension of early transcripts into downstream middle genes and 2) activation of T4 middle promoters through a process called sigma appropriation. In this activation, the T4 co-activator AsiA binds to Region 4 of σ70, the specificity subunit of RNA polymerase. This binding dramatically remodels this portion of σ70, which then allows the T4 activator MotA to also interact with σ70. In addition, AsiA restructuring of σ70 prevents Region 4 from forming its normal contacts with the -35 region of promoter DNA, which in turn allows MotA to interact with its DNA binding site, a MotA box, centered at the -30 region of middle promoter DNA. T4 sigma appropriation reveals how a specific domain within RNA polymerase can be remolded and then exploited to alter promoter specificity

A bacterial genome in transition - an exceptional enrichment of IS elements but lack of evidence for recent transposition in the symbiont Amoebophilus asiaticus

<p>Abstract</p> <p>Background</p> <p>Insertion sequence (IS) elements are important mediators of genome plasticity and are widespread among bacterial and archaeal genomes. The 1.88 Mbp genome of the obligate intracellular amoeba symbiont <it>Amoebophilus asiaticus </it>contains an unusually large number of transposase genes (n = 354; 23% of all genes).</p> <p>Results</p> <p>The transposase genes in the <it>A. asiaticus </it>genome can be assigned to 16 different IS elements termed ISCaa1 to ISCaa16, which are represented by 2 to 24 full-length copies, respectively. Despite this high IS element load, the <it>A. asiaticus </it>genome displays a GC skew pattern typical for most bacterial genomes, indicating that no major rearrangements have occurred recently. Additionally, the high sequence divergence of some IS elements, the high number of truncated IS element copies (n = 143), as well as the absence of direct repeats in most IS elements suggest that the IS elements of <it>A. asiaticus </it>are transpositionally inactive. Although we could show transcription of 13 IS elements, we did not find experimental evidence for transpositional activity, corroborating our results from sequence analyses. However, we detected contiguous transcripts between IS elements and their downstream genes at nine loci in the <it>A. asiaticus </it>genome, indicating that some IS elements influence the transcription of downstream genes, some of which might be important for host cell interaction.</p> <p>Conclusions</p> <p>Taken together, the IS elements in the <it>A. asiaticus </it>genome are currently in the process of degradation and largely represent reflections of the evolutionary past of <it>A. asiaticus </it>in which its genome was shaped by their activity.</p

Steroid receptor coactivator-1 modulates the function of Pomc neurons and energy homeostasis

Hypothalamic neurons expressing the anorectic peptide Pro-opiomelanocortin (Pomc) regulate food intake and body weight. Here, we show that Steroid Receptor Coactivator-1 (SRC-1) interacts with a target of leptin receptor activation, phosphorylated STAT3, to potentiate Pomc transcription. Deletion of SRC-1 in Pomc neurons in mice attenuates their depolarization by leptin, decreases Pomc expression and increases food intake leading to high-fat diet-induced obesity. In humans, fifteen rare heterozygous variants in SRC-1 found in severely obese individuals impair leptin-mediated Pomc reporter activity in cells, whilst four variants found in non-obese controls do not. In a knock-in mouse model of a loss of function human variant (SRC-1L1376P), leptin-induced depolarization of Pomc neurons and Pomc expression are significantly reduced, and food intake and body weight are increased. In summary, we demonstrate that SRC-1 modulates the function of hypothalamic Pomc neurons, and suggest that targeting SRC-1 may represent a useful therapeutic strategy for weight loss.Peer reviewe

Low-frequency variation in TP53 has large effects on head circumference and intracranial volume.

Cranial growth and development is a complex process which affects the closely related traits of head circumference (HC) and intracranial volume (ICV). The underlying genetic influences shaping these traits during the transition from childhood to adulthood are little understood, but might include both age-specific genetic factors and low-frequency genetic variation. Here, we model the developmental genetic architecture of HC, showing this is genetically stable and correlated with genetic determinants of ICV. Investigating up to 46,000 children and adults of European descent, we identify association with final HC and/or final ICV + HC at 9 novel common and low-frequency loci, illustrating that genetic variation from a wide allele frequency spectrum contributes to cranial growth. The largest effects are reported for low-frequency variants within TP53, with 0.5 cm wider heads in increaser-allele carriers versus non-carriers during mid-childhood, suggesting a previously unrecognized role of TP53 transcripts in human cranial development

The UK10K project identifies rare variants in health and disease

M. Kivimäki työryhmäjäsen.The contribution of rare and low-frequency variants to human traits is largely unexplored. Here we describe insights from sequencing whole genomes (low read depth, 7x) or exomes (high read depth, 80x) of nearly 10,000 individuals from population-based and disease collections. In extensively phenotyped cohorts we characterize over 24 million novel sequence variants, generate a highly accurate imputation reference panel and identify novel alleles associated with levels of triglycerides (APOB), adiponectin (ADIPOQ) and low-density lipoprotein cholesterol (LDLR and RGAG1) from single-marker and rare variant aggregation tests. We describe population structure and functional annotation of rare and low-frequency variants, use the data to estimate the benefits of sequencing for association studies, and summarize lessons from disease-specific collections. Finally, we make available an extensive resource, including individual-level genetic and phenotypic data and web-based tools to facilitate the exploration of association results.Peer reviewe