Dysregulation of REST-regulated coding and non-coding RNAs in a cellular model of Huntington's disease

Huntingtin (Htt) protein interacts with many transcriptional regulators, with widespread disruption to the transcriptome in Huntington's disease (HD) brought about by altered interactions with the mutant Htt (muHtt) protein. Repressor Element-1 Silencing Transcription Factor (REST) is a repressor whose association with Htt in the cytoplasm is disrupted in HD, leading to increased nuclear REST and concomitant repression of several neuronal-specific genes, including brain-derived neurotrophic factor (Bdnf). Here, we explored a wide set of HD dysregulated genes to identify direct REST targets whose expression is altered in a cellular model of HD but that can be rescued by knock-down of REST activity. We found many direct REST target genes encoding proteins important for nervous system development, including a cohort involved in synaptic transmission, at least two of which can be rescued at the protein level by REST knock-down. We also identified several microRNAs (miRNAs) whose aberrant repression is directly mediated by REST, including miR-137, which has not previously been shown to be a direct REST target in mouse. These data provide evidence of the contribution of inappropriate REST-mediated transcriptional repression to the widespread changes in coding and non-coding gene expression in a cellular model of HD that may affect normal neuronal function and survival.

Transcriptional dysregulation of coding and non-coding genes in cellular models of Huntington's disease

HD (Huntington's disease) is a late onset heritable neurodegenerative disorder that is characterized by neuronal dysfunction and death, particularly in the cerebral cortex and medium spiny neurons of the striatum. This is followed by progressive chorea, dementia and emotional dysfunction, eventually resulting in death. HD is caused by an expanded CAG repeat in the first exon of the HD gene that results in an abnormally elongated polyQ (polyglutamine) tract in its protein product, Htt (Huntingtin). Wild-type Htt is largely cytoplasmic; however, in HD, proteolytic N-terminal fragments of Htt form insoluble deposits in both the cytoplasm and nucleus, provoking the idea that mutHtt (mutant Htt) causes transcriptional dysfunction. While a number of specific transcription factors and co-factors have been proposed as mediators of mutHtt toxicity, the causal relationship between these Htt/transcription factor interactions and HD pathology remains unknown. Previous work has highlighted REST [RE1 (repressor element 1)-silencing transcription factor] as one such transcription factor. REST is a master regulator of neuronal genes, repressing their expression. Many of its direct target genes are known or suspected to have a role in HD pathogenesis, including BDNF (brain-derived neurotrophic factor). Recent evidence has also shown that REST regulates transcription of regulatory miRNAs (microRNAs), many of which are known to regulate neuronal gene expression and are dysregulated in HD. Thus repression of miRNAs constitutes a second, indirect mechanism by which REST can alter the neuronal transcriptome in HD. We will describe the evidence that disruption to the REST regulon brought about by a loss of interaction between REST and mutHtt may be a key contributory factor in the widespread dysregulation of gene expression in HD.

Origin of avian genome size and structure in non-avian dinosaurs

Avian genomes are small and streamlined compared with those of other amniotes by virtue of having fewer repetitive elements and less non-coding DNA(1,2). This condition has been suggested to represent a key adaptation for flight in birds, by reducing the metabolic costs associated with having large genome and cell sizes(3,4). However, the evolution of genome architecture in birds, or any other lineage, is difficult to study because genomic information is often absent for long-extinct relatives. Here we use a novel bayesian comparative method to show that bone-cell size correlates well with genome size in extant vertebrates, and hence use this relationship to estimate the genome sizes of 31 species of extinct dinosaur, including several species of extinct birds. Our results indicate that the small genomes typically associated with avian flight evolved in the saurischian dinosaur lineage between 230 and 250 million years ago, long before this lineage gave rise to the first birds. By comparison, ornithischian dinosaurs are inferred to have had much larger genomes, which were probably typical for ancestral Dinosauria. Using comparative genomic data, we estimate that genome-wide interspersed mobile elements, a class of repetitive DNA, comprised 5 - 12% of the total genome size in the saurischian dinosaur lineage, but was 7 - 19% of total genome size in ornithischian dinosaurs, suggesting that repetitive elements became less active in the saurischian lineage. These genomic characteristics should be added to the list of attributes previously considered avian but now thought to have arisen in non-avian dinosaurs, such as feathers(5), pulmonary innovations 6, and parental care and nesting

Phylogeographic analysis of the chloroplast DNA variation in wild common bean (Phaseolus vulgaris L.) in the Americas

The wild common bean (Phaseolus vulgaris) is widely but discontinuously distributed from northern Mexico to northern Argentina on both sides of the Isthmus of Panama. Little is known on how the species has reached its current disjunct distribution. In this research, chloroplast DNA polymorphisms in seven non-coding regions were used to study the history of migration of wild P. vulgaris between Mesoamerica and South America. A penalized likelihood analysis was applied to previously published Leguminosae ITS data to estimate divergence times between P. vulgaris and its sister taxa from Mesoamerica, and divergence times of populations within P. vulgaris. Fourteen chloroplast haplotypes were identified by PCR-RFLP and their geographical associations were studied by means of a Nested Clade Analysis and Mantel Tests. The results suggest that the haplotypes are not randomly distributed but occupy discrete parts of the geographic range of the species. The current distribution of haplotypes may be explained by isolation by distance and by at least two migration events between Mesoamerica and South America: one from Mesoamerica to South America and another one from northern South America to Mesoamerica. Age estimates place the divergence of P. vulgaris from its sister taxa from Mesoamerica at or before 1.3 Ma, and divergence of populations from Ecuador-northern Peru at or before 0.6 Ma. As these ages are taken as minimum divergence times, the influence of past events, such as the closure of the Isthmus of Panama and the final uplift of the Andes, on the migration history and population structure of this species cannot be disregarded.

Factors required for the uridylylation of the foot-and-mouth disease virus 3B1, 3B2, and 3B3 peptides by the RNA-dependent RNA polymerase (3D(pol)) in vitro

The 5' terminus of picornavirus genomic RNA is covalently linked to the virus-encoded peptide 313 (VTg). Foot-and-mouth disease virus (FMDV) is unique in encoding and using 3 distinct forms of this peptide. These peptides each act as primers for RNA synthesis by the virus-encoded RNA polymerase 3D(pol). To act as the primer for positive-strand RNA synthesis, the 3B peptides have to be uridylylated to form VPgpU(pU). For certain picornaviruses, it has been shown that this reaction is achieved by the 3D(pol) in the presence of the 3CD precursor plus an internal RNA sequence termed a cis-acting replication element (cre). The FMDV ere has been identified previously to be within the 5' untranslated region, whereas all other picornavirus cre structures are within the viral coding region. The requirements for the in vitro uridylylation of each of the FMDV 313 peptides has now been determined, and the role of the FMDV ere (also known as the 3B-uridylylation site, or bus) in this reaction has been analyzed. The poly(A) tail does not act as a significant template for FMDV 3B uridylylation.

Structure and function analysis of the poliovirus cis-acting replication element (CRE)

The poliovirus cis-acting replication element (CRE) templates the uridylylation of VPg, the protein primer for genome replication. The CRE is a highly conserved structural RNA element in the enteroviruses and located within the polyprotein-coding region of the genome. We have determined the native structure of the CRE, defined the regions of the structure critical for activity, and investigated the influence of genomic location on function. Our results demonstrate that a 14-nucleotide unpaired terminal loop, presented on a suitably stable stem, is all that is required for function. These conclusions complement the recent analysis of the 14-nucleotide terminal loop in the CRE of human rhinovirus type 14. The CRE can be translocated to the 5' noncoding region of the genome, at least 3.7-kb distant from the native location, without adversely influencing activity, and CRE duplications do not adversely influence replication. We do not have evidence for a specific interaction between the CRE and the RNA-binding 3CD(pro) complex, an essential component of the uridylylation reaction, and the mechanism by which the CRE is coordinated and orientated during the reaction remains unclear. These studies provide a detailed overview of the structural determinants required for CRE function, and will facilitate a better understanding of the requirements for picornavirus replication.

Detection of Chlamydia psittaci DNA in avian clinical samples by polymerase chain reaction

A polymerase chain reaction (PCR) assay was developed to detect Chlamydia psittaci DNA in faeces and tissue samples from avian species. Primers were designed to amplify a 264 bp product derived from part of the 5' non-translated region and part of the coding region of the ompA gene which encodes the major outer membrane protein. Amplified sequences were confirmed by Southern hybridization using an internal probe. The sensitivity of the combined assay was found to be between 60 to 600 fg of chlamydial DNA (approximately 6 to 60 genome copies). The specificity of the assay was confirmed since PCR product was not obtained from samples containing several serotypes of C. trachomatis, strains of C. pneumoniae, the type strain of C. pecorum, nor from samples containing microorganisms commonly found in the avian gut flora. In this study, 404 avian faeces and 141 avian tissue samples received by the Central Veterinary Laboratory over a 6 month period were analysed by PCR, antigen detection ELISA and where possible, cell culture isolation. PCR performed favourably compared with ELISA and cell culture, or with ELISA alone. The PCR assay was especially suited to the detection of C. psittaci DNA in avian faeces samples. The test was also useful when applied to tissue samples from small contact birds associated with a case of human psittacosis where ELISA results were negative and chlamydial isolation was a less favourable method due to the need for rapid diagnosis.

Plant U13 orthologues and orphan snoRNAs identified by RNomics of RNA from Arabidopsis nucleoli

Small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs) are non-coding RNAs whose main function in eukaryotes is to guide the modification of nucleotides in ribosomal and spliceosomal small nuclear RNAs, respectively. Full-length sequences of Arabidopsis snoRNAs and scaRNAs have been obtained from cDNA libraries of capped and uncapped small RNAs using RNA from isolated nucleoli from Arabidopsis cell cultures. We have identified 31 novel snoRNA genes (9 box C/D and 22 box H/ACA) and 15 new variants of previously described snoRNAs. Three related capped snoRNAs with a distinct gene organization and structure were identified as orthologues of animal U13snoRNAs. In addition, eight of the novel genes had no complementarity to rRNAs or snRNAs and are therefore putative orphan snoRNAs potentially reflecting wider functions for these RNAs. The nucleolar localization of a number of the snoRNAs and the localization to nuclear bodies of two putative scaRNAs was confirmed by in situ hybridization. The majority of the novel snoRNA genes were found in new gene clusters or as part of previously described clusters. These results expand the repertoire of Arabidopsis snoRNAs to 188 snoRNA genes with 294 gene variants.

Associations between apolipoprotein e genotype and circulating F-2-isoprostane levels in humans

Apolipoprotein E (apoE), an important determinant of plasma lipoprotein metabolism, has three common alleles (ε 2, ε 3, and ε 4). Population studies have shown that the risk of diseases characterized by oxidative damage, such as coronary heart disease and Alzheimer's disease, is significantly higher in ε 4 carriers. We evaluated the association between apoE genotypes and plasma F-2-isoprostane levels, an index of lipid peroxidation, in humans. Two hundred seventy-four healthy subjects (104 males, 170 females; 46.9 ± 13.0 yr; 200 whites, 74 blacks; 81 nonsmokers, 64 passive smokers, and 129 active smokers) recruited for a randomized clinical antioxidant intervention trial were included in this analysis. ApoE genotype was determined by PCR and restriction enzyme digestion. Free plasma F2-isoprostane was measured by GC-MS. Genotype groups were compared using multiple regression analysis with adjustment for sex, age, race, smoking status, body mass index, plasma ascorbic acid, and β-carotene. Subjects with ε 3/ε 4 and ε 4/ε 4 genotype (ε 4-carriers) and with ε 2/ε 3 and ε 3/ε 3 (non-ε 4-carriers) were pooled for analysis. In subjects with high cholesterol levels (total cholesterol above 200 mg/dl), plasma F-2-isoprostane levels were 29% higher in ε 4 carriers than in non-ε 4-carriers (P= 0.0056). High-cholesterol subjects that are ε 4 carriers have significantly higher levels of lipid peroxidation as assessed by circulating F-2-isoprostane levels.

Profiling dehydrin gene sequence and physiological parameters in drought tolerant and susceptible spring wheat cultivars.

Physiological and yield traits such as stomatal conductance (mmol m-2s-1), Leaf relative water content (RWC %) and grain yield per plant were studied in a separate experiment. Results revealed that five out of sixteen cultivars viz. Anmol, Moomal, Sarsabz, Bhitai and Pavan, appeared to be relatively more drought tolerant. Based on morphophysiological results, studies were continued to look at these cultivars for drought tolerance at molecular level. Initially, four well recognized primers for dehydrin genes (DHNs) responsible for drought induction in T. durum L., T. aestivum L. and O. sativa L. were used for profiling gene sequence of sixteen wheat cultivars. The primers amplified the DHN genes variably like Primer WDHN13 (T. aestivum L.) amplified the DHN gene in only seven cultivars whereas primer TdDHN15 (T. durum L.) amplified all the sixteen cultivars with even different DNA banding patterns some showing second weaker DNA bands. Third primer TdDHN16 (T. durum L.) has shown entirely different PCR amplification prototype, specially showing two strong DNA bands while fourth primer RAB16C (O. sativa L.) failed to amplify DHN gene in any of the cultivars. Examination of DNA sequences revealed several interesting features. First, it identified the two exon/one intron structure of this gene (complete sequences were not shown), a feature not previously described in the two database cDNA sequences available from T. aestivum L. (gi|21850). Secondly, the analysis identified several single nucleotide polymorphisms (SNPs), positions in gene sequence. Although complete gene sequence was not obtained for all the cultivars, yet there were a total of 38 variable positions in exonic (coding region) sequence, from a total gene length of 453 nucleotides. Matrix of SNP shows these 37 positions with individual sequence at positions given for each of the 14 cultivars (sequence of two cultivars was not obtained) included in this analysis. It demonstrated a considerable diversity for this gene with only three cultivars i.e. TJ-83, Marvi and TD-1 being similar to the consensus sequence. All other cultivars showed a unique combination of SNPs. In order to prove a functional link between these polymorphisms and drought tolerance in wheat, it would be necessary to conduct a more detailed study involving directed mutation of this gene and DHN gene expression.

Low relative humidity triggers RNA-directed de novo DNA methylation and suppression of genes controlling stomatal development

Environmental cues influence the development of stomata on the leaf epidermis, and allow plants to exert plasticity in leaf stomatal abundance in response to the prevailing growing conditions. It is reported that Arabidopsis thaliana ‘Landsberg erecta’ plants grown under low relative humidity have a reduced stomatal index and that two genes in the stomatal development pathway, SPEECHLESS and FAMA, become de novo cytosine methylated and transcriptionally repressed. These environmentally-induced epigenetic responses were abolished in mutants lacking the capacity for de novo DNA methylation, for the maintenance of CG methylation, and in mutants for the production of short-interfering non-coding RNAs (siRNAs) in the RNA-directed DNA methylation pathway. Induction of methylation was quantitatively related to the induction of local siRNAs under low relative humidity. Our results indicate the involvement of both transcriptional and post-transcriptional gene suppression at these loci in response to environmental stress. Thus, in a physiologically important pathway, a targeted epigenetic response to a specific environmental stress is reported and several of its molecular, mechanistic components are described, providing a tractable platform for future epigenetics experiments. Our findings suggest epigenetic regulation of stomatal development that allows for anatomical and phenotypic plasticity, and may help to explain at least some of the plant’s resilience to fluctuating relative humidity.

The role of REST in transcriptional and epigenetic dysregulation in Huntington's disease

Huntington's disease (HD) is a devastating disorder that affects approximately 1 in 10,000 people and is accompanied by neuronal dysfunction and neurodegeneration. HD manifests as a progressive chorea, a decline in mental abilities accompanied by behavioural, emotional and psychiatric problems followed by, dementia, and ultimately, death. The molecular pathology of HD is complex but includes widespread transcriptional dysregulation. Although many transcriptional regulatory molecules have been implicated in the pathogenesis of HD, a growing body of evidence points to the pivotal role of RE1 Silencing Transcription Factor (REST). In HD, REST, translocates from the cytoplasm to the nucleus in neurons resulting in repression of key target genes such as BDNF. Since these original observations, several thousand direct target genes of REST have been identified, including numerous non-coding RNAs including both microRNAs and long non-coding RNAs, several of which are dysregulated in HD. More recently, evidence is emerging that hints at epigenetic abnormalities in HD brain. This in turn, promotes the notion that targeting the epigenetic machinery may be a useful strategy for treatment of some aspects of HD. REST also recruits a host of histone and chromatin modifying activities that can regulate the local epigenetic signature at REST target genes. Collectively, these observations present REST as a hub that coordinates transcriptional, posttranscriptional and epigenetic programmes, many of which are disrupted in HD. We identify several spokes emanating from this REST hub that may represent useful sites to redress REST dysfunction in HD.

Using coordinated observations in polarized white light and Faraday rotation to probe the spatial position and magnetic field of an interplanetary sheath

Coronal mass ejections (CMEs) can be continuously tracked through a large portion of the inner heliosphere by direct imaging in visible and radio wavebands. White light (WL) signatures of solar wind transients, such as CMEs, result from Thomson scattering of sunlight by free electrons and therefore depend on both viewing geometry and electron density. The Faraday rotation (FR) of radio waves from extragalactic pulsars and quasars, which arises due to the presence of such solar wind features, depends on the line-of-sight magnetic field component B ∥ and the electron density. To understand coordinated WL and FR observations of CMEs, we perform forward magnetohydrodynamic modeling of an Earth-directed shock and synthesize the signatures that would be remotely sensed at a number of widely distributed vantage points in the inner heliosphere. Removal of the background solar wind contribution reveals the shock-associated enhancements in WL and FR. While the efficiency of Thomson scattering depends on scattering angle, WL radiance I decreases with heliocentric distance r roughly according to the expression Ir –3. The sheath region downstream of the Earth-directed shock is well viewed from the L4 and L5 Lagrangian points, demonstrating the benefits of these points in terms of space weather forecasting. The spatial position of the main scattering site r sheath and the mass of plasma at that position M sheath can be inferred from the polarization of the shock-associated enhancement in WL radiance. From the FR measurements, the local B ∥sheath at r sheath can then be estimated. Simultaneous observations in polarized WL and FR can not only be used to detect CMEs, but also to diagnose their plasma and magnetic field properties.

Regulation of Bcl-xL expression by H2O2 in cardiac myocytes.

Oxidative stress promotes cardiac myocyte apoptosis through the mitochondrial death pathway. Since Bcl-2 family proteins are key regulators of apoptosis, we examined the effects of H2O2 on the expression of principal Bcl-2 family proteins (Bcl-2, Bcl-xL, Bax, Bad) in neonatal rat cardiac myocytes. Protein expression was assessed by immunoblotting. Bcl-2, Bax, and Bad were all down-regulated in myocytes exposed to 0.2 mm H2O2, a concentration that induces apoptosis. In contrast, although Bcl-xL levels initially declined, the protein was re-expressed from 4-6 h. Bcl-xL mRNA was up-regulated from 2 to 4 h in neonatal rat or mouse cardiac myocytes exposed to H2O2, consistent with the re-expression of protein. Four different untranslated first exons have been identified for the Bcl-x gene (exons 1, 1B, 1C, and 1D, where exon 1 is the most proximal and exon 1D the most distal to the coding region). All were detected in mouse or rat neonatal cardiac myocytes, but exon 1D was not expressed in adult mouse hearts. In neonatal mouse or rat cardiac myocytes, H2O2 induced the expression of exons 1B, 1C, and 1D, but not exon 1. These data demonstrate that the Bcl-x gene is selectively responsive to oxidative stress, and the response is mediated through distal promoter regions.

Analysis of incoherent scatter radar data from non-thermal F-region plasma

A procedure is presented for fitting incoherent scatter radar data from non-thermal F-region ionospheric plasma, using theoretical spectra previously predicted. It is found that values of the shape distortion factor D∗, associated with deviations of the ion velocity distribution from a Maxwellian distribution, and ion temperatures can be deduced (the results being independent of the path of iteration) if the angle between the line-of-sight and the geomagnetic field is larger than about 15–20°. The procedure can be used with one or both of two sets of assumptions. These concern the validity of the adopted model for the line-of-sight ion velocity distribution in the one case or for the full three-dimensional ion velocity distribution function in the other. The distribution function employed was developed to describe the line-of-sight velocity distribution for large aspect angles, but both experimental data and Monte Carlo simulations indicate that the form of the field-perpendicular distribution can also describe the distribution at more general aspect angles. The assumption of this form for the line-of-sight velocity distribution at a general aspect angle enables rigorous derivation of values of the one-dimensional, line-of-sight ion temperature. With some additional assumptions (principally that the field-parallel distribution is always Maxwellian and there is a simple relationship between the ion temperature anisotropy and the distortion of the field-perpendicular distribution from a Maxwellian), fits to data for large aspect angles enable determination of line-of-sight temperatures at all aspect angles and hence, of the average ion temperature and the ion temperature anisotropy. For small aspect angles, the analysis is restricted to the determination of the line-of-sight ion temperature because the theoretical spectrum is insensitive to non-thermal effects when the plasma is viewed along directions almost parallel to the magnetic field. This limitation is expected to apply to any realistic model of the ion velocity distribution function and its consequences are discussed. Fit strategies which allow for mixed ion composition are also considered. Examples of fits to data from various EISCAT observing programmes are presented.