Hypoxia-induced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype

BACKGROUND: Pulmonary fibrosis is a debilitating and lethal disease with no effective treatment options. Understanding the pathological processes at play will direct the application of novel therapeutic avenues. Hypoxia has been implicated in the pathogenesis of pulmonary fibrosis yet the precise mechanism by which it contributes to disease progression remains to be fully elucidated. It has been shown that chronic hypoxia can alter DNA methylation patterns in tumour-derived cell lines. This epigenetic alteration can induce changes in cellular phenotype with promoter methylation being associated with gene silencing. Of particular relevance to idiopathic pulmonary fibrosis (IPF) is the observation that Thy-1 promoter methylation is associated with a myofibroblast phenotype where loss of Thy-1 occurs alongside increased alpha smooth muscle actin (α-SMA) expression. The initial aim of this study was to determine whether hypoxia regulates DNA methylation in normal human lung fibroblasts (CCD19Lu). As it has been reported that hypoxia suppresses Thy-1 expression during lung development we also studied the effect of hypoxia on Thy-1 promoter methylation and gene expression.

METHODS: CCD19Lu were grown for up to 8 days in hypoxia and assessed for global changes in DNA methylation using flow cytometry. Real-time PCR was used to quantify expression of Thy-1, α-SMA, collagen I and III. Genomic DNA was bisulphite treated and methylation specific PCR (MSPCR) was used to examine the methylation status of the Thy-1 promoter.

RESULTS: Significant global hypermethylation was detected in hypoxic fibroblasts relative to normoxic controls and was accompanied by increased expression of myofibroblast markers. Thy-1 mRNA expression was suppressed in hypoxic cells, which was restored with the demethylating agent 5-aza-2'-deoxycytidine. MSPCR revealed that Thy-1 became methylated following fibroblast exposure to 1% O2.

CONCLUSION: These data suggest that global and gene-specific changes in DNA methylation may play an important role in fibroblast function in hypoxia.

Biallelic ATM inactivation significantly reduces survival in patients treated on the United Kingdom Leukemia Research Fund Chronic Lymphocytic Leukemia 4 trial.

PURPOSE: The prognostic significance of ATM mutations in chronic lymphocytic leukemia (CLL) is unclear. We assessed their impact in the context of a prospective randomized trial. PATIENTS AND METHODS: We analyzed the ATM gene in 224 patients treated on the Leukemia Research Fund Chronic Lymphocytic Leukemia 4 (LRF-CLL4) trial with chlorambucil or fludarabine with and without cyclophosphamide. ATM status was analyzed by denaturing high-performance liquid chromatography and was related to treatment response, survival, and the impact of TP53 alterations for the same patient cohort. RESULTS: We identified 36 ATM mutations in 33 tumors, 16 with and 17 without 11q deletion. Mutations were associated with advanced disease stage and involvement of multiple lymphoid sites. Patients with both ATM mutation and 11q deletion showed significantly reduced progression-free survival (median, 7.4 months) compared with those with ATM wild type (28.6 months), 11q deletion alone (17.1 months), or ATM mutation alone (30.8 months), but survival was similar to that in patients with monoallelic (6.7 months) or biallelic (3.4 months) TP53 alterations. This effect was independent of treatment, immunoglobulin heavy chain variable gene (IGHV) status, age, sex, or disease stage. Overall survival for patients with biallelic ATM alterations was also significantly reduced compared with those with ATM wild type or ATM mutation alone (median, 42.2 v 85.5 v 77.6 months, respectively). CONCLUSION: The combination of 11q deletion and ATM mutation in CLL is associated with significantly shorter progression-free and overall survival following first-line treatment with alkylating agents and purine analogs. Assessment of ATM mutation status in patients with 11q deletion may influence the choice of subsequent therapy.

Repeated evolution of heat responsiveness among Brassicaceae COPIA transposable elements

Eukaryotic genomes contain repetitive DNA sequences. This includes simple repeats and more complex transposable elements (TEs). Many TEs reach high copy numbers in the host genome, owing to their amplification abilities by specific mechanisms. There is growing evidence that TEs contribute to gene transcriptional regulation. However, excess of TE activity may lead to reduced genome stability. Therefore, TEs are suppressed by the transcriptional gene silencing machinery via specific chromatin modifications. In contrary, effectiveness of the epigenetic silencing mechanisms imposes risk for TE survival in the host genome. Therefore, TEs may have evolved specific strategies for bypassing epigenetic control and allowing the emergence of new TE copies. Recent studies suggested that the epigenetic silencing can be, at least transiently, attenuated by heat stress in A. thaliana. Heat stress induced strong transcriptional activation of COPIA78 family LTR-retrotransposons named ONSEN, and even their transposition in mutants deficient in siRNA-biogenesis. ONSEN transcriptional activation was facilitated by the presence of heat responsive elements (HREs) within the long terminal repeats, which serve as a binding platform for the HEAT SHOCK FACTORs (HSFs). This thesis focused on the evolution of ONSEN heat responsiveness in Brassicaceae. By using whole-transcriptome sequencing approach, multiple Arabidopsis lyrata ONSENs with conserved heat response were found and together with ONSENs from other Brassicaceae were used to reconstruct the evolution of ONSEN HREs. This indicated ancestral situation with two, in palindrome organized, HSF binding motifs. In the genera Arabidopsis and Ballantinia, a local duplication of this locus increased number of HSF binding motifs to four, forming a high-efficiency HRE. In addition, whole transcriptome analysis revealed novel heat-responsive TE families COPIA20, COPIA37 and HATE. Notably, HATE represents so far unknown COPIA family which occurs in several Brassicaceae species but is absent in A. thaliana. Putative HREs were identified within the LTRs of COPIA20, COPIA37 and HATE of A. lyrata, and could be preliminarily validated by transcriptional analysis upon heat induction in subsequent survey of Brassicaeae species. Subsequent phylogenetic analysis indicated a repeated evolution of heat responsiveness within Brassicaceae COPIA LTR-retrotransposons. This indicates that acquisition of heat responsiveness may represent a successful strategy for survival of TEs within the host genome.

Genetic manipulation of Anopheles stephensi immunity to increase plasmodium falciparum salivary gland sporozoite infection levels

Human malaria is responsible for over 700,000 deaths a year. To stay abreast of the threat posed by the parasite, a constant stream of new drugs and vector control methods are required. This study focuses on a vaccine that has the potential to protect against parasite infection, but has been hindered by developmental challenges. In malaria prevention, live, attenuated, aseptic, Plasmodium falciparum sporozoites (PfSPZ) can be administered as a highly protective vaccine. PfSPZ are produced using adult female Anopheles stephensi mosquitoes as bioreactors. Production volume and cost of a PfSPZ vaccine for malaria are expected to be directly correlated with Plasmodium falciparum infection intensity in the salivary glands. The sporogonic development of Plasmodium falciparum in A. stephensi to fully infected salivary gland stage sporozoites is dictated by the activities of several known components of the mosquito’s innate immune system. Here I report on the use of genetic technologies that have been rarely, if ever, used in Anopheles stephensi Sda500 to increase the yield of sporozoites per mosquito and enhance vaccine production. By combining the Gal4/UAS bipartite system with in vivo expression of shRNA gene silencing, activity of the IMD signaling pathway downstream effector LRIM1, an antagonist to Plasmodium development, was reduced in the midgut, fat body, and salivary glands of A. stephensi. In infection studies using P. berghei and P. falciparum these transgenic mosquitoes consistently produced significantly more salivary gland stage sporozoites than wildtype controls, with increases in P. falciparum ranging from 2.5 to 10 fold. Using Plasmodium infection assays and qRT-PCR, two novel findings were identified. First, it was shown that 14 days post Plasmodium infection, transcript abundance of the IMD immune effector genes LRIM1, TEP1 and APL1c are elevated, in the salivary glands of A. stephensi, suggesting the salivary glands may play a role in post midgut defense against the parasite. Second, a non-pathogenic IMD signaling pathway response was observed which could suggest an alternative pathway for IMD activation. The information gained from these studies has significantly increased our knowledge of Plasmodium defense in A. stephensi and moreover could significantly improve vaccine production.

REST is a hypoxia-responsive transcriptional repressor

Cellular exposure to hypoxia results in altered gene expression in a range of physiologic and pathophysiologic states. Discrete cohorts of genes can be either up- or down-regulated in response to hypoxia. While the Hypoxia-Inducible Factor (HIF) is the primary driver of hypoxia-induced adaptive gene expression, less is known about the signalling mechanisms regulating hypoxia-dependent gene repression. Using RNA-seq, we demonstrate that equivalent numbers of genes are induced and repressed in human embryonic kidney (HEK293) cells. We demonstrate that nuclear localization of the Repressor Element 1-Silencing Transcription factor (REST) is induced in hypoxia and that REST is responsible for regulating approximately 20% of the hypoxia-repressed genes. Using chromatin immunoprecipitation assays we demonstrate that REST-dependent gene repression is at least in part mediated by direct binding to the promoters of target genes. Based on these data, we propose that REST is a key mediator of gene repression in hypoxia.

Mealybug wilt disease

Mealybug wilt disease (MWD) is a serious field disease of pineapples worldwide that was first described in Hawaii in 1910. MWD is thought to be caused by a complex involving viruses, mealybugs and ants. The viruses are transmitted by mealybugs, which in turn are tended by ants. Although a number of distinct viruses have been associated with the disease, the identity of the causal agent(s) has not been determined unequivocally. This chapter describes the disease symptopms, aetiology and management of MWD. In the last 20 years, significant advances have been achieved in identifying the causal viral agents, and gaining a better understanding of MWD. However, the interactions between the viruses, mealybugs and environmental factors are complicated, and the conditions required for the expression of MWD have only been partially elucidated at this time. The possible role of gene silencing, the identity of the additional ampelovirus(es) and badnavirus(es) that have been detected but not characterized, and the interaction between these disease-inducing factors are fertile areas for future research.

PAMP-triggered immunity against Pseudomonas syringae involves microRNA-mediated regulation of several uncharacterized R genes

Two main types of noncoding small RNA molecules have been found in plants: microRNAs (miRNAs) and small interfering RNAs (siRNAs). They differ in their biogenesis and mode of action, but share similar sizes (20-24 nt). Their precursors are processed by Dicer-Like RNase III (dcl) proteins present in Arabidopsis thaliana, and in their mature form can act as negative regulators of gene expression, being involved in a vast array of plant processes, including plant development, genomic integrity or response to stress. Small-RNA mediated regulation can occurs at transcriptional level (TGS) or at post-transcriptional level (PTGS). In recent years, the role of gene silencing in the regulation of expression of genes related to plant defence responses against bacterial pathogens is becoming clearer. Comparisons carried out in our lab between the expression profiles of different mutants affected in gene silencing, and plants challenged with Pseudomonas syringae pathovar tomato DC3000, led us to identify a set of uncharacterized R genes, belonging to the TIR-NBS-LRR gene family, differentially expressed in these conditions. Through the use of bioinformatics tools, we found a miRNA* of 22 nt putatively responsible for down-regulating expression of these R genes through the generation of siRNAs. We have also found that the corresponding pri-miRNA is down-regulated after PAMP-perception in a SA-dependent manner. We also demonstrate that plants with altered levels of miRNA* (knockdown lines or overexpression lines) exhibit altered PTI-associated phenotypes, suggesting a role for this miRNA* in this defence response against bacteria. In addition we identify one of the target genes as a negative regulator of defence response against Pseudomonas syringae.

Silenciamento do canal de potássio Eag1 potencializa efeitos da temozolomida em células U-87 MG de glioblastoma multiforme

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde, 2015.

Mechanisms of force-mediated regulation of transcription, chromatin remodeling and cell fate decisions

Tissue mechanics and cellular interactions influence every single cell in our bodies to drive morphogenesis. However, little is known about mechanisms by which cells sense physical forces and transduce them from the cytoskeleton to the nucleus to control gene expression and stem cell fate. We have identified a novel nuclear-mechanosensor complex, consisting of the nuclear membrane protein emerin (Emd), actin and non-muscle myosin IIA (NMIIA), that regulates transcription, chromatin remodeling and lineage commitment. Force-induced enrichment of Emd at the outer nuclear membrane leads to a compensation between H3K9me2,3 and H3K27me3 on constitutive heterochromatin. This strain-induced epigenetic switch is accompanied by the global rearrangement of chromatin. In parallel, forces promote local F-actin polymerization at the outer nuclear membrane, which limits the availability of nuclear G-actin. Subsequently, the reduction of nuclear G-actin results in attenuated global transcription and therefore increased H3K27me3 occupancy to reinforce gene silencing. Restoring nuclear actin levels in the presence of mechanical strain counteracts PRC2-mediated silencing of transcribed genes. This mechanosensory circuit is also observed in vivo. Depletion of NMIIA in mouse epidermis leads to decreased H3K27me3 levels and precocious lineage commitment, thus abrogating organ growth and patterning. Our results reveal how mechanical signals regulate nuclear architecture, chromatin organization and transcription to control cell fate decisions.

Using a bespoke 'Omics' approach to devise a flexible new way to control Fusarium in wheat.

2016

A non-invasive method for silencing gene transcription in honeybees maintained under natural conditions

in the Apis mellifera post-genomic era, RNAi protocols have been used in functional approaches. However, sample manipulation and invasive methods such as injection of double-stranded RNA (dsRNA) can compromise physiology and survival. To circumvent these problems, we developed a non-invasive method for honeybee gene knockdown, using a well-established vitellogenin RNAi system as a model. Second instar larvae received dsRNA for vitellogenin (dsVg-RNA) in their natural diet. For exogenous control, larvae received dsRNA for GFP (dsGFP-RNA). Untreated larvae formed another control group. Around 60% of the treated larvae naturally developed until adult emergence when 0.5 mu g of dsVg-RNA or dsGFP-RNA was offered while no larvae that received 3.0 mu g of dsRNA reached pupal stages. Diet dilution did not affect the removal rates. Viability depends not only on the delivered doses but also on the internal conditions of colonies. The weight of treated and untreated groups showed no statistical differences. This showed that RNAi ingestion did not elicit drastic collateral effects. Approximately 90% of vitellogenin transcripts from 7-day-old workers were silenced compared to controls. A large number of samples are handled in a relatively short time and smaller quantities of RNAi molecules are used compared to invasive methods. These advantages culminate in a versatile and a cost-effective approach. (c) 2008 Elsevier Ltd. All rights reserved.

RNAi-mediated silencing of vitellogenin gene function turns honeybee (Apis mellifera) workers into extremely precocious foragers

The switch from within-hive activities to foraging behavior is a major transition in the life cycle of a honeybee (Apis mellifera) worker. A prominent regulatory role in this switch has long been attributed to juvenile hormone (JH), but recent evidence also points to the yolk precursor protein vitellogenin as a major player in behavioral development. In the present study, we injected vitellogenin double-stranded RNA (dsVg) into newly emerged worker bees of Africanized genetic origin and introduced them together with controls into observation hives to record flight behavior. RNA interference-mediated silencing of vitellogenin gene function shifted the onset of long-duration flights (> 10 min) to earlier in life (by 3-4 days) when compared with sham and untreated control bees. In fact, dsVg bees were observed conducting such flights extremely precociously, when only 3 days old. Short-duration flights (< 10 min), which bees usually perform for orientation and cleaning, were not affected. Additionally, we found that the JH titer in dsVg bees collected after 7 days was not significantly different from the controls. The finding that depletion of the vitellogenin titer can drive young bees to become extremely precocious foragers could imply that vitellogenin is the primary switch signal. At this young age, downregulation of vitellogenin gene activity apparently had little effect on the JH titer. As this unexpected finding stands in contrast with previous results on the vitellogenin/JH interaction at a later age, when bees normally become foragers, we propose a three-step sequence in the constellation of physiological parameters underlying behavioral development.

Induction of the ferritin gene (ftnA) of Escherichia coli by Fe2+–Fur is mediated by reversal of H-NS silencing and is RyhB independent

FtnA is the major iron-storage protein of Escherichia coli accounting for < or = 50% of total cellular iron. The FtnA gene (ftnA) is induced by iron in an Fe(2+)-Fur-dependent fashion. This effect is reportedly mediated by RyhB, the Fe(2+)-Fur-repressed, small, regulatory RNA. However, results presented here show that ftnA iron induction is independent of RyhB and instead involves direct interaction of Fe(2+)-Fur with an 'extended' Fur binding site (containing five tandem Fur boxes) located upstream (-83) of the ftnA promoter. In addition, H-NS acts as a direct repressor of ftnA transcription by binding at multiple sites (I-VI) within, and upstream of, the ftnA promoter. Fur directly competes with H-NS binding at upstream sites (II-IV) and consequently displaces H-NS from the ftnA promoter (sites V-VI) which in turn leads to derepression of ftnA transcription. It is proposed that H-NS binding within the ftnA promoter is facilitated by H-NS occupation of the upstream sites through H-NS oligomerization-induced DNA looping. Consequently, Fur displacement of H-NS from the upstream sites prevents cooperative H-NS binding at the downstream sites within the promoter, thus allowing access to RNA polymerase. This direct activation of ftnA transcription by Fe(2+)-Fur through H-NS antisilencing represents a new mechanism for iron-induced gene expression.

A non-invasive method for silencing gene transcription in honeybees maintained under natural conditions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)