Hog1 bypasses stress-mediated down-regulation of transcription by RNA polymerase II redistribution and chromatin remodeling

Cells are subjected to dramatic changes of gene expression upon environmental changes. Stresscauses a general down-regulation of gene expression together with the induction of a set of stress-responsivegenes. The p38-related stress-activated protein kinase Hog1 is an important regulator of transcription uponosmostress in yeast. Genome-wide localization studies of RNA polymerase II (RNA Pol II) and Hog1 showed that stress induced major changes in RNA Pol II localization, with a shift toward stress-responsive genes relative to housekeeping genes. RNA Pol II relocalization required Hog1, which was also localized to stress-responsive loci. In addition to RNA Pol II-bound genes, Hog1 also localized to RNA polymerase III-bound genes, pointing to a wider role for Hog1 in transcriptional control than initially expected. Interestingly, an increasing association of Hog1 with stressresponsive genes was strongly correlated with chromatin remodeling and increased gene expression. Remarkably, MNase-Seq analysis showed that although chromatin structure was not significantly altered at a genome-wide level in response to stress, there was pronounced chromatin remodeling for those genes that displayed Hog1 association. Hog1 serves to bypass the general down-regulation of gene expression that occurs in response to osmostress, and does so both by targeting RNA Pol II machinery and by inducing chromatin remodeling at stressresponsive loci.

Genome-wide chromatin occupancy reveals a role for ASH2 in transcriptional pausing

While it is widely acknowledged that the ubiquitin-proteasome system plays an important role in transcription, little is known concerning the mechanistic basis, in particular the spatial organization of proteasome-dependent proteolysis at the transcription site. Here, we show that proteasomal activity and tetraubiquitinated proteins concentrate to nucleoplasmic microenvironments in the euchromatin. Such proteolytic domains are immobile and distinctly positioned in relation to transcriptional processes. Analysis of gene arrays and early genes in Caenorhabditis elegans embryos reveals that proteasomes and proteasomal activity are distantly located relative to transcriptionally active genes. In contrast, transcriptional inhibition generally induces local overlap of proteolytic microdomains with components of the transcription machinery and degradation of RNA polymerase II. The results establish that spatial organization of proteasomal activity differs with respect to distinct phases of the transcription cycle in at least some genes, and thus might contribute to the plasticity of gene expression in response to environmental stimuli.

Similar and Divergent Effects of ppGpp and DksA Deficiencies on Transcription in Escherichia coli.

The concerted action of ppGpp and DksA in transcription has been widely documented. In disparity with this model, phenotypic studies showed that ppGpp and DksA might also have independent and opposing roles in gene expression in Escherichia coli. In this study we used a transcriptomic approach to compare the global transcriptional patterns of gene expression in strains deficient in ppGpp (ppGpp0) and/or DksA ( dksA). Approximately 6 and 7% of all genes were significantly affected by more than twofold in ppGpp- and DksAdeficient strains, respectively, increasing to 13% of all genes in the ppGpp0 dksA strain. Although the data indicate that most of the affected genes were copositively or conegatively regulated by ppGpp and DksA, some genes that were independently and/or differentially regulated by the two factors were found. The large functional group of chemotaxis and flagellum synthesis genes were notably differentially affected, with all genes being upregulated in the DksA-deficient strain but 60% of them being downregulated in the ppGpp-deficient strain. Revealingly, mutations in the antipausing Gre factors suppress the upregulation observed in the DksA-deficient strain, emphasizing the importance of the secondary channel of the RNA polymerase for regulation and fine-tuning of gene expression in E. coli.

Similar and Divergent Effects of ppGpp and DksA Deficiencies on Transcription in Escherichia coli.

The concerted action of ppGpp and DksA in transcription has been widely documented. In disparity with this model, phenotypic studies showed that ppGpp and DksA might also have independent and opposing roles in gene expression in Escherichia coli. In this study we used a transcriptomic approach to compare the global transcriptional patterns of gene expression in strains deficient in ppGpp (ppGpp0) and/or DksA ( dksA). Approximately 6 and 7% of all genes were significantly affected by more than twofold in ppGpp- and DksAdeficient strains, respectively, increasing to 13% of all genes in the ppGpp0 dksA strain. Although the data indicate that most of the affected genes were copositively or conegatively regulated by ppGpp and DksA, some genes that were independently and/or differentially regulated by the two factors were found. The large functional group of chemotaxis and flagellum synthesis genes were notably differentially affected, with all genes being upregulated in the DksA-deficient strain but 60% of them being downregulated in the ppGpp-deficient strain. Revealingly, mutations in the antipausing Gre factors suppress the upregulation observed in the DksA-deficient strain, emphasizing the importance of the secondary channel of the RNA polymerase for regulation and fine-tuning of gene expression in E. coli.

Estudis funcionals i de plegament dels dominis d'activació de les procarboxipeptidases

Els dominis d’activació (ADs) de les procarboxipeptidases de la subfamília A/B sempre han sorprès ja que representen una quarta part del proenzim. S’han realitzat alguns estudis per intentar descobrir-ne alguna possible funció alternativa, però no han estat fructífers. El descobriment de l’elevada velocitat de plegament del domini d’activació de la procarboxipeptidasa A2 humana, (ADA2h), emperò, va portar a proposar la possibilitat de que realitzessin una funció d’assistència al plegament del domini enzimàtic. Posteriorment, l’anàlisi del plegament d’ADA2h a pH baix va revelar la capacitat d’aquest domini per formar fibres amiloides, a més de demostrar que un increment de l’estabilitat proteica podia prevenir la formació d’aquests agregats. La profunda caracterització del plegament d’ADA2h va fer que aquesta proteïna fos un bon model amiloidogènic, de manera que es van proposar un seguit d’experiments que s’han desenvolupat en el present treball per tal de conèixer millor aquest procés. S’han dut a terme estudis cinètics d’agregació per tal de valorar la contribució dels diferents aminoàcids de la seqüència polipeptídica, utilitzant 29 variants puntuals d’ADA2h. Es va eliminar la contribució de l’estabilitat mitjançant la utilització d’urea, i per dicroïsme circular conjuntament amb un aparell de flux detingut, es van obtenir dues velocitats diferents, v1 i v2, que corresponen a la formació d’un intermediari i a la seva reorganització, respectivament. Experiments complementaris utilitzant espectroscòpia d’infraroig (IR) revelaren la reorganització de l’estat natiu (en aquest cas) per a donar la forma agregada. Les cinètiques d’IR van mostrar que ADA2h forma l’estructura _ típica de les fibres amiloides, previ desplegament les seves hèlixs-_. Finalment, s’han realitzat estudis de biocomputació per tal d’esbrinar possibles funcions alternatives dels ADs. Les superposicions estructurals semblen mostrar similaritat dels ADs amb dominis de reconeixement d’RNA (RRM). Aquesta hipòtesi s’ha comprovat experimentalment amb ADA4h, mostrant una dèbil, però existent, unió a RNA.

Selección de péptidos sintéticos del Virus de la Hepatitis G (GBV-C/HGV) inhibidores del Péptido de Fusión HIV-I

El GB virus C (GBV-C) o virus de l'hepatitis G (HGV) es un virus format per una única cadena de RNA que pertany a la familia Flaviviridae. En els últims anys, s'han publicat nombrosos treballs en els quals s'associa la coinfecció del GBV-C i del virus de la immunodeficiència humana (VIH) amb una menor progressió de l'esmentada malaltia així com amb una major supervivència dels pacients una vegada que la SIDA s'ha desenvolupat. El mecanisme pel qual el virus GBV-C/HGV exerceix un “efecte protector” en els pacients amb VIH encara no està descrit. L’estudi de la interacció entre els virus GBVC/HGV i VIH podria donar lloc al desenvolupament de nous agents terapèutics per al tractament de la SIDA.Treballs recents mostren com la capacitat inhibitòria del virus del GBV-C/HGV és deguda a la seva glicoproteina estructural E2. S’ha vist que aquesta proteina seria capaç d’inhibir la primera fase de replicació de VIH, així com la unió i la fusió amb les membranes cel•lulars. Sobre la base d’aquests estudis, l’objectiu d’aquest treball ha estat seleccionar inhibidors del pèptid de fusió del VIH utilitzant pèptids sintètics de la proteina E2 del GBV-C/HGV. El treball realitzat ha consistit en estudiar, utilitzant assajos biofísics de leakage i de lipid mixing, la capacitat dels pèptids de la proteina estructural del virus del GBV-C/HGV per inhibir la interacció i el procés de desestabilització de membranes induïdes pel pèptid de fusió de la glicoproteina de l’embolcall, GP41, del VIH. Aquests assajos, com es descriu en treballs anteriors, han resultat útils per a la selecció i la identificació de compostos amb activitat específica anti-GP41. Es pot afirmar que efectivament els pèptids seleccionats de la proteina E2 del virus del GBV-C/HGV inhibeixen l’activitat del pèptid de fusió del VIH probablement com a consequència d’un canvi conformacional en aquest darrer.

Anàlisi genètica i molecular de les malalties Niemann-Pick tipus A/B i tipus C. Estratègies terapèutiques.

Aquest treball es basa en l’estudi de dues malalties lisosòmiques: la malaltia de Niemann-Pick A/B (NPAB) i la malaltia de Niemann-Pick tipus C (NPC). En relació a la malaltia de NPAB, s’ha realitzat l’expressió in vitro d’algunes de les mutacions de canvi d’aminoàcid trobades en pacients espanyols per tal de detectar les activitats enzimàtiques residuals. Totes les mutacions presenten una activitat molt baixa, gairebé nul•la, excepte la p.L225P i la R608del que tenen un 11% i 20% d’activitat respectivament. Els resultats obtinguts són coherents amb la severitat del fenotip que presenten els pacients. D’altra banda, s’ha caracteritzat un al•lel amb una mutació que afecta a una posició poc conservada d’un donador de splicing i que produeix la generació de trànscrits aberrants corresponents a trànscrits minoritaris de SMPD1, prèviament descrits, que no codifiquen per proteïna funcional. Respecte a malaltia de NPC, s’ha realitzat una anàlisi molecular de pacients espanyols prèviament estudiats identificant, en la majoria dels casos, la segona mutació responsable de la patologia. S’ha descrit per primer cop per aquesta malaltia una gran deleció que inclou el gen NPC1 i altres gens flanquejants i s’ha estudiat l’efecte que tenen les mutacions de splicing trobades a nivell de RNA. Per una d’aquestes mutacions, c.1554-1009G&A, s’ha assajat amb èxit una estratègia terapèutica basada en la utilització d’oligonuclèotids antisentit. D’altra banda, s’està desenvolupant un model cel•lular neuronal de la malaltia de Niemann-Pick tipus C, basat en la utilització de RNAs d’interferència, sobre el qual es podran assajar possibles estratègies terapèutiques en un futur.

Cirp function and characterisation in RNA and microRNAs

In order to search for novel genes involved in cell proliferation, the hypothesis was that by infecting primary cells with a cDNA library of immortal cells would render immortalizing genes. Consequently it has been discovered CIRP (Cold inducible RNA-binding protein). Mammalian cells exposed to mild hypothermia show a general inhibition of protein synthesis and a concomitant increase in the expression of a small number of cold-shock mRNAs and proteins. Rbm3, another RNA binding protein belonging to the same family, has been postulated to facilitate protein synthesis at mild cold shock. To investigate if the same occurs for CIRP, CIRP was overexpressed in primary cells and protein sintesis was measured. Interestingly, CIRP increased protein synthesis, however, such increase did not involve an increase in the polysome fraction or affected the ribosome profile. In addition, the effect caused by CIRP inhibition or knockdown was also analyzed. Different siRNAs against CIRP were tested. Once checked their efficiency by decreasing CIRP at mRNA and protein levels, proliferation was tested by BrdU, cell number (DAPI) and proliferation curves were performed. Interestingly, CIRP provoke a decreased proliferation in primary cells: MEFs, HMEC; and cancer cells: TERA2 and HeLa. In conclusion, we describe for the first time that CIRP bypasses replicative senescence when over-expressed at physiological temperature (37ºC) by increasing a general protein synthesis. This effect is achieved through ERK1/2 activation in MEFs.The decrease in growth rate found in mammalian cells treated with mild cold stress is not entirely attributable to arrested metabolism. This decrease may also involve an active process in which CIRP and other stress-responsive proteins play a fundamental role in stimulating proliferation. Although most cell proteins are down-regulated or inhibited with cold stress, CIRP is activated to maintain cells in an active proliferative status and its overexpression at 37°C might be potentially oncogenic.

Síntesi de siRNAs (short interfering RNAs) modificats amb nucleobases 5-metil i 5-propinil pirimidíniques i avaluació de la seva activitat in vitro en cultius cel•lulars i de la seva estabilitat en sèrum humà. Disseny de siRNAs dirigits a la inhibició de l'expressió de mutacions de l'oncogen K-ras.

Projecte de recerca elaborat a partir d’una estada a la Stanford University, EEUU, entre 2007 i 2009. El present projecte es basa 1) en la síntesi de cadenes d'ARN dirigides a la inhibició de l'expressió gènica per un mecanisme d'ARN d'interferència (siRNAs o short interefering RNAs) i 2) en l'avaluació de l'activitat in vitro d'aquests oligonucleòtids en cultius cel•lulars. Concretament, la meva recerca ha estat enfocada principalment a l'estudi de cadenes de siRNA modificades amb nucleobases 5-metil i 5-propinil pirimidíniques. Es tractava d'avaluar l'efecte que exerceixen els factors estèrics en el major groove (solc major) dels siRNAs sobre la seva activitat biològica. En aquest sentit, he dut aterme síntesi de fosforamidits de nucleòsis pirimidínics modificats a la posició C-5 de la nucleobase. A continuació he incorporat aquestes unitats nucleosídiques en cadenes d'ARN emprant un sintetitzador d’ADN/ARN i he estudiat l'estabilitat dels corresponents dúplexs d'ARN mitjançant experiments de desnaturalització tèrmica. Finalment he dut a terme experiments d'inhibició de l'expressió gènica en cèl.lules HeLa per tal d'avaluar l'activitat biològia d'aquests siRNAs modificats. Els resultats d'aquests estudis han posat de manifest que la presència de grups voluminosos com el propinil a l'extrem 5' del dúplex de siRNA (definit per la cadena guia o antisense) influeix de forma molt negativa en la seva activitat biològica. En canvi, grups menys voluminosos com el metil hi influeixen positivament, de manera que algunes de les cadenes sintetitzades han resultat ser més actives que els corresponents siRNAs naturals (wild type siRNAs). A més, aquest tipus de modificació contribueix positivament en l'estabilitat de cadenes de siRNA en sèrum humà. Aquest treball ha estat publicat (Terrazas, M.; Kool, E.T. "Major Groove Modifications Improve siRNA Stability and Biological Activity" Nucleic Acids Res. 2009, in press).

RNA interference: a new and powerful tool for functional genomic analysis.

In many species, the introduction of double-stranded RNA induces potent and specific gene silencing, referred to as RNA interference. This phenomenon, which is based on targeted degradation of mRNAs and occurs in almost any eukaryote, from trypanosomes to mice including plants and fungi, has sparked general interest from both applied and fundamental standpoints. RNA interference, which is currently used to investigate gene function in a variety of systems, is linked to natural resistance to viruses and transposon silencing, as if it were a primitive immune system involved in genome surveillance. Here, we review the mechanism of RNA interference in post-transcriptional gene silencing, its function in nature, its value for functional genomic analysis, and the modifications and improvements that may make it more efficient and inheritable. We also discuss the future directions of this versatile technique in both fundamental and applied science.

RNA interference is ineffective as a routine method for gene silencing in chick embryos as monitored by fgf8 silencing.

The in vivo accessibility of the chick embryo makes it a favoured model system for experimental developmental biology. Although the range of available techniques now extends to miss-expression of genes through in ovo electroporation, it remains difficult to knock out individual gene expression. Recently, the possibility of silencing gene expression by RNAi in chick embryos has been reported. However, published studies show only discrete quantitative differences in the expression of the endogenous targeted genes and unclear morphological alterations. To elucidate whether the tools currently available are adequate to silence gene expression sufficiently to produce a clear and specific null-like mutant phenotype, we have performed several experiments with different molecules that trigger RNAi: dsRNA, siRNA, and shRNA produced from a plasmid coexpressing green fluorescent protein as an internal marker. Focussing on fgf8 expression in the developing isthmus, we show that no morphological defects are observed, and that fgf8 expression is neither silenced in embryos microinjected with dsRNA nor in embryos microinjected and electroporated with a pool of siRNAs. Moreover, fgf8 expression was not significantly silenced in most isthmic cells transformed with a plasmid producing engineered shRNAs to fgf8. We also show that siRNA molecules do not spread significantly from cell to cell as reported for invertebrates, suggesting the existence of molecular differences between different model systems that may explain the different responses to RNAi. Although our results are basically in agreement with previously reported studies, we suggest, in contrast to them, that with currently available tools and techniques the number of cells in which fgf8 gene expression is decreased, if any, is not sufficient to generate a detectable mutant phenotype, thus making RNAi useless as a routine method for functional gene analysis in chick embryos.

O6-Methylguanine-DNA methyltransferase protein expression by immunohistochemistry in brain and non-brain systemic tumours: systematic review and meta-analysis of correlation with methylation-specific polymerase chain reaction

Background: The DNA repair protein O6-Methylguanine-DNA methyltransferase (MGMT) confers resistance to alkylating agents. Several methods have been applied to its analysis, with methylation-specific polymerase chain reaction (MSP) the most commonly used for promoter methylation study, while immunohistochemistry (IHC) has become the most frequently used for the detection of MGMT protein expression. Agreement on the best and most reliable technique for evaluating MGMT status remains unsettled. The aim of this study was to perform a systematic review and meta-analysis of the correlation between IHC and MSP. Methods A computer-aided search of MEDLINE (1950-October 2009), EBSCO (1966-October 2009) and EMBASE (1974-October 2009) was performed for relevant publications. Studies meeting inclusion criteria were those comparing MGMT protein expression by IHC with MGMT promoter methylation by MSP in the same cohort of patients. Methodological quality was assessed by using the QUADAS and STARD instruments. Previously published guidelines were followed for meta-analysis performance. Results Of 254 studies identified as eligible for full-text review, 52 (20.5%) met the inclusion criteria. The review showed that results of MGMT protein expression by IHC are not in close agreement with those obtained with MSP. Moreover, type of tumour (primary brain tumour vs others) was an independent covariate of accuracy estimates in the meta-regression analysis beyond the cut-off value. Conclusions Protein expression assessed by IHC alone fails to reflect the promoter methylation status of MGMT. Thus, in attempts at clinical diagnosis the two methods seem to select different groups of patients and should not be used interchangeably.

Interferència mediada per RNA, una nova revolució genètica

Els RNA (o ARN, àcids ribonucleics) són biomolècules lineals de cadena senzilla, com un fil, formades per la unió seqüencial d'altres molècules més senzilles, els nucleòtids. Abans de la descoberta del fenòmen de RNAi es creia que el RNA era només un intermediari silenciós de la maquinària genètica, que transportava cegament les instruccions dels gens, en descodificava el missatge i el convertia en proteïnes, procés que es coneix amb el nom de flux d'informació genètica (del gen, que emmagatzema la informació i és format per ADN, a les proteïnes, que fan la feina especificada pel gen) [...].