51 research outputs found

    ChIPOTle: a user-friendly tool for the analysis of ChIP-chip data

    Get PDF
    ChIPOTle (Chromatin ImmunoPrecipitation On Tiled arrays) takes advantage of two unique properties of ChIP-chip data: the single-tailed nature of the data, caused by specific enrichment but not specific depletion of genomic fragments; and the predictable enrichment of DNA fragments adjacent to sites of direct protein-DNA interaction. Implemented as a Microsoft Excel macro written in Visual Basic, ChIPOTle uses a sliding window approach that yields improvements in the identification of bona fide sites of protein-DNA interaction

    The 19S proteasome subcomplex promotes the targeting of NuA4 HAT to the promoters of ribosomal protein genes to facilitate the recruitment of TFIID for transcriptional initiation in vivo

    Get PDF
    Previous studies have implicated SAGA (Spt-Ada-Gcn5-acetyltransferase) and TFIID (Transcription factor-IID)-dependent mechanisms of transcriptional activation in yeast. SAGA-dependent transcriptional activation is further regulated by the 19S proteasome subcomplex. However, the role of the 19S proteasome subcomplex in transcriptional activation of the TFIID-dependent genes has not been elucidated. Therefore, we have performed a series of chromatin immunoprecipitation, mutational and transcriptional analyses at the TFIID-dependent ribosomal protein genes such as RPS5, RPL2B and RPS11B. We find that the 19S proteasome subcomplex is recruited to the promoters of these ribosomal protein genes, and promotes the association of NuA4 (Nucleosome acetyltransferase of histone H4) co-activator, but not activator Rap1p (repressor-activator protein 1). These observations support that the 19S proteasome subcomplex enhances the targeting of co-activator at the TFIID-dependent promoter. Such an enhanced targeting of NuA4 HAT (histone acetyltransferase) promotes the recruitment of the TFIID complex for transcriptional initiation. Collectively, our data demonstrate that the 19S proteasome subcomplex enhances the targeting of NuA4 HAT to activator Rap1p at the promoters of ribosomal protein genes to facilitate the recruitment of TFIID for transcriptional stimulation, hence providing a new role of the 19S proteasome subcomplex in establishing a specific regulatory network at the TFIID-dependent promoter for productive transcriptional initiation in vivo

    Regulon-Specific Control of Transcription Elongation across the Yeast Genome

    Get PDF
    Transcription elongation by RNA polymerase II was often considered an invariant non-regulated process. However, genome-wide studies have shown that transcriptional pausing during elongation is a frequent phenomenon in tightly-regulated metazoan genes. Using a combination of ChIP-on-chip and genomic run-on approaches, we found that the proportion of transcriptionally active RNA polymerase II (active versus total) present throughout the yeast genome is characteristic of some functional gene classes, like those related to ribosomes and mitochondria. This proportion also responds to regulatory stimuli mediated by protein kinase A and, in relation to cytosolic ribosomal-protein genes, it is mediated by the silencing domain of Rap1. We found that this inactive form of RNA polymerase II, which accumulates along the full length of ribosomal protein genes, is phosphorylated in the Ser5 residue of the CTD, but is hypophosphorylated in Ser2. Using the same experimental approach, we show that the in vivo–depletion of FACT, a chromatin-related elongation factor, also produces a regulon-specific effect on the expression of the yeast genome. This work demonstrates that the regulation of transcription elongation is a widespread, gene class–dependent phenomenon that also affects housekeeping genes

    Transcriptional regulation in eukaryotic ribosomal protein genes

    Get PDF
    AbstractUnderstanding ribosomal protein gene regulation provides a good avenue for understanding gene regulatory networks. Even after 5 decades of research on ribosomal protein gene regulation, little is known about how higher eukaryotic ribosomal protein genes are coordinately regulated at the transcriptional level. However, a few recent papers shed some light on this complicated problem

    Characteristic differences between the promoters of intron-containing and intronless ribosomal protein genes in yeast

    Get PDF
    <p>Abstract</p> <p>Background</p> <p>More than two thirds of the highly expressed ribosomal protein (RP) genes in <it>Saccharomyces cerevisiae </it>contain introns, which is in sharp contrast to the genome-wide five percent intron-containing genes. It is well established that introns carry regulatory sequences and that the transcription of RP genes is extensively and coordinately regulated. Here we test the hypotheses that introns are innately associated with heavily transcribed genes and that introns of RP genes contribute regulatory TF binding sequences. Moreover, we investigate whether promoter features are significantly different between intron-containing and intronless RP genes.</p> <p>Results</p> <p>We find that directly measured transcription rates tend to be lower for intron-containing compared to intronless RP genes. We do not observe any specifically enriched sequence motifs in the introns of RP genes other than those of the branch point and the two splice sites. Comparing the promoters of intron-containing and intronless RP genes, we detect differences in number and position of Rap1-binding and IFHL motifs. Moreover, the analysis of the length distribution and the folding free energies suggest that, at least in a sub-population of RP genes, the 5' untranslated sequences are optimized for regulatory function.</p> <p>Conclusion</p> <p>Our results argue against the direct involvement of introns in the regulation of transcription of highly expressed genes. Moreover, systematic differences in motif distributions suggest that RP transcription factors may act differently on intron-containing and intronless gene promoters. Thus, our findings contribute to the decoding of the RP promoter architecture and may fuel the discussion on the evolution of introns.</p

    Scoring functions for transcription factor binding site prediction

    Get PDF
    BACKGROUND: Transcription factor binding site (TFBS) prediction is a difficult problem, which requires a good scoring function to discriminate between real binding sites and background noise. Many scoring functions have been proposed in the literature, but it is difficult to assess their relative performance, because they are implemented in different software tools using different search methods and different TFBS representations. RESULTS: Here we compare how several scoring functions perform on both real and semi-simulated data sets in a common test environment. We have also developed two new scoring functions and included them in the comparison. The data sets are from the yeast (S. cerevisiae) genome. Our new scoring function LLBG (least likely under the background model) performs best in this study. It achieves the best average rank for the correct motifs. Scoring functions based on positional bias performed quite poorly in this study. CONCLUSION: LLBG may provide an interesting alternative to current scoring functions for TFBS prediction

    Predicting Gene Expression from Sequence: A Reexamination

    Get PDF
    Although much of the information regarding genes' expressions is encoded in the genome, deciphering such information has been very challenging. We reexamined Beer and Tavazoie's (BT) approach to predict mRNA expression patterns of 2,587 genes in Saccharomyces cerevisiae from the information in their respective promoter sequences. Instead of fitting complex Bayesian network models, we trained naïve Bayes classifiers using only the sequence-motif matching scores provided by BT. Our simple models correctly predict expression patterns for 79% of the genes, based on the same criterion and the same cross-validation (CV) procedure as BT, which compares favorably to the 73% accuracy of BT. The fact that our approach did not use position and orientation information of the predicted binding sites but achieved a higher prediction accuracy, motivated us to investigate a few biological predictions made by BT. We found that some of their predictions, especially those related to motif orientations and positions, are at best circumstantial. For example, the combinatorial rules suggested by BT for the PAC and RRPE motifs are not unique to the cluster of genes from which the predictive model was inferred, and there are simpler rules that are statistically more significant than BT's ones. We also show that CV procedure used by BT to estimate their method's prediction accuracy is inappropriate and may have overestimated the prediction accuracy by about 10%

    Overexpression of transcriptional activators in Saccharomyces cerevisiae for improved recombinant protein production

    Get PDF
    Thesis (MSc)--Stellenbosch University, 2019.ENGLISH ABSTRACT: The yeast, Saccharomyces cerevisiae, possesses multiple characteristics that classify it as an excellent host organism for the recombinant production of industrial and medical proteins. This includes its ability to process and secrete heterologous eukaryotic proteins at far higher levels than native proteins, which significantly simplifies downstream protein purification. However, it has had limited success in industry due to limitations in the yeast’s recombinant protein production capability. Gene expression in S. cerevisiae is tightly regulated and metabolic engineering is therefore required to direct cellular resources and metabolic energy towards heterologous gene expression. Promoters are genetic elements that control gene expression and specific DNA sequences within promoters act as binding sites for transcriptional activators. Transcriptional activators upregulate gene expression, but they are also subject to regulation that could limit the level of heterologous gene expression. Increasing the cellular concentration of such activators could further upregulate gene expression and consequently, increase recombinant protein production. The aim of this study was to develop a tool for the overproduction of recombinant proteins in S. cerevisiae and increase its competitiveness as a microbial cell factory in industry. Therefore, the effect of overexpressing two promoter-specific transcriptional activators (RAP1 and GCR1) on the strength of three strong constitutive promoters (ENO1P, ATEF1P and STEF1P) in the S. cerevisiae Y294 laboratory strain, was evaluated. Reporter genes coding for raw starch-degrading enzymes were used as they have a large industrial significance in the food, fermentation, textile, paper, detergent and pharmaceutical industries. The abovementioned transcriptional manipulations were evaluated in terms of the extracellular enzyme activity and protein production under aerobic and fermentative conditions. The best candidates were further tested in raw corn starch fermentations in terms of ethanol production and carbon conversion. While GCR1 overexpression had no effect, RAP1 overexpression resulted in a three-fold increase in the extracellular protein concentration. Under aerobic conditions, the volumetric activity of the recombinant AteA α-amylase was increased by 81%, 28% and 46%, when the gene was expressed under the transcriptional control of the ENO1, ATEF1 and STEF1 promoters, respectively. In addition, improvements of 99%, 29% and 69% were observed under fermentative conditions for the three respective promoters. RAP1-overexpressing strains were subsequently used for the construction of an amylolytic co-culture (S. cerevisiae Y294[ENO1_TemA_RAP1] + Y294[ENO1_TemG_RAP1]) that exhibited an improved fermentation profile during the bioconversion of 200 g/l raw corn starch. The ethanol yield produced after 48 hours was comparable to that obtained from a commercial enzyme preparation (STARGENTM 002) and 54% higher than the benchmark co-culture (S. cerevisiae Y294[ENO1_TemA] + Y294[ENO1_TemG]). More encouraging, was that the ethanol yield from the S. cerevisiae Y294[TemA_TemG_RAP1] strain (co-expressing the TemA and TemG genes) was 111% higher with RAP1 overexpression and 201% higher than that of the S. cerevisiae Y294[AmyA_GlaA] reference strain after 48 hours. Furthermore, RAP1 overexpression resulted in a fermentation profile in the S. cerevisiae Y294[TemA_TemG_RAP1] laboratory strain that was comparable to that of a recombinant S. cerevisiae industrial strain (S. cerevisiae Ethanol Red[TemA_TemG]). This study has confirmed the potential of RAP1 overexpression as a tool to increase recombinant protein production in S. cerevisiae, thus increasing the economic viability of S. cerevisiae as a microbial cell factory.AFRIKAANSE OPSOMMING: Die gis, Saccharomyces cerevisiae, bevat vele eienskappe wat dit 'n uitstekende mikrobiese gasheer vir die produksie van rekombinante industriële en medies-toepaslike proteïene maak. Dit sluit in die vermoë om heteroloë eukariotiese proteïene te prosesseer en teen baie hoër vlakke as eie proteïene uit te skei, wat stroom-af suiwering van proteïene beduidend vereenvoudig. Dit het egter tot dusver beperkte sukses in die industrie gehad weens beperkinge in die gis se rekombinante proteïenproduksievermoë. Geenuitdrukking in S. cerevisiae word streng gereguleer en metaboliese ingenieurswese word dus vereis om sellulêre hulpbronne en metaboliese energie na heteroloë geenuitdrukking te herlei. Promotors is genetiese elemente wat geenuitdrukking beheer en spesifieke DNA- volgordes in promoters funksioneer as bindingsetels vir transkripsionele aktiveerders. Transkripsionele aktiveerders verhoog geenuitdrukking, maar hulle is self ook aan regulering onderworpe wat die vlak van heteroloë geenuitdrukking kan beperk. 'n Verhoging in die sellulêre konsentrasie van sulke aktiveerders kan moontlik geenuitdrukking bevorder en dus die vlakke van rekombinante proteïenproduksie verhoog. Die doel van hierdie studie was om gereedskap vir die oorproduksie van rekombinante proteïene in S. cerevisiae te ontwikkel en sodoende hierdie gis se mededingendheid as 'n mikrobiese selfabriek in die bedryf te verhoog. Die effek van die ooruitdrukking van twee promotor-spesifieke transkripsionele aktiveerders (RAP1 en GCR1) op die sterkte van drie sterk konstitutiewe promotors (ENO1P, ATEF1P en STEF1P) is in die S. cerevisiae Y294 laboratoriumras geëvalueer. Verklikkergene wat vir rou stysel-hidroliserende ensieme kodeer is hiervoor gebruik, aangesien hulle groot industriële waarde in die voedsel-, fermentasie-, tekstiel-, papier-, skoonmaakmiddel- en farmaseutiese bedryf het. Bogenoemde transkripsionele manipulasies is in terme van die ekstrasellulêre ensiemaktiwiteit en proteïenproduksie onder aërobiese en fermentatiewe toestande geëvalueer. Die beste kandidate is verder in rou mieliestysel fermentasies in terme van etanolproduksie en koolstof- omskakeling getoets. Terwyl GCR1-ooruitdrukking geen effek gehad het nie, het RAP1-ooruitdrukking die rekombinante proteïenkonsentrasie tot drievoudig verhoog. Onder aërobiese toestande is die volumetriese aktiwiteit van die rekombinante AteA α-amilase met 81%, 28% en 46% verhoog toe die geen onderskeidelik onder die transkripsionele beheer van die ENO1, ATEF1 en STEF1 promotors uitgedruk is. Verder is verbeterings van 99%, 29% en 69% vir die onderskeie promotors onder fermentatiewe toestande waargeneem. Ooruitdrukking van RAP1 is gevolglik vir die konstruksie van 'n amilolitiese kombinasiekultuur (S. cerevisiae Y294 [ENO1_TemA_RAP1] + Y294[ENO1_TemG_RAP1]) gebruik wat gedurende die bio- omskakeling van 200 g/l rou mieliestysel 'n verbeterde fermentasieprofiel getoon het. Die etanolopbrengs was vergelykbaar met 'n kommersiële ensiempreparaat (STARGENTM 002) en 54% hoër as die maatstaf kombinasiekultuur (S. cerevisiae Y294[ENO1_TemA] + Y294[ENO1_TemG]). Meer bemoedigend, was die feit dat die etanolopbrengs van die S. cerevisiae Y294[TemA-TemG_RAP1] ras (wat die TemA and TemG gene gesamentlik uitdruk) na 48 ure 111% hoër met RAP1 ooruitdrukking en 201% hoër as die S. cerevisiae Y294[AmyA-GlaA] verwysingsras was. Verder het die ooruitdrukking van RAP1 ’n fermentasieprofiel in die S. cerevisiae Y294[TemA_TemG_RAP1] laboratoriumras geskep wat vergelykbaar met 'n industriële ras (S. cerevisiae Ethanol Red[TemA_TemG]) was. Hierdie studie het die potensiaal van RAP1-ooruitdrukking as 'n hulpmiddel vir verhoogde rekombinante proteïenproduksie in S. cerevisiae bevestig, wat sodoende die ekonomiese lewensvatbaarheid van S. cerevisiae as 'n mikrobiese selfabriek kan verhoog.Master
    corecore