Gene expression and splicing alterations analyzed by high throughput RNA sequencing of chronic lymphocytic leukemia specimens.

BackgroundTo determine differentially expressed and spliced RNA transcripts in chronic lymphocytic leukemia specimens a high throughput RNA-sequencing (HTS RNA-seq) analysis was performed.MethodsTen CLL specimens and five normal peripheral blood CD19+ B cells were analyzed by HTS RNA-seq. The library preparation was performed with Illumina TrueSeq RNA kit and analyzed by Illumina HiSeq 2000 sequencing system.ResultsAn average of 48.5 million reads for B cells, and 50.6 million reads for CLL specimens were obtained with 10396 and 10448 assembled transcripts for normal B cells and primary CLL specimens respectively. With the Cuffdiff analysis, 2091 differentially expressed genes (DEG) between B cells and CLL specimens based on FPKM (fragments per kilobase of transcript per million reads and false discovery rate, FDR q < 0.05, fold change >2) were identified. Expression of selected DEGs (n = 32) with up regulated and down regulated expression in CLL from RNA-seq data were also analyzed by qRT-PCR in a test cohort of CLL specimens. Even though there was a variation in fold expression of DEG genes between RNA-seq and qRT-PCR; more than 90 % of analyzed genes were validated by qRT-PCR analysis. Analysis of RNA-seq data for splicing alterations in CLL and B cells was performed by Multivariate Analysis of Transcript Splicing (MATS analysis). Skipped exon was the most frequent splicing alteration in CLL specimens with 128 significant events (P-value <0.05, minimum inclusion level difference >0.1).ConclusionThe RNA-seq analysis of CLL specimens identifies novel DEG and alternatively spliced genes that are potential prognostic markers and therapeutic targets. High level of validation by qRT-PCR for a number of DEG genes supports the accuracy of this analysis. Global comparison of transcriptomes of B cells, IGVH non-mutated CLL (U-CLL) and mutated CLL specimens (M-CLL) with multidimensional scaling analysis was able to segregate CLL and B cell transcriptomes but the M-CLL and U-CLL transcriptomes were indistinguishable. The analysis of HTS RNA-seq data to identify alternative splicing events and other genetic abnormalities specific to CLL is an added advantage of RNA-seq that is not feasible with other genome wide analysis

Cloning of the gene for metallothionein from Artemia and challenges with cloning the promoter

Four metallothionein (MT) isoforms have previously been identified in the brine shrimp Artemia. The goals of this study were to isolate at least one of the MT genes, clone a MT promoter region, and characterize the cis-acting sequences that regulate MT gene expression. PCR of Artemia genomic DNA amplified a product containing a MT gene with no internal introns. Three different approaches were made to clone the MT promoter. Firstly, conventional cloning of size-selected genomic DNA into a dephosphorylated vector produced empty clones or no clones. The use of a novel linear cloning vector produced many recombinant clones, but screening of the DNA library did not identify a MT promoter sequence. Lastly, genome walking by PCR amplified a sequence that lacked a TAT A box and metal response elements. A blast search identified the sequence as a mitochondrial hydroxyacylglutathione hydrolase found in marine organisms

E-cadherin gene re-expression in chronic lymphocytic leukemia cells by HDAC inhibitors

Abstract Background The tumor suppressor gene E-cadherin gene is frequently silenced in chronic lymphocytic leukemia (CLL) cells and results in wnt-pathway activation. We analyzed the role of histone epigenetic modifications in E-cadherin gene silencing. Methods CLL specimens were treated with histone deacetylase inhibitor (HDACi) MS-275 and analyzed for E-cadherin expression with western blot and RT-PCR analysis. The downstream effects of HDACi treated leukemic cells were studied by analyzing the effect on wnt-pathway signaling. HDACi induced alterations in E-cadherin splicing were investigated by transcript specific real time PCR analysis. Results Treatment of CLL specimens with histone deacetylase inhibitors (HDACi) treatment resulted in an increase of the E-cadherin RNA transcript (5 to 119 fold increase, n=10) in eight out of ten CLL specimens indicating that this gene is down regulated by histone hypoacetylation in a majority of CLL specimens. The E-cadherin re-expression in CLL specimens was noted by western blot analysis as well. Besides epigenetic silencing another mechanism of E-cadherin inactivation is aberrant exon 11 splicing resulting in an alternatively spliced transcript that lacks exon 11 and is degraded by the non-sense mediated decay (NMD) pathway. Our chromatin immunoprecipitation experiments show that HDACi increased the acetylation of histones H3 and H4 in the E-cadherin promoter region. This also affected the E-cadherin exon 11 splicing pattern as HDACi treated CLL specimens preferentially expressed the correctly spliced transcript and not the exon 11 skipped aberrant transcript. The re-expressed E- cadherin binds to β-catenin with inhibition of the active wnt-beta-catenin pathway in these cells. This resulted in a down regulation of two wnt target genes, LEF and cyclinD1 and the wnt pathway reporter. Conclusion The E-cadherin gene is epigenetically modified and hypoacetylated in CLL leukemic cells. Treatment of CLL specimens with HDACi MS-275 activates transcription from this silent gene with expression of more correctly spliced E-cadherin transcripts as compared to the aberrant exon11 skipped transcripts that in turn inhibits the wnt signaling pathway. The data highlights the role of epigenetic modifications in altering gene splicing patterns

E-cadherin gene re-expression in chronic lymphocytic leukemia cells by HDAC inhibitors

Abstract Background The tumor suppressor gene E-cadherin gene is frequently silenced in chronic lymphocytic leukemia (CLL) cells and results in wnt-pathway activation. We analyzed the role of histone epigenetic modifications in E-cadherin gene silencing. Methods CLL specimens were treated with histone deacetylase inhibitor (HDACi) MS-275 and analyzed for E-cadherin expression with western blot and RT-PCR analysis. The downstream effects of HDACi treated leukemic cells were studied by analyzing the effect on wnt-pathway signaling. HDACi induced alterations in E-cadherin splicing were investigated by transcript specific real time PCR analysis. Results Treatment of CLL specimens with histone deacetylase inhibitors (HDACi) treatment resulted in an increase of the E-cadherin RNA transcript (5 to 119 fold increase, n=10) in eight out of ten CLL specimens indicating that this gene is down regulated by histone hypoacetylation in a majority of CLL specimens. The E-cadherin re-expression in CLL specimens was noted by western blot analysis as well. Besides epigenetic silencing another mechanism of E-cadherin inactivation is aberrant exon 11 splicing resulting in an alternatively spliced transcript that lacks exon 11 and is degraded by the non-sense mediated decay (NMD) pathway. Our chromatin immunoprecipitation experiments show that HDACi increased the acetylation of histones H3 and H4 in the E-cadherin promoter region. This also affected the E-cadherin exon 11 splicing pattern as HDACi treated CLL specimens preferentially expressed the correctly spliced transcript and not the exon 11 skipped aberrant transcript. The re-expressed E- cadherin binds to β-catenin with inhibition of the active wnt-beta-catenin pathway in these cells. This resulted in a down regulation of two wnt target genes, LEF and cyclinD1 and the wnt pathway reporter. Conclusion The E-cadherin gene is epigenetically modified and hypoacetylated in CLL leukemic cells. Treatment of CLL specimens with HDACi MS-275 activates transcription from this silent gene with expression of more correctly spliced E-cadherin transcripts as compared to the aberrant exon11 skipped transcripts that in turn inhibits the wnt signaling pathway. The data highlights the role of epigenetic modifications in altering gene splicing patterns.</p

Gene expression and splicing alterations analyzed by high throughput RNA sequencing of chronic lymphocytic leukemia specimens.

BackgroundTo determine differentially expressed and spliced RNA transcripts in chronic lymphocytic leukemia specimens a high throughput RNA-sequencing (HTS RNA-seq) analysis was performed.MethodsTen CLL specimens and five normal peripheral blood CD19+ B cells were analyzed by HTS RNA-seq. The library preparation was performed with Illumina TrueSeq RNA kit and analyzed by Illumina HiSeq 2000 sequencing system.ResultsAn average of 48.5 million reads for B cells, and 50.6 million reads for CLL specimens were obtained with 10396 and 10448 assembled transcripts for normal B cells and primary CLL specimens respectively. With the Cuffdiff analysis, 2091 differentially expressed genes (DEG) between B cells and CLL specimens based on FPKM (fragments per kilobase of transcript per million reads and false discovery rate, FDR q &lt; 0.05, fold change &gt;2) were identified. Expression of selected DEGs (n = 32) with up regulated and down regulated expression in CLL from RNA-seq data were also analyzed by qRT-PCR in a test cohort of CLL specimens. Even though there was a variation in fold expression of DEG genes between RNA-seq and qRT-PCR; more than 90 % of analyzed genes were validated by qRT-PCR analysis. Analysis of RNA-seq data for splicing alterations in CLL and B cells was performed by Multivariate Analysis of Transcript Splicing (MATS analysis). Skipped exon was the most frequent splicing alteration in CLL specimens with 128 significant events (P-value &lt;0.05, minimum inclusion level difference &gt;0.1).ConclusionThe RNA-seq analysis of CLL specimens identifies novel DEG and alternatively spliced genes that are potential prognostic markers and therapeutic targets. High level of validation by qRT-PCR for a number of DEG genes supports the accuracy of this analysis. Global comparison of transcriptomes of B cells, IGVH non-mutated CLL (U-CLL) and mutated CLL specimens (M-CLL) with multidimensional scaling analysis was able to segregate CLL and B cell transcriptomes but the M-CLL and U-CLL transcriptomes were indistinguishable. The analysis of HTS RNA-seq data to identify alternative splicing events and other genetic abnormalities specific to CLL is an added advantage of RNA-seq that is not feasible with other genome wide analysis

Additional file 8: of Gene expression and splicing alterations analyzed by high throughput RNA sequencing of chronic lymphocytic leukemia specimens

List of skipped exon events tested in CLL specimens with PCR analysis. (PPTX 119 kb

Stellenbosch Media Forum 2003

Stellenbosch Media Forum is an annual publication written and produced by the BPhil (Journalism) class of that specific year in the Department of Journalism, Stellenbosch University."Die sorgsame inagneming van die geskiedenis is deel van sy verantwoordelikheid. Maar terselfdertyd is vernuwing dwingend deel van sy taak," skryf die kanselier, prof. Elize Botha, in die Universiteit van Stellenbosch se jongste jaarverslag oor die rol van die nuwe rektor in die "Heiligen Hailen" van hierdie besonderse universiteit op hierdie besonderse dorp in hierdie besonderse land -in hierdie besonderse tyd. Die woorde van Prof Elize, wat haar loopbaan as joernalis begin het, en onder andere ook 'n lid van Naspers se moederdireksie is, kan ook op ons departement toegepas word. Ook op die joernalistiek en die media. Die geskiedenis is deel van ons verantwoordelikheid. En dit moet verreken word. Nie verniet nie is ons die "geskiedskrywers op galop" - "history in a hurry". Maar ewe veel is dit die joernalistiek en die media se rol om 'n katalisator te wees vir die dwingende vernuwing op alle terreine in ons gemeenskappe en in ons land. Die Departement Joernalistiek, wat met hierdie jubileumuitgawe 25 jaar van joernalistiek-opleiding aan die US vier, het, soos die media die afgelope 25 jaar, deur sy eie transformasie gegaan. Van mense, tegnologie, kursus-inhoud. Dit was altyd 'n departement aan't morfeer, 'n weerspieeling van die mediabedryf self. Reg van die begin af. Wie van daardie eerste klas in 1978 onthou nie die terugkoms na die winterwegbreek, en die afvaardiging wat die professor in sy eie "Oval Office" gaan spreek het nie? Moet 'n mens ook byvoeg, 'n professor wat vinnig agter teekoppies (en natuurlik, ook die vrug van die omgewing) in 'n Pa Piet getransformeer het. Dit het gegaan oor die Vreeslike Verskrikking, by name Shorthand en Snelskrif. Oulike studente - soos wat die departement nog altyd gehad het - het naamlik in die vakansie 'n groot ontdekking gemaak. Dit staan nie in die boek van die lewe, oftewel die jaarboek, dat die twee S'e deurgekom moet word om die kursus te slaag nie. En: het Sy Eminensie dalk al van bandopnemers gehoor? Die kursus is summier getransformeer na een waar die Piet slegs maar 'n beroep kon doen op ons om tog maar ter wille van die joernalistiek te probeer. Dit was die begin, glo sommige van ons graag, van die tegnologiese revolusie in die media. Een van daardie eerste klas se studente, ons eie "verdwaalde hippie", het immers toe al gedroom van 'n rekenaarprogram waarvoor jy slegs maar die inligting invoer, 'n knoppie druk, en die rekenaar prut voort op die storie. Sonder die pyn en lyding van 'n intro skryf. En herskryf en weer skryf. En dan poleer tot hy blink, en dan die tweede paragraaf, en so voort tot die laaste pynlike paragraaf. But let's take the focus back to the department. It has developed in such a way that it today delivers multi-skilled, multi-media beginner-journalists to the industry in its BPhil programme (the former BHons). In its master's programme it gives practising Journalists an opportunity to "sharpen the sword." And for those who want to take it even further, the DPhil in journalism is in place. At the same time we have literally moved from the old black Olivetti's and "takes" to state of the art computers, complete with digital editing programmes for TV production - shot on digital video cameras. Yet, beyond the practical skills, the department has built a reputation of delivering well-rounded beginners, those with not only exceptional practical skills, but especially with exceptional conceptual skills. Journalists who are able to analyse and contextualise - the most important skills for the profession. This is thanks to the lecturers who over the years contributed to build the department's curriculum. From founder-professor Piet Cillie, to his successor Johannes Grosskopf, to the third head, George Claassen, plus all their co-lecturers. This past 25 years have also seen our media developing from the "most free" oppressed media in the world to a media where freedom of expression as entrenched in our constitution sets an example to the world. The media's role in our so-called "fledgling democracy", however, is more important than ever. And we need to train future Journalists who will have the knowledge, the skills, the passion and the heart to serve our democracy, and to be servants to our people. For the standards set, and the challenges to live up to, I want to thank all who went through the front door of Protea, 26 Crozier Street, over this past 25 years. Thank you for your example. And wishing success and sweet years of learning and discovering to all those who still have to experience Stellenbosch in springtime