0000000000133160
AUTHOR
Antonio Jordán-pla
MOESM8 of Subtracting the sequence bias from partially digested MNase-seq data reveals a general contribution of TFIIS to nucleosome positioning
Additional file 8. Nucleosome positioning of genes with significant changes between wt and dst1∆. Genes were ordered by the number of nucleosomes that changed (in occupancy or fuzziness) between the wt and dst1∆. The nucleosomal profile of the top five genes is presented.
Cytoplasmic 5′-3′ exonuclease Xrn1p is also a genome-wide transcription factor in yeast
The 5′ to 3′ exoribonuclease Xrn1 is a large protein involved in cytoplasmatic mRNA degradation as a critical component of the major decaysome. Its deletion in the yeast Saccharomyces cerevisiae is not lethal, but it has multiple physiological effects. In a previous study, our group showed that deletion of all tested components of the yeast major decaysome, including XRN1, results in a decrease in the synthetic rate and an increase in half-life of most mRNAs in a compensatory manner. Furthermore, the same study showed that the all tested decaysome components are also nuclear proteins that bind to the 5′ region of a number of genes. In the present work, we show that disruption of Xrn1 activi…
Measuring RNA polymerase activity genome-wide with high-resolution run-on-based methods
The biogenesis of RNAs is a multi-layered and highly regulated process that involves a diverse set of players acting in an orchestrated manner throughout the transcription cycle. Transcription initiation, elongation and termination factors act on RNA polymerases to modulate their movement along the DNA template in a very precise manner, more complex than previously anticipated. Genome-scale run-on-based methodologies have been developed to study in detail the position of transcriptionally-engaged RNA polymerases. Genomic run-on (GRO), and its many variants and refinements made over the years, are helping the community to address an increasing amount of scientific questions, spanning an incr…
Subtracting the sequence bias from partially digested MNase-seq data reveals a general contribution of TFIIS to nucleosome positioning.
Background TFIIS stimulates RNA cleavage by RNA polymerase II and promotes the resolution of backtracking events. TFIIS acts in the chromatin context, but its contribution to the chromatin landscape has not yet been investigated. Co-transcriptional chromatin alterations include subtle changes in nucleosome positioning, like those expected to be elicited by TFIIS, which are elusive to detect. The most popular method to map nucleosomes involves intensive chromatin digestion by micrococcal nuclease (MNase). Maps based on these exhaustively digested samples miss any MNase-sensitive nucleosomes caused by transcription. In contrast, partial digestion approaches preserve such nucleosomes, but intr…
A genomic view of mRNA turnover in yeast
The steady-state mRNA level is the result of two opposing processes: transcription and degradation; both of which can provide important points to regulate gene expression. In the model organism yeast Saccharomyces cerevisiae, it is now possible to determine, at the genomic level, the transcription and degradation rates, as well as the mRNA amount, using DNA chip or parallel sequencing technologies. In this way, the contribution of both rates to individual and global gene expressions can be analysed. Here we review the techniques used for the genomic evaluation of the transcription and degradation rates developed for this yeast, and we discuss the integration of the data obtained to fully an…
Defects in the NC2 repressor affect both canonical and non-coding RNA polymerase II transcription initiation in yeast.
BACKGROUND: The formation of the pre-initiation complex in eukaryotic genes is a key step in transcription initiation. The TATA-binding protein (TBP) is a universal component of all pre-initiation complexes for all kinds of RNA polymerase II (RNA pol II) genes, including those with a TATA or a TATA-like element, both those that encode proteins and those that transcribe non-coding RNAs. Mot1 and the negative cofactor 2 (NC2) complex are regulators of TBP, and it has been shown that depletion of these factors in yeast leads to defects in the control of transcription initiation that alter cryptic transcription levels in selected yeast loci. RESULTS: In order to cast light on the molecular func…
Genomic insights into the different layers of gene regulation in yeast.
The model organism Saccharomyces cerevisiae has allowed the development of new functional genomics techniques devoted to the study of transcription in all its stages. With these techniques, it has been possible to find interesting new mechanisms to control gene expression that act at different levels and for different gene sets apart from the known cis-trans regulation in the transcription initiation step. Here we discuss a method developed in our laboratory, Genomic Run-On, and other new methods that have recently appeared with distinct technical features. A comparison between the datasets generated by them provides interesting genomic insights into the different layers of gene regulation …
Xrn1 influence on gene transcription results from the combination of general effects on elongating RNA pol II and gene-specific chromatin configuration.
mRNA homoeostasis is favoured by crosstalk between transcription and degradation machineries. Both the Ccr4-Not and the Xrn1-decaysome complexes have been described to influence transcription. While Ccr4-Not has been shown to directly stimulate transcription elongation, the information available on how Xrn1 influences transcription is scarce and contradictory. In this study we have addressed this issue by mapping RNA polymerase II (RNA pol II) at high resolution, using CRAC and BioGRO-seq techniques in Saccharomyces cerevisiae. We found significant effects of Xrn1 perturbation on RNA pol II profiles across the genome. RNA pol II profiles at 5ʹ exhibited significant alterations that were com…
MOESM11 of Subtracting the sequence bias from partially digested MNase-seq data reveals a general contribution of TFIIS to nucleosome positioning
Additional file 11. Occupancy-versus-fuzziness changes of the TATA-like gene bodies and + 1 nucleosomes. Heat maps of the difference between the mutant dst1∆ and the wt in fuzziness versus occupancy for the gene body nucleosomes of the TATA-like genes (A), and for the + 1 nucleosome (defined as that between the TSS and 200 bp downstream) of each gene (B).
MOESM7 of Subtracting the sequence bias from partially digested MNase-seq data reveals a general contribution of TFIIS to nucleosome positioning
Additional file 7. Nucleotide composition of the sequence of the TATA and TATA-like genes. A) Frequency of each nucleotide in the TATA (red) and TATA-like genes (blue) at each position in relation to the TSS. B) The average nucleotide frequency in the promoter (− 500 to − 100) and the gene body (50–500) of the TATA and TATA-like genes. A Student’s t test was applied to compare the TATA and TATA-like genes. S indicates that the difference is significant (p 0.001).
MOESM6 of Subtracting the sequence bias from partially digested MNase-seq data reveals a general contribution of TFIIS to nucleosome positioning
Additional file 6. A metagene analysis to compare the sequencing data before and after the correction in TATA genes versus TATA-like genes A) The metagene analysis of the chromatin (blue before the correction, red afterward) and the naked DNA signals (green) around the pAS in the TATA (left panel) and TATA-like genes (right panel). Genes were scaled to the same length and then aligned to their pAS. B) Genes were divided into quartiles according to their transcription rate [45] and then further subdivided into TATA or TATA-like genes. All the resulting eight groups were scaled and aligned to their TSS. The chromatin signal before and after correction is shown.
MOESM1 of Subtracting the sequence bias from partially digested MNase-seq data reveals a general contribution of TFIIS to nucleosome positioning
Additional file 1. Overview of the method. A) A diagram with the main protocol steps is shown. The fragments to be sequenced were isolated from an ethidium bromide-stained gel (see the example in the figure). The naked DNA samples were visually matched to the chromatin samples by choosing those with a similar maximum fragment size (arrow). Then, the mononucleosome-sized fragments (squares) were isolated. B) The chromatin (blue and red) and naked DNA signals (green) over the STL1 gene are shown as examples of the results, analyzed by qPCR. The chromatin data are presented before (blue) and after (red) the naked DNA correction. C) The naked DNA signal in the STL1 gene from different Saccharom…
The telomeric Cdc13-Stn1-Ten1 complex regulates RNA polymerase II transcription
Advance article.
Adult Neural Stem Cells Are Alerted by Systemic Inflammation through TNF-α Receptor Signaling.
Summary Adult stem cells (SCs) transit between the cell cycle and a poorly defined quiescent state. Single neural SCs (NSCs) with quiescent, primed-for-activation, and activated cell transcriptomes have been obtained from the subependymal zone (SEZ), but the functional regulation of these states under homeostasis is not understood. Here, we develop a multilevel strategy to analyze these NSC states with the aim to uncover signals that regulate their level of quiescence/activation. We show that transitions between states occur in vivo and that activated and primed, but not quiescent, states can be captured and studied in culture. We also show that peripherally induced inflammation promotes a …
Biotin-Genomic Run-On (Bio-GRO): A High-Resolution Method for the Analysis of Nascent Transcription in Yeast
Transcription is a highly complex biological process, with extensive layers of regulation, some of which remain to be fully unveiled and understood. To be able to discern the particular contributions of the several transcription steps it is crucial to understand RNA polymerase dynamics and regulation throughout the transcription cycle. Here we describe a new nonradioactive run-on based method that maps elongating RNA polymerases along the genome. In contrast with alternative methodologies for the measurement of nascent transcription, the BioGRO method is designed to minimize technical noise that arises from two of the most common sources that affect this type of strategies: contamination wi…
MOESM3 of Subtracting the sequence bias from partially digested MNase-seq data reveals a general contribution of TFIIS to nucleosome positioning
Additional file 3. Comparison with chemical mapping method. A) Center-to-center distance of the nearest nucleosome in: the raw data presented here against a chemical modification-based map [31] (blue line), the corrected data against the same reference map [31] (orange line), or the chemical modification-based map against a map that was generated by extensive digestion with MNase [12]. B) Cladogram showing the distance between the different maps mentioned in A.
MOESM5 of Subtracting the sequence bias from partially digested MNase-seq data reveals a general contribution of TFIIS to nucleosome positioning
Additional file 5. A metagene analysis to compare the sequencing data before and after correction in different groups of genes. A) A 2D plot to compare the log10 signal intensity in the naked DNA sample and the GC content of fragments (normalized by subtracting the genomic average). Pearson’s correlation is shown (p
MOESM2 of Subtracting the sequence bias from partially digested MNase-seq data reveals a general contribution of TFIIS to nucleosome positioning
Additional file 2. Metagene analysis of the chromatin and naked DNA signals. A, B) Genes were scaled to the same length and then aligned to their TSS or their pAS. All the genes in the yeast genome for which a TSS was available were considered. Zoom-in view of the data in Fig. 1a: A) closer to the TSS; B) closer to the pAS. C) Those genes whose pAS was at least 500 bp away from a TSS were selected, scaled to the same length, and represented as in B. D) Difference between the corrected and raw signals. Genes were scaled and aligned as in Fig. 1a, b. The Y-axis represents the logarithm of the p value of the difference. Two different curves are shown: one represents the positive difference val…
Chromatin-dependent regulation of RNA polymerases II and III activity throughout the transcription cycle
The particular behaviour of eukaryotic RNA polymerases along different gene regions and amongst distinct gene functional groups is not totally understood. To cast light onto the alternative active or backtracking states of RNA polymerase II, we have quantitatively mapped active RNA polymerases at a high resolution following a new biotin-based genomic run-on (BioGRO) technique. Compared with conventional profiling with chromatin immunoprecipitation, the analysis of the BioGRO profiles in Saccharomyces cerevisiae shows that RNA polymerase II has unique activity profiles at both gene ends, which are highly dependent on positioned nucleosomes. This is the first demonstration of the in vivo infl…
Aberrations of Genomic Imprinting in Glioblastoma Formation
In human glioblastoma (GBM), the presence of a small population of cells with stem cell characteristics, the glioma stem cells (GSCs), has been described. These cells have GBM potential and are responsible for the origin of the tumors. However, whether GSCs originate from normal neural stem cells (NSCs) as a consequence of genetic and epigenetic changes and/or dedifferentiation from somatic cells remains to be investigated. Genomic imprinting is an epigenetic marking process that causes genes to be expressed depending on their parental origin. The dysregulation of the imprinting pattern or the loss of genomic imprinting (LOI) have been described in different tumors including GBM, being one …
MOESM9 of Subtracting the sequence bias from partially digested MNase-seq data reveals a general contribution of TFIIS to nucleosome positioning
Additional file 9. Nucleosome fuzziness in the wt and dst1∆. A) The metagene analysis of the fuzziness score of the wt (blue) and dst1∆ (red) nucleosomes around the TSS. Genes were scaled to the same length and then aligned to their TSS. B) The change in fuzziness score between the wt and dst1∆. Heat map of the fuzziness score of the gene body nucleosomes in the wt and dst1∆ mutant. Color represents density, which increases from blue to red. The red square highlights those nucleosomes below 40 in the wt and above > 40 in the mutant. C) The fuzziness score distribution of the nucleosomes in the gene bodies of the highly transcribed genes of the wt (blue) and dst1∆ (red). D) The fuzziness …
Prefoldin-like Bud27 influences the transcription of ribosomal components and ribosome biogenesis in Saccharomyces cerevisiae
Understanding the functional connection that occurs for the three nuclear RNA polymerases to synthesize ribosome components during the ribosome biogenesis process has been the focal point of extensive research. To preserve correct homeostasis on the production of ribosomal components, cells might require the existence of proteins that target a common subunit of these RNA polymerases to impact their respective activities. This work describes how the yeast prefoldin-like Bud27 protein, which physically interacts with the Rpb5 common subunit of the three RNA polymerases, is able to modulate the transcription mediated by the RNA polymerase I, likely by influencing transcription elongation, the …
GM-CSF Programs Hematopoietic Stem and Progenitor Cells During Candida albicans Vaccination for Protection Against Reinfection
More mechanistic studies are needed to reveal the hidden details of in vivo-induced trained immunity. Here, using a Candida albicans live vaccine mouse model we show that vaccination protects mice against a secondary infection and increases the number of bone marrow, and especially, splenic trained monocytes. Moreover, vaccination expands and reprograms hematopoietic stem and progenitor cells (HSPCs) early during infection and mobilize them transiently to the spleen to produce trained macrophages. Trained HSPCs are not only primed for myeloid cell production but also reprogramed to produce a greater amount of proinflammatory cytokines in response to a second challenge. Additionally, their a…
MOESM10 of Subtracting the sequence bias from partially digested MNase-seq data reveals a general contribution of TFIIS to nucleosome positioning
Additional file 10. Effect of the absence of TFIIS on the expression of the different types of genes. A) Scatter plot of the nascent transcription rate of each gene in the wt (X-axis) and in dst1∆ (Y-axis). B) Diagram showing the relationship between the group of genes with a weaker GRO signal in dst1∆ compared to the wt (TFIIS-dependent) in the TATA-containing genes and TATA-like genes. The TATA-containing genes are overrepresented in the TFIIS-dependent genes (hypergeometric test, p = 0.006), while the TATA-like genes are under-represented (hypergeometric test, p = 0.028). C) The Bio-GRO signals of the RP genes in the wt and dst1∆. After classification, genes were aligned to their TSS.
The SWI/SNF subunits BRG1 affects alternative splicing by changing RNA binding factor interactions with RNA
AbstractBRG1 and BRM are ATPase core subunits of the human SWI/SNF chromatin remodelling complexes. The function of the SWI/SNF complexes in transcriptional initiation has been well studied, while a function in alternative splicing has only been studied for a few cases for BRM-containing SWI/SNF complexes. Here, we have expressed BRG1 in C33A cells, a BRG1 and BRM-deficient cell line, and we have analysed the effects on the transcriptome by RNA sequencing. We have shown that BRG1 expression affects the splicing of a subset of genes. For some, BRG1 expression favours exon inclusion and for others, exon skipping. Some of the changes in alternative splicing induced by BRG1 expression do not re…
The rates of adult neurogenesis and oligodendrogenesis are linked to cell cycle regulation through p27-dependent gene repression of SOX2
Abstract Cell differentiation involves profound changes in global gene expression that often have to occur in coordination with cell cycle exit. Because cyclin-dependent kinase inhibitor p27 reportedly regulates proliferation of neural progenitor cells in the subependymal neurogenic niche of the adult mouse brain, but can also have effects on gene expression, we decided to molecularly analyze its role in adult neurogenesis and oligodendrogenesis. At the cell level, we show that p27 restricts residual cyclin-dependent kinase activity after mitogen withdrawal to antagonize cycling, but it is not essential for cell cycle exit. By integrating genome-wide gene expression and chromatin accessibil…
The mRNA degradation factor Xrn1 regulates transcription elongation in parallel to Ccr4
Abstract Co-transcriptional imprinting of mRNA by Rpb4 and Rpb7 subunits of RNA polymerase II (RNAPII) and by the Ccr4–Not complex conditions its post-transcriptional fate. In turn, mRNA degradation factors like Xrn1 are able to influence RNAPII-dependent transcription, making a feedback loop that contributes to mRNA homeostasis. In this work, we have used repressible yeast GAL genes to perform accurate measurements of transcription and mRNA degradation in a set of mutants. This genetic analysis uncovered a link from mRNA decay to transcription elongation. We combined this experimental approach with computational multi-agent modelling and tested different possibilities of Xrn1 and Ccr4 acti…
Xrn1 influence on gene transcription results from the combination of general effects on elongating RNA pol II and gene-specific chromatin configuration
mRNA homoeostasis is favoured by crosstalk between transcription and degradation machineries. Both the Ccr4-Not and the Xrn1-decaysome complexes have been described to influence transcription. While Ccr4-Not has been shown to directly stimulate transcription elongation, the information available on how Xrn1 influences transcription is scarce and contradictory. In this study we have addressed this issue by mapping RNA polymerase II (RNA pol II) at high resolution, using CRAC and BioGRO-seq techniques in Saccharomyces cerevisiae. We found significant effects of Xrn1 perturbation on RNA pol II profiles across the genome. RNA pol II profiles at 5ʹ exhibited significant alterations that were com…
MOESM4 of Subtracting the sequence bias from partially digested MNase-seq data reveals a general contribution of TFIIS to nucleosome positioning
Additional file 4. Genes included in the different categories analyzed in this work.