0000000000116196
AUTHOR
Gabriel Gutiérrez
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.
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…
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…
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…
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 …
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.
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.