Resultados de investigación

Figure S1. Evolutionary conservation of newly analysed cell type marker genes: A. Gene conservation. For the cell type marker genes that were analysed in figure 1, closest relatives were identified by BLASTp in a protein sequence library compiled from 47 annotated genomes and transcriptomes, representative of all major and minor taxon groups of Dictyostelia. The locus tags of the genes are colour coded according to the taxon group to which the host species belongs (B) with the more intense colours representing the well annotated D. discoideum, D. purpureum, D. lacteum, P. pallidum and D. fasciculatum genomes. Species belonging to minor taxon groups are colour coded in grey. Protein sequences were aligned with Clustal Omega (Sievers and Higgins, 2014) with five combined iterations and phylogenies were inferred using MrBayes 3.2 (Ronquist and Huelsenbeck, 2003). Posterior probabilities (BIPP) of the nodes are indicated by coloured dots. Full species names and Bioproject or Assembly IDs of the genomes and transcriptomes that were used are listed in SupData1_Genes&Species.xlsx. B. Dictyostelid phylogeny. Phylogeny of the five taxon-group-representative Dictyostelium species for which developmental- and cell type specific transcriptome data are shown in Figure 1B. The phylogeny was inferred from 30 concatenated proteins and rooted on the non-dictyostelid Amoebozoan Physarum polycephalum (Romeralo et al., 2013)., Figure S2. Standardized staining of cells transformed with ecmA- and ecmB-lacZ: Ax2 cells transformed with ecmA-lacZ, ecmA-ile-lacZ , ecmB-lacZ and ecmB-ile-lacZ (Detterbeck et al., 1994; Jermyn and Williams, 1991) were incubated on nitrocellulose filters supported by non-nutrient agar until migrating slugs, mid-culminants and fruiting bodies were formed. After fixation, the structures were stained with X-gal for the period required to strongly but not overstain the culminants and fruiting bodies. Bar: 100 μm. Note that the poor expression in slugs is not a consequence of β-galactosidase accumulating in the later stages, since the unstable ile-gal forms (Detterbeck et al., 1994) also show poor staining in slugs compared to fruiting bodies., Figure S3. Conservation and transcription profiles of previously described marker genes: A. Gene conservation. For the gene induction experiment presented in figure 2, we used additional cell type markers from an earlier study (Kin et al., 2018), the DIF-inducible markers ecmA and ecmB (Jermyn et al., 1987) and two markers identified in a RNAseq study of mutants that cannot synthesize c-diGMP (Chen et al., 2017) . These and other markers from the same studies that were validated using promoter-lacZ constructs were analysed for evolutionary conservation as in Fig. S1. Replicate studies of staE-lacZ showed expression in the basal disc of culminants (image), which was not reported in the earlier study (Kin et al., 2018). B. Transcriptomics. The developmental time courses and cell type specificity data of the markers were retrieved from published RNAseq experiments as described in the legend to Figure 1. When known, gene names are also listed. Identifiers in grey text represent genes not otherwise used in this study., Figure S4. P-values for pair-wise comparison of the differential effects of stimuli on gene induction: The data describing the effects of 15 stimulation regimens on 25 genes that are presented in figure 2 were subjected to Kruskal-Wallis ANOVA on ranks to identify significant differences between treatments on each of the genes. The stringent Tukey test and less stringent Student-Newman-Keuls (SNK) test were used to calculate P-values for the null hypothesis of no difference between treatments. The full analysis of 105 pairwise comparisons is listed with the experimental data for each gene in SupData2_Induction.xlsx. Biologically relevant comparisons are shown here as heatmaps of the P-values obtained with either test. Black squares represent experiments that were only performed once or not at all. Combined treatments used 3 μM c-di-GMP and the same concentrations for the other compounds as used for single treatments., Figure S5. Fold-change differences between treatments annotated with P-values: The table lists the numerical values of the fold-change over control values (left panel) and fold-changes between different treatments (right panel). The values are presented in different fonts to reflect whether the difference between treatments is significant (P<0.05) as assessed by ANOVA on ranks with P-values calculated by either the stringent Tukey test (bold black or white font) or the less stringent SNK test (italic red font). The extent of fold-change is further indicated as a yellow-blue heatmap in the left panel and a blue to red heatmap in the right panel, where a blue background marks a reduced effect and a red background an increased effect of the first over the second treatment, shown at the top of each column. Black boxes represent absent data. Note that small fold-changes can be statistically significant when there is little variation between individual experiments, while large fold-changes may not reach significance because the absolute value of the change varies strongly between experiments. We therefore also show the data and their analysis in Supdata2_induction.xlsx., Figure S6. Gene induction expressed as fold-change of control: For a subset of the gene induction data presented in Figure 2 where relatively small effects of stimuli were observed, data are re-plotted here as means and SE of fold-change over control (no addition). The red dashed line marks the control value of 1., Figure S7. Hierarchical clustering of gene induction data relative to fold change: A. Hierarchical tree. The averaged data for induction of 25 genes by 15 single or combined stimuli as presented in figure 2 are expressed here as fold-change relative to control and subjected to hierarchical clustering as described for figure 3. The hierarchical tree is combined with a heatmap of the gene induction responses as fold-change over control and a heatmap of the cell-type specific and developmental expression of the genes. B. Multidimensional scaling. The induction data relative to fold-change were also used to cluster the genes by multidimensional scaling as in figure 3., Table S1: Oligonucleotide primers used in this work., Cell differentiation is traditionally monitored with a few marker genes, which may bias results. To understand the evolution and regulation of the spore, stalk, cup and basal disc cells in Dictyostelia, we previously performed RNAseq on purified cell-types of taxon-group representative dictyostelids. Using promoter-lacZ constructs in D. discoideum, we here investigate the spatio-temporal expression pattern of 29 cell-type specific genes. Genes selected for spore- or cup-specificity in RNAseq were validated as such by lacZ expression, but genes selected for stalk-specificity showed variable additional expression in basal disc, early cup or prestalk populations. We measured responses of 25 genes to 15 single or combined regimes of induction by stimuli known to regulate cell differentiation. The outcomes of these experiments were subjected to hierarchical clustering to identify whether common modes of regulation were correlated with specific expression patterns. The analysis identified a cluster combining the spore and cup genes, which shared upregulation by 8-bromo cyclic AMP and down-regulation by Differentiation Inducing Factor 1 (DIF-1). Most stalk-expressed genes combined into a single cluster and shared strong upregulation by cyclic di-guanylate (c-di-GMP), and synergistic upregulation by combined DIF-1 and c-di-GMP. There was no clustering of genes expressed in other soma besides the stalk, but two genes that were only expressed in the stalk did not respond to any stimuli. In contrast to current models, the study indicates the existence of a stem-cell like soma population in slugs, whose members only acquire ultimate cell fate after progressing to their terminal location during fruiting body morphogenesis., Supplementary Figures S1-S7 and supplementary Table S1., Peer reviewed

This spreadsheet contains:1. Expression profiles obtained from RNAseq studies, expression patterns obtained from promoter-lacZ studies, protein function and or functional domains and evolutionary conservation of all genes studied in this work. 2. Species names and Genbank or ENA bioproject IDs for all genomes/transcriptomes data used to establish evolutionary conservation of genes., Cell differentiation is traditionally monitored with a few marker genes, which may bias results. To understand the evolution and regulation of the spore, stalk, cup and basal disc cells in Dictyostelia, we previously performed RNAseq on purified cell-types of taxon-group representative dictyostelids. Using promoter-lacZ constructs in D. discoideum, we here investigate the spatio-temporal expression pattern of 29 cell-type specific genes. Genes selected for spore- or cup-specificity in RNAseq were validated as such by lacZ expression, but genes selected for stalk-specificity showed variable additional expression in basal disc, early cup or prestalk populations. We measured responses of 25 genes to 15 single or combined regimes of induction by stimuli known to regulate cell differentiation. The outcomes of these experiments were subjected to hierarchical clustering to identify whether common modes of regulation were correlated with specific expression patterns. The analysis identified a cluster combining the spore and cup genes, which shared upregulation by 8-bromo cyclic AMP and down-regulation by Differentiation Inducing Factor 1 (DIF-1). Most stalk-expressed genes combined into a single cluster and shared strong upregulation by cyclic di-guanylate (c-di-GMP), and synergistic upregulation by combined DIF-1 and c-di-GMP. There was no clustering of genes expressed in other soma besides the stalk, but two genes that were only expressed in the stalk did not respond to any stimuli. In contrast to current models, the study indicates the existence of a stem-cell like soma population in slugs, whose members only acquire ultimate cell fate after progressing to their terminal location during fruiting body morphogenesis., Peer reviewed

Contents: 1. All original data (delta OD/min) for induction of gene expression by various stimuli listed by gene locus tag without DDB_G0 prefix or gene/promoter fragment name. 2. Data standardized as percentage of induction by 3 uM c-di-GMP with basic descriptive statistics (means, SD and SE), used for figure 2. 3. Data standardized as fold-change of control (no addition) with basic descriptive statistics. 4. Results of pairwise comparison of all treatments with the others by ANOVA (Analysis Of Variance) on ranks, with the Tukey or SNK (Student-Newman-Keuls) tests to determine P-values for significant differences between treatments., Cell differentiation is traditionally monitored with a few marker genes, which may bias results. To understand the evolution and regulation of the spore, stalk, cup and basal disc cells in Dictyostelia, we previously performed RNAseq on purified cell-types of taxon-group representative dictyostelids. Using promoter-lacZ constructs in D. discoideum, we here investigate the spatio-temporal expression pattern of 29 cell-type specific genes. Genes selected for spore- or cup-specificity in RNAseq were validated as such by lacZ expression, but genes selected for stalk-specificity showed variable additional expression in basal disc, early cup or prestalk populations. We measured responses of 25 genes to 15 single or combined regimes of induction by stimuli known to regulate cell differentiation. The outcomes of these experiments were subjected to hierarchical clustering to identify whether common modes of regulation were correlated with specific expression patterns. The analysis identified a cluster combining the spore and cup genes, which shared upregulation by 8-bromo cyclic AMP and down-regulation by Differentiation Inducing Factor 1 (DIF-1). Most stalk-expressed genes combined into a single cluster and shared strong upregulation by cyclic di-guanylate (c-di-GMP), and synergistic upregulation by combined DIF-1 and c-di-GMP. There was no clustering of genes expressed in other soma besides the stalk, but two genes that were only expressed in the stalk did not respond to any stimuli. In contrast to current models, the study indicates the existence of a stem-cell like soma population in slugs, whose members only acquire ultimate cell fate after progressing to their terminal location during fruiting body morphogenesis., Peer reviewed

Hierarchical clustering of gene induction data The averaged data for gene induction by stimuli, standardized either as percentage of 3 uM c-di-GMP or as fold-change relative to control (no addition) are shown in the sheet "InductionAverages" Both data matrices were used for cluster analysis using "Orange" datamining software (https://orangedatamining.com/). The columns for t0, 100nM DIF + 0.3mM cAMP and 100 nM DIF+ 8Br-cAMP, which were not tested for all genes and only in 1 or 2 experiments for others, were deleted from the matrices before analysis Distances between the effects of the treatments on the different genes were determined by Pearson correlation and a hierarchical tree was computed using average linkage (the average distance between the elements of two clusters) The full induction data matrices were re-ordered to the gene positions in the hierarchical tree in the sheet "Tree-ordered" Heatmaps of the developmental expression profiles and cell-type specificity profiles of the genes were included for presentation in Figure 3 and supplemental figure S7, Cell differentiation is traditionally monitored with a few marker genes, which may bias results. To understand the evolution and regulation of the spore, stalk, cup and basal disc cells in Dictyostelia, we previously performed RNAseq on purified cell-types of taxon-group representative dictyostelids. Using promoter-lacZ constructs in D. discoideum, we here investigate the spatio-temporal expression pattern of 29 cell-type specific genes. Genes selected for spore- or cup-specificity in RNAseq were validated as such by lacZ expression, but genes selected for stalk-specificity showed variable additional expression in basal disc, early cup or prestalk populations. We measured responses of 25 genes to 15 single or combined regimes of induction by stimuli known to regulate cell differentiation. The outcomes of these experiments were subjected to hierarchical clustering to identify whether common modes of regulation were correlated with specific expression patterns. The analysis identified a cluster combining the spore and cup genes, which shared upregulation by 8-bromo cyclic AMP and down-regulation by Differentiation Inducing Factor 1 (DIF-1). Most stalk-expressed genes combined into a single cluster and shared strong upregulation by cyclic di-guanylate (c-di-GMP), and synergistic upregulation by combined DIF-1 and c-di-GMP. There was no clustering of genes expressed in other soma besides the stalk, but two genes that were only expressed in the stalk did not respond to any stimuli. In contrast to current models, the study indicates the existence of a stem-cell like soma population in slugs, whose members only acquire ultimate cell fate after progressing to their terminal location during fruiting body morphogenesis., Peer reviewed

1 figure., a) with climatic condition (control (O) and heat stress (J) as sample-level covariate and b) with climatic condition and run as sample-level covariates. In both cases K = 2, épsilon = 104, V = 1., Peer reviewed

(I) Listing of the general experimental methods; (II) text and schemes describing the synthetic routes; (III) 1H and 13C NMR spectra; (IV) text, figures and tables illustrating results from ground state, excited state, and time-resolved transient absorption spectroscopy; (V) text, figures and tables describing the theoretical details., Peer reviewed

1 table., Peer reviewed

Experimental and computational methods; Mechanical properties of layered hybrid organic–inorganic metal-halide perovskites; Additional Raman spectroscopy characterization; Characterization of the SiO2 rings and flake/ring structures by SEM and AFM; z-depth penetration of the lasers; Supplementary photoluminescence and Raman spectroscopy data; Evolution of the temperature-dependent photoluminescence and Raman maps with the SiO2 ring dimensions; Power-dependence of the photoluminescence maps; and Additional finite element simulations., Peer reviewed

1 table., Peer reviewed

Mononucleosomal DNA was isolated after MNase treatment and was then prepared for sequencing..-- Organism: Saccharomyces cerevisiae, Resources available on the publisher's site: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE192701, The stability of the genome is occasionally challenged by the formation of DNA-RNA hybrids and R-loops, which can be influenced by the chromatin context. This is mainly due to the fact that DNA-RNA hybrids hamper the progression of replication forks, leading to fork stalling and, ultimately, DNA breaks. Through a specific screening of chromatin modifiers performed in the yeast Saccharomyces cerevisiae, we have found that the Rtt109 histone acetyltransferase is involved in several steps of R-loop-metabolism and their associated genetic instability. On one hand, Rtt109 prevents DNA-RNA hybridization by the acetylation of histone H3 lysines 14 and 23, and on the other hand, it is involved in the repair of replication-born DNA breaks, such as those that can be caused by R-loops, by acetylating lysines 14 and 56. In addition, Rtt109 loss renders cells highly sensitive to replication stress in combination with R-loop-accumulating THO-complex mutants. Our data evidence that the chromatin context simultaneously influences the occurrence of DNA-RNA hybrid-associated DNA damage and its repair, adding complexity to the source of R-loop-associated genetic instability., Peer reviewed