Juan Mata

Methylation of Histone H4 Lysine 20 Controls Recruitment of Crb2 to Sites of DNA Damage

Histone lysine methylation is a key regulator of gene expression and heterochromatin function, but little is known as to how this modification impinges on other chromatin activities. Here we demonstrate that a previously uncharacterized SET domain protein, Set9, is responsible for H4-K20 methylation in the fission yeast Schizosaccharomyces pombe. Surprisingly, H4-K20 methylation does not have any apparent role in the regulation of gene expression or heterochromatin function. Rather, we find the modification has a role in DNA damage response. Loss of Set9 activity or mutation of H4-K20 markedly impairs cell survival after genotoxic challenge and compromises the ability of cells to maintain checkpoint mediated cell cycle arrest. Genetic experiments link Set9 to Crb2, a homolog of the mammalian checkpoint protein 53BP1, and the enzyme is required for Crb2 localization to sites of DNA damage. These results argue that H4-K20 methylation functions as a histone mark required for the recruitment of the checkpoint protein Crb2.

Periodic gene expression program of the fission yeast cell cycle

Cell-cycle control of transcription seems to be universal, but little is known about its global conservation and biological significance. We report on the genome-wide transcriptional program of the Schizosaccharomyces pombe cell cycle, identifying 407 periodically expressed genes of which 136 show high-amplitude changes. These genes cluster in four major waves of expression. The forkhead protein Sep1p regulates mitotic genes in the first cluster, including Ace2p, which activates transcription in the second cluster during the M-G1 transition and cytokinesis. Other genes in the second cluster, which are required for G1-S progression, are regulated by the MBF complex independently of Sep1p and Ace2p. The third cluster coincides with S phase and a fourth cluster contains genes weakly regulated during G2 phase. Despite conserved cell-cycle transcription factors, differences in regulatory circuits between fission and budding yeasts are evident, revealing evolutionary plasticity of transcriptional control. Periodic transcription of most genes is not conserved between the two yeasts, except for a core set of ∼40 genes that seem to be universally regulated during the eukaryotic cell cycle and may have key roles in cell-cycle progression.

The transcriptional program of meiosis and sporulation in fission yeast

Sexual reproduction requires meiosis to produce haploid gametes, which in turn can fuse to regenerate a diploid organism. We have studied the transcriptional program that drives this developmental process in Schizosaccharomyces pombe using DNA microarrays. Here we show that hundreds of genes are regulated in successive waves of transcription that correlate with major biological events of meiosis and sporulation. Each wave is associated with specific promoter motifs. Clusters of neighboring genes (mostly close to telomeres) are co-expressed early in the process, which reflects a more global control of these genes. We find that two Atf-like transcription factors are essential for the expression of late genes and formation of spores, and identify dozens of potential Atf target genes. Comparison with the meiotic program of the distantly related Saccharomyces cerevisiae reveals an unexpectedly small shared meiotic transcriptome, suggesting that the transcriptional regulation of meiosis evolved independently in both species.

tea1 and the Microtubular Cytoskeleton Are Important for Generating Global Spatial Order within the Fission Yeast Cell

Fission yeast cells identify and maintain growing regions exactly opposed at the ends of a cylindrical cell. tea1 mutants disrupt this organization, producing bent and T-shaped cells. We have cloned tea1 and shown that tea1 is located at the cell poles. Microtubules are continuously required to transfer tea1 to the cell ends, and tea1 is located at the ends of microtubules growing toward the cell poles. We suggest that tea1 acts as an end marker, directing the growth machinery to the cell poles. tea1 is down-regulated in cells treated with pheromone that grow toward a mating partner and no longer maintain their ends exactly opposed. tea1 may also influence microtubular organization, affecting the maintenance of a single central axis.

Tribbles Coordinates Mitosis and Morphogenesis in Drosophila by Regulating String/CDC25 Proteolysis

Morphogenesis and cell differentiation in multicellular organisms often require accurate control of cell divisions. We show that a novel cell cycle regulator, tribbles, is critical for this control during Drosophila development. During oogenesis, the level of tribbles affects the number of germ cell divisions as well as oocyte determination. The mesoderm anlage enters mitosis prematurely in tribbles mutant embryos, leading to gastrulation defects. We show that Tribbles acts by specifically inducing degradation of the CDC25 mitotic activators String and Twine via the proteosome pathway. By regulating CDC25, Tribbles serves to coordinate entry into mitosis with morphogenesis and cell fate determination.

Whole-genome microarrays of fission yeast: characteristics, accuracy, reproducibility, and processing of array data

BackgroundThe genome of the fission yeast Schizosaccharomyces pombe has recently been sequenced, setting the stage for the post-genomic era of this increasingly popular model organism. We have built fission yeast microarrays, optimised protocols to improve array performance, and carried out experiments to assess various characteristics of microarrays.ResultsWe designed PCR primers to amplify specific probes (180–500 bp) for all known and predicted fission yeast genes, which are printed in duplicate onto separate regions of glass slides together with control elements (~13,000 spots/slide). Fluorescence signal intensities depended on the size and intragenic position of the array elements, whereas the signal ratios were largely independent of element properties. Only the coding strand is covalently linked to the slides, and our array elements can discriminate transcriptional direction. The microarrays can distinguish sequences with up to 70% identity, above which cross-hybridisation contributes to the signal intensity. We tested the accuracy of signal ratios and measured the reproducibility of array data caused by biological and technical factors. Because the technical variability is lower, it is best to use samples prepared from independent biological experiments to obtain repeated measurements with swapping of fluorochromes to prevent dye bias. We also developed a script that discards unreliable data and performs a normalization to correct spatial artefacts.ConclusionsThis paper provides data for several microarray properties that are rarely measured. The results define critical parameters for microarray design and experiments and provide a framework to optimise and interpret array data. Our arrays give reproducible and accurate expression ratios with high sensitivity. The scripts for primer design and initial data processing as well as primer sequences and detailed protocols are available from our website.

A Network of Multiple Regulatory Layers Shapes Gene Expression in Fission Yeast

Summary Gene expression is controlled at multiple layers, and cells may integrate different regulatory steps for coherent production of proper protein levels. We applied various microarray-based approaches to determine key gene-expression intermediates in exponentially growing fission yeast, providing genome-wide data for translational profiles, mRNA steady-state levels, polyadenylation profiles, start-codon sequence context, mRNA half-lives, and RNA polymerase II occupancy. We uncovered widespread and unexpected relationships between distinct aspects of gene expression. Translation and polyadenylation are aligned on a global scale with both the lengths and levels of mRNAs: efficiently translated mRNAs have longer poly(A) tails and are shorter, more stable, and more efficiently transcribed on average. Transcription and translation may be independently but congruently optimized to streamline protein production. These rich data sets, all acquired under a standardized condition, reveal a substantial coordination between regulatory layers and provide a basis for a systems-level understanding of multilayered gene-expression programs.

Global Effects on Gene Expression in Fission Yeast by Silencing and RNA Interference Machineries

ABSTRACT Histone modifications influence gene expression in complex ways. The RNA interference (RNAi) machinery can repress transcription by recruiting histone-modifying enzymes to chromatin, although it is not clear whether this is a general mechanism for gene silencing or whether it requires repeated sequences such as long terminal repeats (LTRs). We analyzed the global effects of the Clr3 and Clr6 histone deacetylases, the Clr4 methyltransferase, the zinc finger protein Clr1, and the RNAi proteins Dicer, RdRP, and Argonaute on the transcriptome of Schizosaccharomyces pombe (fission yeast). The clr mutants derepressed similar subsets of genes, many of which also became transcriptionally activated in cells that were exposed to environmental stresses such as nitrogen starvation. Many genes that were repressed by the Clr proteins clustered in extended regions close to the telomeres. Surprisingly few genes were repressed by both the silencing and RNAi machineries, with transcripts from centromeric repeats and Tf2 retrotransposons being notable exceptions. We found no correlation between repression by RNAi and proximity to LTRs, and the wtf family of repeated sequences seems to be repressed by histone deacetylation independent of RNAi. Our data indicate that the RNAi and Clr proteins show only a limited functional overlap and that the Clr proteins play more global roles in gene silencing.

Transcriptional regulatory network for sexual differentiation in fission yeast.

BackgroundChanges in gene expression are hallmarks of cellular differentiation. Sexual differentiation in fission yeast (Schizosaccharomyces pombe) provides a model system for gene expression programs accompanying and driving cellular specialization. The expression of hundreds of genes is modulated in successive waves during meiosis and sporulation in S. pombe, and several known transcription factors are critical for these processes.ResultsWe used DNA microarrays to investigate meiotic gene regulation by examining transcriptomes after genetic perturbations (gene deletion and/or overexpression) of rep1, mei4, atf21 and atf31, which encode known transcription factors controlling sexual differentiation. This analysis reveals target genes at a genome-wide scale and uncovers combinatorial control by Atf21p and Atf31p. We also studied two transcription factors not previously implicated in sexual differentiation whose meiotic induction depended on Mei4p: Rsv2p induces stress-related genes during spore formation, while Rsv1p represses glucose-metabolism genes. Our data further reveal negative feedback interactions: both Rep1p and Mei4p not only activate specific gene expression waves (early and middle genes, respectively) but are also required for repression of genes induced in the previous waves (Ste11p-dependent and early genes, respectively).ConclusionThese data give insight into regulatory principles controlling the extensive gene expression program driving sexual differentiation and highlight sophisticated interactions and combinatorial control among transcription factors. Besides triggering simultaneous expression of gene waves, transcription factors also repress genes in the previous wave and induce other factors that in turn regulate a subsequent wave. These dependencies ensure an ordered and timely succession of transcriptional waves during cellular differentiation.

Global roles of Ste11p, cell type, and pheromone in the control of gene expression during early sexual differentiation in fission yeast

Fission yeast cells belong to one of two specialized cell types, M or P. Specific environmental conditions trigger sexual differentiation, which leads to an internal program starting with pheromone signaling between M and P cells, followed by mating, meiosis, and sporulation. The initial steps of this process are controlled by Ste11p, a master transcriptional regulator that activates the expression of cell type-specific genes (only expressed in either M or P cells) as well as genes expressed in both M and P cells. Pheromone signaling is activated by Ste11p-dependent transcription and, in turn, enhances some of this transcription in a positive feedback. To obtain a genomewide view of Ste11p target genes, their cell-type specificity, and their dependence on pheromone, we used DNA microarrays along with different genetic and environmental manipulations of fission yeast cells. We identified 78 Ste11p-dependent genes, 12 and 4 of which are only expressed in M and P cells, respectively. These genes show differing grades of pheromone dependencies for Ste11p-activated transcription, ranging from complete independence to complete dependence on pheromone. We systematically deleted all novel cell type-specific genes and characterized their phenotype during sexual differentiation. A comparison with a similar data set from the distantly related budding yeast reveals striking conservation in both number and types of the proteins that define cell types. Given the divergent mechanisms regulating cell type-specific gene expression, our results highlight the plasticity of regulatory circuits, which evolve to allow adaptation to changing environments and lifestyles.