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Background: The generation of mature mRNAs involves interconnected processes, including transcription by RNA polymerase II (Pol II), modification of histones, and processing of pre-mRNAs through capping, intron splicing, and polyadenylation. These processes are thought to be integrated, both spatially and temporally, but it is unclear how these connections manifest at a global level with respect to chromatin patterns and transcription kinetics. We sought to clarify the relationships between chromatin, transcription and splicing using multiple genome-wide approaches in fission yeast., Results: To investigate these functional interdependencies, we determined Pol II occupancy across all genes using high-density tiling arrays. We also performed ChIP-chip on the same array platform to globally map histone H3 and its H3K36me3 modification, complemented by formaldehyde-assisted isolation of regulatory elements (FAIRE). Surprisingly, Pol II occupancy was higher in introns than in exons, and this difference was inversely correlated with gene expression levels at a global level. Moreover, introns showed distinct distributions of histone H3, H3K36me3 and FAIRE signals, similar to those at promoters and terminators. These distinct transcription and chromatin patterns of intronic regions were most pronounced in poorly expressed genes., Conclusions: Our findings suggest that Pol II accumulates at the 3' ends of introns, leading to substantial transcriptional delays in weakly transcribed genes. We propose that the global relationship between transcription, chromatin remodeling, and splicing may reflect differences in local nuclear environments, with highly expressed genes being associated with abundant processing factors that promote effective intron splicing and transcriptional elongation.

The regulation of the G1- to S-phase transition is critical for cell-cycle progression. This transition is driven by a transient transcriptional wave regulated by transcription factor complexes termed MBF/SBF in yeast and E2F-DP in mammals. Here we apply genomic, genetic, and biochemical approaches to show that the Yox1p homeodomain protein of fission yeast plays a critical role in confining MBF-dependent transcription to the G1/S transition of the cell cycle. The yox1 gene is an MBF target, and Yox1p accumulates and preferentially binds to MBF-regulated promoters, via the MBF components Res2p and Nrm1p, when they are transcriptionally repressed during the cell cycle. Deletion of yox1 results in constitutively high transcription of MBF target genes and loss of their cell cycle-regulated expression, similar to deletion of nrm1. Genome-wide location analyses of Yox1p and the MBF component Cdc10p reveal dozens of genes whose promoters are bound by both factors, including their own genes and histone genes. In addition, Cdc10p shows promiscuous binding to other sites, most notably close to replication origins. This study establishes Yox1p as a new regulatory MBF component in fission yeast, which is transcriptionally induced by MBF and in turn inhibits MBF-dependent transcription. Yox1p may function together with Nrm1p to confine MBF-dependent transcription to the G1/S transition of the cell cycle via negative feedback. Compared to the orthologous budding yeast Yox1p, which indirectly functions in a negative feedback loop for cell-cycle transcription, similarities but also notable differences in the wiring of the regulatory circuits are evident., Competing Interests: The authors have declared that no competing interests exist.

Recent data from several organisms indicate that the transcribed portions of genomes are larger and more complex than expected, and that many functional properties of transcripts are based not on coding sequences but on regulatory sequences in untranslated regions or non-coding RNAs. Alternative start and polyadenylation sites and regulation of intron splicing add additional dimensions to the rich transcriptional output. This transcriptional complexity has been sampled mainly using hybridization-based methods under one or few experimental conditions. Here we applied direct high-throughput sequencing of complementary DNAs (RNA-Seq), supplemented with data from high-density tiling arrays, to globally sample transcripts of the fission yeast Schizosaccharomyces pombe, independently from available gene annotations. We interrogated transcriptomes under multiple conditions, including rapid proliferation, meiotic differentiation and environmental stress, as well as in RNA processing mutants to reveal the dynamic plasticity of the transcriptional landscape as a function of environmental, developmental and genetic factors. High-throughput sequencing proved to be a powerful and quantitative method to sample transcriptomes deeply at maximal resolution. In contrast to hybridization, sequencing showed little, if any, background noise and was sensitive enough to detect widespread transcription in >90% of the genome, including traces of RNAs that were not robustly transcribed or rapidly degraded. The combined sequencing and strand-specific array data provide rich condition-specific information on novel, mostly non-coding transcripts, untranslated regions and gene structures, thus improving the existing genome annotation. Sequence reads spanning exon-exon or exon-intron junctions give unique insight into a surprising variability in splicing efficiency across introns, genes and conditions. Splicing efficiency was largely coordinated with transcript levels, and increased transcription led to increased splicing in test genes. Hundreds of introns showed such regulated splicing during cellular proliferation or differentiation.

The last two years have seen the publication of three genome-wide gene expression studies of the fission yeast cell cycle. While these microarray papers largely agree on the main patterns of cell cycle-regulated transcription and its control, there are discrepancies with regard to the identity and numbers of periodically expressed genes. We present benchmark and reproducibility analyses showing that the main discrepancies do not reflect differences in the data themselves (microarray or synchronization methods seem to lead only to minor biases) but rather in the interpretation of the data. Our reanalysis of the three datasets reveals that combining all independent information leads to an improved identification of periodically expressed genes. These evaluations suggest that the available microarray data do not allow reliable identification of more than about 500 cell cycle-regulated genes. The temporal expression pattern of the top 500 periodically expressed genes is generally consistent across experiments and the three studies, together with our integrated analysis, provide a coherent and rich source of information on cell cycle-regulated gene expression in Schizosaccharomyces pombe. The reanalysed datasets and other supplementary information are available from an accompanying website: http://www.cbs.dtu.dk/cellcycle/. We hope that this paper will resolve the apparent discrepancies between the previous studies and be useful both for wet-lab biologists and for theoretical scientists who wish to take advantage of the data for follow-up work., (Copyright 2006 John Wiley & Sons, Ltd.)