Genome-Wide Studies of Histone Demethylation Catalysed by the Fission Yeast Homologues of Mammalian LSD1

In order to gain a more global view of the activity of histone demethylases, we report here genome-wide studies of the fission yeast SWIRM and polyamine oxidase (PAO) domain homologues of mammalian LSD1. Consistent with previous work we find that the two S. pombe proteins, which we name Swm1 and Swm2 (after SWIRM1 and SWIRM2), associate together in a complex. However, we find that this complex specifically demethylates lysine 9 in histone H3 (H3K9) and both up- and down-regulates expression of different groups of genes. Using chromatin-immunoprecipitation, to isolate fragments of chromatin containing either H3K4me2 or H3K9me2, and DNA microarray analysis (ChIP-chip), we have studied genome-wide changes in patterns of histone methylation, and their correlation with gene expression, upon deletion of the swm1+ gene. Using hyper-geometric probability comparisons we uncover genetic links between lysine-specific demethylases, the histone deacetylase Clr6, and the chromatin remodeller Hrp1. The data presented here demonstrate that in fission yeast the SWIRM/PAO domain proteins Swm1 and Swm2 are associated in complexes that can remove methyl groups from lysine 9 methylated histone H3. In vitro, we show that bacterially expressed Swm1 also possesses lysine 9 demethylase activity. In vivo, loss of Swm1 increases the global levels of both H3K9me2 and H3K4me2. A significant accumulation of H3K4me2 is observed at genes that are up-regulated in a swm1 deletion strain. In addition, H3K9me2 accumulates at some genes known to be direct Swm1/2 targets that are down-regulated in the swm1¿ strain. The in vivo data indicate that Swm1 acts in concert with the HDAC Clr6 and the chromatin remodeller Hrp1 to repress gene expression. In addition, our in vitro analyses suggest that the H3K9 demethylase activity requires an unidentified post-translational modification to allow it to act. Thus, our results highlight complex interactions between histone demethylase, deacetylase and chromatin remodelling activities in the regulation of gene expression

The fission yeast Rpb4 subunit of RNA polymerase II plays a specialized role in cell separation

RNA polymerase II is a complex of 12 subunits, Rpb1 to Rpb12, whose specific roles are only partly understood. Rpb4 is essential in mammals and fission yeast, but not in budding yeast. To learn more about the roles of Rpb4, we expressed the rpb4 gene under the control of regulatable promoters of different strength in fission yeast. We demonstrate that below a critical level of transcription, Rpb4 affects cellular growth proportional to its expression levels: cells expressing lower levels of rpb4 grew slower compared to cells expressing higher levels. Lowered rpb4 expression did not affect cell survival under several stress conditions, but it caused specific defects in cell separation similar to sep mutants. Microarray analysis revealed that lowered rpb4 expression causes a global reduction in gene expression, but the transcript levels of a distinct subset of genes were particularly responsive to changes in rpb4 expression. These genes show some overlap with those regulated by the Sep1-Ace2 transcriptional cascade required for cell separation. Most notably, the gene expression signature of cells with lowered rpb4 expression was highly similar to those of mcs6, pmh1, sep10 and sep15 mutants. Mcs6 and Pmh1 encode orthologs of metazoan TFIIH-associated cyclin-dependent kinase (CDK)-activating kinase (Cdk7-cyclin H-Mat1), while Sep10 and Sep15 encode mediator components. Our results suggest that Rpb4, along with some other general transcription factors, plays a specialized role in a transcriptional pathway that controls the cell cycle-regulated transcription of a specific subset of genes involved in cell division. ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00438-006-0161-5 and is accessible for authorized users

Recruitment of the mitotic exit network to yeast centrosomes couples septin displacement to actomyosin constriction

The Mitotic Exit Network (MEN) promotes mitotic exit and cytokinesis but if and how MEN independently controls these two processes is unclear. Here, the authors report that MEN displaces septins from the cell division site to promote actomyosin ring constriction, independently of MEN control of mitotic exit

Systematic Two-Hybrid and Comparative Proteomic Analyses Reveal Novel Yeast Pre-mRNA Splicing Factors Connected to Prp19

Prp19 is the founding member of the NineTeen Complex, or NTC, which is a spliceosomal subcomplex essential for spliceosome activation. To define Prp19 connectivity and dynamic protein interactions within the spliceosome, we systematically queried the Saccharomyces cerevisiae proteome for Prp19 WD40 domain interaction partners by two-hybrid analysis. We report that in addition to S. cerevisiae Cwc2, the splicing factor Prp17 binds directly to the Prp19 WD40 domain in a 1∶1 ratio. Prp17 binds simultaneously with Cwc2 indicating that it is part of the core NTC complex. We also find that the previously uncharacterized protein Urn1 (Dre4 in Schizosaccharomyces pombe) directly interacts with Prp19, and that Dre4 is conditionally required for pre-mRNA splicing in S. pombe. S. pombe Dre4 and S. cerevisiae Urn1 co-purify U2, U5, and U6 snRNAs and multiple splicing factors, and dre4Δ and urn1Δ strains display numerous negative genetic interactions with known splicing mutants. The S. pombe Prp19-containing Dre4 complex co-purifies three previously uncharacterized proteins that participate in pre-mRNA splicing, likely before spliceosome activation. Our multi-faceted approach has revealed new low abundance splicing factors connected to NTC function, provides evidence for distinct Prp19 containing complexes, and underscores the role of the Prp19 WD40 domain as a splicing scaffold

Retro-trochanteric sciatica-like pain: current concept

The aim of this manuscript is to review the current knowledge in terms of retro-trochanteric pain syndrome, make recommendations for diagnosis and differential diagnosis and offer suggestions for treatment options. The terminology in the literature is confusing and these symptoms can be referred to as ‘greater trochanteric pain syndrome’, ‘trochanteric bursitis’ and ‘trochanteritis’, among other denominations. The authors focus on a special type of sciatica, i.e. retro-trochanteric pain radiating down to the lower extremity. The impact of different radiographic assessments is discussed. The authors recommend excluding pathology in the spine and pelvic area before following their suggested treatment algorithm for sciatica-like retro-trochanteric pain. Level of evidence II

Molecular evolution of cyclin proteins in animals and fungi

<p>Abstract</p> <p>Background</p> <p>The passage through the cell cycle is controlled by complexes of cyclins, the regulatory units, with cyclin-dependent kinases, the catalytic units. It is also known that cyclins form several families, which differ considerably in primary structure from one eukaryotic organism to another. Despite these lines of evidence, the relationship between the evolution of cyclins and their function is an open issue. Here we present the results of our study on the molecular evolution of A-, B-, D-, E-type cyclin proteins in animals and fungi.</p> <p>Results</p> <p>We constructed phylogenetic trees for these proteins, their ancestral sequences and analyzed patterns of amino acid replacements. The analysis of infrequently fixed atypical amino acid replacements in cyclins evidenced that accelerated evolution proceeded predominantly during paralog duplication or after it in animals and fungi and that it was related to aromorphic changes in animals. It was shown also that evolutionary flexibility of cyclin function may be provided by consequential reorganization of regions on protein surface remote from CDK binding sites in animal and fungal cyclins and by functional differentiation of paralogous cyclins formed in animal evolution.</p> <p>Conclusions</p> <p>The results suggested that changes in the number and/or nature of cyclin-binding proteins may underlie the evolutionary role of the alterations in the molecular structure of cyclins and their involvement in diverse molecular-genetic events.</p

SUMOylation is required for normal development of linear elements and wild-type meiotic recombination in Schizosaccharomyces pombe

In the fission yeast, Schizosaccharomyces pombe, synaptonemal complexes (SCs) are not formed during meiotic prophase. However, structures resembling the axial elements of SCs, the so-called linear elements (LinEs) appear. By in situ immunostaining, we found Pmt3 (S. pombe's SUMO protein) transiently along LinEs, suggesting that SUMOylation of some component(s) of LinEs occurs during meiosis. Mutation of the SUMO ligase Pli1 caused aberrant LinE formation and reduced genetic recombination indicating a role for SUMOylation of LinEs for the regulation of meiotic recombination. Western blot analysis of TAP-tagged Rec10 demonstrated that there is a Pli1-dependent posttranslational modification of this protein, which is a major LinE component and a distant homolog of the SC protein Red1. Mass spectrometry (MS) analysis revealed that Rec10 is both phosphorylated and ubiquitylated, but no evidence for SUMOylation of Rec10 was found. These findings indicate that the regulation of LinE and Rec10 function is modulated by Pli1-dependent SUMOylation of LinE protein(s) which directly or indirectly regulates Rec10 modification. On the side, MS analysis confirmed the interaction of Rec10 with the known LinE components Rec25, Rec27, and Hop1 and identified the meiotically upregulated protein Mug20 as a novel putative LinE-associated protein