12 research outputs found
Genome wide analysis of the Ssn6-Tup11/Tup12 co-repressor complex in the fission yeast Schizosaccharomyces pombe
In this study, we have investigated the fission yeast Ssn6-Tup11 /Tup 12
transcriptional corepressor which is involved in regulation of many genes
important for a wide variety of processes. In contrast to the well
characterised budding yeast Tup1 protein there are two paralogous
proteins present in fission yeast, namely Tup11 and Tup12. We have shown
that the two proteins can interact with each other and are expressed at
similar levels, which is in line with a reported redundant function.
Sequence analysis shows that the intermediate proposed histone
interacting domain is highly variable between Tup11 and Tup12 indicating
a diversification. Interestingly, we show that tup11 and tup12 mutants
have different phenotypes on media containing KC1 and CaC12. Consistent
with this functional difference, we identify a number of target genes by
genome wide expression profiling that are differentially affected by
tup11 - and tup12. Many of these genes are Tup12 dependent and correlate
with genes that have previously been shown to respond to a range of
different environmental stress conditions. The observed different
physiological roles of Tup11 and Tup12 can not be explained by
differential recruitment of Ssn6 which can interact independently with
both Tup11 and Tup12. Most interestingly we show that the Ssn6 protein is
essential in fission yeast and therefore must have a distinct role
separated from Tup11 and Tup12. Surprisingly, a conditional ssn6HA-ts
mutant displays the same growth phenotype as tup12, indicating a role in
Tup12 dependent stress response. Consistent with the diverse phenotypes
of the individual co-repressor proteins, we identify a group of genes
that requires Ssn6 for their regulation which is overlapping but distinct
from the group of genes that depend on Tup11 or Tup12. Genome wide
chromatin immunoprecipitation shows that Ssn6 is almost invariably found
in the same genomic locations as Tup11 and/or Tup12. All three
co-repressor subunits are generally bound to genes that are selectively
regulated by Ssn6 or Tup11/12, and thus, likely in the context of a
co-repressor complex containing all three subunits. The co-repressor
binds to both the intergenic and coding regions of genes, but
differential localization of the co-repressor within genes does not
appear to account for the selective dependence of target genes on the
Ssn6 or Tup11/12 subunits. Ssn6, Tup11, and Tup12 are preferentially
found at genomic locations at which histones are deacetylated, primarily
by the Clr6 class I HDAC. A subset of co-repressor target genes,
including direct target genes affected by Ssn6 overexpression, is in
addition associated with the function of class II (Clr3) and III (Hst4
and Sir2) HDACs. Interestingly, many specific Hst4 repressed ORF targets
involved in amino acid biosynthesis are also direct targets for the
Ssn6-Tup11/12 co-repressor, suggesting an association with the class ill
sirtuins which has not been reported previously
WD40 Domain Divergence Is Important for Functional Differences between the Fission Yeast Tup11 and Tup12 Co-Repressor Proteins
We have previously demonstrated that subsets of Ssn6/Tup target genes have distinct requirements for the Schizosaccharomyces pombe homologs of the Tup1/Groucho/TLE co-repressor proteins, Tup11 and Tup12. The very high level of divergence in the histone interacting repression domains of the two proteins suggested that determinants distinguishing Tup11 and Tup12 might be located in this domain. Here we have combined phylogenetic and structural analysis as well as phenotypic characterization, under stress conditions that specifically require Tup12, to identify and characterize the domains involved in Tup12-specific action. The results indicate that divergence in the repression domain is not generally relevant for Tup12-specific function. Instead, we show that the more highly conserved C-terminal WD40 repeat domain of Tup12 is important for Tup12-specific function. Surface amino acid residues specific for the WD40 repeat domain of Tup12 proteins in different fission yeasts are clustered in blade 3 of the propeller-like structure that is characteristic of WD40 repeat domains. The Tup11 and Tup12 proteins in fission yeasts thus provide an excellent model system for studying the functional divergence of WD40 repeat domains
An evaluation of analysis pipelines for DNA methylation profiling using the Illumina HumanMethylation450 BeadChip platform
The proper identification of differentially methylated CpGs is central in most epigenetic studies. The Illumina HumanMethylation450 BeadChip is widely used to quantify DNA methylation; nevertheless, the design of an appropriate analysis pipeline faces severe challenges due to the convolution of biological and technical variability and the presence of a signal bias between Infinium I and II probe design types. Despite recent attempts to investigate how to analyze DNA methylation data with such an array design, it has not been possible to perform a comprehensive comparison between different bioinformatics pipelines due to the lack of appropriate data sets having both large sample size and sufficient number of technical replicates. Here we perform such a comparative analysis, targeting the problems of reducing the technical variability, eliminating the probe design bias and reducing the batch effect by exploiting two unpublished data sets, which included technical replicates and were profiled for DNA methylation either on peripheral blood, monocytes or muscle biopsies. We evaluated the performance of different analysis pipelines and demonstrated that: (1) it is critical to correct for the probe design type, since the amplitude of the measured methylation change depends on the underlying chemistry; (2) the effect of different normalization schemes is mixed, and the most effective method in our hands were quantile normalization and Beta Mixture Quantile dilation (BMIQ); (3) it is beneficial to correct for batch effects. In conclusion, our comparative analysis using a comprehensive data set suggests an efficient pipeline for proper identification of differentially methylated CpGs using the Illumina 450K arrays
Functional Comparison of the Tup11 and Tup12 Transcriptional Corepressors in Fission Yeast
Gene duplication is considered an important evolutionary mechanism. Unlike many characterized species, the fission yeast Schizosaccharomyces pombe contains two paralogous genes, tup11(+) and tup12(+), that encode transcriptional corepressors similar to the well-characterized budding yeast Tup1 protein. Previous reports have suggested that Tup11 and Tup12 proteins play redundant roles. Consistently, we show that the two Tup proteins can interact together when expressed at normal levels and that each can independently interact with the Ssn6 protein, as seen for Tup1 in budding yeast. However, tup11(−) and tup12(−) mutants have different phenotypes on media containing KCl and CaCl(2). Consistent with the functional difference between tup11(−) and tup12(−) mutants, we identified a number of genes in genome-wide gene expression experiments that are differentially affected by mutations in the tup11(+) and tup12(+) genes. Many of these genes are differentially derepressed in tup11(−) mutants and are over-represented in genes that have previously been shown to respond to a range of different stress conditions. Genes specifically derepressed in tup12(−) mutants require the Ssn6 protein for their repression. As for Tup12, Ssn6 is also required for efficient adaptation to KCl- and CaCl(2)-mediated stress. We conclude that Tup11 and Tup12 are at least partly functionally diverged and suggest that the Tup12 and Ssn6 proteins have adopted a specific role in regulation of the stress response
The LAMMER Kinase Homolog, Lkh1, Regulates Tup Transcriptional Repressors through Phosphorylation in Schizosaccharomyces pombe*
Disruption of the fission yeast LAMMER kinase, Lkh1, gene resulted in diverse phenotypes, including adhesive filamentous growth and oxidative stress sensitivity, but an exact cellular function had not been assigned to Lkh1. Through an in vitro pull-down approach, a transcriptional repressor, Tup12, was identified as an Lkh1 binding partner. Interactions between Lkh1 and Tup11 or Tup12 were confirmed by in vitro and in vivo binding assays. Tup proteins were phosphorylated by Lkh1 in a LAMMER motif-dependent manner. The LAMMER motif was also necessary for substrate recognition in vitro and cellular function in vivo. Transcriptional activity assays using promoters negatively regulated by Tup11 and Tup12 showed 6 or 2 times higher activity in the Δlkh1 mutant than the wild type, respectively. Northern analysis revealed derepressed expression of the fbp1+ mRNA in Δlkh1 and in Δtup11Δtup12 mutant cells under repressed conditions. Δlkh1 and Δtup11Δtup12 mutant cells showed flocculation, which was reversed by co-expression of Tup11 and -12 with Ssn6. Here, we presented a new aspect of the LAMMER kinase by demonstrating that the activities of global transcriptional repressors, Tup11 and Tup12, were positively regulated by Lkh1-mediated phosphorylation
Specific functions for the fission yeast Sirtuins Hst2 and Hst4 in gene regulation and retrotransposon silencing
Expression profiling, ChiP–CHIP and phenotypic analysis were used to investigate the functional relationships of class III NAD+-dependent HDACs (Sirtuins) in fission yeast. We detected significant histone acetylation increases in Sirtuin mutants at their specific genomic binding targets and were thus able to identify an in vivo substrate preference for each Sirtuin. At heterochromatic loci, we demonstrate that although Hst2 is mainly cytoplasmic, a nuclear pool of Hst2 colocalizes with the other Sirtuins at silent regions (cen, mat, tel, rDNA), and that like the other Sirtuins, Hst2 is required for rDNA and centromeric silencing. Interestingly we found specific functions for the fission yeast Sirtuins Hst2 and Hst4 in gene regulation. Hst2 directly represses genes involved in transport and membrane function, whereas Hst4 represses amino-acid biosynthesis genes and Tf2 retrotransposons. A specific role for Hst4 in Tf2 5′ mRNA processing was revealed. Thus, Sirtuins share functions at many genomic targets, but Hst2 and Hst4 have also evolved unique functions in gene regulation