| 1. |
- Appelgren, Henrik, et al.
(author)
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Distinct centromere domain structures with separate functions demonstrated in live fission yeast cells
- 2003
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In: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 116:19, s. 4035-4042
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Journal article (peer-reviewed)abstract
- Fission yeast (Saccharomyces pombe) centromere DNA is organized in a central core region flanked on either side by a region of outer repeat (otr) sequences. The otr region is known to be heterochromatic and bound by the Swi6 protein whereas the central core region contains an unusual chromatin structure involving the histone H3 variant Cnp1 (S. pombe CENP-A). The central core is the base for formation of the kinetochore structure whereas the flanking region is important for sister centromere cohesion. We have previously shown that the ultrastructural domain structure of S. pombe centromeres in interphase is similar to that of human centromeres. Here we demonstrate that S. pombe centromeres are organized in cytologically distinct domains even in mitosis. Fluorescence in situ hybridization of fixed metaphase cells revealed that the otr regions of the centromere were still held together by cohesion even after the sister kinetochores had separated. In live cells, the central cores and kinetochores of sister chromosomes could be distinguished from one Another when they were subjected to mitotic tension. The function of the different centromeric domains was addressed. Transacting mutations affecting the kinetochore (nuf2) central core domain (mis6) and the heterochromatin domain (rik1) were analyzed in live cells. In interphase, both nuf2 and mis6 caused declustering of centromeres from the spindle pole body whereas centromere clustering was normal in rik1 despite an apparent decondensation defect. The declustering of centromeres in mis6 cells correlated with loss the Ndc80 kinetochore marker protein from the centromeres. Interestingly the declustered centromeres were still restricted to the nuclear periphery thus revealing a kinetochore-independent peripheral localization mechanism for heterochromatin. Time-lapse microscopy of live mis6 and nuf2-1 mutant cells in mitosis showed similar severe misaggregation phenotypes whereas the rik1 mutants showed a mild cohesion defect. Thus, S. pombe centromeres have two distinguishable domains even during mitosis, and our functional analyses support the previous observations that the kinetochore/central core and the heterochromatin domains have distinct functions both in interphase and mitosis.
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| 2. |
- Bayne, Elizabeth H., et al.
(author)
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Splicing factors facilitate RNAi-directed silencing in fission yeast
- 2008
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In: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 322:5901, s. 602-606
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Journal article (peer-reviewed)abstract
- Heterochromatin formation at fission yeast centromeres is directed by RNA interference (RNAi). Noncoding transcripts derived from centromeric repeats are processed into small interfering RNAs (siRNAs) that direct the RNA-induced transcriptional silencing (RITS) effector complex to engage centromer transcripts, resulting in recruitment of the histone H3 lysine 9 methyltransferase Clr4, and hence silencing. We have found that defects in specific splicing factors, but not splicing itself, affect the generation of centromeric siRNAs and consequently centromeric heterochromatin integrity. Moreover, splicing factors physically associate with Cid12, a component of the RNAi machinery, and with centromeric chromatin, consistent with a direct role in RNAi. We propose that spliceosomal complexes provide a platform for siRNA generation and hence facilitate effective centromere repeat silencing.
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| 3. |
- Bernard, Pascal, et al.
(author)
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Cell-cycle regulation of cohesin stability along fission yeast chromosomes
- 2008
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In: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 27:1, s. 111-121
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Journal article (peer-reviewed)abstract
- Sister chromatid cohesion is mediated by cohesin, but the process of cohesion establishment during S-phase is still enigmatic. In mammalian cells, cohesin binding to chromatin is dynamic in G1, but becomes stabilized during S-phase. Whether the regulation of cohesin stability is integral to the process of cohesion establishment is unknown. Here, we provide evidence that fission yeast cohesin also displays dynamic behavior. Cohesin association with G1 chromosomes requires continued activity of the cohesin loader Mis4/Ssl3, suggesting that repeated loading cycles maintain cohesin binding. Cohesin instability in G1 depends on wpl1, the fission yeast ortholog of mammalian Wapl, suggestive of a conserved mechanism that controls cohesin stability on chromosomes. wpl1 is nonessential, indicating that a change in wpl1-dependent cohesin dynamics is dispensable for cohesion establishment. Instead, we find that cohesin stability increases at the time of S-phase in a reaction that can be uncoupled from DNA replication. Hence, cohesin stabilization might be a pre-requisite for cohesion establishment rather than its consequence.
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| 4. |
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| 5. |
- Bjerling, Pernilla, et al.
(author)
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Centromere domain organization and histone modifications
- 2002
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In: Brazilian journal of medical and biological research. - 0100-879X .- 1414-431X. ; 35:5, s. 499-507
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Journal article (peer-reviewed)abstract
- Centromere function requires the proper coordination of several subfunctions, such as kinetochore assembly, sister chromatid cohesion, binding of kinetochore microtubules, orientation of sister kinetochores to opposite spindle poles, and their movement towards the spindle poles. Centromere structure appears to be organized in different, separable domains in order to accomplish these functions. Despite the conserved nature of centromere functions, the molecular genetic definition of the DNA sequences that form a centromere in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, in the fruit fly Drosophila melanogaster, and in humans has revealed little conservation at the level of centromere DNA sequences. Also at the protein level few centromere proteins are conserved in all of these four organisms and many are unique to the different organisms. The recent analysis of the centromere structure in the yeast S. pombe by electron microscopy and detailed immunofluorescence microscopy of Drosophila centromeres have brought to light striking similarities at the overall structural level between these centromeres and the human centromere. The structural organization of the centromere is generally multilayered with a heterochromaun domain and a central core/inner plate region, which harbors the outer plate structures of the kinetochore. It is becoming increasingly clear that the key factors for assembly and function of the centromere structure are the specialized histories and modified histones which are present in the centromeric heterochromatin and in the chromatin of the central core. Thus, despite the differences in the DNA sequences and the proteins that define a centromere, there is an overall structural similarity between centromeres in evolutionarily diverse eukaryotes.
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| 6. |
- Bjerling, Pernilla, et al.
(author)
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Functional divergence between histone deacetylases in fission yeast by distinct cellular localization and in vivo specificity
- 2002
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In: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 22:7, s. 2170-2181
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Journal article (peer-reviewed)abstract
- Histone deacetylases (HDACs) are important for gene regulation and the maintenance of heterochromatin in eukaryotes. Schizosaccharomyces pombe was used as a model system to investigate the functional divergence within this conserved enzyme family. S. pombe has three HDACs encoded by the hda1(+), clr(3+), and clr6(+) genes. Strains mutated in these genes have previously been shown to display strikingly different phenotypes when assayed for viability, chromosome loss, and silencing. Here, conserved differences in the substrate binding pocket identify Clr6 and Hda1 as class I HDACs, while Clr3 belongs in the class II family. Furthermore, these HDACs were shown to have strikingly different subcellular localization patterns. Hda1 was localized to the cytoplasm, while most of Clr3 resided throughout the nucleus. Finally, Clr6 was localized exclusively on the chromosomes in a spotted pattern. Interestingly, Clr3, the only HDAC present in the nucleolus, was required for ribosomal DNA (rDNA) silencing. Clr3 presumably acts directly on heterochromatin, since it colocalized with the centromere, mating-type region, and rDNA as visualized by in situ hybridization. In addition, Clr3 could be cross-linked to mat3 in chromatin immunoprecipitation experiments. Western analysis of bulk histone preparations indicated that Hda1 (class I) had a generally low level of activity in vivo and Clr6 (class 1) had a high level of activity and broad in vivo substrate specificity, whereas Clr3 (class II) displayed its main activity on acetylated lysine 14 of histone H3. Thus, the distinct functions of the S. pombe HDACs are likely explained by their distinct cellular localization and their different in vivo specificities.
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| 7. |
- Carmichael, J B, et al.
(author)
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Ago1 and Dcr1, two core components of the RNA interference pathway, functionally diverge from Rdp1 in regulating cell cycle events in Schizosaccharomyces pombe
- 2004
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In: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 15:3, s. 1425-1435
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Journal article (peer-reviewed)abstract
- In the fission yeast Schizosaccharomyces pombe, three genes that function in the RNA interference (RNAi) pathway, ago1(+), dcr1(+), and rdp1(+), have recently been shown to be important for timely formation of heterochromatin and accurate chromosome segregation. In the present study, we present evidence that null mutants for ago1(+) and dcr1(+) but not rdp1(+), exhibit abnormal cytokinesis, cell cycle arrest deficiencies, and mating defects. Subsequent analyses showed that ago1(+) and dcr1(+) are required for regulated hyperphosphorylation of Cdc2 when encountering genotoxic insults. Because rdp1(+) is dispensable for this process, the functions of ago1(+) and dcr1(+) in this pathway are presumably independent of their roles in RNAi-mediated heterochromatin formation and chromosome segregation. This was further supported by the finding that ago1(+) is a multicopy suppressor of the S-M checkpoint deficiency and cytokinesis defects associated with loss of Dcr1 function, but not for the chromosome segregation defects of this mutant. Accordingly, we conclude that Dcr1-dependent production of small interfering RNAs is not required for enactment and/or maintenance of certain cell cycle checkpoints and that Ago1 and Dcr1 functionally diverge from Rdp1 to control cell cycle events in fission yeast. Finally, exogenous expression of hGERp95/EIF2C2/hAgo2, a human Ago1 homolog implicated in posttranscriptional gene silencing, compensated for the loss of ago1(+) function in S. pombe. This suggests that PPD proteins may also be important for regulation of cell cycle events in higher eukaryotes.
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| 8. |
- Deneberg, Stefan, et al.
(author)
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Prognostic DNA methylation patterns in cytogenetically normal acute myeloid leukemia are predefined by stem cell chromatin marks
- 2011
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In: Blood. - : American Society of Hematology. - 0006-4971 .- 1528-0020. ; 118:20, s. 5573-5582
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Journal article (peer-reviewed)abstract
- Cytogenetically normal acute myeloid leukemia (CN-AML) comprise between forty and fifty percent of all adult acute myeloid leukemia (AML) cases. In this clinically diverse group molecular aberrations such as FLT3ITD, NPM1 and CEBPA mutations recently have added to the prognostic accuracy. Aberrant DNA methylation is a hallmark of cancer including AML. We investigated in total 118 CN-AML samples in a test and a validation cohort for genome-wide promoter DNA methylation with Illumina Methylation Bead arrays and compared them to normal myeloid precursors and global gene expression. IDH and NPM1 mutations were associated with different methylation patterns (p=0.0004 and 0.04, respectively). Genome-wide methylation levels were elevated in IDH mutated samples (p=0.006). We observed a negative impact of DNA methylation on transcription. Genes targeted by Polycomb group (PcG) proteins and genes associated with bivalent histone marks in stem cells showed increased aberrant methylation in AML (p<0.0001). Furthermore, high methylation levels of PcG target genes were independently associated with better progression free (OR 0.47, p=0.01) and overall survival (OR 0.36, p=0.001). In summary, genome wide methylation patterns show preferential methylation of PcG targets with prognostic impact in CN-AML.
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| 9. |
- Djupedal, Ingela, et al.
(author)
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Analysis of small RNA in fission yeast; centromeric siRNAs are potentially generated through a structured RNA
- 2009
-
In: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 28:24, s. 3832-3844
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Journal article (peer-reviewed)abstract
- The formation of heterochromatin at the centromeres in fission yeast depends on transcription of the outer repeats. These transcripts are processed into siRNAs that target homologous loci for heterochromatin formation. Here, high throughput sequencing of small RNA provides a comprehensive analysis of centromere-derived small RNAs. We found that the centromeric small RNAs are Dcr1 dependent, carry 50-monophosphates and are associated with Ago1. The majority of centromeric small RNAs originate from two remarkably well-conserved sequences that are present in all centromeres. The high degree of similarity suggests that this non-coding sequence in itself may be of importance. Consistent with this, secondary structure-probing experiments indicate that this centromeric RNA is partially double-stranded and is processed by Dicer in vitro. We further demonstrate the existence of small centromeric RNA in rdp1D cells. Our data suggest a pathway for siRNA generation that is distinct from the well-documented model involving RITS/RDRC. We propose that primary transcripts fold into hairpin-like structures that may be processed by Dcr1 into siRNAs, and that these siRNAs may initiate heterochromatin formation independent of RDRC activity. The EMBO Journal (2009) 28, 3832-3844. doi: 10.1038/emboj.2009.351; Published online 26 November 2009
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| 10. |
- Djupedal, Ingela
(author)
-
Characterization of RNA polymerase II subunit Rpb7 in silencing and transcription
- 2009
-
Doctoral thesis (other academic/artistic)abstract
- The DNA in eukaryotes is arranged in fibres of chromatin. The chromatin may be more or less compacted and the degree of condensation of the chromatin affects the accessibility of the DNA. The accessibility of the DNA, in turn, affects transcription and gene regulation. Genes within inaccessible DNA are commonly repressed whereasgenes within accessible DNA are active and expressed. This thesis concerns the interplay between chromatin and transcription with focus on the function of the RNA polymerase II (pol II) subunit Rpb7. We have demonstrated that processing of centromeric transcripts by the ribonuclease III family protein Dcr1 is required for heterochromatin formation at the centromeres of Schizosaccharomyces pombe. A point mutation in the pol II subunit Rpb7 caused a specific defect in centromeric heterochromatin formation. We have shown i) that the centromeric transcripts that accumulate in dcr1delta cells are products of pol II, ii) the rpbG150D mutation is deficient in recognition and/or initiation of transcription from the centromeric promoter. Transcription by pol II within the centromeres was surprising since insertion of marker genes within these loci normally results in repression of pol II transcription. Here, paradoxically, pol II transcription was required for the construction of the inaccessible heterochromatin structure. Our analysis of sRNA in S. pombe revealed that most centromeric siRNA are originating from two clusters, which are repeated several times within the centromeres. This lead us to propose a model in which centromeric transcripts fold into double stranded structures that are processed by Dcr1. The resulting siRNAs may contribute with the starting signal for the RNAi feedback loop required for heterochromatin formation at the centromeres. Finally, we demonstrate that the genome-wide association of Rpb7 is nearly identical to that of the core pol II subunit Rpb2, indicating a general role for Rpb7 in transcription. We further show that the occupancy pattern of Rpb4, a pol II subunit that forms a subcomplex together with Rpb7, differs from those of Rpb2 and Rpb7. Rpb4 may therefore have a less general function in transcription than Rpb7. Hence, transcription by pol II is required not only for gene expression but also for repression via formation of inaccessible heterochromatin.
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| 11. |
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| 12. |
- Djupedal, Ingela, et al.
(author)
-
Epigenetics : heterochromatin meets RNAi
- 2009
-
In: Cell Research. - : Springer Science and Business Media LLC. - 1001-0602 .- 1748-7838. ; 19:3, s. 282-295
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Journal article (peer-reviewed)abstract
- The term epigenetics refers to heritable changes not encoded by DNA. The organization of DNA into chromatin fibers affects gene expression in a heritable manner and is therefore one mechanism of epigenetic inheritance. Large parts of eukaryotic genomes consist of constitutively highly condensed heterochromatin, important for maintaining genome integrity but also for silencing of genes within. Small RNA, together with factors typically associated with RNA interference (RNAi) targets homologous DNA sequences and recruits factors that modify the chromatin, commonly resulting in formation of heterochromatin and silencing of target genes. The scope of this review is to provide an overview of the roles of small RNA and the RNAi components, Dicer, Argonaute and RNA dependent polymerases in epigenetic inheritance via heterochromatin formation, exemplified with pathways from unicellular eukaryotes, plants and animals.
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| 13. |
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| 14. |
- Djupedal, Ingela, et al.
(author)
-
RNA Pol II subunit Rpb7 promotes centromeric transcription and RNAi-directed chromatin silencing
- 2005
-
In: Genes & Development. - : Cold Spring Harbor Laboratory. - 0890-9369 .- 1549-5477. ; 19:19, s. 2301-2306
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Journal article (peer-reviewed)abstract
- Fission yeast centromeric repeats are transcribed into small interfering RNA (siRNA) precursors (pre-siRNAs), which are processed by Dicer to direct heterochromatin formation. Recently, Rpb1 and Rpb2 subunits of RNA polymerase II (RNA Pol II) were shown to mediate RNA interference (RNAi)-directed chromatin modification but did not affect pre-siRNA levels. Here we show that another Pol II subunit, Rpb7 has a specific role in presiRNA transcription. We define a centromeric presiRNA promoter from which initiation is exquisitely sensitive to the rpb7-G150D mutation. In contrast to other Pol II subunits, Rpb7 promotes pre-siRNA transcription required for RNAi-directed chromatin silencing.
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| 15. |
- Dreja, Karl, et al.
(author)
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Njursjukdomar
- 2016
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In: Omvårdnad & medicin. - 9789144076645 ; , s. 457-482
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Book chapter (pop. science, debate, etc.)
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| 16. |
- Dunleavy, Elaine M., et al.
(author)
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A NASP (N1/N2)-related protein, Sim3, binds CENP-A and is required for its deposition at fission yeast Centromeres
- 2007
-
In: Molecular Cell. - : Elsevier BV. - 1097-2765 .- 1097-4164. ; 28:6, s. 1029-1044
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Journal article (peer-reviewed)abstract
- A defining feature of centromeres is the presence of the histone H3 variant CENP-A(Cnp1). It is not known how CENP-A(Cnp1) is specifically delivered to, and assembled into, centromeric chromatin. Through a screen for factors involved in kinetochore integrity in fission yeast, we identified Sim3. Sim3 is homologous to known histone binding proteins NASP(Human) and N1/N2(Xenopus) and aligns with Hif1(S. cerevisiae), defining the SHNi-TPR family. Sim3 is distributed throughout the nucleoplasm, yet it associates with CENP-A(Cnp1) and also binds H3. Cells defective in Sim3 function have reduced levels of CENP-A(CnP1) at centromeres (and increased H3) and display chromosome segregation defects. Sim3 is required to allow newly synthesized CENP-A(Cnp1) to accumulate at centromeres in S and G2 phase-arrested cells in a replication-independent mechanism. We propose that one function of Sim3 is to act as an escort that hands off CENP-A(Cnp1) to chromatin assembly factors, allowing its incorporation into centromeric chromatin.
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| 17. |
- Durand-Dubief, Mickael, et al.
(author)
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Heterochromatin tells CENP-A where to go
- 2008
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In: Bioessays. - : Wiley. - 0265-9247 .- 1521-1878. ; 30:6, s. 526-529
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Journal article (peer-reviewed)abstract
- The centromere is the region of the chromosome where the kinetochore forms. Kinetochores are the attachment sites for spindle microtubules that separate duplicated chromosomes in mitosis and meiosis. Kinetochore formation depends on a special chromatin structure containing the histone H3 variant CENP-A. The epigenetic mechanisms that maintain CENP-A chromatin throughout the cell cycle have been studied extensively but little is known about the mechanism that targets CENP-A to naked centromeric DNA templates. In a recent report published in Science,((1)) such de novo centromere assembly of CENP-A is shown to be dependent on heterochromatin and the RNA interference pathway.
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| 18. |
- Durand-Dubief, Mickael, et al.
(author)
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Specific functions for the fission yeast Sirtuins Hst2 and Hst4 in gene regulation and retrotransposon silencing
- 2007
-
In: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 26:10, s. 2477-2488
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Journal article (peer-reviewed)abstract
- 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 50 mRNA processing was revealed. Thus, Sirtuins share functions at many genomic targets, but Hst2 and Hst4 have also evolved unique functions in gene regulation.
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| 19. |
- Ekwall, Karl
(author)
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'Arc' escorts siRNAs in heterochromatin assembly
- 2007
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In: Nature Structural & Molecular Biology. - : Springer Science and Business Media LLC. - 1545-9993 .- 1545-9985. ; 14:3, s. 178-179
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Journal article (other academic/artistic)abstract
- RNA interference (RNAi) is important in directing heterochromatin assembly at centromeres in fission yeast, which is crucial for maintaining a stable genome through mitotic and meiotic divisions. In this issue, Buker et al. describe a new Argonaute siRNA chaperone (ARC) that converts duplex RNA to single-stranded RNA. This is a previously unknown step in the RNAi-directed heterochromatin-formation pathway.
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| 20. |
- Ekwall, Karl
(author)
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Epigenetic control of centromere behavior
- 2007
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In: Annual review of genetics. - Palo Atlo : Annual Reviews. - 1545-2948 .- 0066-4197. - 9780824312411 ; , s. 63-81
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Book chapter (peer-reviewed)abstract
- The centromere is the DNA region that ensures genetic stability and is therefore of vital importance. Paradoxically, centromere proteins and centromeric structural domains are conserved despite that fact that centromere DNA sequences are highly variable and are not conserved. Remarkably, heritable states at the centromere can be propagated independent of the underlying centromeric DNA sequences. This review describes the epigenetic mechanisms governing centromere behavior, i.e., the mechanisms that control centromere assembly and propagation. A centromeric histone variant, CenH3, and histone modifications play key roles at centromeric chromatin. Histone modifications and RNA interference are important in assembly of pericentric heterochromatin structures. The molecular machinery that is directly involved in epigenetic control of centromeres is shared with regulation of gene expression. Nucleosome remodeling factors, histone chaperones, histone-modifying enzymes, transcription factors, and even RNA polymerase II itself control epigenetic states at centromeres.
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| 21. |
- Ekwall, Karl
(author)
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Genome-wide analysis of HDAC function
- 2005
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In: Trends in Genetics. - : Elsevier BV. - 0168-9525 .- 1362-4555. ; 21:11, s. 608-615
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Journal article (peer-reviewed)abstract
- This article focuses on new developments in the genome-wide analysis of histone deacetylase (HDAC) function in yeast. HDACs are highly conserved in many organisms; therefore, their basic functions can be investigated using experimentally tractable model organisms, such as the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. New microarray techniques have enabled the systematic study of HDACs by identifying their direct and indirect gene targets in addition to their physiological functions and enzymatic specificity. These new approaches have already provided new surprising insights into the basic function of HDACs.
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| 22. |
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| 23. |
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| 24. |
- Ekwall, Karl
(author)
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The roles of histone modifications and small RNA in centromere function
- 2004
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In: Chromosome Research. - 0967-3849 .- 1573-6849. ; 12:6, s. 535-542
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Journal article (peer-reviewed)abstract
- Here, epigenetic regulation of centromeric chromatin in fission yeast (Schizosaccharomyces pombe) is reviewed, focussing on the role of histone modifications and the link to RNA interference (RNAi). Fission yeast centromeres are organized into two structurally and functionally distinct domains, both of which are required for centromere function. The central core domain anchors the kinetochore structure while the flanking heterochromatin domain is important for sister centromere cohesion. The chromatin structure of both domains is regulated epigenetically. In the central core domain, the histone H3 variant Cnp1(CENP-A) plays a key role. In the flanking heterochromatin domain, histones are kept underacetylated by the histone deacetylases (HDACs) Clr3, Clr6 and Sir2, and methylated by Clr4 methyltransferase (HMTase) to create a specific binding site for the Swi6 protein. Swi6 then directly mediates cohesin binding to the centromeric heterochromatin. Recently, a surprising link was made between heterochromatin formation and RNAi. Centromeric flanking repeats are transcribed and the transcripts processed by the RNAse III-like enzyme, Dicer (Dcr1), to produce small interfering RNAs ( siRNA), which direct formation of heterochromatin via the RNA-induced Initiation of Transcriptional Silencing (RITS) protein complex. Consequently Dicer, Argonaute (Ago1), an RNA-dependent RNA polymerase (Rdp1) and several hitherto uncharacterized Csp ( centromere suppressor of position effect) gene products implicated in the RNAi pathway at centromeres are required for sister chromatid cohesion.
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| 25. |
- Facanha, A L O, et al.
(author)
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The endoplasmic reticulum cation P-type ATPase Cta4p is required for control of cell shape and microtubule dynamics
- 2002
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In: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 157:6, s. 1029-1039
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Journal article (peer-reviewed)abstract
- Here we describe the phenotypic characterization of the cta(4+) gene, encoding a novel member of the P4 family of P-type ATPases of fission yeast. The cta4Delta mutant is temperature sensitive and cold sensitive lethal and displays several morphological defects in cell polarity and cytokinesis. Microtubules are generally destabilized in cells lacking Cta4p. The microtubule length is decreased, and the number of microtubules per cell is increased. This is concomitant with an increase in the number of microtubule catastrophe events in the midzone of the cell. These defects are likely due to a general imbalance in cation homeostasis. Immunofluorescence microscopy and membrane fractionation experiments revealed that green fluorescent protein-tagged Cta4 localizes to the ER. Fluorescence resonance energy transfer experiments in living cells using the yellow cameleon indicator for Ca2+ indicated that Cta4p regulates the cellular Ca2+ concentration. Thus, our results reveal a link between cation homeostasis and the control of cell shape, microtubule dynamics, and cytokinesis, and appoint Ca2+ as a key ion in controlling these processes.
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| 26. |
- Fagerström-Billai, Fredrik
(author)
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Genome wide analysis of the Ssn6-Tup11/Tup12 co-repressor complex in the fission yeast Schizosaccharomyces pombe
- 2007
-
Doctoral thesis (other academic/artistic)abstract
- 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.
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| 27. |
- Fagerström-Billai, Fredrik, et al.
(author)
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Individual Subunits of the Ssn6-Tup11/12 corepressor are selectively required for repression of different target genes
- 2007
-
In: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 27:3, s. 1069-1082
-
Journal article (peer-reviewed)abstract
- The Saccharomyces cerevisiae Ssn6 and Tup1 proteins form a corepressor complex that is recruited to target genes by DNA-bound repressor proteins. Repression occurs via several mechanisms, including interaction with hypoacetylated N termini of histones, recruitment of histone deacetylases (HDACs), and interactions with the RNA polymerase II holoenzyme. The distantly related fission yeast, Schizosaccharomyces pombe, has two partially redundant Tup1-like proteins that are dispensable during normal growth. In contrast, we show that Ssn6 is an essential protein in S. pombe, suggesting a function that is independent of Tup11 and Tup12. Consistently, the group of genes that requires Ssn6 for their regulation overlaps but is distinct from the group of genes that depend on Tup11 or Tup12. Global chip-on-chip analysis shows that Ssn6 is almost invariably found in the same genomic locations as Tup11 and/or Tup12. All three corepressor subunits are generally bound to genes that are selectively regulated by Ssn6 or Tup11/12, and thus, the subunit specificity is probably manifested in the context of a corepressor complex containing all three subunits. The corepressor binds to both the intergenic and coding regions of genes, but differential localization of the corepressor 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. Clr6 is also important for the repression of corepressor target genes. Interestingly, a subset of corepressor target genes, including direct target genes affected by Ssn6 overexpression, is associated with the function of class II (CIr3) and III (Hst4 and Sir2) HDACs.
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| 28. |
- Hallberg, Magnus, 1974-
(author)
-
Studies of Functional Interactions within Yeast Mediator and a Proposed Novel Mechanism for Regulation of Gene Expression
- 2004
-
Doctoral thesis (other academic/artistic)abstract
- The yeast Mediator complex is required for transcriptional regulation both in vivo and in vitro and the identification of similar complexes from metazoans indicates that its function is conserved through evolution. Mediator subunit composition and structure is well characterized both by biochemical, genetic and biophysical methods. In contrast, little is known about the mechanisms by which Mediator operates and how the complex is regulated. The aim of my thesis was to elucidate how Mediator functions at the molecular level and to investigate functional interactions within Mediator. It is possible to recruit RNA polymerase II to a target promoter and thus to activate transcription by fusing Mediator subunits to a DNA binding domain. In order to investigate functional interactions within Mediator, we made such fusion proteins where different Mediator subunits were fused to the DNA binding domain of lexA. The expression of a reporter gene containing binding sites for lexA was subsequently measured in both a wild type strain and in strains where genes encoding specific Mediator subunits had been disrupted. We found that lexA-Med2 and lexA-Gal11 are strong activators that function independently of all Mediator subunits tested. On the other hand, lexA-Srb10 is a weak activator that depends on Srb8 and Srb11 and lexA-Med1 and lexA-Srb7 are both cryptic activators that become active in the absence of Srb8, Srb10, Srb11, or Sin4. Both lexA-Med1 and lexA-Srb7 proteins showed a stable association with the Mediator subunits Med4 and Med8 in wild type cells and in all deletion strains tested, indicating that they were functionally incorporated into the Mediator complex. We also showed that both Med4 and Med8 exist in two forms that differed in electrophoretic mobility and that these forms differed in their ability to associate with Mediator immuno-purified from the LEXA-SRB7 and LEXA-MED1 strains. Dephosphorylation assays of purified Mediator indicated that the two mobility forms of Med4 corresponded to the phosphorylated and unphosphorylated forms of the Med4 protein respectively. Some of the data presented in this study as well as previous genetic and biochemical data obtained in our lab suggested a functional link between the Med1, Med2, Srb10 and Srb11 proteins. We extended these findings by showing that the Srb10 kinase phosphorylates the Med2 protein at residue serine 208, both in vitro and in vivo. We also showed that a point mutation of the single phosphorylation site to an alanine or to an aspartic acid residue altered the gene expression of a specific set of genes. Taken together, these data indicate that posttranslational modification of Mediator subunits is a so far uncharacterized mechanism for regulation of gene expression. In order to study the function of the Srb7 subunit of Mediator, we isolated a temperature sensitive strain where the amino acids 2 to 8 of srb7 were deleted. The Mediator subunits Nut2 and Med7 were isolated as high copy suppressor of srb7-∆(2-8) and we were also able to show that Srb7 interacted with Nut2 and Med7 both in a 2-hybrid system and in co-immuno precipitation experiments using recombinantly expressed proteins. Interestingly, a deletion of amino acids 2 to 8 of Srb7 abolishes its interaction with both Med7 and Nut2 in vitro. Med4 also interacted with Srb7 in the 2-hybrid system and surprisingly, the first eight amino acids of Srb7 were shown to be sufficient for this interaction.
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| 29. |
- Hogan, C. J., et al.
(author)
-
Fission yeast Iec1-Ino80-mediated nucleosome eviction regulates nucleotide and phosphate metabolism
- 2010
-
In: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 30:3, s. 657-674
-
Journal article (peer-reviewed)abstract
- Ino80 is an ATP-dependent nucleosome-remodeling enzyme involved in transcription, replication, and the DNA damage response. Here, we characterize the fission yeast Ino80 and find that it is essential for cell viability. We show that the Ino80 complex from fission yeast mediates ATP-dependent nucleosome remodeling in vitro. The purification of the Ino80-associated complex identified a highly conserved complex and the presence of a novel zinc finger protein with similarities to the mammalian transcriptional regulator Yin Yang 1 (YY1) and other members of the GLI-Krüppel family of proteins. Deletion of this Iec1 protein or the Ino80 complex subunit arp8, ies6, or ies2 causes defects in DNA damage repair, the response to replication stress, and nucleotide metabolism. We show that Iec1 is important for the correct expression of genes involved in nucleotide metabolism, including the ribonucleotide reductase subunit cdc22 and phosphate- and adenineresponsive genes. We find that Ino80 is recruited to a large number of promoter regions on phosphate starvation, including those of phosphate- and adenine-responsive genes that depend on Iec1 for correct expression. Iec1 is required for the binding of Ino80 to target genes and subsequent histone loss at the promoter and throughout the body of these genes on phosphate starvation. This suggests that the Iec1-Ino80 complex promotes transcription through nucleosome eviction.
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| 30. |
- Isaac, Sara, et al.
(author)
-
Interaction of Epe1 with the heterochromatin assembly pathway in Schizosaccharomyces pombe
- 2007
-
In: Genetics. - : Oxford University Press (OUP). - 0016-6731 .- 1943-2631. ; 175:4, s. 1549-1560
-
Journal article (peer-reviewed)abstract
- Epe1 is a JmjC domain protein that antagonizes heterochromatization in Schizosaccharomyces pombe. Related JmjC domain proteins catalyze a histone demethylation reaction that depends on Fe(II) and alpha-ketoglutarate. However, no detectable demethylase activity is associated with Epe1, and its JmjC domain lacks conservation of Fe(II)-binding residues. We report that Swi6 recruits Epe1 to heterochromatin and that overexpression of epe1(+), like mutations in silencing genes or overexpression of swi6(+), upregulates expression of certain genes. A significant overlap was observed between the lists of genes that are upregulated by overexpression of epe1(+) and those that are upregulated by mutations in histone deacetylase genes. However, most of the common genes are not regulated by Clr4 histone methyltransferase. This suggests that Epe1 interacts with the heterochromatin assembly pathway at the stage of histone deacetylation. Mutational inactivation of Epe1 downregulates similar to 12% of S. pombe genes, and the list of these genes overlaps significantly with the lists of genes that are upregulated by mutations in silencing genes and genes that are hyperacetylated at their promoter regions in clr6-1 mutants. We propose that an interplay between the repressive HDACs activity and Epe1 helps to regulate gene expression in S. pombe.
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| 31. |
- Johnsson, Anna, et al.
(author)
-
HAT-HDAC interplay modulates global histone H3K14 acetylation in gene-coding regions during stress
- 2009
-
In: EMBO Reports. - : EMBO. - 1469-221X .- 1469-3178. ; 10:9, s. 1009-1014
-
Journal article (peer-reviewed)abstract
- Histone acetylation and deacetylation are important for gene regulation. The histone acetyltransferase, Gcn5, is an activator of transcriptional initiation that is recruited to gene promoters. Here, we map genome-wide Gcn5 occupancy and histone H3K14ac at high resolution. Gcn5 is predominantly localized to coding regions of highly transcribed genes, where it collaborates antagonistically with the class-II histone deacetylase, Clr3, to modulate H3K14ac levels and transcriptional elongation. An interplay between Gcn5 and Clr3 is crucial for the regulation of many stress-response genes. Our findings suggest a new role for Gcn5 during transcriptional elongation, in addition to its known role in transcriptional initiation.
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| 32. |
- Kanduri, Meena, 1974-
(author)
-
The Functional Significance and Chromatin Organisation of the Imprinting Control Regions of the H19 and Kcnq1 Genes
- 2004
-
Doctoral thesis (other academic/artistic)abstract
- Genomic imprinting is a phenomenon through which a subset of genes are epigenetically marked during gemtogenisis. This mark is maintained in the soma to often manifest parent of origin-specific monoalleleic expresson patterns. Genetics evidence suggests that gene expression patterns in mprinted genes, which are frequently organised in clusters, are regulated by the imprinting control regions (ICR). This thesis is mainly focused on the mechanisms through which the ICRs control the imprinting in the cluster, containing the Kcnq1, Igf2 and H19 genes, located at the distal end of mouse chromosome 7. The H19 ICR, located in the 5' flank of the H19 gene represses paternal H19 and maternal Igf2 expression, respectively, but has no effect on Kcnq1 expression, which is controlled by another ICR located at the intron 10 of the Kcnq1 gene. This thesis demonstrates that the maternal H19 ICR allele contains several DNase I hypersensitive sites, which map to target sites for the chromatin insulator protein CTCF at the linker regions between the positioned nucleosomes. The thesis demonstrates that the H19 ICR acts as a unidirectional insulator and that this property invovles three nucleosome positioning sites facilitating interaction between the H19 ICR and CTCF. The Kcnq1 ICR function is much more complex, since it horbours both lineage-specific silencing functions and a methylation sensitive unidirectional chromatin insulator function. Importantly, the thesis demonstrates that the Kcnq1 ICR spreads DNA methylation into flanking region only when it is itself unmethylated. Both the methylation spreading and silencing functions map to the same regions. In conclusion, the thesis has unraveled and unrivalled complexity of the epigenetic control and function of short strtches of sequences. The epigenetic status of these cis elements conspires to control long-range silencing and insulation. The manner these imprinting control regions can cause havoc in expresson domains in human diseases is hence emerging.
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| 33. |
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| 34. |
- Kniola, Barbara, et al.
(author)
-
The domain structure of centromeres is conserved from fission yeast to humans
- 2001
-
In: Molecular Biology of the Cell. - : American Society for Cell Biology (ASCB). - 1059-1524 .- 1939-4586. ; 12:9, s. 2767-2775
-
Journal article (peer-reviewed)abstract
- The centromeric DNA of fission yeast is arranged with a central core flanked by repeated sequences. The centromere-associated proteins, Mis6p and Cnp1p (SpCENP-A), associate exclusively with central core DNA, whereas the Swi6 protein binds the surrounding repeats. Here, electron microscopy and immunofluorescence light microscopy reveal that the central core and flanking regions occupy distinct positions within a heterochromatic domain. An "anchor" structure containing the Ndc80 protein resides between this heterochromatic domain and the spindle pole body. The organization of centromere-associated proteins in fission yeast is reminiscent of the multilayered structures of human kinetochores, indicating that such domain structure is conserved in eukaryotes.
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| 35. |
- Kärblane, Kairi, et al.
(author)
-
ABCE1 is a highly conserved RNA silencing suppressor
- 2015
-
In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 10:2
-
Journal article (peer-reviewed)abstract
- ATP-binding cassette sub-family E member 1 (ABCE1) is a highly conserved protein among eukaryotes and archaea. Recent studies have identified ABCE1 as a ribosomerecycling factor important for translation termination in mammalian cells, yeast and also archaea. Here we report another conserved function of ABCE1. We have previously described AtRLI2, the homolog of ABCE1 in the plant Arabidopsis thaliana, as an endogenous suppressor of RNA silencing. In this study we show that this function is conserved: human ABCE1 is able to suppress RNA silencing in Nicotiana benthamiana plants, in mammalian HEK293 cells and in the worm Caenorhabditis elegans. Using co-immunoprecipitation and mass spectrometry, we found a number of potential ABCE1-interacting proteins that might support its function as an endogenous suppressor of RNA interference. The interactor candidates are associated with epigenetic regulation, transcription, RNA processing and mRNA surveillance. In addition, one of the identified proteins is translin, which together with its binding partner TRAX supports RNA interference.
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| 36. |
- Larsson, Chatarina, 1979-, et al.
(author)
-
Restoration of KMT2C/MLL3 in human colorectal cancer cells reinforces genome-wide H3K4me1 profiles and influences cell growth and gene expression
- 2020
-
In: Clinical Epigenetics. - : Springer Nature. - 1868-7083 .- 1868-7075. ; 12:1
-
Journal article (peer-reviewed)abstract
- Background The histone 3 lysine 4 (H3K4) monomethylase KMT2C is mutated across several cancer types; however, the effects of mutations on epigenome organization, gene expression, and cell growth are not clear. A frequently recurring mutation in colorectal cancer (CRC) with microsatellite instability is a single nucleotide deletion within the exon 38 poly-A(9) repeat (c.8390delA) which results in frameshift preceding the functional carboxy-terminal SET domain. To study effects ofKMT2Cexpression in CRC cells, we restored one allele to wild typeKMT2Cin the two CRC cell lines RKO and HCT116, which both are homozygous c.8390delA mutant. Results Gene editing resulted in increasedKMT2Cexpression, increased H3K4me1 levels, altered gene expression profiles, and subtle negative effects on cell growth, where higher dependence and stronger effects ofKMT2Cexpression were observed in RKO compared to HCT116 cells. Surprisingly, we found that the two RKO and HCT116 CRC cell lines have distinct baseline H3K4me1 epigenomic profiles. In RKO cells, a flatter genome-wide H3K4me1 profile was associated with more increased H3K4me1 deposition at enhancers, reduced cell growth, and more differential gene expression relative to HCT116 cells when KMT2C was restored. Profiling of H3K4me1 did not indicate a highly specific regulation of gene expression as KMT2C-induced H3K4me1 deposition was found globally and not at a specific enhancer sub-set in the engineered cells. Although we observed variation in differentially regulated gene sets between cell lines and individual clones, differentially expressed genes in both cell lines included genes linked to known cancer signaling pathways, estrogen response, hypoxia response, and aspects of immune system regulation. Conclusions Here, KMT2C restoration reduced CRC cell growth and reinforced genome-wide H3K4me1 deposition at enhancers; however, the effects varied depending upon the H3K4me1 status of KMT2C deficient cells. Results indicate that KMT2C inactivation may promote colorectal cancer development through transcriptional dysregulation in several pathways with known cancer relevance.
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| 37. |
- Lejeune, Erwan, et al.
(author)
-
The chromatin-remodeling factor FACT contributes to centromeric heterochromatin independently of RNAi
- 2007
-
In: Current Biology. - : Elsevier BV. - 0960-9822 .- 1879-0445. ; 17:14, s. 1219-1224
-
Journal article (peer-reviewed)abstract
- Centromeres exert vital cellular functions in mitosis and meiosis. A specialized histone and other chromatin-bound factors nucleate a dynamic protein assembly that is required for the proper segregation of sister chromatids. In several organisms, including the fission yeast, Schizosaccharomyces pombe, the RNAi pathway contributes to the formation of silent chromatin in pericentromeric regions. Little is known about how chromatin-remodeling factors contribute to heterochromatic integrity and centromere function. Here we show that the histone chaperone and remodeling complex FACT is required for centromeric-heterochromatin integrity and accurate chromosome segregation. We show that Spt16 and Pob3 are two subunits of the S. pombe FACT complex. Surprisingly, yeast strains deleted for pob3+ are viable and alleviate gene silencing at centromeric repeats and at the silent mating-type locus. Importantly, like heterochromatin and RNAi pathway mutants, Pob3 null strains exhibit lagging chromosomes on anaphase spindles. Whereas the processing of centromeric RNA transcripts into siRNAs is maintained in Pob3 mutants, Swi6-association with the centromere is reduced. Our studies provide the first experimental evidence for a role of the RNA polymerase II cofactor FACT in heterochromatin integrity and in centromere function.
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| 38. |
- Lloyd, Katy A., et al.
(author)
-
Differential ACPA Binding to Nuclear Antigens Reveals a PAD-Independent Pathway and a Distinct Subset of Acetylation Cross-Reactive Autoantibodies in Rheumatoid Arthritis
- 2019
-
In: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 9, s. 1-22
-
Journal article (peer-reviewed)abstract
- Rheumatoid arthritis (RA) associated anti-citrullinated protein autoantibodies (ACPA) target a wide range of modified proteins. Citrullination occurs during physiological processes such as apoptosis, yet little is known about the interaction of ACPA with nuclear antigens or apoptotic cells. Since uncleared apoptotic cells and neutrophil extracellular trap (NET) products have been postulated to be central sources of autoantigen and immunostimulation in autoimmune disease, we sought to characterize the anti-nuclear and anti-neutrophil reactivities of ACFA. Serology showed that a subset of anti-CCP2 seropositive RA patients had high reactivity to full-length citrullinated histones. In contrast, seronegative RA patients displayed elevated IgG reactivity to native histone compared to controls, but no citrulline-specific reactivity. Screening of 10 single B-cell derived monoclonal AGFA from RA patients revealed that four ACPA exhibited strong binding to apoptotic cells and three of these had anti-nuclear (ANA) autoantibody reactivity. Modified histones were confirmed to be the primary targets of this anti-nuclear ACPA subset following immunoprecipitation from apoptotic cell lysates. Monoclonal ACPA were also screened for reactivities against stimulated murine and human neutrophils, and all the nuclear-reactive monoclonal ACPA bound to NETs. Intriguingly, one ACPA mAb displayed a contrasting cytoplasmic perinuclear neutrophil binding and may represent a different NET-reactive ACPA subset. Notably, studies of CRISPR-Cas9 PAD4 KO cells and cells from PAD KO mice showed that the cytoplasmic NET-binding was fully dependent on PAD4, whilst nuclear- and histone-mediated NEI reactivity was largely PAD-independent. Our further analysis revealed that the nuclear binding could be explained by consensus-motif driven ACPA cross-reactivity to acetylated histones. Specific acetylated histone peptides targeted by the monoclonal antibodies were identified and the anti-modified protein autoantibody (AMPA) profile of the ACPA was found to correlate with the functional activity of the antibodies. In conclusion, when investigating monoclonal ACPA, we could group ACPA into distinct subsets based on their nuclear binding-patterns and acetylation-mediated binding to apoptotic cells, neutrophils, and NETs. Differential anti-modified protein reactivities of RA-autoantibody subsets could have an important functional impact and provide insights in RA pathogenesis.
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| 39. |
- Maksimov, Vladimir, et al.
(author)
-
The binding of Chp2's chromodomain to methylated H3K9 is essential for Chp2's role in heterochromatin assembly in fission yeast
- 2018
-
In: PLOS ONE. - : PUBLIC LIBRARY SCIENCE. - 1932-6203. ; 13:8
-
Journal article (peer-reviewed)abstract
- The binding of heterochromatin protein 1 (HP1) to lysine 9-methylated histone H3 (H3K9me) is an essential step in heterochromatin assembly. Chp2, an HP1-family protein in the fission yeast Schizosaccharomyces pombe, is required for heterochromatic silencing. Chp2 recruits SHREC, a multifunctional protein complex containing the nucleosome remodeler Mit1 and the histone deacetylase Clr3. Although the targeting of SHREC to chromatin is thought to occur via two distinct modules regulated by the SHREC components Chp2 and Clr2, it is not clear how Chp2's chromatin binding regulates SHREC function. Here, we show that H3K9me binding by Chp2's chromodomain (CD) is essential for Chp2's silencing function and for SHREC's targeting to chromatin. Cells expressing a Chp2 mutant with defective H3K9me binding (Chp2-W199A) have a silencing defect, with a phenotype similar to that of chp2-null cells. Genetic analysis using a synthetic silencing system revealed that a Chp2 mutant and SHREC-component mutants had similar phenotypes, suggesting that Chp2's function also affects SHREC's chromatin binding. Size-exclusion chromatography of native protein complexes showed that Chp2-CD's binding of H3K9me3 ensures Clr3's chromatin binding, and suggested that SHREC's chromatin binding is mediated by separable functional modules. Interestingly, we found that the stability of the Chp2 protein depended on the Clr3 protein's histone deacetylase activity. Our findings demonstrate that Chp2's H3K9me binding is critical for SHREC function and that the two modules within the SHREC complex are interdependent.
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| 40. |
- Mariano, Piero, 1966-
(author)
-
Epigenetic Regulation and Reprogramming of the H19 Imprinting Control Region
- 2006
-
Doctoral thesis (other academic/artistic)abstract
- The development of a new individual from the fertilized oocyte can ultimately be seen as the consequence of the establishment and maintenance of specific patterns of gene expression. Although regulation of gene activity occurs at different levels, cellular specialization and differentiation are the results of developmental cues that essentially take place at the transcriptional level. The involvement of epigenetics in this process has become increasingly clear during the last decade. Imprinted genes constitute an excellent example as monoallelic expression seems to reflect differential epigenetic marks on the two alleles. This is the case of the imprinted H19 and Igf2 genes were the monoallelic expression is coordinated through a differentially methylated region (hypermethylated on the paternal allele), known as ICR (imprinted control region). In the mouse the ICR harbours four binding sites for the methylation sensitive insulator protein CTCF. Previous studies with episomal constructs had shown that this region behaved as an insulator and that CTCF is required for the insulator activity of the H19 ICR This thesis establish a clear link between the insulator function and the chromatin structure at the H19 ICR and indicates that the precise allocation of the CTCF target sites in the linker regions can play a critical role in this process. The importance of the CTCF interaction at the ICR was also confirmed in vivo using a mouse model that showed how intact CTCF target sites are needed to manifest insulator activity and methylation protection. We have investigated the role of CTCF and a related protein BORIS in establishing the maternal to paternal imprint transition in chromatin structure at the H19/Igf2 locus in the male germline. This thesis also describe the development of a new technique for the localization of chromatin associated factors and modifications with higher sensitivity and resolution compared to existing approaches.
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| 41. |
- Mujahed, Huthayfa, et al.
(author)
-
AML displays increased CTCF occupancy associated with aberrant gene expression and transcription factor binding
- 2020
-
In: Blood. - : American Society of Hematology. - 0006-4971 .- 1528-0020. ; 136:3, s. 339-352
-
Journal article (peer-reviewed)abstract
- CCTC-binding factor (CTCF) is a key regulator of gene expression through organization of the chromatin structure. Still, it is unclear how CTCF binding is perturbed in leukemia or in cancer in general. We studied CTCF binding by chromatin immunoprecipitation sequencing in cells from patients with acute myeloid leukemia (AML) and in normal bone marrow (NBM) in the context of gene expression, DNA methylation, and azacitidine exposure. CTCF binding was increased in AML compared with NBM. Aberrant CTCF binding was enriched for motifs for key myeloid transcription factors such as CEBPA, PU.1, and RUNX1. AML with TET2 mutations was characterized by a particularly strong gain of CTCF binding, highly enriched for gain in promoter regions, while AML in general was enriched for changes at enhancers. There was a strong anticorrelation between CTCF binding and DNA methylation. Gain of CTCF occupancy was associated with increased gene expression; however, the genomic location (promoter vs distal regions) and enrichment of motifs (for repressing vs activating cofactors) were decisive for the gene expression pattern. Knockdown of CTCF in K562 cells caused loss of CTCF binding and transcriptional repression of genes with changed CTCF binding in AML, as well as loss of RUNX1 binding at RUNX1/CTCF-binding sites. In addition, CTCF knockdown caused increased differentiation. Azacitidine exposure caused major changes in CTCF occupancy in AML patient cells, partly by restoring a CTCF-binding pattern similar to NBM. We conclude that AML displays an aberrant increase in CTCF occupancy that targets key genes for AML development and impacts gene expression.
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| 42. |
- Okorokova-Facanha, A L, et al.
(author)
-
An inventory of the P-type ATPases in the fission yeast Schizosaccharomyces pombe
- 2003
-
In: Current Genetics. - : Springer Science and Business Media LLC. - 0172-8083 .- 1432-0983. ; 43:4, s. 273-280
-
Journal article (peer-reviewed)abstract
- The analysis of the Schizosaccharomyces pombe genome revealed the presence of 14 putative P-type ATPases. The clustering of ATPases resembles that of Saccharomyces cerevisiae, indicating that the main classes of pumps were already present before the split of the Archiascomycetes from the other Ascomycota. The overall amino acid identity between fission and budding yeast P-type ATPases is generally low (30-50%). This is similar to the fungus-plant and fungus-animal comparisons.. suggesting that fungal ATPases underwent an extensive process of diversification. Unlike Sac. cerevisiae. fission yeast lacks Na+-ATPases, has a single heavy-metal ATPase and three ATPases of unknown specificity. The observed divergence within these fungi might reflect physiological differences, including adaptation to environmental stresses.
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| 43. |
- Olsson, T G S, et al.
(author)
-
Transient inhibition of histone deacetylase activity overcomes silencing in the mating-type region in fission yeast
- 1999
-
In: Current Genetics. - : Springer Science and Business Media LLC. - 0172-8083 .- 1432-0983. ; 35:2, s. 82-87
-
Journal article (peer-reviewed)abstract
- We have investigated the effects of inhibition of histone de-acetylase activity on silencing at the silent mating-type loci in fission yeast. Treatment of exponentially growing cells with the histone deacetylase inhibitor, trichostatin A (TSA), resulted in derepression of a marker gene inserted 150 bp distal from the silent mat3-M locus. The natural targets for the silencing mechanism in this region were only partially derepressed and the activation appeared to be asymmetric. i.e. the mat2-P cassette remained silent at concentrations that clearly partially derepressed the mat3-M cassette. We further noted that treatment of wild-type h(90) cells resulted in the generation of altered sporulation phenotypes, indicating that the treatment affected the expression of mating-type genes and/or mating-type switching. The results are discussed in the light of recent accumulated data regarding the role of deacetylation for silencing in other species.
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| 44. |
- Opel, Michael, et al.
(author)
-
Genome-Wide Studies of Histone Demethylation Catalysed by the Fission Yeast Homologues of Mammalian LSD1
- 2007
-
In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 2:4
-
Journal article (peer-reviewed)abstract
- 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 Delta 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.
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| 45. |
- Pandey, Gaurav Kumar
(author)
-
Regulatory Roles of Noncoding RNA in Development and Disease
- 2013
-
Doctoral thesis (other academic/artistic)abstract
- Long noncoding RNAs (lncRNAs) are being realized as important players in gene regulation and their misregulation has been considered as one of the underlying causes for tumor initiation and progression in many human pathologies. In the current thesis, I have addressed the functional role of lncRNAs in development and disease model systems.Genomic imprinting is an epigenetic phenomenon by which subset of genes are expressed in a parent of origin-specific manner. The Kcnq1 imprinted locus is epigenetically regulated by Kcnq1ot1 lncRNA. Deletion of an 890bp region at the 5’ end of Kcnq1ot1 in mouse resulted in the loss of silencing of neighboring ubiqui-tously imprinted genes (UIGs). In addition, we observed loss of DNA methylation at the UIG promoters. We have shown that Kcnq1ot1 RNA establishes CpG methylation by interacting with DNMT1. To explore the stability of lncRNA mediated silencing pathways, we have conditionally deleted Kcnq1ot1 in the mouse in a stage and tissue-specific manner. We have shown that Kcnq1ot1 is continuously required for maintaining the silencing of UIGs, whereas the silencing of the placental im-printed genes is maintained in an RNA independent manner. To identify chromatin-associated lncRNA (CARs) on a genome-wide scale, we purified RNA from the sucrose gradient fractionated chromatin and subjected it to RNA sequencing. Our study has identified 141 intronic and 74 long intergenic CARs. Characterization of one of the CARs revealed that it regulates the expression of neighboring genes in cis by modulating the chromatin structure. We have explored the functional role of lncRNA in tumor progression and initiation by using pediatric neuroblastoma. By transcriptional profiling of low- and high-risk tumors, we have identified several lncRNAs differentially expressed between these subtypes. We report an uncharacterized RNA NBAT-1, expressed at lower levels in high-risk tumors relative to low-risk tumors. Using neuroblastoma cell culture system, we demonstrated that NBAT-1 has anti-cell proliferative and anti-invasive properties. In addition, it promotes differentiation of neurons from undifferentiated neuroblastoma cell lines. In summary, by employing mouse genetics, cell culture based model system and expression profiling in tumors, we have uncovered new roles of lncRNA in gene regulation.
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| 46. |
- Pandey, Radha Raman, 1978-
(author)
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Molecular Insights into Kcnq1ot1 Noncoding Antisense RNA Mediated Long Range Transcriptional Gene Silencing
- 2008
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Doctoral thesis (other academic/artistic)abstract
- Non-coding antisense RNAs have been implicated in the epigenetic silencing of individual gene as well as chromosomal domains. While silencing of the overlapping gene by antisense RNAs has been well investigated, their functional role in silencing of chromosomal domains remains enigmatic. To elucidate mechanisms underlying the non-coding RNA mediated epigenetic silencing of chromosomal domains, we have chosen an antisense non-coding RNA, Kcnq1ot1, as a model system. Previously, a functional role of Kcnq1ot1 RNA and/or its transcriptional process has been implicated in silencing of multiple genes in the Kcnq1 imprinted cluster. However, these studies could not rule out the mechanisms involving other than Kcnq1ot1 RNA. Furthermore, it was also unclear how the Kcnq1ot1 promoter escapes silencing when its encoded RNA is capable of silencing flanking genes in cis.We have shown that NF-Y transcription factor plays a central role in the Kcnq1ot1 promoter activity, and that mutation of the NF-Y binding sites not only resulted in loss of silencing of flanking genes but also the ability of the Kcnq1ot1 promoter to protect against repressive chromatin marks, indicating that NF-Y maintains transcription-competent chromatin at the promoter through resisting the strong silencing effects of Kcnq1ot1 RNA.The Kcnq1ot1 RNA is an RNA Polymerase II encoded 91 kb long moderately stable nuclear transcript. We have demonstrated that it is the RNA not the act of transcription responsible for silencing and that the degree of silencing was proportional to the length of Kcnq1ot1 RNA. The kinetics of heterochromatin formation in relation to Kcnq1ot1 transcription revealed that overlapping gene was silenced initially by occlusion of basal transcription machinery and heterochromatin formation, whereas nonoverlapping gene was silenced subsequently by Kcnq1ot1-mediated heterochromatin spreading. This transcriptional silencing by Kcnq1ot1 RNA is mediated by an 890 bp region through promoting its interaction with the chromatin. Interestingly, we show that Kcnq1ot1 RNA establishes heterochromatin structures in a lineage-specific fashion by interacting with chromatin and chromatin remodelling complexes such as G9a and PRC2 complexes. More importantly, one of the parental chromosomes comprising Kcnq1 domain always found in the vicinity of perinucleolar region. Based on these data we proposed a mechanism whereby Kcnq1ot1 RNA establishes transcriptional silencing through recruitment of chromatin remodelling machinery and the maintenance of silencing achieved via targeting to the perinucleolar region.
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| 47. |
- Persson, Jenna, et al.
(author)
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Chd1 remodelers maintain open chromatin and regulate the epigenetics of differentiation
- 2010
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In: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 316:8, s. 1316-1323
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Journal article (peer-reviewed)abstract
- Eukaryotic DNA is packaged around octamers of histone proteins into nucleosomes, the basic unit of chromatin. In addition to enabling meters of DNA to fit within the confines of a nucleus, the structure of chromatin has functional implications for cell identity. Covalent chemical modifications to the DNA and to histones, histone variants, ATP-dependent chromatin remodelers, small noncoding RNAs and the level of chromatin compaction all contribute to chromosomal structure and to the activity or silencing of genes. These chromatin-level alterations are defined as epigenetic when they are heritable from mother to daughter cell. The great diversity of epigenomes that can arise from a single genome permits a single, totipotent cell to generate the hundreds of distinct cell types found in humans. Two recent studies in mouse and in fly have highlighted the importance of Chd1 chromatin remodelers for maintaining an open, active chromatin state. Based on evidence from fission yeast as a model system, we speculate that Chd1 remodelers are involved in the disassembly of nucleosomes at promoter regions, thus promoting active transcription and open chromatin. It is likely that these nucleosomes are specifically marked for disassembly by the histone variant H2A.Z.
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| 48. |
- Provost, P, et al.
(author)
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Dicer is required for chromosome segregation and gene silencing in fission yeast cells
- 2002
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In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 99:26, s. 16648-16653
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Journal article (peer-reviewed)abstract
- RNA interference is a form of gene silencing in which the nuclease Dicer cleaves double-stranded RNA into small interfering RNAs. Here we report a role for Dicer in chromosome segregation of fission yeast. Deletion of the Dicer (dcr1(+)) gene caused slow growth, sensitivity to thiabendazole, lagging chromosomes during anaphase, and abrogated silencing of centromeric repeats. As Dicer in other species, Dcr1p degraded double-stranded RNA into approximate to23 nucleotide fragments in vitro, and dcr1Delta cells were partially rescued by expression of human Dicer, indicating evolutionarily conserved functions. Expression profiling demonstrated that dcr1(+) was required for silencing of two genes containing a conserved motif.
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| 49. |
- Qu, Ying, et al.
(author)
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Cancer specific changes in DNA methylation reveal aberrant silencing and activation of enhancers in leukemia
- 2017
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In: Blood. - : American Society of Hematology. - 0006-4971 .- 1528-0020. ; 129:7, s. e13-e25
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Journal article (peer-reviewed)abstract
- Acute myeloid leukemia (AML) is characterized by an impaired differentiation process leading to an accumulation of immature blasts in the blood. One feature of cytogenetically normal AML is alterations to the DNA methylome. Here we have analyzed 57 AML patients with normal karyotype using Illuminas 450 k array and show that aberrant DNA methylation is significantly altered at enhancer regions and that the methylation levels at specific enhancers predict overall survival of AML patients. The majority of sites that become differentially methylated in AML occur in regulatory elements of the human genome. Hypermethylation associates with enhancer silencing. In addition, ChIP-seq analyses showed that a subset of hypomethylated sites correlate with enhancer activation, indicated by increased H3K27 acetylation. DNA hypomethylation is not therefore sufficient for enhancer activation. Some sites of hypomethylation occur at weak / poised enhancers marked with H3K4 monomethylation in hematopoietic progenitor cells. Other hypomethylated regions occur at sites inactive in progenitors and reflect the de novo acquisition of AML specific enhancers. Altered enhancer dynamics are reflected in the gene expression of enhancer target genes including genes involved in oncogenesis and blood cell development. This study demonstrates that histone variants and different histone modifications interact with aberrant DNA methylation, causing perturbed enhancer activity in CN-AML that contributes to a leukemic transcriptome.
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| 50. |
- Rhind, Nicholas, et al.
(author)
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Comparative Functional Genomics of the Fission Yeasts
- 2011
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In: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 332:6032, s. 930-936
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Journal article (peer-reviewed)abstract
- The fission yeast clade-comprising Schizosaccharomyces pombe, S. octosporus, S. cryophilus, and S. japonicus-occupies the basal branch of Ascomycete fungi and is an important model of eukaryote biology. A comparative annotation of these genomes identified a near extinction of transposons and the associated innovation of transposon-free centromeres. Expression analysis established that meiotic genes are subject to antisense transcription during vegetative growth, which suggests a mechanism for their tight regulation. In addition, trans-acting regulators control new genes within the context of expanded functional modules for meiosis and stress response. Differences in gene content and regulation also explain why, unlike the budding yeast of Saccharomycotina, fission yeasts cannot use ethanol as a primary carbon source. These analyses elucidate the genome structure and gene regulation of fission yeast and provide tools for investigation across the Schizosaccharomyces clade.
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