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Robin Allshire

Publications and source records attributed to Robin Allshire.

13 recordsLinked to original sources
PubMed2025

ChIPmentation for Epigenomic Analysis in Fission Yeast.

Histone modifications and transcription factor-DNA interactions regulate vital processes such as transcription, recombination, repair, and accurate chromosome segregation. Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) has been instrumental in studying genome-wide distribution of DNA-bound or chromatin-associated factors and histone posttranslational modifications (PTMs). Here, we describe a ChIPmentation protocol adapted for fission yeast, Schizosaccharomyces pombe. This method merges Tn5 mediated tagmentation with existing ChIP protocols, resulting in lower sample input requirements with significant reduction in hands-on time and sample preparation costs.

Schizosaccharomyces↗
PubMed2025

Genome-Wide Profiling of Histone Modifications in Fission Yeast Using CUT&Tag.

Eukaryotic DNA is organized in the nucleus in the form of chromatin. Nucleosomes, the fundamental unit of chromatin, are subject to many posttranslational modifications (PTMs) as well as compositional variations through incorporation of histone variants. These alterations play important roles in regulation of genome structure and activity. Genome-wide profiling of these regulatory features is essential for understanding of genome function. Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-Seq) is a widely used method to assay genome-wide localization in fission yeast but suffers from the requirement for a large amount of input chromatin, antibodies, and a cumbersome experimental pipeline. New methods such as Cleavage Under Targets and Tagmentation (CUT&Tag), which combine the specificity of targeted cleavage and adapter insertion with the sensitivity of next-generation sequencing, enable identification and characterization of various epigenetic marks affording low input requirement as well as more streamlined protocols. However, these approaches have not been adapted for use in fission yeast, Schizosaccharomyces pombe. Here, we describe an adapted CUT&Tag protocol for epigenomic profiling in fission yeast using the heterochromatin-associated histone H3K9 methylation PTM for benchmarking.

Schizosaccharomyces↗
PubMed2006

Fta2, an essential fission yeast kinetochore component, interacts closely with the conserved Mal2 protein.

The fission yeast multiprotein-component Sim4 complex plays a fundamental role in the assembly of a functional kinetochore. It affects centromere association of the histone H3 variant CENP-A as well as kinetochore association of the DASH complex. Here, multicopy suppressor analysis of a mutant version of the Sim4 complex component Mal2 identified the essential Fta2 kinetochore protein, which is required for bipolar chromosome attachment. Kinetochore localization of Mal2 and Fta2 depends on each other, and overexpression of one protein can rescue the phenotype of the mutant version of the other protein. fta2 mal2 double mutants were inviable, implying that the two proteins have an overlapping function. This close interaction with Fta2 is not shared by other Sim4 complex components, indicating the existence of functional subgroups within this complex. The Sim4 complex seems to be assembled in a hierarchical way, because Fta2 is localized correctly in a sim4 mutant. However, Fta2 kinetochore localization is reduced in a spc7 mutant. Spc7, a suppressor of the EB1 family member Mal3, is part of the conserved Ndc80-MIND-Spc7 kinetochore complex.

Chromosomal Proteins, Non-Histone↗
PubMed2005

Centromeric chromatin makes its mark.

Post-translational modifications of core histones, particularly in the N terminus, seem to define different chromatin states in the genome. For instance, actively transcribed regions of euchromatin have a completely different set of 'marks' compared with silent heterochromatin. Recent analyses surprisingly demonstrate that, in addition to containing the histone H3 variant CENP-A, the centromeric chromatin that underlies the kinetochore bears a distinct combination of histone H3 modifications.

Amino Acid Sequence↗
PubMed2004

Those interfering little RNAs! Silencing and eliminating chromatin.

RNA interference (RNAi) is widely used for knocking down expression of genes of interest and in systematic screens for desired phenotypes. In post-transcriptional gene silencing, double-stranded RNA triggers are processed to small interfering RNAs, which act to seek out and destroy homologous transcripts. A variety of organisms utilise the RNAi pathway to silence expression of potentially harmful endogenous mobile elements and to eliminate unnecessary sequences. In plants and fission yeast, RNAi can also mediate chromatin-based silencing resulting in transcriptional shutdown of homologous transcription units (transcriptional gene silencing) and the formation of centromeric heterochromatin. In metazoans, the expression of non-coding RNAs is often associated with the formation of silent chromatin domains but it remains to be determined if RNAi is involved.

Animals↗
PubMed2004

Analysis of chromatin in fission yeast.

The use of fission yeast as a model system for studies of chromosome biology has contributed to several key advances in the last few years. The structure of its large complex centromeres and composition of its transcriptionally silent heterochromatin resemble those of metazoa. The application of chromatin immunoprecipitation to fission yeast has been instrumental in these advances and we describe an improved version of this technique in detail. In addition, we describe several other techniques, which are useful in the analysis of chromatin in fission yeast.

Chromatin↗
PubMed2003

RETRACTED: Hairpin RNAs and retrotransposon LTRs effect RNAi and chromatin-based gene silencing.

The expression of short hairpin RNAs in several organisms silences gene expression by targeted mRNA degradation. This RNA interference (RNAi) pathway can also affect the genome, as DNA methylation arises at loci homologous to the target RNA in plants. We demonstrate in fission yeast that expression of a synthetic hairpin RNA is sufficient to silence the homologous locus in trans and causes the assembly of a patch of silent Swi6 chromatin with cohesin. This requires components of the RNAi machinery and Clr4 histone methyltransferase for small interfering RNA generation. A similar process represses several meiotic genes through nearby retrotransposon long terminal repeats (LTRs). These analyses directly implicate interspersed LTRs in regulating gene expression during cellular differentiation.

Cell Cycle Proteins↗
PubMed2003

Chromosome segregation: clamping down on deviant orientations.

Chromosome segregation depends on proper orientation of sister kinetochores. The protein Csm1 is required for mono-orientation of sister kinetochores at meiosis I in budding yeast. Surprisingly, its homologue in fission yeast appears instead of clamp micro-tubule binding sites together on single mitotic kinetochores so that they all face one spindle pole.

Cell Cycle Proteins↗
PubMed2003

A new role for the transcriptional corepressor SIN3; regulation of centromeres.

Centromeres play a vital role in maintaining the genomic stability of eukaryotes by coordinating the equal distribution of chromosomes to daughter cells during mitosis and meiosis. Fission yeast (S. pombe) centromeres consist of a 4-9 kb central core region and 30-100 kb of flanking inner (imr/B) and outer (otr/K) repeats. These sequences direct a laminar kinetochore structure similar to that of human centromeres. Centromeric heterochromatin is generally underacetylated. We have previously shown that inhibition of histone deacetylases (HDACs) caused hyperacetylation of centromeres and defective chromosome segregation. SIN3 is a HDAC corepressor that has the ability to mediate HDAC targeting in the repression of promoters. In this study, we have characterized S. pombe sin three corepressors (Pst1p and Pst2p) to investigate whether SIN3-HDAC is required in the regulation of centromeres. We show that only pst1-1 and not pst2Delta cells displayed anaphase defects and thiabendazole sensitivity. pst1-1 cells showed reduced centromeric silencing, increased histone acetylation in centromeric chromatin, and defective centromeric sister chromatid cohesion. The HDAC Clr6p and Pst1p coimmunoprecipitated, and Pst1p colocalized with centromeres, particularly in binucleate cells. These data are consistent with a model in which Pst1p-Clr6p temporally associate with centromeres to carry out the initial deacetylation necessary for subsequent steps in heterochromatin formation.

Acetylation↗
PubMed2002

Centromeres become unstuck without heterochromatin.

In most if not all eukaryotes, sister-chromatid cohesion, which is mediated by the chromosomal complex Cohesin, is destroyed by proteolysis at the transition from metaphase to anaphase. In metazoans, Cohesin is removed from chromosomes in two steps, and the centromere and its associated pericentric heterochromatin constitute the last point of linkage between sister chromatids at metaphase. Mechanistic insight is now emerging on the way in which cells distinguish cohesion at the centromere from cohesion along chromosome arms. We discuss recent advances in our understanding of the role of centromeric heterochromatin in sister-chromatid cohesion and propose a causal relationship between this specialized type of chromatin and the removal by proteolysis of Cohesins that are associated with it.

Animals↗