Epigenetic inheritance: establishment and transmission of specialised chromatin domains
Chromosomal DNA is wrapped around nucleosomes containing core histones (H3/H4/H2A/ H2B). However, at centromeres a specific histone H3 variant, CENP-A, replaces histone H3 to form specialized CENP-A nucleosomes. CENP-A chromatin is critical for assembly of the chromosome segregation machinery – kinetochores – at these specific chromosomal locations and is flanked by histone H3 lysine 9 methylated heterochromatin.
Our goal is to decipher conserved mechanisms that establish, maintain and regulate the assembly of heterochromatin and CENP-A chromatin domains. Heterochromatin is required for the establishment of CENP-A chromatin on centromere DNA. One objective is to provide further insight into mechanisms that promote heterochromatin formation on pericentromeric repeats. Heterochromatin might also silence genes throughout the genome, we therefore also investigate how heterochromatin formation is regulated and whether such mechanisms influence phenotype. We endeavour to determine how heterochromatin, spatial nuclear organisation and non-coding RNAPII transcription combine to mediate CENP-A incorporation at centromeres.
Our main questions are:
1. How do DNA, RNA and chromatin signatures instigate the assembly of specialised chromatin domains?
2. How does chromatin architecture and subnuclear compartmentalization affect specialised chromatin domains?
3. How does heterochromatin influence gene expression?
Long-read sequencing allowed the de novo assembly of the genomes of two fission yeast species that are evolutionarily distinct from Schizosaccharomyces pombe. Our assemblies are contiguous across all three centromeres, and other heterochromatin regions, of both species and permits comparison of centromere organization between these divergent species (Figure a). Centromeres from all three species retain an overall structural resemblance, however, no sequence similarity is detected between repetitive elements and central regions of even the closest two species, apart from the presence of similarly ordered tRNA genes (Figure b). Interspecies functional tests reveal that non-homologous S. octosporus centromere DNA is recognized and promotes centromere assembly in S. pombe. We surmise that conserved processes recognize features associated with non-conserved sequences allowing preservation of centromere identity and location over evolutionary time. Principal Component Analysis detect a distinct pattern of all possible nucleotide pentamers enriched in CENP-A-associated DNA from all three species (Figure c). Conserved processes, such as transcription, may promote recognition of these centromeric DNAs and the replacement of histone H3 chromatin with CENP-A chromatin. Consistent with this, other analyses indicate that H3 nucleosomes turnover at a high rate on centromere DNA and de novo CENP-A assembly requires H2A.Z deposition by the Swr1C chromatin remodeling complex.
*Subramanian, L., *Medina-Pritchard, B., Barton, R., Spiller, F., Kulasegaran-Shylini, R., Radaviciute, G., Allshire, R.C., and Jeyaprakash A. A. (2016). Centromere Localization and Function of Mis18 Requires 'Yippee-like' Domain-Mediated Oligomerization. EMBO Rep 17, 496-507. * joint first authors
Ard, R., and Allshire, R.C. (2016). Transcription-coupled changes to chromatin underpin gene silencing by transcriptional interference. Nucleic Acids Res. 44, 10619–10630.
Yadav, R.K., Jablonowski, C.M., Fernandez, A.G., Lowe, B.R., Henry, R.A., Finkelstein, D., Barnum, K.J., Pidoux, A.L., Kuo, Y.M., Huang, J., O'Connell, M.J., Andrews, A.J., Onar-Thomas, A., Allshire, R.C., Partridge, J.F. (2017). Histone H3G34R mutation causes replication stress, homologous recombination defects and genomic instability in S. pombe. Elife 6, pii: e27406.