Epigenetic inheritance: establishment and transmission of specialized chromatin domains
Most 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 specialised CENP-A nucleosomes. CENP-A chromatin is critical for kinetochore assembly and chromosome segregation and is flanked by histone H3 lysine 9 methylated heterochromatin. 2
Our goal is to decipher conserved mechanisms that establish, maintain and regulate 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 determine how heterochromatin is formed on canonical pericentromeric repeats. Heterochromatin could also promote the silencing of genes throughout the genome, thus we investigate how heterochromatin formation is regulated and the mechanisms that might allow it to influence phenotype. Overall we endeavour to understand 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?
To determine if conserved sequences are involved in forming centromeres we employed long-read sequencing to de novo assemble the genomes of two fission yeast species that are evolutionarily distinct from Schizosaccharomyces pombe. This allowed sequence comparison across entire centromeres from S. pombe, S. octosporus and S. cryophilus (Figure 1). Our analyses show that centromeres from all three species retain an overall structural resemblance with repetitive elements surrounding unique central regions. No sequence similarity is detected between repetitive elements and central regions of these different species. However, in all species heterochromatin is located over flanking repeats whereas CENP-A chromatin is restricted to central regions (Figure 2). We propose that conserved processes may recognise features associated with non-conserved sequences allowing preservation of centromere identity and location over evolutionary time. In other words, provided these DNA elements retain key features, such as transcription to non-coding RNA, their sequence can drift. For example, processing of non-coding RNA transcribed from flanking repeats ensures siRNA production and RNAi-directed heterochromatin formation on these elements. Other processes may destabilize H3 chromatin and favour CENP-A assembly on non-conserved central regions. Remarkably, central region DNA from S. octosporus promotes CENP-A and functional kinetochore assembly in S. pombe (Figure 3). We conclude that conserved cryptic features must drive processes that assemble CENP-A chromatin in place of H3 nucleosomes.
Audergon, P.N.C.B., Catania, S., Kagansky, A., Tong, P., Shukla, M., Pidoux, A., and Allshire, R.C. (2015) Restricted epigenetic inheritance of H3K9 methylation. Science 348, 132-135.
*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.