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Robin Allshire


Co-workers: Ryan Ard, Tatsiana Auchynnikava, Pauline Audergon, Max Fitz-James, Alison Pidoux, Manu Shukla, Raghavendran Kulasegaran Shylini, Puneet Singh, Lakxmi Subramanian, Nick Toda, Pin Tong, Sharon White, Conor Young
Allshire lab website

Epigenetic inheritance: establishment and transmission of specialized chromatin domains

Robin gives a brief overview of his research.

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 H3to form specialized CENP-A nucleosomes. CENP-A chromatin is critical for kinetochore assemblyand chromosome segregation. What determines where on chromosomes CENP-A rather thanhistone H3 is assembled? Primary DNA sequence is not absolute in dictating where activeregional centromeres are formed. Instead, ‘epigenetic’ features provide cues that promote theassembly of CENP-A chromatin. Our objective is to understand the nature of these features andhow they ensure deposition of CENP-A instead of H3 to form active centromeres.

Post-translational modifications on histones are frequently referred to as ‘epigenetic marks’,however, there was no definitive demonstration that H3K9me-dependent heterochromatinexhibits epigenetic heritability. Fission yeast has a single H3K9 methyltransferase (Clr4)required to form all heterochromatin. By tethering Clr4 we generated domains ofconditional synthetic heterochromatin at euchromatic loci in fission yeast and directly tested if H3K9-methylation-dependent heterochromatin persists following Clr4 release. Ouranalyses revealed that a read-write mechanism permits inheritance of heterochromatin,through mitotic and meiotic cell divisions. However, transmission of this heterochromatinis only observed in cells lacking Epe1 (JmjC/demethylase) which otherwise erases H3K9-methylation and heterochromatin from these euchromatic loci.

We have developed a powerful approach CREAM (Chromatin Region Enrichment and Measurement) to affinity select specific chromatin regions allowing protein identification and quantification with state-of-the-art mass-spectrometry. Application of CREAM to isolated heterochromatic centromere repeats enriches most known core RNAi/heterochromatin components along with additional proteins not previously reported to be heterochromatin associated (Figure 1). By implementing quantitative methods we simultaneously measure 100-250 target proteins, detecting substantial differences in centromere repeat chromatin composition between genetically identical cells derived by the distinct routes of establishment or maintenance in RNAi/heterochromatin mutants (Epi-Proteomics).

Long-read sequencing has allowed accurate sequence assembly across centromeres of two additional fission yeast species that are evolutionarily distinct from S. pombe. ChIP-seq shows that the overall organization of heterochromatin and CENP-A chromatin domains is preserved, despite the lack of sequence similarity between these centromeres. Conserved processes may recognize properties encoded by these non-conserved sequences, allowing preservation of centromere identity and location (Figure 2). Indeed, in S. pombe we find that H3 is deposited as a placeholder for CENP-A in S phase. This transient H3 is subsequently replaced by CENP-A in G2 (Figure 3). The process driving this cell-cycle regulated H3→CENP-A exchange may involve transcriptional stalling programmed by centromeric DNA.

Selected publications:

Cantania, S., Pidoux, A.L., Allshire, R.C. (2015) Sequence features and stalled transcription within centromere DNA promote establishment of CENP-A chromatin. PLOS Genetics 11, e1004986.
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 Feb 26. pii: e201541520.
* joint first authors

1. Proteins enriched on fission yeast (S. pombe) heterochromatin relative to control chromatin, detected and quantified by mass spectrometry.
2. S. octosporus centromeres de novo assembled from PacBio long reads; CENP-A chromatin and H3K9me2-heterochromatin domains mapped by ChIP-seq.
3. RITE tag exchange indicates that new S. pombe CENP-A is incorporated during G2, replacing H3 deposited in Sphase as a placeholder.