Co-workers:

The centromere is the region on a chromosome where the kinetochore, a large protein complex, assembles; it enables chromosomes to bind spindle microtubules so that during cell division cells receive a complete set of chromosomes.

Most chromosomal DNA is wrapped around nucleosomes, containing core histones H3, H4, H2A and H2B. Strikingly, at the foundation of all kinetochores a centromere specific histone, CENP-A, replaces histone H3 forming CENP-A nucleosomes. This CENP-A chromatin is critical for kinetochore assembly; chromosome segregation is aberrant in cells with defective CENP-A. What determines where on a chromosome CENP-A is assembled instead of histone H3? Primary DNA sequence is not absolute in dictating where active regional centromeres are formed. Instead, ‘epigenetic’ features provide cues that promote the assembly of CENP-A chromatin and the foundations to build kinetochores. Our objective is to understand the nature of these features and how they mediate the replacement of histone H3 with CENP-A to form active centromeres.

We utilize the model organism fission yeast; as with human cells their centromeres are regional, composed of repeats surrounding a central domain with CENP-A chromatin. We discovered that heterochromatin and RNAi components are required to allow establishment of CENP-A chromatin and kinetochore proteins. Synthetic heterochromatin made by tethering Clr4 methyltransferase to DNA binding sites bypasses the need for repetitive DNA and RNAi to form functional centromeres. Ongoing analyses allows identification of key features of heterochromatin required to direct CENP-A chromatin and kinetochore assembly.

Central CENP-A chromatin was thought to be transcriptionally inert but analyses in mutants with defective RNA degradation (e.g. Exosome/Dis3) or which disrupt CENP-A chromatin reveal the presence of transcripts underlying kinetochores (TUKs). We speculate that adjacent heterochromatin constrains RNAPII transcription-coupled events to allow replacement of H3 with CENP-A and are testing this idea.

In related Schizosaccharomyces, gene order adjacent to centromeres is preserved but centromeric DNA has rapidly diverged so that it is not homologous. We are testing the idea that conserved processing of non-coding transcripts from these DNAs allows centromeres to form by a similar manner to that observed in S. pombe.
 

Selected publications:

Bayne, E.H., White, S.A., Kagansky, A., Bijos, D.A., Sanchez- Pulido, L., Hoe, K.L., Kim, D.U., Park, H.O., Ponting, C.P., Rappsilber, J., Allshire, R.C. (2010) Stc1: a critical link between RNAi and chromatin modification required for heterochromatin integrity. Cell 140, 666-677.

Choi, E.S., Stralfors, A., Castillo, A.G., Durand-Dubief, M., Ekwall, K., and Allshire, R.C. (2011) Identification of non-coding transcripts from within CENP-A chromatin at fission yeast centromeres. J. Biol. Chem. Apr 28. [Epub ahead of print].

Rhind, N., Chen, Z., Yassour, M., Thompson, D.A., Haas, B.J., Habib, N., Wapinski, I., Roy, S., Lin, M.F., Heiman, D.I., et al.(2011) Comparative functional genomics of the fission yeasts. Science 332, 930-936.