How centromeres are specified: the interplay between heterochromatin, CENP-A chromatin, and kinetochore assembly.
Our overall objective is to understand the structure and function of centromeric chromatin and in particular the relationship between heterochromatin and the kinetochore.
How are centromeres made?
How is the site for building a centromere on a chromosome chosen?
Robin Allshire explains his research in this short movie:
This movie from Science 2010 discusses : What is Epigenetics?
The centromere is the chromosomal site where kinetochores assemble, allowing chromosomes to attach to spindle microtubules and segregate. Human centromeres are recognizable as the primary constriction. This distinct architecture is partly due to packaging of centromeric DNA in unusual chromatin – termed heterochromatin – that remains condensed throughout the cell cycle. Genes placed within heterochromatin are transcriptionally silenced. Image from BetaBlog.
Specific post-translational modifications are a characteristic of centromeric heterochromatin. For instance, methylation of lysine 9 on histone H3 (H3K9me2) allows binding of Heterochromatin protein 1 (HP1) via its chromodomain. Embedded within heterochromatin, the kinetochore is assembled on specialized nucleosomes where histone H3 is replaced by the centromere specific histone H3-like protein CENP-A to form ‘CENP-A chromatin’. This CENP-A chromatin is critical in determining the site of kinetochore assembly.
It is not known how the site of CENP-A assembly is selected or how CENP-A is deposited and maintained. Is it determined by DNA sequence? Specific DNA sequences, such as alpha satellite repeats, may be a preferred substrate for kinetochore assembly but it is not an absolute requirement. The prevailing view is that an epigenetic component may be involved in establishing and maintaining the site of kinetochore assembly. CENP-A is an excellent candidate since it is only deposited and maintained at active centromeres.
There are many outstanding questions, including: What is the mechanism of heterochromatin formation on centromeric repeats and in what ways does it contribute to centromere functions? How is the site of CENP-A chromatin and thus kinetochore assembly chosen and maintained? Does flanking heterochromatin play a role in specifying the site of CENP-A chromatin assembly?
To understand the process of centromere-kinetochore formation we utilise the fission yeast, Schizosaccharomyces pombe, as a model organism. Fission yeast centromeres are complex, with repetitive outer repeat DNA flanking the central core domain.
We first discovered that expression of marker genes placed within the central or outer repeat domains is silenced. Silencing remains the bedrock of our research and we utilise silencing as a read out to identify factors involved in heterochromatin formation or kintochore assembly and develop assays for their role in vivo. Silencing reflects the fact that both the outer repeats and central domain are assembled in chromatin that is incompatible with normal gene expression. Our analyses showed that the central and outer repeat domains are distinct: different factors affect transcriptional silencing and different proteins associate.
CENP-A/Cnp1 and other kinetochore proteins associate exclusively with the central domain. In contrast, the outer repeats are packaged in heterochromatin with H3K9me2 nucleosomes allowing binding of HP1-like chromodomain proteins (e.g. Swi6). This overall arrangement of CENP-A chromatin flanked by heterochromatin is astonishingly similar to that of metazoan centromeres. Thus fission yeast provides an excellent model system in which to dissect and understand the mechanism of centromeric heterochromatin formation and kinetochore assembly.
Our research focuses on inter-related areas:
- histone modifications associated with centromeric chromatin.
- how the RNAi pathway brings about heterochromatin formation and understanding the role of heterochromatin in centromere-kinetochore function.
- how the centromere specific H3 variant CENP-A is delivered to and assembled into centromeric chromatin.
- investigating the relationship between heterochromatin, CENP-A and kinetochore assembly.
- utilizing synthethic biological approaches to investigate centromere function.