Heterochromatin represents a significant but mostly unexplored portion of the mammalian genome. Although its composition and architecture are largely unknown it has been implied in several fundamental cellular processes, including cell division, genomic stability and expression.
One of the most conserved hallmarks of heterochromatic regions is a high density of cytosine methylation. Methylation of cytosine residues at CpG dinucleotides is recognized and read in mammals by the methyl CpG binding domain (MBD) protein family. This protein family comprises five members MBD1-4 and MeCP2. All members (except for MBD3) have a conserved MBD domain that targets them to methylated DNA (1
) and consequently accumulate at constitutive heterochromatin in vivo
). We have previously shown that MeCP2, the founding member of the MBD protein family, induces aggregation of pericentric heterochromatin in a dose dependent manner in vivo
with the MBD domain being necessary and sufficient for this function (3
). MeCP2 has also been shown to cluster polynucleosomes in vitro
). Mutations within the MECP2
gene have been linked to the neurological disease Rett syndrome (RTT), a post-natal disorder with an incidence of 1/10 000 female birth (7
). Whereas missense mutations are mostly clustered within the MBD domain, nonsense mutations occur frequently after the MBD domain. We have recently analyzed the effect of 22 MBD missense mutations on MeCP2 ability to bind and cluster heterochromatin and could show that half of the mutants were significantly affected in their heterochromatin binding and two-third of all mutants exhibited significantly decreased clustering ability of pericentric heterochromatin (9
). The majority of mutants tested were affected in both, their binding to and clustering of heterochromatin (9
). As most drastic examples, two mutants (MeCP2 R111G and F155S) showed the lowest binding to heterochromatin resulting in their mislocalization to nucleoli and concomitantly lacked any capability to aggregate pericentric heterochromatin (9
). These findings raised the question whether in such Rett mutations heterochromatin clustering is impaired as a consequence of the inability to bind methylated DNA or whether these mutants are independently affected in heterochromatin clustering.
In this study, we have developed and validated molecular tools to target proteins to heterochromatin regions and follow their impact on heterochromatin composition, architecture and dynamics in living cells. This approach allowed us to discriminate whether Rett mutations affecting chromatin binding consequently decrease the ability of the MeCP2 mutant to aggregate heterochromatin, or alternatively whether forcing Rett mutants to bind to chromocenters can rescue their clustering ability. The addition of an estrogen receptor domain to our targeting tool further enabled us to temporally control the intra-nuclear re-localization of a MeCP2 Rett mutant. Thus, we were able to monitor in living cells the dynamics of large-scale chromatin reorganization. The targeting of Rett mutants to pericentric heterochromatin constitutes a very clear and feasible in vivo assay to reveal the effect (or phenotype) of various Rett mutations on MeCP2 clustering properties independently of chromatin binding. Furthermore, these novel molecular tools are readily applicable to other chromatin regulators to dissect their functional effects on heterochromatin dynamics and organization.