Heterochromatin is localized at the nuclear periphery in most eukaryotic organisms. A distinct compartmentalization of euchromatin and heterochromatin occurs upon cellular differentiation. The functional relevance of sequestration of chromatin at the nuclear periphery is unknown. While a variety of studies have begun to identify factors involved in anchoring chromatin at the nuclear periphery, the functional characterization of this mechanism, particularly in differentiated tissues, has yet to be described. In C. elegans embryos, we have identified the histone H3K9 methylation reader CEC-4 as a factor that maintains chromatin positioning at the nuclear periphery. CEC-4 binds selectively histone H3 bearing mono-, di- or tri-methylation on lysine 9 and is localized independently of chromatin to the nuclear periphery. In cec-4 mutant strains the anchoring of chromatin is perturbed both for an integrated heterochromatic array and for endogenous chromosomes, although gene expression is not affected. Beyond larval stages, additional anchoring pathways compensate for the loss of CEC-4. We carried out a forced muscle cell transdifferentiation assay by heat-shock induction of the MyoD homologue, HLH-1, to address the function of reduced anchoring in early embryos. cec-4 mutant embryos had the ability to continue developing into a larva-like stage, unlike the wild-type embryos. Therefore, we propose that the anchorage of chromatin at the nuclear periphery in early C. elegans embryos helps to stabilize cell fate decisions.
CEC-4 is expressed throughout development with variable expression levels across differentiated tissues and muscle cells in larvae and adult worms. Muscles exhibit particularly high levels of CEC-4. Live cell imaging of cec-4 mutants harboring a heterochromatic reporter suggests that CEC-4 contributes to chromatin anchoring in differentiated muscle and gut cells. H3K9 methylation has been implicated in nuclear organization across species, although CEC-4 is not conserved. We are currently investigating the additional mechanisms that tether chromatin to the nuclear periphery in order to find other factors that are involved. RNAi of all known nuclear envelope proteins in worms, individually and in pairwise combinations, did not disrupt CEC-4 localization. Mutations that act synergistically with CEC-4 to delocalize chromatin include other histone modifying enzymes and readers. We are currently exploiting BioID (proximity-dependent biotin identification) to screen for protein- protein interactions of CEC-4 to characterize its role in perinuclear anchoring throughout development.