The Hinge and Chromo Shadow Domain Impart Distinct Targeting of HP1-Like Proteins

Abstract

Drosophila heterochromatin-associated protein 1 (HP1) is an abundant component of heterochromatin, a highly condensed compartment of the nucleus that comprises a major fraction of complex genomes. Some organisms have been shown to harbor multiple HP1-like proteins, each exhibiting spatially distinct localization patterns within interphase nuclei. We have characterized the subnuclear localization patterns of two newly discovered Drosophila HP1-like proteins (HP1b and HP1c), comparing them with that of the originally described fly HP1 protein (here designated HP1a). While HP1a targets heterochromatin, HP1b localizes to both heterochromatin and euchromatin and HP1c is restricted exclusively to euchromatin. All HP1-like proteins contain an amino-terminal chromo domain, a connecting hinge, and a carboxyl-terminal chromo shadow domain. We expressed truncated and chimeric HP1 proteins in vivo to determine which of these segments might be responsible for heterochromatin-specific and euchromatin-specific localization. Both the HP1a hinge and chromo shadow domain independently target heterochromatin, while the HP1c chromo shadow domain is implicated solely in euchromatin localization. Comparative sequence analyses of HP1 homologs reveal a conserved sequence block within the hinge that contains an invariant sequence (KRK) and a nuclear localization motif. This block is not conserved in the HP1c hinge, possibly accounting for its failure to function as an independent targeting segment. We conclude that sequence variations within the hinge and shadow account for HP1 targeting distinctions. We propose that these targeting features allow different HP1 complexes to be distinctly sequestered in organisms that harbor multiple HP1-like proteins.

Drosophila HP1c isoform interacts with the zinc-finger proteins WOC and Relative-of-WOC to regulate gene expression

“…ROW also contains several variant PxVxL motifs, suggesting that it might also bind directly to HP1c. In agreement with these observations, euchromatic localization of HP1c depends on the C-terminal chromo-shadow domain (Smothers and Henikoff 2001), strongly suggesting that it mediates interaction with WOC and ROW.…”

“…1B), indicating that HP1c preferentially localizes to euchromatin being mostly excluded from heterochromatin. Similar results were reported earlier by others (Smothers and Henikoff 2001). Similarly, in polytene chromosomes, HP1a and HP1c also show drastically different patterns of localization, as HP1a localizes to the heterochromatic chromocentre (Fig.…”

“…Altogether, these observations indicate that recognition of H3K9me2,3 is not a major determinant of the association of HP1c with chromatin in vivo. Consistent with this interpretation, replacing the chromodomain of HP1c by that of HP1a does not alter its chromosomal distribution (Smothers and Henikoff 2001). The interaction with WOC and ROW does not appear to hinder the chromodomain from binding H3K9me2,3 since overexpression of HP1c-lacI brings both HP1c and WOC to heterochromatin.…”

“…That binding of HP1c to chromatin is strictly dependent on the zinc-finger proteins WOC and ROW was unexpected, as HP1c contains an N-terminal chromodomain that, being homologous to that of HP1a, could mediate binding to H3K9me2,3 (Smothers and Henikoff 2001). In fact, all residues directly involved in this interaction are conserved in the chromodomain of HP1c.…”

“…For instance, in Drosophila, three of the five HP1 isoforms (HP1a, HP1b, and HP1c) are ubiquitously expressed, while the other two (HP1d/Rhino and HP1e) are predominantly expressed in the germline (Vermaak et al 2005). Moreover, ubiquitously expressed HP1 isoforms show differential chromosomal distributions, as HP1a is mainly associated to heterochromatin, while HP1c is excluded from centromeric heterochromatin and HP1b is found both at euchromatic and heterochromatic domains (Smothers and Henikoff 2001). A similar situation is observed in mammals, where the patterns of localiza-tion of the three HP1 isoforms (HP1␣, HP1␤, and HP1␥) overlap only partially and show differential dynamics during differentiation and cell cycle progression (Minc et al 1999;Hayakawa et al 2003;Dialynas et al 2007).…”