N-Terminal Phosphorylation of HP1α Promotes Its Chromatin Binding

Abstract

The phosphorylation of heterochromatin protein 1 (HP1) has been previously described in studies of mammals, but the biological implications of this modification remain largely elusive. Here, we show that the N-terminal phosphorylation of HP1␣ plays a central role in its targeting to chromatin. Recombinant HP1␣ prepared from mammalian cultured cells exhibited a stronger binding affinity for K9-methylated histone H3 (H3K9me) than that produced in Escherichia coli. Biochemical analyses revealed that HP1␣ was multiply phosphorylated at N-terminal serine residues (S11-14) in human and mouse cells and that this phosphorylation enhanced HP1␣'s affinity for H3K9me. Importantly, the N-terminal phosphorylation appeared to facilitate the initial binding of HP1␣ to H3K9me by mediating the interaction between HP1␣ and a part of the H3 tail that was distinct from the methylated K9. Unphosphorylatable mutant HP1␣ exhibited severe heterochromatin localization defects in vivo, and its prolonged expression led to increased chromosomal instability. Our results suggest that HP1␣'s N-terminal phosphorylation is essential for its proper targeting to heterochromatin and that its binding to the methylated histone tail is achieved by the cooperative action of the chromodomain and neighboring posttranslational modifications.The formation of the higher-order chromatin structure, the so-called heterochromatin, is critical for genomic stability and transcriptional silencing (19,21,30). Heterochromatin protein 1 (HP1) is a nonhistone chromosomal protein that was initially discovered in Drosophila melanogaster as a factor that is enriched at highly condensed, transcriptionally inert regions of polytene chromosomes (24,25). HP1 is an evolutionarily conserved molecule whose homologues and isoforms have been previously identified in a wide range of eukaryotic organisms (9). As well as being an architectural component of heterochromatin, HP1 is known to play crucial roles in heterochromatin-mediated gene silencing. Profound effects of HP1 deletion, mutation, or overexpression on transcriptional silencing have been previously demonstrated in Drosophila (11, 12), Schizosaccharomyces pombe (13), and mammals (17). Moreover, an ever-expanding list of HP1-interacting proteins indicates that HP1 may also be involved in the regulation of several biological processes, including transcriptional activation, faithful chromosome segregation, and DNA damage repair (8,19,21,30).Structurally, all HP1 homologues possess two functionally distinct globular domains, the N-terminal chromodomain (CD) (39) and the C-terminal chromoshadow domain (CSD) (1), which are linked by an unstructured hinge region. The CD recognizes and interacts with methylated lysine 9 of the histone H3 N-terminal tail, which is a characteristic modification in heterochromatic regions (4,27,35). The CSD, on the other hand, is responsible for dimer formation and is involved in HP1's interactions with a large variety of proteins (5,44,46). Because of its abilities to bind directly to chromatin an...

Phosphorylation–dephosphorylation cycle of HP1α governs accurate mitotic progression

“…40,[50][51][52] We have recently demonstrated that mammalian HP1α is a mitotic substrate for NDR (nuclear Dbf2-related) kinase and undergoes a hinge specific phosphorylation as the cells progress from G 2 to mitosis. 27 Constitutive phosphorylation within the N terminus 12,27 of HP1α strengthens its binding to chromatin. 12 We have observed that depletion of NDR1 causes the reduction of hinge-specific phosphorylation of HP1α and impaired Sgo1 loading to mitotic centromere.…”

“…6,11 Phosphorylation at the N-terminal serine residues (S11-14) in human and mouse cells have been implicated in enhancing the binding affinity of HP1α for the H3K9 trimethyl chromatin mark. 12 On the other hand, casein-kinase-II (CKII)-mediated phosphorylation of HP1β at Thr-51 occurs during DNA damage response, and this modification facilitates the release of HP1-β from the chromatin. 13 The HP1γ isoform phosphorylated at Ser-83 demonstrates impaired silencing activity and is associated with transcriptional elongation.…”

Physical and Functional Interactions between the Histone H3K4 Demethylase KDM5A and the Nucleosome Remodeling and Deacetylase (NuRD) Complex

“…Other cDNAs were introduced into pCDNA4/TO/3F/puro, a pcDNA4/TO derivative containing the 3ϫFLAG tag sequences, and the puromycin resistance gene. To express full-length or truncated proteins in HEK293T cells, corresponding cDNAs were introduced into pFLAG-C1 (36). Plasmids were introduced into human cultured cells using the Polyfect transfection reagent (Qiagen) or Lipofectamine 2000 reagent (Invitrogen).…”

Unliganded progesterone receptor-mediated targeting of an RNA-containing repressive complex silences a subset of hormone-inducible genes

“…However, we cannot rule out that post-translational modifications of HP1g itself influences the interactions with partner proteins, as phosphorylation of HP1g on S83 impairs its silencing activity and serves as a marker for transcriptional elongation (Lomberk et al 2006). In addition, phosphorylation of HP1a (S11-S14) and HP1b (S51) plays a critical role in modulating efficient binding to the methylated histone H3 tail and regulates mobilization during the initiation of the DNA damage response (Ayoub et al 2008;Hiragami-Hamada et al 2011). Nevertheless, the biological functions of HP1 phosphorylation in mammals remain largely unclear.…”