The HP1 chromo shadow domain binds a consensus peptide pentamer

Heterochromatin-associated protein 1 (HP1) is thought to affect chromatin structure through interactions with other proteins in heterochromatin. Chromo domains located near the amino (amino chromo) and carboxy (chromo shadow) termini of HP1 may mediate such interactions, as suggested by domain swapping, in vitro binding and 3D structural studies . Several HP1-associated proteins have been reported, providing candidates that might specifically complex with the chromo domains of HP1. However, such association studies provide little mechanistic insight and explore only a limited set of potential interactions in a largely non-competitive setting. To determine how chromo domains can selectively interact with other proteins, we probed random peptide phage display libraries using chromo domains from HP1. Our results demonstrate that a consensus pentapeptide is suffident for specific interaction with the HP1 chromo shadow domain. The pentapeptide is found in the amino acid sequence of reported HP1-associated proteins, including the shadow domain itself. Peptides that bind the shadow domain also disrupt shadow domain dimers. Our results suggest that HP1 dimerization, which is thought to mediate heterochromatin compaction and cohesion, occurs via pentapeptide binding. In general, chromo domains may function by avidly binding short peptides at the surface of chromatin-associated proteins.     

Structural basis of HP1/PXVXL motif peptide interactions and HP1 localisation to heterochromatin

HP1 family proteins are adaptor molecules, containing two related chromo domains that are required for chromatin packaging and gene silencing. Here we present the structure of the chromo shadow domain from mouse HP1beta bound to a peptide containing a consensus PXVXL motif found in many HP1 binding partners. The shadow domain exhibits a novel mode of peptide recognition, where the peptide binds across the dimer interface, sandwiched in a beta-sheet between strands from each monomer. The structure allows us to predict which other shadow domains bind similar PXVXL motif-containing peptides and provides a framework for predicting the sequence specificity of the others. We show that targeting of HP1beta to heterochromatin requires shadow domain interactions with PXVXL-containing proteins in addition to chromo domain recognition of Lys-9-methylated histone H3. Interestingly, it also appears to require the simultaneous recognition of two Lys-9-methylated histone H3 molecules. This finding implies a further complexity to the histone code for regulation of chromatin structure and suggests how binding of HP1 family proteins may lead to its condensation.     

Heterochromatin protein 1 binds to nucleosomes and DNA in vitro

Heterochromatin protein 1 (HP1) is a nonhistone chromosomal protein primarily associated with the pericentric heterochromatin and telomeres in Drosophila. The molecular mechanism by which HP1 specifically recognizes and binds to chromatin is unknown. The purpose of this study was to test whether HP1 can bind directly to nucleosomes. HP1 binds nucleosome core particles and naked DNA. HP1-DNA complex formation is length-dependent and cooperative but relatively sequence-independent. We show that histone H4 amino-terminal peptides bind to monomeric and dimeric HP1 in vitro. Acetylation of lysine residues had no significant effect on in vitro binding. The C-terminal chromo shadow domain of HP1 specifically binds H4 N-terminal peptide. Neither the chromo domain nor chromo shadow domain alone binds DNA; intact native HP1 is required for such interactions. Together, these observations suggest that HP1 may serve as a cross-linker in chromatin, linking nucleosomal DNA and nonhistone protein complexes to form higher order chromatin structures.     

Diversification of HP1‐like Chromo Domain Proteins in Tetrahymena thermophila

Proteins that possess a chromo domain are well‐known for their roles in heterochromatin assembly and maintenance. The Heterochromatin Protein 1 (HP1) family, with a chromo domain and carboxy‐terminal chromo shadow domain, targets heterochromatin through interaction with histone H3 methylated on lysine 9 (H3K9me2/3). The structural and functional diversity of these proteins observed in both fission yeast and metazoans correlate with chromatin specialization. To expand these studies, we examined chromo domain proteins in the ciliate Tetrahymena thermophila, which has functionally diverse and developmentally regulated heterochromatin domains. We identified thirteen proteins similar to HP1. Together they possess only a fraction of the possible chromo domain subtypes and most lack a recognizable chromo shadow domain. Using fluorescence microscopy to track chromatin localization of tagged proteins through the life cycle, we show evidence that in T. thermophila this family has diversified with biological roles in RNAi‐directed DNA elimination, germline genome structure, and somatic heterochromatin. Those proteins with H3K27me3 binding sequence characteristics localize to chromatin in mature nuclei, whereas those with H3K9me2/3 binding characteristics localize to developing nuclei undergoing DNA elimination. Findings point to an expanded and diversified family of chromo domain proteins that parallels heterochromatin diversity in ciliates.     

The hinge and chromo shadow domain impart distinct targeting of HP1-like proteins

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.     

Crystal structure of the HP1-EMSY complex reveals an unusual mode of HP1 binding

Heterochromatin protein-1 (HP1) plays an essential role in both the assembly of higher-order chromatin structure and epigenetic inheritance. The C-terminal chromo shadow domain (CSD) of HP1 is responsible for homodimerization and interaction with a number of chromatin-associated nonhistone proteins, including EMSY, which is a BRCA2-interacting protein that has been implicated in the development of breast and ovarian cancer. We have determined the crystal structure of the HP1beta CSD in complex with the N-terminal domain of EMSY at 1.8 A resolution. Surprisingly, the structure reveals that EMSY is bound by two HP1 CSD homodimers, and the binding sequences differ from the consensus HP1 binding motif PXVXL. This structural information expands our understanding of HP1 binding specificity and provides insights into interactions between HP1 homodimers that are likely to be important for heterochromatin formation.     

Localization and organization of protein factors involved in chromosome inheritance in Dictyostelium discoideum

Heterochromatin protein 1 (HP1) proteins are highly conserved heterochromatin components required for genomic integrity. We have previously shown that the two HP1 isoforms expressed in Dictyostelium, HcpA and HcpB, are mainly localized to (peri-)centromeric heterochromatin and have largely overlapping functions. However, they cause distinct phenotypes when overexpressed. We show here that these isoforms display quantitative differences in dimerization behavior. Dimerization preference, as well as the mutant phenotype in overexpression strains, depends on the C-terminus containing the hinge and chromo shadow domains. Both Hcp proteins are targeted to distinct subnuclear regions by different chromo shadow domain-dependent and -independent mechanisms. In addition, both proteins bind to DNA and RNA in vitro and binding is independent of the chromo shadow domain. Thus, this DNA and/or RNA binding activity may contribute to protein targeting. To further characterize heterochromatin, we cloned the Dictyostelium homolog of the origin recognition complex subunit 2 (OrcB). OrcB localizes to distinct subnuclear foci that were also targeted by HcpA. In addition, it is associated with the centrosome throughout the cell cycle. The results indicate that, similar to Orc2 homologs from other organisms, it is required for different processes in chromosome inheritance.     

Functional domain analysis of human HP1 isoforms in Drosophila

Three subtypes of HP1, a conserved non-histone chromosomal protein enriched in heterochromatin, have been identified in humans, HP1alpha, beta and gamma. In the present study, we utilized a Drosophila system to characterize human HP1 functions. Over-expression of HP1beta in eye imaginal discs caused abnormally patterned eyes, with reduced numbers of ommatidia, and over-expression of HP1gamma in wing imaginal discs caused abnormal wings, in which L4 veins were gapped. These phenotypes were specific to the HP1 subtypes and appear to reflect suppressed gene expression. To determine the molecular domains of HP1 required for each specific phenotype, we constructed a series of chimeric molecules with HP1beta and HP1gamma. Our data show that the C-terminal chromo shadow domain (CSD) of HP1gamma is necessary for HP1gamma-type phenotype, whereas for the HP1beta-type phenotype both the chromo domain and the CSD are required. These results suggest human HP1 subtypes use different domains to suppress gene expression in Drosophila cells.     

Coordinated methyl and RNA binding is required for heterochromatin localization of mammalian HP1alpha

In mammalian cells, as in Schizosaccharomyces pombe and Drosophila, HP1 proteins bind histone H3 tails methylated on lysine 9 (K9). However, whereas K9-methylated H3 histones are distributed throughout the nucleus, HP1 proteins are enriched in pericentromeric heterochromatin. This observation suggests that the methyl-binding property of HP1 may not be sufficient for its heterochromatin targeting. We show that the association of HP1α with pericentromeric heterochromatin depends not only on its methyl-binding chromo domain but also on an RNA-binding activity present in the hinge region of the protein that connects the conserved chromo and chromoshadow domains. Our data suggest the existence of complex heterochromatin binding sites composed of methylated histone H3 tails and RNA, with each being recognized by a separate domain of HP1α.     

Positive selection drives the evolution of rhino, a member of the heterochromatin protein 1 family in Drosophila

Heterochromatin comprises a significant component of many eukaryotic genomes. In comparison to euchromatin, heterochromatin is gene poor, transposon rich, and late replicating. It serves many important biological roles, from gene silencing to accurate chromosome segregation, yet little is known about the evolutionary constraints that shape heterochromatin. A complementary approach to the traditional one of directly studying heterochromatic DNA sequence is to study the evolution of proteins that bind and define heterochromatin. One of the best markers for heterochromatin is the heterochromatin protein 1 (HP1), which is an essential, nonhistone chromosomal protein. Here we investigate the molecular evolution of five HP1 paralogs present in Drosophila melanogaster. Three of these paralogs have ubiquitous expression patterns in adult Drosophila tissues, whereas HP1D/rhino and HP1E are expressed predominantly in ovaries and testes respectively. The HP1 paralogs also have distinct localization preferences in Drosophila cells. Thus, Rhino localizes to the heterochromatic compartment in Drosophila tissue culture cells, but in a pattern distinct from HP1A and lysine-9 dimethylated H3. Using molecular evolution and population genetic analyses, we find that rhino has been subject to positive selection in all three domains of the protein: the N-terminal chromo domain, the C-terminal chromo-shadow domain, and the hinge region that connects these two modules. Maximum likelihood analysis of rhino sequences from 20 species of Drosophila reveals that a small number of residues of the chromo and shadow domains have been subject to repeated positive selection. The rapid and positive selection of rhino is highly unusual for a gene encoding a chromosomal protein and suggests that rhino is involved in a genetic conflict that affects the germline, belying the notion that heterochromatin is simply a passive recipient of "junk DNA" in eukaryotic genomes.     

Pericentromeric heterochromatin domains are maintained without accumulation of HP1

The heterochromatin protein 1 (HP1) family is thought to be an important structural component of heterochromatin. HP1 proteins bind via their chromodomain to nucleosomes methylated at lysine 9 of histone H3 (H3K9me). To investigate the role of HP1 in maintaining heterochromatin structure, we used a dominant negative approach by expressing truncated HP1alpha or HP1beta proteins lacking a functional chromodomain. Expression of these truncated HP1 proteins individually or in combination resulted in a strong reduction of the accumulation of HP1alpha, HP1beta, and HP1gamma in pericentromeric heterochromatin domains in mouse 3T3 fibroblasts. The expression levels of HP1 did not change. The apparent displacement of HP1alpha, HP1beta, and HP1gamma from pericentromeric heterochromatin did not result in visible changes in the structure of pericentromeric heterochromatin domains, as visualized by DAPI staining and immunofluorescent labeling of H3K9me. Our results show that the accumulation of HP1alpha, HP1beta, and HP1gamma at pericentromeric heterochromatin domains is not required to maintain DAPI-stained pericentromeric heterochromatin domains and the methylated state of histone H3 at lysine 9 in such heterochromatin domains.     

Functional analysis of the chromo domain of HP1.

Heterochromatin protein 1 (HP1) is a non-histone chromosomal protein in Drosophila with dosage-dependent effects on heterochromatin-mediated gene silencing. An evolutionarily conserved amino acid sequence in the N-terminal half of HP1 (the 'chromo domain') shares > 60% sequence identity with a motif found in the Polycomb protein, a silencer of homeotic genes. We report here that point mutations in the HP1 chromo domain abolish the ability of HP1 to promote gene silencing. We show that the HP1 chromo domain, like the Polycomb chromo domain, has chromosome binding activity, but to distinct chromosomal sites. We constructed a chimeric HP1-Polycomb protein, consisting of the chromo domain of Polycomb in the context of HP1, and show that it binds to both heterochromatin and Polycomb binding sites in polytene chromosomes. In flies expressing chimeric HP1-Polycomb protein, endogenous HP1 is mislocalized to Polycomb binding sites, and endogenous polycomb is misdirected to the heterochromatic chromocenter, suggesting that both proteins are recruited to their distinct chromosomal binding sites through protein-protein contacts. Chimeric HP1-Polycomb protein expression in transgenic flies promotes heterochromatin-mediated gene silencing, supporting the view that the chromo domain homology reflects a common mechanistic basis for homeotic and heterochromatic silencing.     

Self-interaction of heterochromatin protein 1 is required for direct binding to histone methyltransferase,SUV39H1

Heterochromatin protein 1 (HP1) binds to the nucleosome via a methylated lysine residue 9 of histone H3 which is catalyzed by a histone methyltransferase such as SUV39H1. Although co-localization of HP1 and SUV39H1 has been evident in immunostaining and immunoprecipitation experiments, direct protein-protein interactions have remained to be characterized. We examined interactions between mouse HP1 alpha (mHP1 alpha) and SUV39H1 in yeast and in vitro. A yeast two-hybrid and a glutathione S-transferase pull-down study indicated that the chromo shadow domain of mHP1 alpha directly interacts with the N-terminal 39 amino acid stretch of SUV39H1. The IY165/168EE mutation in the chromo shadow domain of mHP1 alpha abrogated a self-interaction and this mutant did not interact with SUV39H1. The 13-mer peptide containing a consensus sequence for binding to the dimer surface formed by the chromo shadow domains inhibited interaction between mHP1 alpha and SUV39H1. It seems that self-interaction through the chromo shadow domain of HP1 is crucial for recruitment of SUV39H1 onto nucleosomes.     

Functional domain structure of human heterochromatin protein HP1(Hsalpha): involvement of internal DNA-binding and C-terminal self-association domains in the formation of discrete dots in interphase nuclei

Human heterochromatin protein HP1(Hsalpha) possesses two evolutionarily conserved regions in the N- and C-terminal halves, so-called chromo and chromo-shadow domains, and DNA-binding domain in the internal non-conserved region. Here, to examine its in vivo properties, we expressed HP1(Hsalpha) as a fusion product with green fluorescent protein in human cells. HP1(Hsalpha) was observed to form discrete dots in interphase nuclei and to localize in the centromeric region of metaphase chromosomes by fluorescence microscopy. Interestingly, this dot-forming activity was also found in the N-terminal half retaining the chromo and DNA-binding domains and in the C-terminal chromo-shadow domain. However, the chromo domain alone stained nuclei homogeneously. To correlate this dot-forming activity with self-associating activity in vitro, the chromo and chromo-shadow domain peptides were independently expressed in Escherichia coli, affinity purified, and chemically cross-linked with glutaraldehyde. In a SDS-polyacrylamide gel, the former mainly produced a dimer, while the latter produced a ladder of bands up to a tetramer. When passed through a gel filtration column in a native state, these peptides were exclusively separated as a dimer and a tetramer, respectively. These results suggested that the internal DNA-binding and C-terminal chromo-shadow domains are both involved in heterochromatin formation in vivo.     

In vivo HP1 targeting causes large-scale chromatin condensation and enhanced histone lysine methylation

Changes in chromatin structure are a key aspect in the epigenetic regulation of gene expression. We have used a lac operator array system to visualize by light microscopy the effect of heterochromatin protein 1 (HP1) alpha (HP1alpha) and HP1beta on large-scale chromatin structure in living mammalian cells. The structure of HP1, containing a chromodomain, a chromoshadow domain, and a hinge domain, allows it to bind to a variety of proteins. In vivo targeting of an enhanced green fluorescent protein-tagged HP1-lac repressor fusion to a lac operator-containing, gene-amplified chromosome region causes local condensation of the higher-order chromatin structure, recruitment of the histone methyltransferase SETDB1, and enhanced trimethylation of histone H3 lysine 9. Polycomb group proteins of both the HPC/HPH and the EED/EZH2 complexes, which are involved in the heritable repression of gene activity, are not recruited to the amplified chromosome region by HP1alpha and HP1beta in vivo targeting. HP1alpha targeting causes the recruitment of endogenous HP1beta to the chromatin region and vice versa, indicating a direct interaction between the two HP1 homologous proteins. Our findings indicate that HP1alpha and HP1beta targeting is sufficient to induce heterochromatin formation.