The structure of mouse HP1 suggests a unique mode of single peptide recognition by the shadow chromo domain dimer

The heterochromatin protein 1 (HP1) family of proteins is involved in gene silencing via the formation of heterochromatic structures. They are composed of two related domains: an N-terminal chromo domain and a C-terminal shadow chromo domain. Present results suggest that chromo domains may function as protein interaction motifs, bringing together different proteins in multi-protein complexes and locating them in heterochromatin. We have previously determined the structure of the chromo domain from the mouse HP1beta protein, MOD1. We show here that, in contrast to the chromo domain, the shadow chromo domain is a homodimer. The intact HP1beta protein is also dimeric, where the interaction is mediated by the shadow chromo domain, with the chromo domains moving independently of each other at the end of flexible linkers. Mapping studies, with fragments of the CAF1 and TIF1beta proteins, show that an intact, dimeric, shadow chromo domain structure is required for complex formation.     

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.     

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.     

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.     

Plasmodium falciparum heterochromatin protein 1 binds to tri-methylated histone 3 lysine 9 and is linked to mutually exclusive expression of var genes

Increasing experimental evidence shows a prominent role of histone modifications in the coordinated control of gene expression in the human malaria parasite Plasmodium falciparum. The search for the histone-mark-reading machinery that translates histone modifications into biological processes, such as formation of heterochromatin and antigenic variation is of foremost importance. In this work, we identified the first member of a histone modification specific recognition protein, an orthologue of heterochromatin protein 1 (PfHP1). Analysis of the PfHP1 amino-acid sequence revealed the presence of the two characteristic HP1 domains: a chromodomain (CD) and a chromo shadow domain (CSD). Recombinant CD binds to di- and tri-methylated lysine 9 from histone H3, but not to unmodified or methylated histone H3 in lysine 4. PfHP1 is able to interact with itself to form dimers, underlying its potential role in aggregating nucleosomes to form heterochromatin. Antibodies raised against PfHP1 detect this molecule in foci at the perinuclear region. ChIP analysis using anti-PfHP1 shows that this protein is linked to heterochromatin of subtelomeric non-coding repeat regions and monoallelic expression of the major virulence var gene family. This is the first report implicating an HP1 protein in the control of antigenic variation of a protozoan parasite.     

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.     

嗜热四膜虫Tcd1蛋白功能结构域的突变分析

有性生殖过程特异表达的Tcd1在四膜虫大核基因组重排和修复中起到重要调节作用, Tcd1含有进化中保守的chromodomain(CD)结构域以及chromo shadow domain (CSD)结构域, 然而不同结构域的具体功能并不清楚。本研究首先鉴定了TCD1基因仅含有1个CD结构域的选择性剪切本TCD1β,免疫荧光定位表明,Tcd1β定位在胞质中。定点突变Tcd1中CD1内159位色氨酸为丙氨酸, Tcd1W159A点状定位在亲本大核,然后转移到新发育的大核上围绕核膜致密分布,发育的晚期逐步消失。进一步突变CD2中437位的色氨酸为丙氨酸后,Tcd1W159AW437A在早期亲本大核形成异常的环状分布, 而在新发育大核中形成点状分布。截短CSD结构域C端35个氨基酸后, Tcd1Δ35在亲本大核和新大核上的定位不受影响。 然而,截短CSD结构域C端的53个氨基酸后, Tcd1Δ53定位在细胞质中, 无核内定位。结果表明, Tcd1中的CD1和CD2结构域决定了Tcd1蛋白在核内的分布, CSD结构域决定了Tcd1入核转运, Tcd1的3个功能结构域共同决定了Tcd1在四膜虫中的功能定位。     

The Levels of the bancal Product, a Drosophila Homologue of Vertebrate hnRNP K Protein, Affect Cell Proliferation and Apoptosis in Imaginal Disc Cells

We have characterized the Drosophila bancal gene, which encodes a Drosophila homologue of the vertebrate hnRNP K protein. The bancal gene is essential for the correct size of adult appendages. Reduction of appendage size in bancal mutant flies appears to be due mainly to a reduction in the number of cell divisions in the imaginal discs. Transgenes expressing Drosophila or human hnRNP K are able to rescue weak bancal phenotype, showing the functional similarity of these proteins in vivo. High levels of either human or Drosophila hnRNP K protein in imaginal discs induces programmed cell death. Expression of the antiapoptotic P35 protein suppresses this phenotype in the eye, suggesting that apoptosis is the major cellular defect caused by overexpression of K protein. Finally, the human K protein acts as a negative regulator of bancal gene expression. We propose that negative autoregulation limits the level of Bancal protein produced in vivo.     

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 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.     

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.     

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.     

Specificity of the HP1 chromo domain for the methylated N-terminus of histone H3

Recent studies show that heterochromatin-associated protein-1 (HP1) recognizes a 'histone code' involving methylated Lys9 (methyl-K9) in histone H3. Using in situ immunofluorescence, we demonstrate that methyl-K9 H3 and HP1 co-localize to the heterochromatic regions of Drosophila polytene chromosomes. NMR spectra show that methyl-K9 binding of HP1 occurs via its chromo (chromosome organization modifier) domain. This interaction requires methyl-K9 to reside within the proper context of H3 sequence. NMR studies indicate that the methylated H3 tail binds in a groove of HP1 consisting of conserved residues. Using fluorescence anisotropy and isothermal titration calorimetry, we determined that this interaction occurs with a K(D) of approximately 100 microM, with the binding enthalpically driven. A V26M mutation in HP1, which disrupts its gene silencing function, severely destabilizes the H3-binding interface, and abolishes methyl-K9 H3 tail binding. Finally, we note that sequence diversity in chromo domains may lead to diverse functions in eukaryotic gene regulation. For example, the chromo domain of the yeast histone acetyltransferase Esa1 does not interact with methyl- K9 H3, but instead shows preference for unmodified H3 tail.     

The fat tumor suppressor gene in Drosophila encodes a novel member of the cadherin gene superfamily

Recessive lethal mutations in the fat locus of Drosophila cause hyperplastic, tumor-like overgrowth of larval imaginal discs, defects in differentiation and morphogenesis, and death during the pupal stage. Clones of mutant cells induced by mitotic recombination demonstrate that the overgrowth phenotype is cell autonomous. Here we show that the fat locus encodes a novel member of the cadherin gene superfamily: an enormous transmembrane protein of over 5000 amino acids with a putative signal sequence, 34 tandem cadherin domains, four EGF-like repeats, a transmembrane domain, and a novel cytoplasmic domain. Two recessive lethal alleles contain alterations in the fat coding sequence, and the dominant fat allele, Gull, contains an insertion of a transposable element in the 33rd cadherin domain. Thus, this novel member of the cadherin gene superfamily functions as a tumor suppressor gene and is required for correct morphogenesis.