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C- 0 oqulum Does heterochromatin protein ~ always follow code? Yuhong Li*, Dawn A. Kirschmannt, and Lori L. Wallrath*t Departments of *Biochemistry and "Anatomy and Cell Biology, University of Iowa, Iowa City, IA 52242 Heterochromatin protein 1 (HP1) is a conserved chromosomal protein that participates in chromatin packaging and gene silenc- ing. A loss of HP1 leads to lethality- in Drosophila and correlates with metastasis in human breast cancer cells. On Drosophila polytene chromosomes HP1 is localized to centric regions, telo- meric regions, in a banded pattern along the fourth chromosome, and at many sites scattered throughout the euchromatic arms. Recently, one mechanism of HP1 chromosome association was revealed; the amino-terminal chrome domain of HP1 interacts with methylated Iysine nine of histone H3, consistent with the histone code hypothesis. Compelling data support this mechanism of HP1 association at centric regions. Is this the only mechanism by which HP1 associates with chromosomes? Interest is now shifting toward the role of HP1 within euchromatic domains. Accumulating evi- dence in Drosophila and mammals suggests that HP1 associates with chromosomes through interactions with nonhistone chromo- somal proteins at locations other than centric heterochromatin. Does HP1 play a similar role in chromatin packaging and gene regulation at these sites as it does in centric heterochromatin? Does HP1 associate with the same proteins at these sites as it does in centric heterochromatin? A first step toward answering these questions is the identification of sequences associated with HP1 within euchromatic domains. Such sequences are likely to include HP1 "target genes" whose discovery will aid in our understanding of HP1 lethality in Drosophila and metastasis of breast cancer cells. n eukaryotes, there are two major types of chromatin: hetero- ~ chromatin and euchromatin (1~. Heterochromatin corre- sponds to the relatively gene-poor, late-replicating, repetitious sequences found near centric and telomeric locations. In con- trast, euchromatin replicates relatively early in the cell cycle and contains single copy sequences, including the majority of genes. Both euchromatin and heterochromatin are packaged into nu- cleosomes, the fundamental packaging unit consisting of a histone octamer. Euchromatin and heterochromatin can be distinguished by specific histone tail modifications. In general, the histone tails in heterochromatin are relatively hypoacety- lated; however, acetylation of lysine twelve of histone H4 is a distinguishing mark for heterochromatin (2-4~. In contrast, histone H3 and H4 tails found in euchromatin are generally acetylated (4~. Histone H3 acetylation is often linked to H3 phosphorylation and is likely to represent a two-component code for high levels of gene expression (5, 6~. In addition to distinct differences in histone modification, euchromatin and heterochromatin show differences in nonhis- tone chromosomal protein constituents. One of the best-studied examples is heterochromatin protein 1 (HP1) first discovered in Drosophila and named for its predominant localization to centric heterochromatin (7) (Fig. Lid. Consistent with this localization, the gene encoding HP1, Su~varJ2-5, was isolated as a dominant suppressor of position effect variegation (PEV) (8, 9~. PEV is the mosaic pattern of expression exhibited by genes placed near centric heterochromatin by chromosomal rearrangements or transposition events (10~. Overexpression of HP1 leads to en- hanced silencing of variegating genes. Conversely, a decreased level of HP1 leads to reduced silencing of variegating genes. A 16462-16469 1 PNAS 1 December 10, 2002 1 vol. 99 1 suppl. 4 complete loss of HP1, as in homozygous Su~var>2-5 null mu- tants, results in lethality. Larvae survive until the late third instar stage because of maternally supplied HP1 (11, 12~. The cause of lethality is unknown. Given the centric localization of HP1, and the interaction between the Schizosaccharomyces pombe HP1- like protein Swi6 and a cohesion protein, chromosome segre- gation might be affected (13, 14~. Thus, HP1 levels are critical for regulating the extent of heterochromatization within centric regions that is required for proper chromosome segregation. In addition to centric regions, HP1 is observed at other regions of the genome known to be heterochromatin The small fourth chromosome of Drosophila melanogaster, interspersed with het- erochromatic domains, shows a banded pattern of HP1 local- ization (7, 15~. Consistent with HP1 having a packaging function at these locations, transgenes inserted along the fourth chromo- some exhibit PEV that is suppressed by Su~varJ2-5 mutations (15, 16~. HP1 localization is also observed et Drosophila telo- meres that terminate in repetitive arrays of retrotransposons (17~. Telomeric association, however, appears to be independent of primary DNA sequence as broken chromosomes lack- ing terminal retrotransposons retain HP1 association (12~. Telomere-telomere fusions occur in larval neuralblasts of Su~var)2-5 mutants, suggesting HP1 plays a role in telomere capping (12~. In contrast to these chromosomal domains rich in repetitive DNA sequences, HP1 is present at approximately 200 sites within the euchromatic arms of polytene chromosomes that are relatively poor in repetitious DNA sequences. Do these sites represent small domains of repressive chromatin? Are there genes at these sites that are regulated by HP1? These questions are currently under investigation. Here we describe current studies on the role of HP1 in gene regulation at both euchromatic and heterochromatin domains. We summarize the results from reports that have identified HP1 partner proteins and discuss implications for these findings. Last, we hypothesize about multiple mechanisms of HP1 chromosome association and their impact on gene expression. HP1 Follows Code HP1 is a highly conserved protein with family members found in a variety of eukaryotic organisms ranging from S. pombe to humans (18-214. In Drosophila, two additional HP1-like pro- teins, HPlb and HPlc, sharing amino acid sequence similarity and domain structure, have been identified (22) (Fig. 2~. Whereas HPlb shows localization to both euchromatin and heterochromatin just as HP1, HPlc is found only in euchromatin (22~. Mice and humans each have three HP1-like proteins that possess similarities in amino acid sequence, domain structure, This paper results from the Arthur M. Sackier Colloquium of the National Academy of Sciences, "Self-Perpetuating Structurai States in Biology, Disease, and Genetics," held March 22-24, 2002, at the National Academy of Sciences in Washington, DC. Abbreviations: CAF1, chromatin assembly factor 1; CD, chrome domain; CSD, chrome shaclow domain; HP1, heterochromatin protein 1; ORC, origin recognition complex; HOAP, HP1/ORC-associated protein; PEV, position effect variegation; Rb, retinobiastoma. tTo whom reprint requests should be acicdressecl. E-maii: iori-wailrath@?ulowa.edu. www.pnas.org/cgi/cloi/10.1073/pnas.162371699

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Fig. 1. (A) Pattern of HP1 distribution on Drosophila polytene chromosomes. D. melanogaster larval polytene chromosomes were stained with mouse monoclonal C1A9 antibodies against HP1 (gin of Sarah C. R. Elgin) and a secondary antibody conjugated with rhodamine. The chromocenter (C), the fourth chromosome (indicated by 4), telomeres (T), and euchromatic sites associated with HP1. (B) The pattern of HP1 and methylated Iysine nine of histone H3 on Drosophila polytene chromosomes. D. melanogaster larval polytene chromosomes were stained with mouse monoclonal C1A9 antibody against HP1 and a rabbit polyclonal antibody that recognizes methylated Iysine nine of histone H3 (gin of C. David Allis, University of Virginia, Char- lottesville). A Cy5-conjugated rabbit secondary antibody and a FITC- conjugated mouse secondary antibody were used for detection. The chromo- center (C) and the fourth chromosome (indicated by 4) show strong colocalization (yellow). Example locations enriched in HP1 are denoted by green arrows; example locations enriched in methylated Iysine nine of histone H3 are indicated by red arrows. (C) Same as in B. showing a closer view of the chromocenter region. (D) Same as in B. showing a closer view of a telomeric region. and centric chromosomal localization properties as Drosophila HP1 (Fig. 2~. Although there are minor differences in chromo- somal localization and protein interaction partners for HP1-like proteins within a given species (23), it is not clear whether these proteins have specific or redundant functions. In flies, mice, and humans, the HP1-like proteins are small in size, ranging from 173 to 240 aa (Fig. 24. Overall the percent identity of HP1-like proteins to Drosophila HP1 is approximately 50% for mamma- lian HP1-like proteins. The majority of conserved amino acids are concentrated in two domains. The structure of HP1-like proteins can be simplified as two conserved domains separated by a less conserved hinge region (Fig. 2~. The conserved amino-terminal region of HP1-like proteins is termed the chromo domain (CD) (24~. This domain is present in 20 proteins in Flybase (www.ebi.ac.uk/proteome/DROME/interpro/ stat.html), many of which play roles in gene regulation. The conserved carboxyl-terminal region, termed the chromo shadow Li et a/. domain (CSD), is related to the CD in primary amino acid sequence (25~. Both the CD and the CSD have been the subject of extensive structural analysis (26-30~. Each domain forms a hydrophobic pocket. The CSD dimerizes (18, 27, 28, 31) as well as interacts with a wide variety of nuclear proteins (see below). Cross-species functional studies in which the mouse HP1-like protein M31 was expressed in S. pombe indicate that species- specific functions of HP1 reside within the CSD (32~. The CD is required for chromosome association (33~. The mechanism~s) by which HP1 establishes the complex localization pattern on chromosomes remained a mystery for over a decade since its discovery. For many chromosomal proteins, localization is achieved through direct interaction with DNA sequences. Attempts to identify specific interactions be- tween HP1 and DNA sequences, particularly repetitive DNA sequences found within heterochromatin, were not particularly revealing (344. For some chromosomal proteins, localization is achieved through interactions with DNA binding proteins. Therefore, a search for HP1 partner proteins might reveal the "missin~ link" between HP1 and the chromosome. A phage display assay was performed to identify peptides that interact with the CD and CSD (31~. This assay revealed peptide se- quences that showed a specific interaction with the CSD. Com- parison of the peptide sequences allowed a consensus pentapep- tide to be generated (31~. Supporting these results, the consensus pentapeptide was found in several proteins shown to interact with HP1 by other types of assays (35-37~. To date, the local- ization pattern of candidate interacting proteins cannot explain the entire localization pattern observed for HP1. In contrast to the results obtained for the CSD, no peptides were identified from the phage display assay that specifically interacted with the CD. These results were surprising because a point mutation within the CD of Drosophila HP1 eliminates the majority of chromosome association, suppresses PEV, and is homozygous lethal (9~. The mystery surrounding interactions of the CD was solved by studies of the murine Suv39hl protein, a homologue of the Drosophila SU(VAR)3-9 protein (38, 39~. A comparative genomic approach in combination with biochemical studies revealed that the SET ta conserved motif in Drosophila Su~rar'3-9, Enhancer of Zeste, and trithora~c] domain of Suv39hl contains methyltransferase activity specific for lysine nine of histone H3. This methylation mark on the histone H3 tail serves as a specific recognition code for the CD of HP1. This discovery supports the histone code hypothesis that proposes histone tail modifications serve as specific recognition motifs for chromatin proteins (40~. The HP1 CD, but not the CD of several other proteins, binds methylated lysine nine of histone H3 (41~. Therefore, the substrate specificity is likely caused by minor differences in the amino acid sequences of CDs from different proteins. The connection between Suv39hl and HP1 is consis- tent with Drosophila research showing that the genes encoding HP1 and SU(VAR)3-9 genetically interact with the heterochro- matin silencing system (8) and that the proteins physically interact (42~. The relationship between HP1 and Suv39hl has been maintained by the S. pombe homologues, Swi6 and Clr4, respectively (43, 44), suggesting evolutionary conservation in this mechanism of chromosome association and heterochromatic . . gene S1 encing. In summary, HP1 serves as a bridging protein, connecting histones, through interactions with the CD, to nonhistone chro- mosomal proteins, through interactions with the CSD (Fig. 3A). In this case, Suv39hl sets the histone code for HP1 association. Based on these findings, mechanisms for heterochromatin spreading have been proposed to involve recruitment of Suv39hl by HP1 and propagation of the methylation mark along the chromosome (39~. Details of such spreading mechanisms remain to be elucidated. ~_ ~ ~ _ ~ ~ - PNAS 1 December 10, 2002 1 vol. 99 1 suppl. 4 1 16463

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Drosophila: HP1 _ 18 76 141 203 HP1 b _ _ 3 52 94 153 HP1c 8 5~ 79 134 Human: HP1 HI _ 14 71 116 176 HP1 Us, __ 15 72 112 172 p1Hs' 14 71 106 166 Mouse: mHP1a _ _ _ 14 71 116 176 _ 1 __ 15 72 112 172 M32 __ 14 71 106 166 S. pombe: 75 136 199 252 JO Identity Total AA Total CD Hinge CSD 206 1 00 240 51 237 44 191 49 185 191 49 185 54 328 100 58 100 100 42 58 53 33 36 ~0 54 67 52 59 60 67 52 59 25 48 27 54 24 26 56 52 27 54 56 26 24 52 13 17 Fig. 2. Diagram of HP1 proteins in Drosophila, mouse, human, and S. pombe. Total length of each protein is indicated and drawn to relative scale. Percent identity when compared with Drosophila HP1 over the full length (total), or the CD, CSD, or the hinge region was calculated according to ref. 85. Are There Multiple Mechanisms for HP1 Association? HP1 localizes to distinctly different environments throughout the genome. Has the discovery of the interaction with the methylated lysine nine of histone H3 cracked the code, or are there alternative mechanisms of HP1 chromosomal association? The importance of this question is evident when reviewing data on the Su~varJ2-502 allele of the gene encoding HP1. This allele contains the amino acid substitution of a highly conserved valine to a methionine in the CD. Structural analysis indicates that this residue plays a critical role in the formation of the hydrophobic pocket of the CD (30~. In most genetic silencing assays this allele behaves as an HP1 null; however, an important distinction between this allele and null alleles was revealed by a cytological analysis of HP1 staining on chromosomes from HP1 mutants. Whereas null alleles show no HP1 chromosome association, the Su~varJ2-502 allele shows diminished HP1 localization to centric regions, but retains association at euchromatic and telomeric sites (12~. These data suggest an alternative mechanism of HP1 association might be operating at concentric locations. Further evidence for alternate mechanisms of association comes from cytological experiments on polytene chromosomes in wild-type flies. The pattern of staining observed by antibodies to HP1 and methylated lysine nine of histone H3 is not com- pletely coincident. Both antibodies show colocalization to the chromocenter and along the fourth chromosome, but not throughout the euchromatic arms and at telomeric regions (Fig. 1 B-D) (45~. One technical explanation for incomplete colocal- ization is that the epitopes recognized by either antibody are masked by fixation at specific genomic locations. However, if this is not the case, sites within the euchromatic arms that stain with only the HP1 antibody could be generated by HP1 associations through mechanisms independent of SU(VAR)3-9. Interactions of HP1 with unmodified histone tails, the histone-fold domain, and histone H1 might account for the staining pattern observed (34, 46~. Such possibilities are diagrammed in Fig. 3B. Alterna- tively, interactions with nonhistone chromosomal proteins might 16464 1 www.pnas.org/cgi/doi/10.1073/pnas.162371699 serve as an additional mechanism of association (Fig. 3C). Interactions between HP1 and transcriptional corepressors that associate with DNA binding proteins (see below) (47, 48) support this hypothesis. The double staining also revealed sites within the euchromatic arms that are detected only by the methyl lysine nine histone H3 antibody. These sites could correspond to different degrees of methylation because the antibody recog- nizes only dimethylated lysine (Upstate Biotechnology, Lake Placid, NY); HP1 is thought to recognize both methylated states with relatively equal affinity (30~. Another explanation for lack of complete colocalization of HP1 and the methyl lysine nine histone H3 antibody is that additional histone modifications might be present that do not permit HP1 association (Fig. 3D). Clearly the code for chromosomal protein association might have multiple components. HP1 Interacts with a Myriad of Proteins Does the identification of proteins that associate with HP1 provide clues about the mechanisms of silencing? Genetic analysis of PEV in Drosophila provided a collection of mutations that encode candidate HP1 interaction partners: for example, SU(VAR)3-9, the histone methylase discussed above interacts with HP1 by two-hybrid analysis (42) (Table 1~. A second example is SU(VAR)3-7, a zinc finger protein that associates with satellite DNA sequences (49~. HP1 and SU(VAR)3-7 colocalize in the Drosophila embryo and on polytene chromo- somes (50, 51) and interactions between the two proteins have been demonstrated by yeast two-hybrid analysis and coimmu- noprecipitation from embryonic extracts (35, 50~. More specif- ically, the CSD of HP1 interacts with multiple regions of SU(VAR)3-7, but it is not yet clear how these two proteins collaborate to form and/or spread heterochromatin. In addition to a gene silencing function, HP1 is thought to play a role in nuclear organization. This hypothesis is based on the discovery that HP1 interacts with lamin B receptor, either directly (52, 53) or indirectly through interactions with histones ~i et a/.

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A) Interaction with methylated histones B) Interactions with nucleosomes ~ ,~ C) Interaction with DNA binding proteins _. D) Exclusion by histone modifications __ ~ ark 43 Me Ac Me Ac 1 1 Fig. 3. Models for HP1 association and nonassociation with chromosomes. (A) Interaction between the HP1 CD with the methylated Iysine nine of histone H3. HP1 serves as a bridge for partner proteins. (B) HP1 interacts with histones in a nonmethylated-dependent fashion. (C) HP1 associates with chromosomes through interactions of the CSD and DNA binding proteins, such as zinc-finger proteins (ZNF). (D) HP1 does not associate with methylated histones that have additional modifications such as acetylation or phosphorylation. (54~. In addition, experimental data support an interaction between HP1 and B-type lamin and Lap2f, lamin-associated protein, located within the nuclear envelope. In vitro, these interactions foster nuclear envelope assembly, suggesting HP1 plays a role in organizing nuclear architecture (55~. Given that heterochromatin localizes to the nuclear periphery in many eukaryotic cell types, HP1 might tether heterochromatin to the Li et a/. nuclear envelope, leaving active regions of the genome free to coalesce into transcription factories within the interior of the nucleus (56~. The localization of HP1 to many sites throughout the Dro- sophila euchromatic arms brings to question the role of HP1 in the regulation of gene expression. Supporting a role for HP1 in transcriptional regulation, HP1 has been shown to interact with numerous proteins involved in modulating chromatin structure and gene expression (Table 1~. In mammals, association of HP1, the retinoblastoma (Rb) protein and SUV39H1 with the cyclin E promoter correlates with gene silencing (57~. Furthermore, HP1 has been implicated in gene repression mediated by Kruppel-associated box (KRAB) zinc finger proteins (47, 48~. Taken together, these findings suggest that HP1 is recruited to specific genes by protein-protein interactions, resulting in gene silencing by an unknown mechanism. In addition to transcriptional regulators, HP1 interacts with proteins involved in DNA replication and repair. Chromatin assembly factor 1 (CAF1) is a three-subunit complex that assembles histones H3 and H4 onto newly replicated DNA in both euchromatic and heterochromatic regions of the genome. In mammals, the large subunit, pl50, contains a domain that interacts with the CSD of HP1 (37~. Deletion of this domain does not alter CAF1-mediated chromatin assembly after replication in vitro or targeting of HP1 to heterochromatin in vivo during DNA replication. However, deletion of this domain reduces the amount of CAF1 present in heterochromatin outside of S phase. Although the significance of CAF1-HP1 interaction is not clear, the data suggest that CAF1 might stabilize heterochromatin structure during times of chromosome decondensation and transcription. HP1 associates with origin recognition complex (ORC) pro- teins (58, 59~. This presents an intriguing parallel to the situation in Saccharomyces cerevisiae where ORC proteins associate with silent information regulator (SIR) proteins to generate silent chromatin (60, 61~. A high molecular weight complex isolated from Drosophila embryos was recently shown to contain HP1/ ORC-associated protein (HOAP) in addition to HP1 and ORCs. HOAP has sequence similarity to high mobility group proteins and binds to satellite sequences in vitro (62~. Mutations in the genes encoding ORC proteins and HOAP are suppressors of PEV, suggesting a role in heterochromatin formation (624. What regulates HP1 association with protein partners? Post- translational modifications are likely to govern interactions between HP1 and partner proteins and/or itself. In Drosophila, HP1 is multiply phosphorylated giving rise to at least eight differently charged isoforms (63~. Drosophila embryonic extracts possess HP1-containing complexes that differ in HP1 phosphor- ylation status (58, 62~. For example, hypophosphorylated HP1 is found in a complex containing ORC and HOAP (58, 62~. In Drosophila, casein kinase II (CKII) is credited for the phosphor- ylation of serine residues at the amino and carboxyl termini of HP1. Mutation of these serine residues to alanine reduces the amount of HP1 localized to centric heterochromatin and reduces gene silencing, suggesting phosphorylation plays a role in chro- mosome association and/or complex stability (63, 64~. In mammals, HP1 phosphorylation changes through the cell cycle. HPlHSa and HPlHS~ exhibit increased levels of phosphor- ylation during mitosis (23~. HPlHS~ is a substrate for Pim-1 kinase that phosphorylates a serine cluster in the center of the protein (65~. Phosphorylation of HPlHS~ is thought to disrupt protein-protein interactions that are necessary to maintain most of the centric localization. In G2, phosphorylated HPlHS~ shifts from a centric location to being dispersed throughout the nucleus. Clearly, the role of phosphorylation needs further investigation to understand the biological significance of this dynamic process. PNAS I December ~o, 2002 I vol. 99 | supp~. 4 | 16465

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Table 1. HP1 interacting partners and candidate partners Protein Organism HP1 variant Methodology HP1 domain Ref(s). Transcription regulation/ chromatin modifying proteins H1 Drosophila HP1 rPD nd 46 HP1-BP74 H1-like Mouse mHP1a Y2H, FW, rPD Hinge region 46, 86 H3 Mouse mHP1cz, M31, M32 FW, rPD, exlP CD 46, 54 H3 Drosophila HP1 rPD nd 46 Methylated K9 of H3 S. pombe Swi6 rPD, ChlP CD 38, 43 Methylated K9 of H3 Drosophila HP1 IF, FAITC, NMR CD 26, 41 Methylated K9 of H3 Mouse mHP1a, M31, M32 rPD CD 39 Methylated K9 of H3 Human HP1HS~, HP1Hs0, HPlHsY rPD, SPRA CD 38 H4 Mouse M31 rPD nd 54 H4 Drosophila HP1 /n vitro cross-linking CSD 34 MacroH2A1.2* Mouse M31 IF nd 87 SUVAR3-9 Drosophila HP1 IF, Y2H, exlP CSD 42 Suv39h1 Mouse M31 IF, exlP, SED nd 88, 89 SUV39H1 Human HP1Hsf IF, exlP, SED nd 88, 89 Suvar3-7 Drosophila HP1 IF, Y2H, exlP CSD 35, 50 KAP-1/TIF1,8 Human HP1Hsa, HPlHs~ IF, rPD, exlP, SPRA, GFC CSD 28, 46~8 KAP-1/TIF1,B Mouse mHP1`x, M31, M32 IF, rPD, Y2H, exlP, GFC CSD 28, 37, 47, 86, 90 TRF1 /PIN2 Mouse M31 IF nd 91 TAF''130 Human HP1Hs~, HP1HsY Y2H, transPD, exPD CSD 36 TIF1a Mouse mHP1a~, M31, M32 Y2H, rPD CSD 86, 90 mSNF2,B Mouse mHP1 cr Y2H CSD 86 Rb Human HP1, HP1Hs' Y2H, exPD, exlP, ChlP nd 57, 82 Rb Maize HP1> rPD, Y2H nd 82 Dnmt3a Mouse cells mHP1c~ IF nd 92 Dnmt3b Mouse cells mHP1Oe IF nd 92 ATRX/HP1-BP38 Mouse mHP1c~, M31 Y2H, IF CSD 86, 93 Pim-1 Human HP1Hs~ Y2H, exlP, rPD CSD 65 CKII Drosophila HP1 In vitro phosphorylation nd 63 dAF10 Drosophila HP1 transPD CSD 94 DNA replication and repair CAF-1 p150 Mouse mHP1a, M31 IF, Y2H, rPD, GFC, NMR CSD 28, 37 CAF-1 p150 Human HP1Hs~ rPD CSD 48 Ku70 Human HP1Hs~ Y2H, rPD, exlP CSD 95 BRCA-1 * Human HP1 Hsa IF nd 96 ORC1 Drosophila HP1 translP CD, CSD 58 ORC2 Drosophila HP1 IF, exPD, exlP, translP CD, CSD 58 ORC3 Drosophila HP1 translP CD, CSD 58 ORC4 Drosophila HP1 translP CD, CSD 58 ORC5 Drosophila HP1 exlP, translP CD, CSD 58 ORC6 Drosophila HP1 exlP, translP CD, CSD 58 Xorc1 Xenopus XHP1~, XHP17 Y2H nd 58 HOAP Drosophi/a HP1 IF, exlP nd 62 Nuclear architecture Lamin B receptor Human HP1Hs~, HP1Hs0, HP1Hs~ Y2H, rPD, exPD, transPD, exlP CSD 48, 52-54 HP1-BP84 Mouse mHP1ar, M31 Y2H CSD 86 Lamin B Mouse M31 BA CD 55 LAP2,B Mouse M31 BA CD 55 Nuclear envelope Mouse mHP1a~, M31, M32 IF, BA CD 55 Other chromosome-associated proteins Psc3 S. pombe Swi6 IF, Y2H, exPD, ChlP CD+Glu-rich 13 DDP1 Drosophila HP1 IF nd 97 Arp4/dArp6 Drosophila HP1 IF nd 98, 99 INCENP Human HP1Hs~, HP1HsY Y2H, transPD Hinge region 100 Ki-67 Human mHP1a, M31, M32 Y2H, exPD, IF CSD 101 SP100B Human HP1Hs~, HP1Hs0, HPlHsY IF, Y2H, rPD, transPD CSD 48, 102, 103 EST AA153281 Mouse mHP1a, M31 Y2H, rPD CSD 37 EST AA003533 Mouse mHP1cY, M31 Y2H, rPD CSD 37 BA, o~nu~ng assay; ChlP, chromatin immunoprecipitation; exlP, co-immunoprecipitation using extract; exPD, pull-down assay using extracts; FAITC, fluorescence anisotropy, isothermal titration calorimetry; FW, farWestern analysis; GFC, gel filtration chromatography; IF, immunofluorescence colocalization; nd, not determined; rlP, coprecipitation using recombinant proteins; rPD, pull-down assay using recombinant proteins; translP, immunoprecipitation with in vitro-translated protein; transPD, pull-down assay using in vitro translated protein; SED, sedimentation assay; SPRA, surface plasmon resonance analysis; Y2H, yeast two-hybrid assay. *Denotes cell cycle-dependent association. 16466 1 www.pnas.org/cgi/doi/10. 1 073/pnas. 162371699 Li et al.

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HP1 Regulates Gene Expression Effects on Gene Expression Near Centric Heterochromatin. The gene encoding HP1, Su~var)2-5, was isolated in a screen for suppres- sors and enhancers of PEV of the white+ gene brought into juxtaposition with heterochromatic through a chromosomal rearrangement (8~. To determine the effects of HP1 on chro- matin packaging, stocks containing the well-characterized Drosophila hsp26 gene inserted within centric heterochromatic were used for chromatin structure analysis (16~. These trans- genes exhibit less accessibility to nucleases and are packaged into a more regular nucleosome array than euchromatic insertions. In an HP1 mutant background the transgenes become more acces- sible to restriction enzyme digestion, indicating a more "open" chromatin configuration (664. To determine the transcriptional mechanism impaired by packaging with HP1, high-resolution chromatin structure analysis was performed (67~. The results indicated that general transcription factors such as TFIID and RNA polymerase II are not associated with heterochromatic transgenes exhibiting silencing. Thus, association of HP1 corre- lates with a "closed" chromatin configuration that limits the accessibility of regulatory sites to trans-acting factors. This is similar to the mechanism of X-chromosome inactivation in mammals where transcription factors are also absent from genes on the inactive X (68), but contrasts the mechanism of silencing at the mating type loci in S. cerevisiae and Polycomb-mediated silencing in Drosophila in which general transcription factors and RNA polymerase II are found in association with silenced promoters (69-71~. Differences between these systems and HP1 silencing could reflect fundamentally different properties of the silencing systems or developmentally different stages of silent chromatin maturation. In contrast to the silencing effects HP1 has on euchromatic genes, HP1 is required for the expression of genes that naturally reside within heterochromatic Over the years genetic and molecular analyses have revealed genes that reside within centric heterochromatic (72, 73~. Two well-characterized genes are light, an essential gene encoding a protein involved in the vesicle transport pathway (74, 75), and rolled, an essential mitogen- activated protein kinase (76~. Heterochromatic genes are not specific for Drosophila; they also have been discovered in Arabidopsis (77) and are likely to be found in other organisms as genomic sequence analysis becomes more complete. In Drosoph- ila, heterochromatic genes appear to be unrelated to each other in function, however, they do share some common properties. Structurally, many heterochromatic genes have introns contain- ing middle repetitive DNA sequences (74) (D. E. Cryderman and L.L.W., unpublished data). Heterochromatic genes are inefficiently expressed and sometimes exhibit PEV when trans- located to euchromatin (78, 79~. In addition, heterochromatic genes require heterochromatic proteins such as HP1 for expression (11~. How does HP1 establish a chromatin configuration that hinders the expression of euchromatic genes while fostering the expression of heterochromatic genes? This question will be better addressed as the promoter regions of heterochromatic genes are analyzed. Assuming a role for HP1 in chromatin compaction, HP1 might bring distant regulatory elements in association with the promoter region of heterochromatic genes. Alternatively, HP1 may be required to set up a favorable chromatin configuration within the promoter proximal region and/or be involved in the recruitment of general transcription factors as suggested by a recent report showing an interaction between HP1 and the general transcription factor TF~130 (36~. Effects of Tethering HP1. What are the effects of HP1 on gene expression at locations other than centric heterochromatin? One approach taken to address this question has been to generate Li et a/. HP1 fusion proteins containing heterologous DNA binding domains. In mammalian cell transient transfection assays, effects of HP1 fusion proteins on the expression of reporter genes possessing the appropriate DNA binding sequences are assayed. In these experiments both murine and human HP1 family members repress transcription when tethered to a small number of sites located in close proximity to the promoter (36~. Tran- scriptional repression no longer occurs as the binding sites are moved to distances more than 2 kb from the promoter (80~. Does this indicate that HP1-mediated repression has only short-range capabilities, perhaps operating on a gene-by-gene basis? If this is the case, HP1 located at euchromatic sites (Fig. 1) might play a role in the regulation of individual euchromatic genes, rather than entire domains. The effects of tethering HP1 in a chromosomal context, as in transgenic Drosophila, demonstrate the complexities of gene silencing. A Gal4-HP1 fusion protein tethered upstream of a reporter gene caused silencing at only one of six genomic locations tested (51, 81~. Interestingly, the site that supported silencing was flanked by middle repetitive DNA sequences, reminiscent of heterochromatic domains. In this case, silencing could spread in bans to a homologue lacking the tethering sites. These results suggest that not all chromosomal contexts will support the formation of silent chromatin by HP1. The inability to silence at certain locations might depend on the chromatin of the neighboring region, including the types of histone modifi- cations, as well as gene density and transcriptional status of the region. Identification of HP1 Target Genes. As a second approach to determine the effects of HP1 on gene expression at locations other than centric heterochromatin, investigators have identified potential target genes by their response to HP1 dosage. Repre- sentational difference analysis identified two genes that are up-regulated in homozygous HP1 mutant larvae (59~. Interest- ingly, one of the genes misregulated in the HP1 homozygous mutant maps to cytological region 31, a chromosome division that stains intensely with antibodies to HP1 (7~. Two additional randomly selected genes within region 31 show up-regulation in HP1 homozygous mutants (59~. For all four candidate HP1 target genes, mutations in additional modifiers of PEV, includ- ing Su~var)3-9, cause increases in gene expression. These results suggest that HP1 might function to silence genes located within euchromatic domains in a mechanism similar to that operating . . . In centric regions. A microarray approach has also been used to identify candi- date genes regulated by HP1. Several hundred genes mapping within the euchromatic arms are up-regulated in an HP1 mutant background (D. E. Cryderman and L.L.W., unpublished data). In addition, several hundred genes were down-regulated, a pattern similar to that of heterochromatic genes. For all of the candidate target genes identified in Drosophila to date, it remains to be determined whether misregulation is caused by a direct interaction with HP1. It will be of interest to determine whether interactions between HP1 and specific genes are conserved through evolution. In mammals, it is also likely that HP1 plays a role in the regulation of genes at concentric locations. HP1 has been identified as a partner protein for many promoter-associated factors involved in control of gene repression. These include the transcription intermediate factor TIF1,B that interacts with zinc finger proteins containing Kruppel-associated box (KRAB) domains known to be involved in transcriptional repression. The role of HP1 in KRAB-mediated repression is not clear, but might involve recruitment of histone deacetylases (47, 48~. The first example of a direct association of HP1 with a promoter region came from studies on the cyclin E gene (574. Binding of the Rb protein upstream of the cyclin E promoter PNAS | December 10, 2002 | vol. 99 | suppl. 4 | 16467

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causes gene silencing, partly through the recruitment of histone deacetylases. In addition, chromatin cross-linking and immuno- precipitation experiments place Rb at the promoter with HP1 and methylated lysine nine of histone H3 (57~. Consistent with the finding, an "Rb binding motif" is present within the amino acid sequences of HP1 from a variety of species (82~. It is unclear whether HP1 is recruited to the cyclin E promoter by Rb, methylated lysine nine of histone H3, SUV39H1, or any com- bination of these interacting molecules. In support of such interactions, HP1 possesses the ability to simultaneously interact with the methylated histone H3 tail and Rb (57~. One hypothesis is that Rb recruits histone deacetylases first, because the histone H3 methyltansferase cannot use an acetylated lysine as a sub- strate for methylation (43), then SUV39H1 methylates the histone tail which serves as the substrate for HP1 binding. Interestingly, Drosophila SU(VAR)3-9 was recently purified in a complex with histone deacetylase HDAC1, suggesting that the two proteins might cooperate to methylate previously acetylated histone tails (83~. The identification of HP1 target genes has implications for understanding breast cancer metastasis in humans. HPlHS~, but not HPlHSf or HPlHS7, is down-regulated in highly invasive/ metastatic breast cancer cells compared with poorly invasive/ nonmetastatic breast cancer cells (84~. Introduction of a tagged HPlHS~ into the highly invasive/metastatic cells, which normally have low levels of HPlHS~, lead to a less in vitro invasive phenotype. These results imply that modulation of the levels of 1. Richards, E. J. & Elgin, S. C. (2002) Cell 108, 489-500. 2. Jeppesen, P., Mitchell, A., Turner, B. & Perry, P. (1992) Chromosoma 101, 322-332. 3. Braunstein, M., Sobel, R. E., Allis, C. D., Turner, B. M. & Broach, J. R. (1996) Mol. Cell. Biol. 16, 4349-4356. 4. Turner, B. M., Birley, A. 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Since the discovery of HP1 over 12 years ago by the laboratory of Sarah C. R. Elgin (Washington University, St. Louis), HP1 has grown in popularity. In part, this has been caused by the fact that HP1 has unexpectedly been identified as an interacting partner for a wide variety of proteins with diverse nuclear functions. Dissecting the function of HP1 in association with its partner proteins lies ahead. These experiments will shed light on the connections between chromatin structure, gene expression, DNA replication and repair, and nuclear organization. We thank Sarah C. R. Elgin for the Drosophila HP1 antibody and C. David Allis for the gift of histone H3 methyl K9 antibodies and the histone code hypothesis. We thank Pamela Geyer and members of the Wallrath lab for suggestions regarding this manuscript. Work in the laboratory of L.L.W. is supported by American Cancer Society Grant GMC-100527 and National Institutes of Health Grant GM61513. 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