HP1 Is Essential for DNA Methylation in Neurospora

Molecular Cell, Vol. 13, 427–434, February 13, 2004, Copyright 2004 by Cell Press

HP1 Is Essential for DNA Methylation in Neurospora

Michael Freitag,1 Patrick C. Hickey,2 Tamir K. Khlafallah,1 Nick D. Read,2 and Eric U. Selker1,* 1 Department of Biology and Institute of Molecular Biology University of Oregon Eugene, Oregon 97403 2 Institute of Cell and Molecular Biology University of Edinburgh Edinburgh EH9 3JH United Kingdom

Summary Methylation of cytosines silences transposable elements and selected cellular genes in mammals, plants, and some fungi. Recent findings have revealed mechanistic connections between DNA methylation and features of specialized condensed chromatin, “heterochromatin.” In Neurospora crassa, DNA methylation depends on trimethylation of Lys9 in histone H3 by DIM-5. Heterochromatin protein HP1 binds methylated Lys9 in vitro. We therefore investigated the possibility that a Neurospora HP1 homolog reads the methylLys9 mark to signal DNA methylation. We identified an HP1 homolog and showed that it is essential for DNA methylation, is localized to heterochromatic foci, and that this localization is dependent on the catalytic activity of DIM-5. We conclude that HP1 serves as an adaptor between methylated H3 Lys9 and the DNA methylation machinery. Unlike mutants that lack DNA methyltransferase, mutants with defects in the HP1 gene hpo exhibit severe growth defects, suggesting that HP1 is required for processes besides DNA methylation. Introduction Control of DNA methylation in eukaryotes has been mysterious, but recent studies have advanced our understanding. In plants, some DNA methylation is triggered by homologous RNA molecules (see Aufsatz et al., 2002), and, in several eukaryotes, connections have been found between histone modifications, methyl-DNA binding proteins, and DNA methylation (reviewed in Dobosy and Selker, 2001; Lachner et al., 2003). Most strikingly, DNA methylation in the fungus N. crassa depends entirely on the histone H3 Lys9 methyltransferase DIM-5 (Tamaru and Selker, 2001; Tamaru et al., 2003). At least some DNA methylation in plants and animals also depends on histone H3 Lys9 methylation (Jackson et al., 2002; Lehnertz et al., 2003; Malagnac et al., 2002). Biochemical studies have revealed that methylated Lys9 is bound by HP1, a heterochromatin protein originally identified in Drosophila and implicated in silencing in Drosophila, fission yeast, and mammals (Bannister et al., 2001; Eis*Correspondence: [email protected]

senberg and Elgin, 2000; Jacobs et al., 2001; Lachner et al., 2001). HP1 has been reported to interact with a large variety of nuclear proteins, including components of the transcriptional machinery, architectural proteins, and proteins involved in DNA replication and repair (see Li et al., 2002). Recent studies revealed interactions and showed colocalization of HP1 homologs with SUVAR3-9 histone methyltransferases, DNA methyltransferases, and a methyl binding domain protein (Bachman et al., 2001; Fujita et al., 2003; Fuks et al., 2003; Schotta et al., 2002). We and others suggested that HP1 homologs might link histone methylation to DNA methylation (Jackson et al., 2002; Selker et al., 2002). It has been recently reported, however, that the Arabidopsis HP1 homolog TFL2/LHP1, which was first identified in screens for developmental mutants (Gaudin et al., 2001; Larsson et al., 1998), is dispensable for DNA methylation (Malagnac et al., 2002). In our study, we identified and disrupted a Neurospora HP1 homolog and found that it is essential for DNA methylation and required for normal growth. Results and Discussion Identification and Mutagenesis of a Neurospora HP1 Homolog We searched the Neurospora genome sequence with D. melanogaster HP1 and its closest S. pombe homolog, Swi6, in tBlastn searches and found a single, 985 bp candidate gene, hpo (Figure 1A). We isolated and sequenced a corresponding cDNA and confirmed the presence of three predicted introns (Figure 1A). Conceptual translation showed that HP1 contains an amino-terminal chromodomain (CD) and a carboxy-terminal chromoshadow domain (CSD), requirements for bona fide HP1 homologs (Eissenberg and Elgin, 2000) (Figure 1B). The Neurospora HP1 chromodomain contains three conserved aromatic residues (Tyr76, Trp98, and Tyr100) that in Drosophila and mouse HP1 form a binding pocket or “aromatic cage” to accept the N-methyl groups of the histone H3 tail (Nielsen et al., 2002; Jacobs and Khorasanizadeh, 2002). Relative to previously identified HP1 homologs, HP1 is most closely related to the S. pombe proteins Chp2/Clo2 (Thon and Verhein-Hansen, 2000; Halverson et al., 2000) and Swi6 (Lorentz et al., 1994) (Figure 1C). We also found HP1 homologs in five other filamentous fungi: the human pathogens Aspergillus fumigatus and Cryptococcus neoformans, the plant pathogens Fusarium graminearum and Magnaporthe grisea, and A. nidulans (Figure 1; Supplemental Figure S1, available online at http://www.molecule.org/cgi/content/full/ 13/3/427/DC1; and data not shown). To test directly whether HP1 is required for cytosine methylation, we used RIP (repeat-induced point mutation) to generate hpo mutants. RIP is a genome defense system that operates during the sexual phase and mutates duplicated genes by C:G to T:A mutations and usually results in DNA methylation of the mutated copies (Selker et al., 2003). Several independent candidate mu-

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Figure 1. Structure of the hpo Gene (A) The hpo gene on linkage group VI consists of four exons (black boxes), interrupted by three introns. The positions of the chromodomain (CD) and chromoshadow domain (CSD) are indicated. The red asterisk, circle, and squares indicate the positions of nonsense mutations in the hpoRIP1, hpoRIP2, and hpoRIP3 alleles, respectively (see Supplemental Figure S2 on Molecular Cell’s website). Primers are indicated by arrows. (B) Comparison of the CD and CSD of HP1 proteins from Neurospora crassa (Nc), Magnaporthe grisea (Mg), Schizosaccharomyces pombe (Sp), Drosophila melanogaster (Dm), Homo sapiens (Hs), and Arabidopsis thaliana (At). Residues identical in all HP1 proteins are indicated by blue asterisks. Colons and periods indicate strong and weak conservation, respectively. (C) All HP1 proteins share the same domain structure, an amino-terminal chromodomain (CD, dark blue) and carboxy-terminal chromoshadow domain (CSD, light blue) interrupted by a hinge region of varying length. The amino terminus of all HP1 proteins contains a run of acidic residues. The CD and CSD are the most conserved regions within HP1 proteins.

tant strains identified by Southern hybridizations were selected among progeny from crosses of hpo duplication strains. We describe three alleles with early nonsense mutations in conserved domains of the protein; all three should be nonfunctional (Figure 1; Supplemental Figure S2, available on Molecular Cell’s website). Allele hpoRIP1 (N2537) contains a single G to A mutation at position 109 in a conserved TWE motif within the CD (Figure 1B). Allele hpoRIP2 (N2538) has a nonsense mutation (Q144*) in the hinge region as well as one silent and two missense mutations. Allele hpoRIP3 (N2539) has 44 C to T substitutions, including three nonsense mutations (Q144*, Q201*, and Q238*) within the hinge region and

the chromoshadow domain, plus 17 missense mutations that result in predominantly conserved substitutions (see Supplemental Figure S2 on Molecular Cell’s website). Neurospora HP1 Is Essential for DNA Methylation Visual comparison of ethidium bromide-stained genomic DNA of wild-type strains and hpo mutants digested with the 5mC-sensitive restriction endonuclease Sau3AI or its 5mC-insensitive isoschizomer DpnII revealed global demethylation in hpo mutants, as observed with dim-2 and dim-5 strains (Figure 2). Most methylated regions in Neurospora are relics of transposons that were inactivated by RIP (Selker et al., 2003). We used Southern

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Figure 2. HP1 Is Essential for DNA Methylation Samples of genomic DNA (0.5 ␮g/lane) of a wildtype strain (wt), a hpo pre-RIP duplication strain (dup), an hpoRIP3 strain (hpo), a dim-2 strain, and a dim-5 strain were digested with 5mC-sensitive Sau3AI (S) or a 5mC-insensitive isoschizomer, DpnII (D), fractionated by agarose gel electrophoresis, stained with ethidium bromide (EtBr), blotted to nylon membrane, and probed sequentially for known methylated regions (8:F10, 9:E1, 11:E5, 9a20, 1d21, 8:G3, 5:B8, rDNA) (see Selker et al., 2003). High-molecular weight fragments indicative of DNA methylation were only detected in Sau3AI digests of genomic DNA from wild-type and pre-RIP strains. Complete digestion was verified by probing with unmethylated regions (e.g., Bml). All samples were loaded on a single gel; replicate center lanes were removed for clarity. In some hpo and dim-5 strain backgrounds, RFLPs can be detected with certain probes when compared to the wild-type strain (e.g., 11:E5, 1d21, and 9a20 for dim-5, and 1d21 and rDNA for hpo). We also observed no DNA methylation in hpoRIP strains in control digests with additional enzymes that can be affected by DNA methylation in Neurospora (data not shown). Positions of size standards (kb) are shown on left.

hybridization to examine the methylation status of representative methylated regions, including a single-copy transposon relic (8:F10), several low-copy number transposon relics (9:E1; 1d21; 9a20; Nogo, 5:B8), several moderately repeated products of RIP (PuntRIP1, ␺63; 11:E5; Dodo1, 8:A6; Dodo3, 8:G3), and some highly repetitive regions (Tcen; Tad; Tcl1; CEN-VII; Tcen-adjacent region, 8:F8; rDNA). Several of these regions have been demonstrated to be enriched for methylated H3 lysine 9 (Tamaru et al., 2003). All regions tested showed complete loss of DNA methylation (Figure 2). We conclude that HP1 is essential for DNA methylation in Neurospora. HP1 apparently acts both in heterochromatic regions associated with the centromeres and in shorter methylated regions within euchromatin. This is reminiscent of the situation in Drosophila, where HP1␣ is found in ⵑ200 euchromatic sites (Li et al., 2002). Neurospora HP1 Is Localized in Heterochromatic Regions Methylated regions are well characterized in Neurospora (Selker et al., 2003), but heterochromatin is cytologically ill-defined in this organism. To investigate whether HP1 is associated with heterochromatic regions in Neurospora, we created a carboxy-terminal translational fusion of the hpo gene to sgfp, an enhanced version of the gene for Green Fluorescent Protein (GFP) that we adapted for use in Neurospora (Freitag et al., 2001). A single copy of an hpo-sgfp fusion gene under the control of the inducible ccg-1 promoter was intro-

duced at the his-3 locus (Freitag et al., 2001). We examined asexual spores and hyphal fragments by fluorescence microscopy. HP1-GFP was localized in a few distinct foci (Figure 3A, GFP image). These foci are likely heterochromatic regions, because the same densely staining foci were found by staining with the DNA dye Hoechst 33258 (Figure 3A, DNA and merged image). We conclude that Neurospora HP1 is specifically localized to heterochromatic regions. Hoechst staining of hpo strains revealed an apparent reduction of heterochromatic foci (Supplemental Figure S4 on Molecular Cell’s website), suggesting that HP1 is required for formation of some but not all heterochromatin in this organism. We used confocal microscopy to further examine heterochromatic foci in live hyphae of HP1-GFP strains and compared the distribution of HP1-GFP and GFP-tagged histone H1, another well-studied chromatin-associated protein. HP1-GFP was found localized to a small number of nuclear foci (Figure 3C). Each nucleus showed at least one prominent HP1-GFP-stained region that may represent a cluster of Neurospora’s seven centromeres (Figure 3C), analogous to “chromocenters” described in Drosophila (Li et al., 2002) and S. pombe (Kniola et al., 2001). We also observed ring-shaped structures, possibly heterochromatic regions associated with the nucleolus, and smaller foci that may reflect short heterochromatic regions associated with telomeres and dispersed relics of RIP (Figure 3C). In contrast, H1-GFP was more widely distributed in the nucleus (Figure 3G), presumably in euchromatic regions (Folco et al., 2003).

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strains, HP1-GFP was found in heterochromatic foci, as described above (Figures 3A and 3C). In contrast, most of the HP1-GFP remained diffuse throughout the nuclei of dim-5 strains (Figures 3B and 3D). We also observed mislocalization of HP1-GFP in Neurospora strains with a catalytically nonfunctional DIM-5 protein, DIM-5Y178V (N2570 and N2571, data not shown; Zhang et al., 2003). We conclude that proper localization of Neurospora HP1 depends on methylation of H3 Lys9 catalyzed by DIM-5, consistent with in vitro observations that HP1 recognizes methylated Lys9 of histone H3 (Bannister et al., 2001; Jacobs et al., 2001; Lachner et al., 2001). Interestingly, we found that dim-5 nuclei retained some heterochromatic foci visualized with Hoechst 33258 (Figure 3B). Confocal microscopy confirmed that dim-5 strains have reduced heterochromatin but retain one weaker heterochromatic spot (Figure 3D). Apparently HP1 and DIM-5 are required for most but not all heterochromatin in Neurospora. It will be interesting to identify the DNA sequences associated with HP1-dependent and -independent heterochromatin. Because HP1 and HP1 homologs apparently serve as adaptor proteins by connecting histone H3 to nonhistone effector proteins (Li et al., 2002; Smothers and Henikoff, 2000), we also tested the possibility that the localization of HP1 depends on the DNA methyltransferase DIM-2. Deletion of dim-2 did not noticeably affect HP1 localization (Figure 3E). Thus DIM-2 is not essential for targeting of HP1 to heterochromatic foci and/or for recognition of methylated H3 Lys9 by HP1.

Figure 3. HP1-GFP Is Localized to Heterochromatin (A) Fluorescence microscopy on hpo⫹ strains N2534 (top), N2540 (bottom left), and N2559 (bottom right) reveals that HP1-GFP (GFP) localizes to foci that coincide with regions of dense staining with the DNA dye Hoechst 33258 (DNA and merge). (B) Fluorescence microscopy on dim-5 strains N2541 (left) and N2542 (right) shows that HP1-GFP is largely mislocalized in dim-5 strains. Confocal microscopy (C–G) reveals strong foci of HP1-GFP in hpo⫹ strain N2540 (C), loss of HP1-GFP localization in dim-5 strain N2541 (D), but retention of the localization in dim-2 strain N2543 (E), and reestablishment of localization in the complemented hpoRIP2 mutant strain N2559 (F). (G) Histone H1-GFP localization is punctate but different from that of HP1-GFP. Scale bar shown in (C) applies also to (D)–(G); scale bar, 1 ␮m.

Localization of Neurospora HP1 Is Dependent on the Catalytic Activity of DIM-5 To determine whether the localization of HP1 requires DIM-5, the enzyme responsible for histone H3 K9 methylation (Tamaru et al., 2003), we crossed dim-5 and hpo⫹-sgfp⫹ strains and compared the distribution of HP1-GFP in dim-5 and dim-5⫹ sibling strains. In dim-5⫹

Neurospora HP1 Is Required for Normal Growth We considered the possibility that HP1 in Neurospora plays roles in addition to its essential function in DNA methylation. HP1 plays important roles in Drosophila and S. pombe even though these organisms have little or no DNA methylation. Null mutants of Drosophila HP1 result in lethality at the third instar larval stage (Eissenberg et al., 1992; Kellum and Alberts, 1995; Lu et al., 2000). In S. pombe, the HP1 homologs swi6 and chp2/ clo2 are important for normal growth but are not essential for viability (Halverson et al., 2000; Lorentz et al., 1994); even double mutants are viable (Halverson et al., 2000). Chp2/Clo2 is localized to centromeres and is involved in heterochromatin silencing at rDNA, telomeres, mating type regions, and—to a lesser degree— centromeres (Thon and Verhein-Hansen, 2000). While mutation of chp2 only slightly increases chromosome loss (Thon and Verhein-Hansen, 2000), overexpression of this gene results in a dramatic increase of minichromosome loss (Halverson et al., 2000). Swi6 is required for the establishment and spreading of silenced heterochromatin (Grewal and Moazed, 2003) as well as for recruitment of cohesin complexes to heterochromatic regions, which is important for chromosome segregation (Bernard et al., 2001; Nonaka et al., 2002). In Neurospora, loss of all DNA methylation by mutation of the DNA methyltransferase gene dim-2 does not result in noticeable growth defects (Kouzminova and Selker, 2001). In contrast, mutation of the histone methyltransferase gene dim-5 causes growth abnormalities, suggesting that DIM-5 is involved in processes other than DNA methylation (Tamaru and Selker, 2001). We found

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Figure 5. DNA Methylation in the hpoRIP2 Mutant N2556 Is Restored to Wild-Type Levels by Complementation with the hpo-sgfp Fusion Gene The hpo-sgfp gene under control of the ccg-1 promoter was targeted to the his-3 locus. Genomic DNA (0.5 or 1 ␮g/lane) of wild-type strain N150 (wt), N1877 (dim-2), N2556 (hpo), and the complemented hpo strain N2559 (hpo⫹-sgfp⫹) was digested with 5mC-sensitive Sau3AI (S) or the 5mC-insensitive isoschizomer DpnII (D), fractionated by agarose gel electrophoresis, stained with ethidium bromide (EtBr), blotted to nylon membranes, and probed for known methylated regions (8:F10 and 9:E1; compare to Figure 2 and see Selker et al., 2003). We used an unmethylated control region (Bml) to ascertain complete digestion (data not shown). The complemented transformant also exhibits normal growth (data not shown) and HP1-GFP at heterochromatic foci (see Figures 3A and 3G). Position of size standards (kb) is shown on left. Figure 4. HP1 Is Essential for Normal Growth (A) Radial growth at 32⬚C of wild-type (blue; n ⫽ 6), dim-5 (green; n ⫽ 8), and hpo (n ⫽ 16) strains. Growth of hpo strains was more variable than that of the other strains, as indicated by the standard deviations (bars). (B) Formation of asexual spores and aerial hyphae observed in a wild-type strain (N150) and hpo⫹ siblings from hpo ⫻ wild-type crosses (N2547, N2549) is greatly retarded in hpo mutants (N2537, N2538, N2552) and somewhat delayed in dim-5 strains (N2264, N2229, N2231). Both hpo and dim-5 strains show irregular radial growth. Cultures were grown 4 days at 32⬚C.

that hpo mutants, like dim-5 mutants, show marked growth defects. Formation of asexual spores and carotenoid production were delayed several days in all three of our original mutants (N2537 to N2539). Eventually, dense, cauliflower-like growth associated with abnormally few spores was formed. Growth of the mycelium was also unusually slow and variable in hpo mutants, reminiscent of dim-5 strains (Tamaru and Selker, 2001). Comparisons of radial growth (Figure 4A) and accumulation of mass (data not shown) of hpo and control (wildtype, dim-5, and dim-2) strains showed that growth defects of hpo strains are even more severe than in dim-5 strains (Figure 4; data not shown). As observed with dim-5 strains (Tamaru and Selker, 2001), hpo mutants showed “stop-start” growth resulting in irregular growth margins on solid medium (Figure 4B). Transformation of his-3 hpoRIP2 strains with the hpo⫹-sgfp⫹ fusion gene

resulted in complementation of the growth phenotypes (data not shown) and DNA methylation defects (Figure 5) and normal localization of HP1-GFP (Figure 3F). We conclude that hpo and dim-5 mutants show similar growth defects that cannot be simply due to absence of DNA methylation. Both hpo and dim-5 are required for the normal complement of heterochromatin, which presumably functions in important cellular processes, including chromosome segregation. The more severe phenotype of hpo mutants, relative to dim-5 mutants, implies that HP1 plays additional roles in the cell, consistent with expectations based on studies with Drosophila and mammals (see Li et al., 2002). Conclusions Our demonstration that HP1 is required for DNA methylation in Neurospora extends our observations that the Lys9 methyltransferase DIM-5 is required for DNA methylation (Tamaru and Selker, 2001) and that trimethylLys9 is associated with cytosine-methylated chromosomal regions (Tamaru et al., 2003). Our working model for control of DNA methylation in Neurospora proposes that the regional specificity of DIM-5 reflects a combination of recruitment by proteins that recognize features of DNA destined to be methylated––most notably sequences modified by RIP––and its inherent sensitivity to certain histone modifications (Selker et al., 2002; Tamaru

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and Selker, 2003). Chromatin with trimethyl-Lys9 histone H3 then binds HP1, which in turn directly or indirectly recruits the DNA methyltransferase DIM-2. In contrast to the situation in Neurospora, where all DNA methylation is dependent on one histone methyltransferase (DIM-5) and one DNA methyltransferase (DIM-2), plants and animals depend on multiple DNA methyltransferases, and perhaps multiple histone methyltransferases, complicating mechanistic analyses. Mutation of one Arabidopsis histone methyltransferase SUVH4 (or “KRYPTONITE”) reduces DNA methylation by the DNA methyltransferase CMT3 (Malagnac et al., 2002; Jackson et al., 2002). Similarly, mutations in the mouse histone methyltransferase Suv39h1 causes selective loss of DNA methylation in pericentric heterochromatin (Lehnertz et al., 2003). No protein directly linking histone methylation to DNA methylation has been identified in either plants or mammals. Curiously, mutation of the only known Arabidopsis HP1 homolog TFL2/ LHP1 does not noticeably affect DNA methylation (Malagnac et al., 2002) even though this protein has been shown to interact with CMT3 in vitro (Jackson et al., 2002). Perhaps transduction of the signal for DNA methylation from histones to DNA can be accomplished by different pathways in eukaryotes. In mammals, colocalization of HP1 with Suv39h1 and the DNA methyltransferases Dnmt3a and Dnmt3b has been observed (Bachman et al., 2001). In addition, direct or indirect interactions have been reported between HP1 and DNA methyltransferases, Suv39h1, and a methyl binding domain protein (Fuks et al., 2003; Lehnertz et al., 2003; Fujita et al., 2003). It will be interesting to learn whether HP1 homologs in mammals are involved in DNA methylation. Experimental Procedures Neurospora Strains and Isolation of Genomic DNA Neurospora strains were grown, maintained, and crossed according to standard procedures (Davis, 2000). Strains used in this study are listed in Supplemental Table S1, available on Molecular Cell’s website. For isolation of genomic DNA, Neurospora strains were grown with shaking in Vogel’s minimal medium N at 32⬚C until saturation (3 days); genomic DNA was isolated and used for PCR and Southern analyses as described (Freitag et al., 2002). Isolation of hpo, Creation of an hpo-sgfp Fusion, and Neurospora Transformation The coding region of the hpo gene (NCU04017.1; http://www. genome.wi.mit.edu/annotation/fungi/neurospora/) was amplified by PCR with primers crd33 (5⬘-GCCGTCTAGAAAATGCCGTACGATC CATCGGCTCTCAGC-3⬘) and crd34 (5⬘-GCGGGATCCTTTGCGAG ACGCTGCCCTCGCGATCCTCGG-3⬘). Fragments were digested with BamHI and XbaI and inserted into BamHI ⫹ XbaI ⫺ digested pMF272 to yield pMF308, a translational fusion of hpo and sgfp. Plasmid pMF308 was inserted at the his-3 locus of N623, N2240, and N2257 by gene replacement as described (Freitag et al., 2002). Generation of hpo Mutants by RIP Primary, heterokaryotic His⫹ transformants were screened for stable expression of hpo-sgfp with an Olympus SZX12 microscope (460– 490 nm excitation, 505 nm dichroic, 510–550 nm emission). GFP⫹ strains N2532 to N2536 were selected and crossed to induce mutation by RIP. Two GFP⫺, presumably RIP-mutated strains (N2537 and N2538), from independent crosses (N2532 ⫻ N2535 and N2533 ⫻ N2536, respectively) were selected for further analyses. Homokaryotic GFP⫹ progeny from N2534 ⫻ N2264 were backcrossed (N2540 ⫻ N2542) and one additional hpo-sgfp RIP mutant (N2539) was selected. Because N2537 to N2539 contain the second

hpo-sgfp copy integrated at his-3, we amplified the mutated endogenous hpo allele by PCR with primers crd31 (5⬘-GCCGGATCCAGCT TGTACAGTAGTAGTAGAGCTTCC-3⬘) and crd32 (5⬘-ATGACTTCTA GATGTTAAGTTCTGTAGTACGAAAAG-3⬘), which hybridize only to the endogenous hpo flanking regions outside of the duplication (Figure 1A). The mutated hpo alleles of N2537 to N2539 were sequenced at the University of Oregon Genomics and Proteomics Facility. Generation of Dim⫺ HP1-GFP Strains To obtain hpo⫹-sgfp⫹ strains in Dim⫺ backgrounds, we crossed N2264 (dim-5) to N2534 and N1877 (dim-2) to N2540. Strains were genetically marked for the dim loci and genotypes were verified by Southern analyses. Sibling Dim⫹ progeny from the respective crosses were used for growth analyses and microscopy (see Supplemental Table S1, available on Molecular Cell’s website).

Visualization of HP1-GFP by Fluorescence Microscopy Neurospora asexual spores and hyphal fragments were spotted on microscope slides in droplets containing 1 ␮g/ml of the DNA dye Hoechst 33258 (Molecular Probes, Eugene, OR) and examined under a Zeiss Axioplan 2 microscope equipped with UV and FITC filtersets. Images were captured with an Orca II digital camera (Hamamatsu, San Jose, CA) and Openlab software (Improvision, Coventry, UK). Images were processed with Image J (NIH, USA) and Photoshop (version 4.0; Adobe, San Jose, CA) software. Neurospora strains were grown overnight at 32⬚C on malt extract agar, and nuclei labeled with GFP in living hyphae were imaged by confocal microscopy. Confocal laser scanning microscopy was performed using a Bio-Rad Radiance 2100 system equipped with an argon ion laser and mounted on a Nikon TE300 inverted microscope (all supplied by Bio-Rad Microscience, Hemel Hempstead, UK). GFP and FM4-64 were imaged simultaneously by excitation with the 488 nm laser line and fluorescence detection at 515/30 nm (for GFP) and ⬎570 nm (for FM4-64). Oil immersion 60⫻ (N.A. 1.4) or dry 20⫻ (N.A. 0.75) plan apochromatic objective lenses were used for imaging. The laser intensity and laser scanning of individual hyphae were kept to a minimum to reduce dye photobleaching and phototoxic effects. Time-lapse imaging was performed at scan intervals of 3–10 s for periods up to 15 min. Confocal scanning speeds of 50 and 166 lines per s were used to collect images. Confocal images were captured using Lasersharp 2000 software (version 4.3; Bio-Rad) and initially viewed using Confocal Assistant software (version 4.02, freeware). These images were transferred into Paintshop Pro software (version 7.0; JASC, Inc.) for further processing.

Computer Analyses The Neurospora (http://www-genome.wi.mit.edu/annotation/fungi/ neurospora/) and Fungal Genome Initiative genome sequences (http:// www-genome.wi.mit.edu/annotation/fungi/fgi/) were searched with tBLASTn and BLASTp (Altschul et al., 1997) for the presence of an HP1 homolog with Drosophila HP1␣ (GenBank accession number AAA28620), S. pombe Swi6 (P40381), and S. pombe Chp2 (NP_596808) as baits. Protein alignments with the preceding sequences and protein sequences of human HP1␣ (NP_036249), A. thaliana TFL2 (LHP1; BAB70689 and AAL04059), and M. grisea HP1 were performed with ClustalW at the Biology Workbench website (http://workbench. sdsc.edu/).

Acknowledgments We thank Hisashi Tamaru for the construction of strains N2264, N2659, and N2660; and Melissa Rolls, Paul Cullen, and Hillary Kemp for advice on fluorescence microscopy at the University of Oregon. We also thank Greg Kothe, Kristina Smith, and Hisashi Tamaru for comments on the manuscript. This work was supported by U.S. Public Health Service grant GM35690 (to E.U.S.). Confocal microscopy at the University of Edinburgh was performed in the Collaborative Optical, Spectroscopy, Micromanipulation, and Imaging Centre (COSMIC) facility, which is a Nikon-Partners-in-Research Lab.

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