genome86231 427..437

Letter

Relics of repeat-induced point mutation direct

heterochromatin formation in Neurospora crassa

Zachary A. Lewis,

Shinji Honda,

Tamir K. Khlafallah,

Jennifer K. Jeffress,

Michael Freitag,

Fabio Mohn,

Dirk Schu

beler,

and Eric U. Selker

1,4

Institute of Molecular Biology, University of Oregon, Eugene, Oregon, 97403-1229, USA;

Center for Genome Research and

Biocomputing, Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, USA;

Friedrich

Miescher Institute for Biomedical Research, 4058 Basel, Switzerland

Both RNAi-dependent and -independent mechanisms have been implicated in the establishment of heterochromatin

domains, which may be stabilized by feedback loops involving chromatin proteins and modifications of histones and

DNA. Neurospora crassa sports features of heterochromatin found in higher eukaryotes, namely cytosine methylation

(5mC), methylation of histone H3 lysine 9 (H3K9me), and heterochromatin protein 1 (HP1), and is a model to investigate

heterochromatin establishment and maintenance. We mapped the distribution of HP1, 5mC, H3K9me3, and H3K4me2 at

100 bp resolution and explored their interplay. HP1, H3K9me3, and 5mC were extensively co-localized and defined 44

heterochromatic domains on linkage group VII, all relics of repeat-induced point mutation. Interestingly, the centromere

was found in an ;350 kb heterochromatic domain with no detectable H3K4me2. 5mC was not found in genes, in contrast

to the situation in plants and animals. H3K9me3 is required for HP1 localization and DNA methylation in N. crassa.In

contrast, we found that localization of H3K9me3 was independent of 5mC or HP1 at virtually all heterochromatin regions.

In addition, we observed complete restoration of DNA methylation patterns after depletion and reintroduction of the

H3K9 methylation machinery. These data show that A:T-rich RIP’d DNA efficiently directs methylation of H3K9, which

in turn, directs methylation of associated cytosines.

[Supplemental material is available online at www.genome.org. The microarray data reported in this work have been

submitted to Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) under accession no. GSE12690.]

Chromatin is the relevant substrate for all DNA-mediated pro-

cesses in eukaryotes. Arrays of nucleosomes consisting of ;146 bp

of DNA wrapped around an octamer of four histone proteins (H3,

H4, H2A, and H2B) represent the lowest level of chromatin orga-

nization. Interactions of nucleosomes with each other and with

nonhistone chromatin proteins and RNAs are thought to mediate

functionally important higher-order chromatin structures. Gene-

rich ‘‘euchromatin’’ exists in an open conformation during much

of the cell cycle, facilitating DNA transactions such as transcrip-

tion. In contrast, the densely staining ‘‘heterochromatin’’ remains

highly condensed throughout the cell cycle, exhibits low levels of

transcription, and contains relatively few genes (Luger 2006;

Grewal and Jia 2007).

The core histones are subject to extensive covalent mod-

ifications (e.g., by phosphorylation, acetylation, methylation, and

ubiquitination) that can impact chromatin structure by pro-

moting or inhibiting nucleosome interactions, or by serving as

binding sites for proteins or protein complexes such as chromatin

remodelers. Generally, euchromatin is enriched for methylated

H3K4 within active genes and is rich in acetylated histones. Some

histone modifications in euchromatin are essential for transcrip-

tional memory during development or for mounting appropriate

and timely transcriptional responses to environmental stimuli.

Conversely, heterochromatin is typically hypoacetylated and

enriched for methylated H3K9 (Bhaumik et al. 2007). Though

generally transcriptionally silent, heterochromatin is essential for

proper centromere function and promotes genome stability by

preventing illegitimate recombination between repeated DNA

(Grewal and Jia 2007; Peng and Karpen 2008).

In addition to modification of histones, many organisms

methylate some cytosines in DNA. Such DNA methylation in

eukaryotes plays roles in development, genomic imprinting, X-

chromosome inactivation, silencing of transposons, and gene

regulation (Miura et al. 2001; Reik and Walter 2001; Reik et al. 2001;

Heard and Disteche 2006; Weber and Schubeler 2007). In animal

genomes, 5mC is restricted to CpG dinucleotides whereas in plants

and some fungi (e.g., Neurospora crassa), DNA methylation occurs

in both symmetric (CpG, CpHpG; H = A, C, or T) and asymmetric

contexts (CHH) (Suzuki and Bird 2008). In plants and animals,

hemimethylated symmetrical sites produced during replication are

recognized and methylated to propagate methylation patterns

(Saze et al. 2003; Bostick et al. 2007; Sharif et al. 2007).

The mechanisms that contribute to the maintenance of

asymmetric DNA methylation and those that establish DNA

methylation remain ill-defined; however, in some cases it is clear

that histone methylation directs DNA methylation. The first, and

most clear-cut example came from studies with the filamentous

fungus N. crassa, which showed that tri-methylation of H3K9 by

the lysine methyltransferase (KMT) defective in methylation-5

(DIM-5) is essential for DNA methylation (Tamaru and Selker

2001). The N. crassa homolog of heterochromatin protein 1 (HP1),

which binds methylated H3K9, is also essential for DNA methyl-

ation (Freitag et al. 2004a). HP1 recruits DIM-2, the DNA meth-

yltransferase (DNMT) responsible for all 5mC in vegetative cells

(Kouzminova and Selker 2001; Honda and Selker 2008). Similarly,

H3K9 methylation is important for some DNA methylation in

both plants and animals (Jackson et al. 2002; Lehnertz et al. 2003).

In addition, plants carry out RNA-directed DNA methylation,

Corresponding author.

E-mail [email protected]; fax (541) 346-5891.

Article published online before print. Article and publication date are at

http://www.genome.org/cgi/doi/10.1101/gr.086231.108.

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a process that involves the DNMT DRM2 and components of the

RNAi machinery (Henderson and Jacobsen 2007). Components of

the RNAi pathway are dispensable for DNA methylation in N.

crassa (Freitag et al. 2004b).

To develop a more complete understanding of how 5mC is

controlled, it is essential to determine the genomic location of this

modification. In plants, Methylated DNA ImmunoPrecipitation

(MeDIP) coupled with microarray analysis (Zhang et al. 2006;

Zilberman et al. 2007), and more recently whole genome shotgun

sequencing of bisulfite-treated DNA (Cokus et al. 2008; Lister et al.

2008), revealed dense methylation of repeated sequences and

transposons that are highly concentrated within the pericentric

heterochromatin domains. Methylation was also found in the

coding regions of genes, but not in the promoters, of over 30% of

expressed genes, indicating that transcriptional repression is not

necessarily an outcome of DNA methylation. In contrast, most

CpG dinucleotides within the mammalian genome are methyl-

ated, though many promoters contain C:G rich ‘‘CpG islands’’

that lack DNA methylation (Suzuki and Bird 2008).

Isolation and characterization of a fraction of the methylated

DNA from N. crassa suggested that most methylated sequences are

relics of repeat-induced point (RIP) mutation, a premeiotic genome

defense system that results in C to T changes within duplicated

sequences (Selker et al. 2003). Moreover, introduction of RIP’d or

A:T rich DNA into N. crassa triggered DNA methylation de novo,

suggesting that positive signals target methylation to specific DNA

sequences (Selker and Stevens 1987; Miao et al. 2000; Tamaru and

Selker 2003). Interestingly, lightly RIP’d sequences were unable to

trigger de novo methylation, but were able to maintain previously

established methylation (Singer et al. 1995; Selker et al. 2002). This

revealed that N. crassa has the capacity to perform both de novo and

maintenance methylation. Still, the extent to which each pathway

contributes to total methylation levels is not clear.

Since chromatin modifications impact DNA methylation, it

is important to determine what other features comprise the

chromatin environments marked by 5mC. Analysis of H3K9me3

and HP1 localization at a small number of RIP’d regions suggested

that these components are co-localized with 5mC in N. crassa

(Tamaru et al. 2003; Honda and Selker 2008), but the extent of co-

localization throughout the genome is unknown. Recent work in

mammalian cells revealed a strong inverse correlation between

H3K4 methylation and 5mC (Weber et al. 2007; Meissner et al.

2008). Despite advances, much remains to be learned about the

chromosomal contexts that promote or inhibit DNA methylation.

Knowledge of the distribution of chromatin marks should

also help elucidate interrelationships between them. Positive

feedback loops have already been implicated in establishment and

maintenance of heterochromatin domains in several organisms.

In Schizosaccharomyces pombe, domains of H3K9 methylation are

found at centromeres, telomeres, and the silent mating type loci

(Cam et al. 2005). Maintenance of heterochromatin domains in

this yeast involves the H3K9 binding protein Swi6 (homolog of

HP1), which is essential for the spread of H3K9 methylation be-

yond RNAi-dependent nucleation sites (Hall et al. 2002). In addi-

tion, the Clr4 H3K9 KMT binds methylated H3K9 via its chromo

domain, thereby facilitating methylation of adjacent nucleosomes

(Zhang et al. 2008). Similarly, Drosophila and mammalian

SU(VAR)3-9 H3K9 KMTs interact with the methyl H3K9 binding

protein HP1 (Aagaard et al. 1999; Schotta et al. 2002). In Arabi-

dopsis thaliana, the chromo domain of the CMT3 DNMT interacts

with H3 methylated at K9 and K27 to target 5methyl-cytosine

(5mC) and KRYPTONITE (also known as SUVH4), an H3K9 KMT,

binds 5mC via its SRA domain to target K9 methylation (Lindroth

et al. 2004; Johnson et al. 2007).

We mapped the chromosomal distribution of methylated

H3K9, HP1, and 5mC and found that these co-localize to form

discrete heterochromatin domains within the N. crassa genome.

We also found that while positive feedback occurs within the DNA

methylation pathway, it is not required for maintenance of most

heterochromatin domains in Neurospora. Finally, we determined

that relics of repeat-induced point mutation trigger efficient de

novo H3K9 and cytosine methylation.

Results

Distribution of 5mC in N. crassa

To investigate heterochromatin organization within the N. crassa

genome, we mapped the distribution of 5mC, H3K9me3, and HP1

across an entire chromosome. The N. crassa genome is composed of

seven chromosomes covering ;42 Mb. We chose LGVII for these

analyses because its current chromosome assembly contains few gaps,

covers ;10 % of the genome, and optical mapping data for LGVI I

suggest that the current genome assembly covers almost the entire

genetically mapped centromere (Centola and Carbon 1994) (http://

www.broad.mit.edu /anno tatio n/g enome/neurospora/Home.html).

We designed a custom microarray containing oligonucleotide

probes covering N. crassa LGVII and 37 previously identified

methylated regions (representing regions from all seven chromo-

somes) at 100 bp resolution and determined the distribution of 5

mC in wild-type N. crassa strain 74-OR-23IV by MeDIP coupled

with microarray analysis (Weber et al. 2005). Because most ‘‘re-

peated’’ sequences in the N. crassa genome have been subjected to

RIP, which typically causes 10%–20% sequence divergence, virtu-

ally all of our oligos were unique sequences (see Methods). In-

spection of the MeDIP data revealed that DNA from all 37

previously identified methylated regions was enriched in the

immunoprecipitated fraction, validating our experimental ap-

proach (Fig. 1). We showed previously that these methylated

sequences have been altered by RIP (Selker et al. 2003). The RIP

machinery, which produces C to T mutations, exhibits a preference

for CpA dinucleotides. Indeed, analyses of the frequencies of TpA

and CpA, relative to the frequencies of control dinucleotides, can

identify sequences that have been subjected to RIP. Typically,

sequences that have not been subjected to RIP exhibit values less

than 0.8 for the ‘‘RIP product index’’ (TpA / ApT) and values greater

than 1.1 for the ‘‘RIP substrate index’’ (CpA + TpG/ ApC + GpT). In

contrast, RIP’d regions typically exhibit values greater than 1.1 and

less than 0.9 for RIP product and substrate indices, respectively

(Margolin et al. 1998; Selker et al. 2003). A composite RIP index

(CRI) can be determined by subtractingthe substrate index from the

product index; thus, a positive CRI value implies that the DNA has

been subjected to RIP. We plotted the %GC and the CRI across each

previously identified region. 5mC was enriched throughout the

RIP’d regions and did not extend significantly into unRIP’d DNA.

We next examined the distribution of 5mC across N. crassa

LGVII (Fig. 2). Interestingly, a large region (;346 kb) comprising

the putative pericentric/centromeric DNA was enriched in the

MeDIP fraction (Fig. 3A). In addition, we observed numerous

methylated domains along both chromosome arms including

short methylated subtelomeric domains (Fig. 3B). To determine

the location of 5mC in an unbiased fashion, peaks of enrichment

were identified using ChIPOTle (

Chromatin Immuno Pre-

cipitation On Tiled microarrays) ChIP-chip data analysis software

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(Buck et al. 2005). Forty-six peaks were identified by ChIPOTle on

LGVII ranging in size from 0.6 to 138.5 kb (Supplemental Table 1).

Consistent with visual inspection of the data, peaks 29, 30, and 31

covered virtually all 346 kb of the putative pericentromere/cen-

tromere. The remaining 43 peaks were distributed on both arms of

LGVII and ranged in size from 0.6 to 21.4 kb (average of 7.1 kb

excluding centromeric region). Overall, methylated regions on

LGVII included 652 kb, or 16.5% of the 3.9 Mb chromosome. This

is somewhat higher than expected since previous studies indicated

that ;2% of cytosines are methylated in N. crassa (Foss et al.

1993), but is consistent with previous reports of heterogeneous

methylation within N. crassa cultures (Selker et al. 1993).

Co-localization of 5mC and H3K9me3

H3K9 methylation in wild-type N. crassa exists predominantly, if

not exclusively, as tri-methyl K9 (Tamaru et al. 2003) and this

chromatin modification has been shown to co-localize with 5mC

at several loci (Honda and Selker 2008). We determined the extent

of co-localization by examining the distribution of H3K9me3 us-

ing our high-resolution tiled microarray. Enrichment of H3K9me3

was found within all regions that had been previously examined,

once again validating the experimental approach (8:B1, 5:H8,

8:F10; Fig. 1). Importantly, experiments using two different anti-

bodies that recognize H3K9me3 yielded virtually identical results

(see Methods). Visual inspection of the data revealed striking co-

localization of 5mC and H3K9me3 across LGVII (Fig. 2). As with

5mC, H3K9me3 was enriched within the putative centromeric

region (Fig. 3A) and in dispersed regions along both arms, in-

cluding subtelomeric domains (Fig. 3B). We used ChIPOTle to

identify peaks of H3K9me3 enrichment; 45 peaks were identified

ranging in size from 0.5 to 151.1 kb (Supplemental Table 2). Peaks

27–31 covered virtually all 346 kb of the putative centromere,

whereas the remaining 40 peaks were distributed on both arms of

Figure 2. Chromatin modification profile for N. crassa LGVII. Base composition of the 3.9 Mb LGVII is shown as the moving average of %GC and the

CRI calculated for 500 bp windows with 100 bp steps at the top of the plot. Enrichment values for MeDIP and ChIP-chip experiments are shown as log

values indicated on the y-axis (right) for immunoprecipitation experiments with antibodies to 5mC, H3K9me3, green fluorescent protein for HP1-GFP

(HP1) and H3K4me2. The positions of predicted open reading frames (predicted genes) and repeats are indicated below. The scale bar on the top left

indicates 0.5 Mb.

Figure 1. Chromatin modification profile of previously identified methylated regions. Data from five representative control regions are shown. A scale

bar indicating 2 kb is shown at the top right of each plot. The base composition (%GC) and a composite RIP index (CRI) of each region, plotted as the

moving average for 500 bp windows with 100 bp steps, are shown at the top of each plot. Enrichment values for MeDIP and ChIP-chip experiments are

shown as log

values (y-axis) for immunoprecipitation experiments with antibodies to 5mC, H3K9me3, green fluorescent protein for HP1-GFP (HP1) and

H3K4me2 for each region. The positions of predicted open reading frames (genes) and the previously identified methylated DNA clones, with their

identification numbers, are shown at the bottom.

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LGVII, ranging in size from 0.5 to 21.6 kb. H3K9me3 regions in-

cluded 646 kb, or 16.4% of the 3.9-Mb chromosome, close to that

detected by MeDIP (see above).

We examined the degree of overlap between the 5mC peaks

and the H3K9me3 peaks. Of the 46 5mC peaks, 41 of these co-

incided with an H3K9me3 peak (Supplemental Table 1). It is likely

that the five 5mC regions that did not overlap with H3K9me3

peaks contain low levels of the modified histone that were not

detected by ChIPOTle. Indeed, three of these 5mC regions (peaks

10, 27, and 44; Supplemental Table 1) appeared to contain

H3K9me3 by visual inspection and were identified as H3K9me3

peaks in one experiment, suggesting that low levels of H3K9me3

are present in these 5mC regions (data not shown). The fourth

peak that did not overlap with an H3K9me3 peak displayed ob-

vious enrichment of HP1 and appeared to have low levels of

H3K9me3 by visual inspection and conventional ChIP (peak 34;

Fig. 4; Supplemental Table 1; see below). The fifth peak that did

not overlap with H3K9me3 had low levels of 5mC enrichment and

may contain H3K9me3 that was not detectable under our exper-

imental conditions. Notably, all 45 H3K9me3 peaks coincided

with 5mC peaks (Supplemental Table 2). Taken together, these

data show that 5mC and H3K9me3 are co-localized within the

N. crassa genome.

Complex localization of HP1

HP1 interacts directly with both H3K9me3 and the DIM-2 DNMT via

its chromo domain and chromo shadow domain, respectively

(Freitag et al. 2004a; Honda and Selker 2008). These interactions are

essential for DNA methylation in N. crassa. To determine if HP1 is

present at all methylated regions and if HP1 is localized to both

methylated and unmethylated regions, we examined the distribu-

tion of an HP1-GFP fusion protein expressed from the native HP1

locus and compared its localization to that of 5mC and H3K9me3 by

ChIP-chip. HP1 was generally co-localized with these two mod-

ifications (Figs. 1–3). Surprisingly, however, variable enrichment of

HP1-GFP was detected, both among H3K9me3 regions and within

some regions. It seems unlikely that this distribution resulted from

altered protein function due to the GFP tag because the strain

expressing this fusion protein displays wild-type levels of DNA

methylation (Honda and Selker 2008). Moreover, we performed

conventional ChIP experiments at four heterochromatic regions

using a strain expressing FLAG-tagged HP1. Like HP1-GFP, the HP1-

33 FLAG fusion protein was expressed from the native HP1 locus

and this strain displayed wild-type levels of DNA methylation (S.

Honda and E.U. Selker, unpubl.). Peak 35 (Supplemental Table 1)

exhibited high levels of both H3K9me3 and HP1 throughout the

RIP’d region (Fig. 4). In contrast, HP1 exhibited significantly higher

enrichment near the edges of peak 33 (Supplemental Table 1),

whereas H3K9me3 was relatively evenly distributed (Fig. 4). Peak 34

(Supplemental Table 1) was significantly enriched for HP1 even

though H3K9me3 levels were low at this region (Fig. 4); the enrich-

ment values for H3K9me3 and HP1 were of 1.9 and 2.6, respectively.

Although theseenrichment valuesare similar, HP1 levelswere;45%

of the maximum enrichment (sixfold), while H3K9me3 levels were

less than 20% of the maximal enrichment (10-fold) suggesting that

while HP1 and H3K9me3 co-localize, they differ quantitatively.

Peak 19 had low levels of HP1 even though H3K9me3 enrichment

levels were high at this region (Fig. 4). Thus, conventional ChIP

experiments confirmed the results seen in the microarray experi-

ments. In addition, similar results were obtained for ChIP-chip

experiments using strains expressing GFP-tagged and FLAG-tagged

HP1 (data not shown), confirming that the location and amount of

HP1 binding is not a direct function of H3K9me3.

DNA sequences in heterochromatic regions are products of RIP

A plot of the CRI for LGVII revealed that heterochromatin, defined

by H3K9me3 and 5mC, is predominantly associated with RIP’d

DNA. Major peaks of 5mC and H3K9me3 enrichment coincided

with CRI peaks (Fig. 2). We next asked if all methylated DNA on

LGVII is present within relics of RIP. We calculated the CRI for all 46

methylated regions described above. The average CRI for all 46

methylated regions was 0.69 and 44 regions had a positive CRI. Both

observations suggest that methylated DNA, in general, had been

extensively RIP’d. We asked if methylated regions also had low levels

of G:C, also expected of sequences exposed to RIP. Indeed, 80% of

Figure 3. Chromatin modification profile for the LGVII centromere and

telomeres. For (A) N. crassa LGVII centromere and (B) N. crassa chromo-

some ends, nucleotide composition is shown as the moving average of

%GC and CRI calculated for 500 bp windows with 100 bp steps at the top

of the plot. Enrichment values for MeDIP and ChIP-chip experiments are

shown as log

values indicated on the y -axis (right) for immunoprecipi-

tation experiments using antibodies to 5mC, H3K9me3, green fluores-

cent protein for HP1-GFP (HP1) and H3K4me2. The position of predicted

open reading frames (genes) and repeats are indicated below. The scale bars

on the top indicate 30 kb and 2 kb for (A)and(B), respectively. The black

dots above each plot in (B) indicate the position of the telomere. The broken

line above telVIIR indicates that sequences containing the telomere repeats

for telVIIR are missing from the current LGVII sequence assembly.

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methylated regions had a G:C content of 40% or lower and the av-

erage GC contentforall 46 methylated regionswas34.4%(compared

to an average G:C content of 54.7% for predicted LGVII genes).

We next asked if all RIP’d DNA is associated with DNA

methylation. We used ChIPOTle to call peaks of RIP’d DNA,

yielding a total of 587 kb of DNA for LGVII. 96% of this predicted

RIP’d DNA overlapped with a peak of 5mC. Interestingly, peaks of

RIP’d DNA that did not overlap with 5mC exhibited one or more

of the following properties: (1) The RIP’d region was less than 300

bp in length, (2) the RIP’d region overlapped with a region that

contained H3K4me2, and/or (3) the RIP’d region had a G:C con-

tent higher than 45%. These data indicate that in N. crassa, A:T

rich, RIP’d sequences that lack H3K4me2 and are greater than 300

bp in length are targeted for H3K9me3 and 5mC.

In contrast to the A:T -rich, RIP’d sequences, N. crassa genes are

G:C-rich. We found that only 0.5% of the predicted ORFs on LGVII

(5/1008) significantly overlap (>90%)with5mCregionsandthese

appeared to be pseudogenes that resulted from RIP. They show

a positive CRI, their predicted coding regions are short (<100 amino

acids) and BLAST searches failed to identify similar sequences in the

NCBI nonredundant database. Other apparent overlap between

genes and 5mC turned out to be simply a consequence of the limited

resolution MeDIP assay, which uses DNA sheared to ;500 bp. For

example, the gene to the left of the 8:B1 region (Fig. 1) appears to

overlap with the edge of the 5mC region, but Southern hybridizations

demonstrated that it is not methylated (data not shown). Taken to-

gether, our data show that genes are not targets of DNA methylation

in Neurospora, unlike the situation in plants and animals.

DNA methylation is a widespread feature of repeated

sequences of plants and animals, and this is also true for Neuros-

pora. N. crassa repeated sequences are divergent, with numerous

C:G to T:A nucleotide changes indicative of RIP. We found that

89% of the annotated repeats on LGVII overlapped with called

5mC peaks. Figure 5 summarizes the distribution of 5mC and

H3K9me3 within LGVII genes and repeated sequences.

DNA methylation is not affected in a double

Argonaute mutant

Some DNA methylation in plants depends on RNAi machinery

(Henderson and Jacobsen 2007), but this does not appear to be the

case in N. crassa. Both maintenance and de novo DNA methyla-

tion occurred normally in a dicer double mutant, a triple RNA-

dependent RNA polymerase mutant, and two single Argonaute

mutants (Freitag et al. 2004b; Chicas et al. 2005). It remained

a formal possibility, however, that the two Argonaute proteins

have redundant roles in DNA methylation. To test this, we per-

formed a MeDIP experiment with a double Argonaute mutant and

determined the distribution of 5mC using our high-resolution

microarray. DNA methylation was detected at all methylated

regions identified in the wild-type strain (Supplemental Fig. 1).

Southern blots also revealed that DNA methylation was equivalent

to that in wild type at all the heterochromatic loci examined (data

not shown). Thus, our findings strengthen the conclusion that the

RNAi machinery is not required for DNA methylation in N. crassa.

Distribution of a euchromatic mark

In contrast to heterochromatin, actively transcribed genes are

methylated at K4 of histone H3 in eukaryotes that have been

examined (Bhaumik et al. 2007). Because levels of H3K4me2

are inversely correlated with DNA methylation in mammals

(Weber et al. 2007; Meissner et al. 2008), we performed ChIP-chip

Figure 4. Complex localization patterns of HP1. Microarray data from immunoprecipitation experiments using antibodies to 5mC, H3K9me3, green

fluorescent protein (for HP1-GFP; HP1), and H3K4me2 are shown as log

values for 5mC peaks 35, 33, 34, and 19 (Supplemental Table 1). The position of

predicted open reading frames (genes) and repeats are indicated below. Conventional ChIP was performed for each region using antibodies to H3K4me2,

H3K9me3, and FLAG (for HP1-FLAG) to validate the microarray data. PCR products obtained using whole cell extracts (WCE), or the indicated immu-

noprecipitate fraction as the template, were resolved by gel electrophoresis. The black bars labeled P1–P5 depict the position of the expected PCR

products within each region. The coding region of histone H4 was used as an internal euchromatin control. The labels to the right of each autoradiograph

identify specific PCR products. The enrichment of each PCR product relative to the PCR product for H4 to is shown below.

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experiments to examine the distribution of this modification in N.

crassa. Similar to the case in other organisms, H3K4me2 was

enriched in the coding regions of genes. Of 1008 predicted ORFs on

LGVII, 42.5%were enriched for H3K4me2 within the genebodies.In

contrast, 5mC enrichment peaks displayed negative log

values,

indicative of H3K4me2 depletion (Figs. 2, 5C). Indeed, the average

log

enrichment value for H3K4me2 within the 46 5mC regions was

0.61 suggesting that heterochromatin and H3K4me2 are mutually

exclusive in N. crassa. In many organisms, the centromeric core is

enriched for H3K4me2 and for a centromere-specific H3 variant

(CENPA, also known as CENH3) (Sullivan and Karpen 2004; Cam

et al. 2005). Interestingly, we did not detect any enrichment of

H3K4me2 within the centromeric core of N. crassa LGVII (Fig. 3A).

Limited positive feedback from HP1 contributes to normal

H3K9 methylation

In mammals, DNA methyl-binding domain (MBD) proteins in-

teract with H3K9 KMTs (Fujita et al. 2003; Sarraf and Stancheva

2004), whereas in plants, the SRA domain present in the KRYP-

TONITE/SUVH4 H3K9 KMT binds 5mC (Johnson et al. 2007). To

examine whether DNA methylation might direct chromatin

modification in N. crassa, we compared the distribution of

H3K9me3 in the dim-2 DNMT mutant with that in a wild-type

strain. As a negative control, we examined the distribution of

H3K9me3 in the dim-5 KMT mutant (strain N3436). We found that

H3K9me3 persists at all LGVII heterochromatic regions in the dim-2

mutant (Fig. 6). We also examined the distribution of H3K9me3 in

the hpo strain because HP1 is known to interact with numerous

proteins that modify chromatin. Most of the H3K9me3 detected in

wild-type strains was also present in the hpo mutant (Fig. 6A), but

we did identify three regions that lost H3K9me3 in the hpo mutant.

These included one region on LGVII (peak 33) and two previously

identified methylated clone sequences (8:A6 and 2:C9). HP1-

sensitive regions were short (<2.0 kbp) and had higher G:C content

than HP1-insensitive regions, although these regions had lower

G:C contents than gene sequences. The data for a typical hetero-

chromatin region (HP1-insensitive) and two of these atypical (HP1-

sensitive) regions are shown in Figure 6B. We conclude that 5mC

and HP1 are dispensable for normal H3K9me3 at most regions of

the genome but that HP1 is required for H3K9me3 at a minority of

heterochromatic regions.

Efficient de novo methylation in N. crassa

Previous studies demonstrated that both maintenance and de novo

DNA methylation occur in N. crassa (Selker et al. 2002). To assess the

fraction of methylated regions that are capable of rapid de novo

methylation, we crossed different mutant strains that lack all DNA

methylation and isolated a wild-type daughter strain. Thus, all 5mC

present in the daughter strain must be a result of de novo methyl-

ation. The distributions of 5mC from wild type, the two parental

strains (dim-2 and hpo) and the wild-type daughter strain (strain

N3668) are shown for LGVII (Fig. 7). As expected, 5mC was not

detected in the two parental strains. In contrast, extensive meth-

ylation was detected in the daughter strain. Indeed, 5mC was

detected at all of the methylated regions detected in the wild-type

strain. This indicates that de novo methylation occurs rapidly in

Neurospora and that DNA methylation does not, in general,

depending on preexisting methylation.

We carried out a similar experiment to determine whether

H3K9me3 is efficiently reestablished de novo. In particular, we

crossed dim-5 (Tamaru and Selker 2001) and dim-8 (Z.A. Lewis, K.K.

Adhvaryu, S. Honda, and E.U. Selker, unpubl.) strains, which are

both defective in H3K9me3 (and 5mC), isolated a wild-type

recombinant and performed a MeDIP experiment to determine

which regions were able to undergo de novo H3K9me3 and sub-

sequent DNA methylation. Remarkably, DNA methylation was

restored at all LGVII heterochromatin regions described above.

Southern analyses were fully consistent with the MeDIP data (Sup-

plemental Fig.2).We concludethat bothH3K9me3 methylation and

DNA methylation are efficiently re-established de novo in N. crassa.

Discussion

Co-localization of 5mC and H3K9me3

We examined the detailed genomic distribution and hierarchical

relationships of DNA methylation, H3K9 methylation, and HP1 in

the filamentous fungus N. crassa. H3K9 methylation is required for

DNA methylation in this fungus and consistent with this obser-

vation, we found that the location of DNA methylation was highly

Figure 5. Genes and repeats reside in distinct chromatin environ-

ments. Scatter plots depicting the relationship between RIP and chro-

matin modifications are shown for LGVII genes (gray dots) and repeats

(black dots). The CRI value calculated for each predicted open reading

frame and annotated repeat is plotted on the y-axis. The median en-

richment value for (A) 5mC, (B) H3K9me3, and (C ) H3K4me2 for each

gene and annotated repeat is plotted on the x-axis.

Lewis et al.

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correlated with the location of H3K9me3. Indeed, all regions that

were enriched for H3K9me3 were also enriched for 5mC. This

suggests that H3K9 methylation is sufficient to trigger DNA

methylation in N. crassa.

Complex localization of HP1

In N. crassa, HP1 appears to directly link H3K9me3 with 5mC

(Honda and Selker 2008). Two PXVXL-related motifs in DIM-2

interact directly with the HP1 chromo shadow domain. In-

teraction of HP1 with methylated H3K9 is well established

(Bannister et al. 2001; Lachner et al. 2001). Consistent with this,

we found that HP1 was generally co-localized with H3K9 meth-

ylation at N. crassa heterochromatic domains. Interestingly, HP1

was not highly enriched at all H3K9me3 regions, however, and

HP1 enrichment was found to be high at one region that showed

low levels of H3K9me3. Furthermore, in some regions HP1 dis-

played a nonuniform distribution relative to H3K9me3 and 5mC

(Fig. 4). Several possible models could account for the complex

pattern of HP1 localization observed in our experiments. (1) HP1-

interacting proteins may contribute to the pattern of HP1 locali-

zation by recognizing additional structural features such as

histone modifications or DNA sequences. Accessory factors that

promote chromatin association of HP1 have been identified in

mammals and Drosophila (Nielsen et al. 2001; Shi et al. 2008). (2)

Additional chromo domain proteins could compete for binding of

H3K9me3 and thereby influence the distribution of HP1. Mam-

mals encode multiple HP1 proteins that show distinct localization

patterns and the fission yeast S. pombe encodes several H3K9-

binding, chromo domain proteins that each display preferential

binding at distinct genomic locations (Maison and Almouzni

2004; Grewal and Jia 2007). N. crassa encodes several chromo

domain proteins (Borkovich et al. 2004). (3) The ChIP procedure

may preferentially enrich for sites where HP1 forms a stable

complex with the chromatin fiber. Both high and low mobility

populations of HP1 exist within cells (Cheutin et al. 2004;

Schmiedeberg et al. 2004). Importantly, the nonuniform distri-

bution of HP1 relative to that of H3K9me3 (and 5mC) may in-

dicate that only transient association with chromatin is required

for HP1-directed DNA methylation by DIM-2. Alternatively, HP1

may be more uniformly distributed with H3K9me3 during a spe-

cific phase of the cell cycle, such as during replication. It will be

important to determine how a somewhat variable pattern of HP1

localization can lead to the observed pattern of DNA methylation.

Figure 6. Distribution of H3K9 methylation in wild-type, hpo, and dim-2 strains. (A) The distribution of H3K9me3 across LGVII is shown for wild-type,

dim-5, hpo, and dim- 2 strains. (B) The distribution of H3K9me3 is plotted for wild-type, hpo,anddim-2 strains for LGVII 5mC peak 40, (C ) the previously

identified methylated region 8:B1, and (D) LGVII 5mC peak 33 (Supplemental Table 1 and Selker et al. 2003). Conventional ChIP was performed using

antibodies to H3K9me3 for the indicated strains. For each strain, PCR products obtained using the immunoprecipitated fraction (H3K9me3, top)and

whole cell extract (WCE, bottom) as a template were resolved by gel electrophoresis. The black bars numbe red P2–P7 depict the position of the expected

PCR products within each region. The coding region of histone H4 (hH4) was used as an internal euchromatic control. The labels to the right of each

autoradiogram indicate specific PCR products. The relative enrichment of each PCR product is shown below.

Heterochromatin in N. crassa

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RIP and DNA methylation

Previous studies revealed a close association between the process

of RIP and DNA methylation (Selker et al. 1993, 2003). The

remaining cytosines within a duplicated sequence are typically

methylated following RIP and isolation of methylated DNA

revealed that most methylated sequences appear to be relics of RIP.

Our results strongly support and extend prior evidence of a tight

connection between RIP and DNA methylation. In this study, we

have shown that both 5mC and H3K9me3 are exclusively asso-

ciated with relics of RIP all along LGVII and in other previously

identified methylated regions.

Structure of LGVII centromere and telomeres

The most notable chromosomal domain of RIP’d DNA includes

the genetically mapped centromere. In S. pombe, Drosophila, and

humans, the centromeric core domain is comprised of CENPA

containing nucleosomes interspersed with nucleosomes that

contain H3K4me2 (Sullivan and Karpen 2004; Cam et al. 2005).

In contrast, pericentric domains in these organisms typically

contain H3 methylated at K9, and in plants and animals, peri-

centric sequences contain 5mC (Amor et al. 2004; Suzuki and Bird

2008). Recent work in S. pombe revealed that heterochromatic

modifications within the pericentric domains are required for

CENP-A deposition (Folco et al. 2008). Interestingly, we failed to

identify any H3K4 methylation within the putative LGVII cen-

tromere. Moreover, 5mC and H3K9me3 were distributed broadly

throughout the regions believed to comprise the centromeric core

suggesting that N. crassa possesses a novel centromere organiza-

tion. Although it is conceivable that the current genome sequence

annotation lacks the bona fide LGVII centromeric core sequence,

this is unlikely since ChIP-sequencing experiments with the N.

crassa CENP-A protein show enrichment of the H3 variant in

sequences included on our microarray (K.M. Smith and M. Freitag,

unpubl.). Thus, our data indicate that enrichment of H3K4me2

within the centromeric core may not be a general feature of

eukaryotic centromeres. Future work to determine the distribution

of N. crassa centromere proteins should provide additional insight

into centromere organization in N. crassa.

Silencing of genes that reside near telomeres, termed the

‘‘telomere position effect,’’ has been attributed to repressive

chromatin that spreads from telomere-associated sequences into

adjacent subtelomeric chromatin (Perrod and Gasser 2003). In S.

pombe, a cenH-like repeat near telomere IL is believed to nucleate

a nearly 40 kb telomeric heterochromatin domain containing

H3K9 methylation and Swi6/HP1 (Cam et al. 2005). Similarly, we

observed localization of H3K9me3 and HP1, as well as 5mC, at the

chromosome ends in N. crassa. In contrast to the extended

domains of subtelomeric heterochromatin in S. pombe, the N.

crassa, LGVIIL 5mC/H3K9me3 telomeric domain was short and

highly correlated with A:T rich sequences. These data suggest that

heterochromatin does not spread beyond the short RIP’d

sequences found at telomeres in N. crassa (Wu et al. 2008).

Moreover, genes that are located close to subtelomeric hetero-

chromatin were found enriched for H3K4 methylation suggesting

that gene expression is not silenced in these regions.

5mC is not found in N. crassa genes

In addition to centromeres and telomeres, H3K9me3, HP1, and

5mC mapped to over 40 dispersed sites across LGVII that also show

hallmarks of RIP. None of the heterochromatic domains on LGVII

appear to contain functional genes. Conversely, coding sequences

and virtually all promoters were found to be free of 5mC, H3K9me3,

and HP1. Thus, our data do not support an extensive role for DNA

methylation, H3K9 methylation, or HP1 in regulation of tran-

scription under standard growth conditions. It is also notable that

H3K4 methylation was found in several heterochromatin-adjacent

genes (see Fig. 4), suggesting that heterochromatic domains do not

necessarily suppress expression of flanking genes. This observation

is consistent with transcript profiling experiments that do not

support a role for 5mC in regulating gene expression in N. crassa (A.

Shiver, Z.A. Lewis, and E.U. Selker, unpubl.). In plants and animals,

DNA methylation within gene bodies has been proposed to prevent

initiation at cry ptic promoters, a function apparently relegated to

methylation of H3K36 in the yeast Saccharomyces cerevisiae (Carrozza

et al. 2005; Zilberman et al. 2007). N. crassa does not appear to rely

on DNA methylation to prevent such aberrant transcription initi-

ation (Rountree and Selker 1997), but does perform H3K36 meth-

ylation (Adhvaryu et al. 2005). Perhaps gene body methylation is

important for inhibiting aberrant transcription initiation in

organisms with larger coding sequences, while H3K36 methylation

may be sufficient to provide this function in organisms with shorter

genes.

Limited feedback within the N. crassa 5mC pathway

Although we found that DNA methylation was dispensable for

H3K9 methylation at all RIP’d regions examined, it is possible that

Figure 7. N. crassa performs efficient de novo methylation of heterochromatic regions. The distribution of 5mC is shown across LGV II for wild type

(wt), the methylation deficient strains dim-2 and hpo, and wild-type daughter strains from two crosses between methylation deficient strains. Enrichment

values for MeDIP experiments are shown as log

values on the y-axis. The wild-type strain N3668 was obtained by crossing two strains that lack 5mC but

retain H3K9me3, dim-2,andhpo. The wild-type strain N3669 was obtained by crossing two strains that lack both H3K9me3 and 5mC, dim-5,anddim- 8.

Thus, all methylation in these strains is a result of de novo methylation.

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DNA methylation recruits factors that reinforce a repressive

chromatin environment. A methylated-DNA binding activity has

been identified in N. crassa nuclear extracts, consistent with this

possibility (Selker et al. 2002). Interestingly, transcriptional acti-

vation of a silent copy of the bacterial hph gene was observed

following depletion of DNA methylation (Irelan and Selker 1997).

Moreover, loss of H3K9 methylation at a RIP’d allele of the N.

crassa am gene (am

RIP8

) was observed in a dim-2 mutant (Tamaru

et al. 2003). This may indicate that DNA methylation is required

for H3K9 methylation of transcriptionally competent sequences,

but not at sequences that are not typically transcribed. The ex-

tensive RIP of LGVII heterochromatic sequences presumably

inactivated promoter sequences within these domains.

Similarly, HP1 had only a modest influence on the distribu-

tion of H3K9me3. Although we found that HP1 is required to

maintain H3K9 methylation at three loci (i.e., one on LGVII and

two previously identified regions), most heterochromatin do-

mains remain unaffected in an hpo mutant. Specifically, 99.7% of

H3K9me3 on LGVII persists in the hpo mutant. This observation

was surprising because current models implicate HP1 in the

spreading and maintenance of heterochromatin domains in other

organisms. In S. pombe, HP1 is required for spreading of H3K9

methylation across the silent mating type locus and Drosophila

HP1 is required for normal localization of the SU(VAR)3-9 H3K9

KMT (Hall et al. 2002; Schotta et al. 2002). HP1 homologs in

Drosophila and mammals interact with H3K9 KMTs in vivo

(Aagaard et al. 1999; Schotta et al. 2002). Unlike its homologs in S.

pombe, Drosophila, and mammals, DIM-5, the N. crassa H3K9 KMT,

does not have a chromo domain and apparently does not interact

directly with HP1 (Tamaru and Selker 2001; Honda and Selker

2008). Therefore, it is likely that the limited effects of HP1 on

H3K9 methylation are indirect. HP1 may act to recruit histone

deacetylases since treatment of N. crassa with the histone deace-

tylase inhibitor Trichostatin-A results in limited loss of DNA

methylation (Selker 1998).

Epigenetic inheritance reflects the stable maintenance of

a chromatin state through replication cycles independently of the

associated DNA sequence. Reporter genes inserted into the S.

pombe mat locus are stably inherited in either the ‘‘on’’ or ‘‘off’’

state through mitosis and meiosis (Grewal and Klar 1996). In

plants and animals, faithful inheritance of DNA methylation

patterns requires maintenance methylation of hemimethylated

symmetrical sites, such as CpGs, following replication. Indeed,

loss of methylation at CpG sites in A. thaliana met1 mutants is not

completely restored following reintroduction of a wild-type met1

gene, highlighting the importance of existing methylation (Saze

et al. 2003). In contrast, we found no chromosomal regions of N.

crassa that rely on maintenance methylation. This result fits nicely

with our observation that H3K9me3 is normally distributed in

a dim-2 mutant (Fig. 6). Interestingly, we observed complete res-

toration of DNA methylation patterns after depletion and rein-

troduction of the H3K9 methylation machinery, indicating that

the signals for de novo heterochromatin formation lie upstream of

H3K9 methylation. These data indicate that A:T-rich RIP’d DNA

efficiently directs methylation of H3K9 and in turn, directs

methylation of associated cytosines.

Taken together, these results support a model for heterochro-

matin formation in N. crassa that involves efficient targeting of

H3K9 methylation and DNA methylation to A:T-rich relics of RIP.

In contrast to heterochromatin formation in S. pombe and some

DNA methylation in plants, this process appears to be independent

of RNAi and probably relies on DNA binding factors that specifi-

cally recognize A:T-rich DNA. Indeed, the AT-hook analog Dis-

tamycin can inhibit DNA methylation in N. crassa (Tamaru and

Selker 2003). Identification of the factors that direct heterochro-

matin formation at these regions will be critical for developing

a complete model of heterochromatin formation in N. crassa.

Methods

Microarray design

Custom microarrays produced by Agilent Technologies (http://

www.agilent.com) contained 39,386 probes that span LGVII and

2622 control probes corresponding to previously identified

methylated regions. A complete LGVII sequence file was assem-

bled based on the physical map assembled by the Neurospora crassa

genome project (http://www.broad.mit.edu/annotation/genome/

neurospora/Home.html). The relevant contig numbers, contig

orientations, and position on the LGVII sequence are given in

Supplemental Table 3. This assembly includes sequences for the

telomere repeats and the adjacent subtelomeric heterochromatin

for telVIIL, but the telomere repeats and adjacent sequences are

currently not available for telVIIR. Isothermal probes between 45

and 60 bp were designed at intervals of ;100 bp from start to start

as previously described (Thibaud-Nissen et al. 2006; Zilberman

et al. 2007). The microarray contained probes corresponding to

both unique and repeated sequences. It is important to note that

virtually all repeated DNA within the N. crassa genome has been

subjected to RIP. Therefore, repeated sequences typically display

<80% similarity (Galagan and Selker 2004). Of the 39,386 probes

covering LGVII, 38,350 contained at least two or more mis-

matches when compared to the most similar repeated sequence.

Our expectation that the vast majority of our microarray oligo-

nucleotides would not cross hybridize with other regions of the

genome was supported by preliminary results of massive se-

quencing of DNA isolated by MeDIP or ChIP with the antibodies

used in this study (K.M. Smith and M. Freitag, unpubl.).

MeDIP and ChIP

Genomic DNA was isolated for MeDIP as previously described

(Freitag et al. 2004a). MeDIP was performed essentially as de-

scribed except typically 1 mL of antibody specific for 5mC (Dia-

genode) was used to precipitate methylated DNA from 4 mg of total

genomic DNA (Weber et al. 2005). The procedure for ChIP analysis

and antibodies that recognize H3K4me2 (#07-030 Upstate) and

H3K9me3 (a gift from Prim Singh) were previously described

(Tamaru et al. 2003). In addition, ChIP was performed using

a second antibody specific for H3K9me3 (#39161 Active Motif). All

primers used for conventional ChIP analysis are listed in Supple-

mental Table 4.

Sample labeling and microarray hybridization

For MeDIP experiments, ;200 ng of sheared input DNA was la-

beled with Cy3 and 25% of the immunoprecipitated fraction

(from 4 mg input) was labeled with Cy5 using the Bioprime Array

CGH Genomic Labeling system (Invitrogen) as described (Lee et al.

2006). For ChIP-chip experiments, 10 ng of input DNA and 50% of

the immunoprecipitated fraction was amplified using a whole

genome amplification as described (O’Geen et al. 2006). After two

rounds of amplification, 300 ng of input DNA was labeled with

Cy3 and 1 mL of amplified immunoprecipitated DNA was labeled

with Cy5 using the Bioprime Array CGH Genomic Labeling sys-

tem. Microarray hybridizations were performed for ;40 h at 42°C

in hybridization buffer (Lee et al. 2006).

Heterochromatin in N. crassa

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Data analysis

Microarrays were scanned at 5 mm resolution using a Genepix

4000a scanner (Axon) and pixel intensities were calculated using

Genepix Pro 6.0 (Axon). The data were median normalized and

the log

[Cy5/Cy3] ratio was calculated for each spot. Data were

plotted as log

values for each spot covering the entire LGVII se-

quence using the Argo genome browser available at http://

www.broad.mit.edu/annotation/argo/. The positions of predicted

open reading frames and repeats were obtained from the Neuros-

pora Genome Project (http://www.broad.mit.edu/annotation/

fungi/neurospora/). To identify statistically significant peaks of

enrichment, we first calculated median values for 500 bp sliding

windows (five spots) to reduce noise. We then utilized the ChIP-

chip data analysis program ChIPOTle v1.11 (Buck et al. 2005) to

call peaks of enrichment. Peaks of 5mC and H3K9me3 were de-

termined for two biological replicate experiments using a window

size of 500 bp and a step size of 100 bp. Replicate experiments for

H3K9me3 were performed with two different antibodies that

recognize H3K9me3 (see above). Only peaks that were detected by

ChIPOTle in both replicate experiments were considered for sub-

sequent analysis. Similarly, we used ChIPOTle to identify peaks of

RIP’d DNA (i.e., regions with high CRI values) using a window size

of 500 bp and a step size of 100 bp. For all analyses, only peaks

with a P-value of 0.05 or better were considered significant.

Strains and culture conditions

N. crassa strains were grown and crossed using standard culture

conditions (http://www.fgsc.net/Neurospora/neurospora.html).

Wild-type strain 74-OR-23-IVA (Selker laboratory strain N150),

dim-2 (strain N1850), hpo (strain N2556), dim-5 hpo-gfp dim-2-

3xflag (strain N3436), hpo-gfp (N3415), and hpo-flag (N3320)

strains were described previously (Kouzminova and Selker 2001;

Freitag et al. 2004a; Honda and Selker 2008). The double Argo-

naute mutant was generously provided by Dr. Yi Liu (UT South-

western). A dim-5 knockout strain (N3074) was created by

replacing the dim-5 gene with a basta-resistance cassette by ho-

mologous recombination as described (Colot et al. 2006). Primers

used to amplify the dim-5 flanking sequence and create a knockout

vector using yeast homologous recombination are listed in Sup-

plemental Table 4. The dim-8 gene was isolated by selecting for

mutants that are defective in methylation (Z.A. Lewis, K.K. Adh-

varyu, S. Honda, and E.U. Selker, unpubl.). Since dim-5 strains are

female sterile, a forced heterokaryon between dim-5 and the

‘‘helper 2’’ strain (FGSC #8745) was used as a female parent in

crosses with dim-8. Helper 2 harbors a deletion of the mating

type locus and therefore does not undergo productive meiosis

(Metzenberg and Sachs 2002).

Acknowledgments

We thank Nicholas Stiffler (UO) for microarray oligo design, Scott

Givan (OSU) for help with oligo design, Reinhard Engels (MIT) for

help with the Argo browser, and Yi Liu (UT Southwestern) for the

double Argonaute mutant. This work was supported by

GM025690-22 to E.U.S. from the National Institutes of Health.

Z.A.L. was supported by fellowships from the American Cancer

Society (PF-04-122-01-GMC) and the Leukemia and Lymphoma

Society (3295-09).

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Received September 8, 2008; accepted in revised form December 8, 2008.

Heterochromatin in N. crassa

Genome Research 437

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2009 19: 427-437 originally published online December 17, 2008Genome Res.

Zachary A. Lewis, Shinji Honda, Tamir K. Khlafallah, et al.

Neurospora crassaformation in

Relics of repeat-induced point mutation direct heterochromatin

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