1

1

2

A Light-Inducible Strain for Genome-Wide Histone Turnover Profiling 3

in Neurospora crassa 4

5

6

7

William K. Storck*, Sabrina Z. Abdulla

1, Michael R. Rountree

2, 8

Vincent T. Bicocca3, and Eric U. Selker

*,† 9

10

11

*Institute of Molecular Biology, University of Oregon, Eugene, OR 97403 12

13

Present Addresses: 14

1Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801 15

2Nzumbe, Inc., 3439 NE Sandy Blvd. #330, Portland, OR 97232 16

3Convergent Genomics, 425 Eccles Ave, South San Francisco, CA 94080 17

18

†Correspondence: selker@uoregon.edu 19

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Genetics: Early Online, published on May 1, 2020 as 10.1534/genetics.120.303217

Copyright 2020.

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Running Title: Histone Turnover in Neurospora 21

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Key Words: Chromatin Profiling; Histone Exchange; Neurospora crassa; Heterochromatin; 23

Light Induction 24

25

Corresponding Author 26

Eric Selker 27

Institute of Molecular Biology 28

University of Oregon 29

1370 Franklin Bvld, Eugene, OR, 97403-1229 30

Phone: 541-346-5193 31

Email: selker@uoregon.edu 32

33

3

ABSTRACT 34

In chromatin, nucleosomes are composed of about 146 base pairs of DNA wrapped around a 35

histone octamer, and are highly dynamic structures subject to remodeling and exchange. Histone 36

turnover has previously been implicated in various processes including regulation of chromatin 37

accessibility, segregation of chromatin domains, and dilution of histone marks. Histones in 38

different chromatin environments may turnover at different rates, possibly with functional 39

consequences. Neurospora crassa sports a chromatin environment that is more similar to that of 40

higher eukaryotes than yeasts, which have been utilized in the past to explore histone exchange. 41

We constructed a simple light-inducible system to profile histone exchange in N. crassa on a 42

3xFLAG-tagged histone H3 under the control of the rapidly inducible vvd promoter. After 43

induction with blue light, incorporation of tagged H3 into chromatin occurred within 20 minutes. 44

Previous studies of histone turnover involved considerably longer incubation periods and relied 45

on a potentially disruptive change of medium for induction. We used this reporter to explore 46

replication-independent histone turnover at genes and examine changes in histone turnover at 47

heterochromatin domains in different heterochromatin mutant strains. In euchromatin, H3-48

3xFLAG patterns were almost indistinguishable from that observed in wild type in all mutant 49

backgrounds tested, suggesting that loss of heterochromatin machinery has little effect on histone 50

turnover in euchromatin. However, turnover at heterochromatin domains increased with loss of 51

H3K9me3 or HP1, but did not depend on DNA methylation. Our reporter strain provides a 52

simple yet powerful tool to assess histone exchange across multiple chromatin contexts. 53

54

4

INTRODUCTION 55

The eukaryotic genome is packaged in the nucleus by wrapping DNA around histone octamers, 56

forming the basic organizational unit called the nucleosome. Nucleosomes form the basis of 57

higher-order organization of the genome, which can impact the function of underlying genetic 58

elements (Van Steensel 2011). The positions of key nucleosomes within chromatin are subject to 59

regulation, and underlying histones are subject to exchange (Venkatesh and Workman 2015). 60

Histone exchange can be independent of DNA replication, and in at least in some cases, levels of 61

histone turnover appear to correlate with levels of gene expression (Venkatesh and Workman 62

2015). High levels of turnover are thought to allow for greater accessibility to DNA for 63

transcriptional machinery such as transcription factors and RNA polymerases, and may play a 64

role in the regulation and distribution of histone marks and histone variants (Venkatesh and 65

Workman 2015). Histone turnover may also play a role as boundary elements to contain 66

heterochromatin spreading (Allshire and Madhani 2018). Heterochromatin is generally thought 67

of as a compacted form of chromatin, rendering its DNA relatively inaccessible to transcriptional 68

factors and suppressing the activity of selfish genetic elements (Allshire and Madhani 2018). 69

Consistent with this view, heterochromatin has been found to be refractory towards histone 70

exchange, and disruption of heterochromatin leads to increased turnover in fission yeast (Aygün, 71

Mehta, and Grewal 2013; Choi et al. 2005). Potentially, suppression of histone turnover could be 72

a general a feature of heterochromatin, which might contribute to epigenetic inheritance and 73

other functions of this genome compartment. 74

In metazoans, replication-independent histone turnover, such as during transcription, 75

results in replacement of hH3.1 deposited during DNA replication with a closely related histone 76

variant, hH3.3. This exchange is thought to help expose DNA binding sites and is apparently 77

crucial for development (Venkatesh and Workman 2015). Investigations of histone turnover have 78

been performed in the yeasts S. cerevisiae and S. pombe, which offer a simpler complement of 79

histone proteins; yeasts have only one hH3 isoform, a homolog of hH3.3 (Venkatesh and 80

Workman 2015). Like yeasts, the filamentous fungus Neurospora crassa possesses only one 81

histone H3 isoform, which is also homologous to metazoan hH3.3. However, N. crassa possesses 82

chromatin features more similar to those in higher eukaryotes. For example, like higher 83

organisms, N. crassa possesses two functionally distinct methyltransferases for lysine 36 on 84

histone H3 (H3K36), SET-2 and ASH1, while yeasts possess just SET-2. SET-2 is responsible 85

5

for nearly all H3K36me3 in N. crassa and is implicated in regulating histone exchange in the 86

wake of Pol II during its passage over gene bodies; ASH1-deposited H3K36me2 appears to mark 87

transcriptionally silent genes (Bicocca et al. 2018). N. crassa also sports DNA methylation at 88

constitutive heterochromatin as well as the facultative heterochromatin mark, methylation of 89

lysine 27 of histone H3 (H3K27me), both of which are absent from yeasts (Aramayo and Selker 90

2013). 91

Here we report the construction and validation of a histone turnover reporter strain of N. 92

crassa. Our strain utilizes 3xFLAG-tagged histone H3 under the control of a light-inducible 93

promoter. Inducible tagged histones have been previously used to assay histone exchange in 94

other models, but required the addition of an inducing agent – often through a change of growth 95

medium – and a lengthy incubation period, typically on the order of hours (Aygün, Mehta, and 96

Grewal 2013; Choi et al. 2005; Rufiange et al. 2007; Dion et al. 2007; Kraushaar et al. 2013; 97

Ahmad and Henikoff 2002). Our use of light as the inducing signal is less disruptive than a 98

change of medium and provides greater control and more rapid induction. We used our system to 99

explore possible effects on histone turnover of mutants defective in heterochromatin machinery. 100

101

MATERIALS AND METHODS 102

N. crassa strains and molecular analyses 103

Strains are listed in Table S1 and were grown, crossed, and maintained according to standard 104

procedures (Davis 2000). Primers used are listed in Table S2. DNA isolation and western 105

blotting were performed as previously described (Honda and Selker 2008). The following 106

antibodies were used in western blot analyses: anti-FLAG-conjugated peroxidase (FLAG-HRP) 107

(A8592; Sigma) and anti-phosphoglycerate kinase 1 (PGK1) (ab113687; Abcam). 108

Chemiluminescence from treatment with SuperSignal West Pico Substrate (34080; Thermo 109

Fisher Scientific) was used for anti-FLAG-HRP. Fluorescence of IRDye 680RD Goat anti-110

Rabbit (926-68071; Licor) was used for anti-PGK1. Both were imaged using a LI-COR Odyssey 111

Fc imaging system. 112

113

Generation of Pvvd::hH3-3xFLAG::his-3+ reporter strain 114

The promoter region of the vvd gene was amplified by PCR using wild-type genomic DNA as 115

template using primers VVD-3000F_EcoRI\NotI and VVD-R1_XbaI containing NotI and XbaI 116

6

sites, respectively. The PCR product and pCCG::C-Gly::3xFLAG (Honda and Selker 2009) were 117

digested with NotI and XbaI and ligated together to create pVVD::C-Gly::3xFLAG. pVVD::C-118

Gly::3xFLAG and pIDTSMART-AMP containing N. crassa codon-optimized hH3 (NEB) were 119

digested with XbaI and PacI and ligated together to yield pVVD:hH3::C-Gly::3xFLAG. The 120

ligation product was verified by restriction analysis and Sanger sequencing. pVVD::hH3::C-121

Gly::3xFLAG was then linearized and transformed into strains N2930 and N3012. The resulting 122

his-3+ strains (N6049 and N6054) were verified by Southern hybridization. 123

124

Histone exchange profiling and analysis 125

Fresh conidia (106/mL) were inoculated in Vogel’s medium N with 1.5% sucrose, and grown for 126

18 hours at 32˚C with shaking (150 rpm) in a dark room. Low-intensity red light was used for 127

working during all culturing steps to prevent light stimulation (Aronson et al. 1994). To block 128

DNA replication, cultures were spiked with hydroxyurea to a final concentration of 100 mM, and 129

incubated for 3 hours (Sachs et al. 1997; E. Martegani, F. Tome 1981). We affixed five, 2 ft long 130

strips of blue light LED lights (LEDs: EL-BVRIB12V, power supply: EL-12VADPT, dimmer 131

switch: EL-SC12DIM, and DC 5-way splitter: EL-SJDCSPLIT; Elemental LED) to the bottom 132

of a New Brunswick G25 incubator lid, which placed them about 16 cm from the top of the 133

growth medium of cultures. To induce H3-3xFLAG expression, the LED strips were activated to 134

100% power, and cultures were exposed to blue light (465 nm; 30 µmole photons/m2/s) for 2 135

minutes. Cultures were incubated for 30 minutes (unless noted otherwise) to allow incorporation 136

of H3-3xFLAG into chromatin and then immediately harvested, washed with PBS buffer, and 137

transferred to 125-mL Erlenmeyer flasks with 10 mL PBS buffer. For chromatin fixation, 138

formaldehyde was added to a final concentration of 0.5% and incubated for 30 minutes at room 139

temperature. The crosslinking reaction was quenched with glycine (final concentration of 0.2 M) 140

and then incubated an additional 5 minutes at room temperature. Tissue was disrupted by 141

sonication, and chromatin was sheared with a Bioruptor (Diagenode) for 20 minutes with 30-142

second on/off intervals at high power. Subsequent chromatin immunoprecipitation (ChIP) was 143

performed as previously described (Tamaru et al. 2003) using anti-FLAG-conjugated agarose 144

beads (A2220; Millipore) and anti-hH3 (Ab1791; Abcam). 145

For ChIP-qPCR analyses, independent experimental replicates were performed in 146

triplicate using PerfeCTa SYBR Green Fastmix ROX (Quantabio) with the listed primers (Table 147

7

S2), and analyzed using a StepOnePlus Real-Time PCR system (Life Technologies). Relative 148

enrichment was determined by calculating enrichment as a percent of the total input. 149

H3-3xFLAG ChIP samples were prepared for sequencing as previously described 150

(Jamieson et al. 2016). Sequencing was performed using an Illumina HiSeq 4000 system with 151

single-end 75-nt or 100-nt reads. Sequencing data were processed and analyzed using the Galaxy 152

platform (https://usegalaxy.org/) (Afgan et al. 2016). Sequences were aligned to the corrected N. 153

crassa OR74A (NC12) genome (Galazka et al. 2016) using Bowtie2 (Langmead and Salzberg 154

2012). For visualization, BedGraph files were generated from mapped read data using HOMER 155

(Heinz et al. 2010), converted into bigWig files using the Wig/BedGraph-to-bigWig program via 156

Galaxy, and visualized with the Integrative Genomics Viewer (IGV) (Thorvaldsdóttir, Robinson, 157

and Mesirov 2013). Meta-analyses were carried out using the computeMatrix and plotProfile 158

programs from deepTools (Ramírez et al. 2016) via Galaxy. 159

160

Light characterization 161

The light spectral range was measured using an ILT350 chroma meter (International Light 162

Technologies) and intensity was measured using an MQ-200 quanta meter (Apogee Instruments). 163

164

Data Availability 165

Complete ChIP-seq and bisulfite-seq files, ChIP-seq intensity values, and gene expression 166

quartile lists and constitutive heterochromatin region coordinate files have been deposited in 167

NCBI’s Gene Expression Omnibus (GEO; http://ncbi.nlm.nih.gov/geo) and are accessible 168

through GEO Series accession number GSE143608 and as part of a previously reported series 169

GSE81129. Supplemental material has been uploaded to Figshare. 170

171

Strain Availability 172

The histone turnover reporter strains used in this study are available by request from the FGSC. 173

174

RESULTS AND DISCUSSION 175

Expression of hH3-3xFLAG under control of the vvd promoter 176

In order to assay histone turnover genome-wide in N. crassa, we constructed a strain expressing 177

3xFLAG-tagged histone H3 at the his-3 locus, regulated by the vvd promoter (Figure 1A). N. 178

8

crassa possesses a well-characterized circadian rhythm, and over 5% of its genes are expressed 179

in response to light exposure (Chen and Loros 2009). The vvd promoter allows for light-180

inducible expression of genes; in dark conditions, vvd is weakly expressed but is rapidly induced 181

by up to 300-fold within minutes of light exposure (Elvin et al. 2005; Heintzen, Loros, and 182

Dunlap 2001). Indeed, the vvd promoter has already been successfully utilized for the inducible 183

and tunable control of gene expression (Hurley et al. 2012). Since complete histone eviction 184

requires removal of H2A-H2B dimers, and the H3-H4 tetramer is incorporated into chromatin 185

prior to other histones, the incorporation of H3-3xFLAG should represent turnover of entire 186

nucleosomes (Luger, Dechassa, and Tremethick 2012). 187

To examine the kinetics of light-inducible expression of H3-3xFLAG and to characterize 188

its expression dynamics post induction, we grew cultures of the reporter strain in complete 189

darkness for 18 hours, exposed them to a short period (two minutes) of blue light, and assessed 190

H3-3xFLAG expression by immunoblotting in a time course experiment (Figure 1B). Consistent 191

with previous characterization of gene expression controlled by the vvd promoter, we observed a 192

low basal level of H3-3xFLAG expression at the pre-induction time point (Figure 1B, DD). By 193

20 minutes there was a marked accumulation of H3-3xFLAG, which appeared to culminate by 194

40 minutes (Figure 1B). This level of H3-3xFLAG persisted for at least two hours after 195

induction. Because slants used to inoculate the dark cultures were grown in light, Pvvd:hH3-196

3xFLAG may have been active at the start of the incubation period. The low level of H3-197

3xFLAG observed in the uninduced strains suggests that high levels brought on by light 198

induction was effectively reduced to this low basal level within 18 hours. Conceivably, longer 199

growth periods in the dark might reduce background levels further. 200

201

H3-3xFLAG is incorporated into chromatin 202

To verify incorporation of light-induced H3-3xFLAG into chromatin, we immunoprecipitated 203

formaldehyde cross-linked FLAG-tagged histones after various periods of incubation to allow 204

for histone incorporation, and tested for association with DNA by quantitative PCR (qPCR). We 205

expected that longer incorporation times would give greater enrichment of immunoprecipitated 206

chromatin. To assess whether FLAG-tagged histones are effectively incorporated in a 207

replication-independent manner, we blocked DNA replication by treatment with hydroxyurea 208

(HU). Cultures were spiked with 100 mM HU and incubated for three hours, a regimen 209

9

previously shown to effectively arrest replication without compromising viability (Sachs et al. 210

1997; E. Martegani, F. Tome 1981). Strains were then exposed to a two-minute light pulse to 211

induce H3-3xFLAG expression and incubated for 20 minutes or two hours to allow for 212

incorporation of FLAG-tagged histones before cross-linking, chromatin shearing, 213

immunoprecipitation, and isolation of associated DNA (Figure 2). 214

We performed qPCR using primers for different chromatin contexts: active genes (actin, 215

fkr-5, and csr-1), constitutive heterochromatin (Cen IIIL, 8:A6, and 8:F10), and facultative 216

heterochromatin (Tel VIIL). We observed similarly low levels of relative enrichment across all 217

regions in the uninduced control. After only 20 minutes of incorporation after induction, 218

enrichment increased at all regions, and further enrichment was found after two hours of 219

incorporation (Figure 3A, left panel). These results indicate that light-induced H3-3xFLAG was 220

readily incorporated into chromatin, and that longer incubation periods following induction result 221

in increased incorporation of H3-3xFLAG. 222

Considering the rapid induction and magnitude of H3-3xFLAG expression, we were 223

concerned about the possibility that this would lead to abnormal levels of histones within 224

chromatin. We therefore immunoprecipitated total hH3 after each incorporation period, and 225

compared relative enrichment of associated DNA from each region across all incorporation time 226

points by qPCR. For all regions examined, we found similar levels of enrichment for both 227

incorporation time points as our uninduced control, suggesting that nucleosome concentration 228

within chromatin was unchanged for at least two hours after induced expression of H3-3xFLAG 229

(Figure 3A, right panel). 230

231

Replication-independent histone turnover in N. crassa 232

We examined replication-independent histone turnover genome-wide in N. crassa by performing 233

next-generation sequencing on H3-3xFLAG-associated chromatin. We were concerned that a 234

lengthy incorporation period might lead to saturation of FLAG-tagged histones within chromatin, 235

resulting in enrichment values that might be more representative of histone occupancy than rates 236

of histone turnover. In order to assess the incorporation of a “pulse” of labeled histones, we 237

allowed only 30 minutes for incorporation (Figure 2). Based on our qPCR results, increasing 238

amounts of H3-3xFLAG were still incorporated between 20 minutes and two hours after 239

10

induction (Figure 3A). We reasoned that at 30 minutes, the size of any observed peaks of 240

enrichment would reflect relative histone turnover rates rather than occupancy. 241

We sequenced DNA pulled down in two replicate turnover experiments and observed 242

reproducible H3-3xFLAG enrichment patterns genome-wide (Figure 3B). The most prominent 243

peaks were present over promoter regions of genes, which were typically adjacent to a region 244

showing low turnover in the corresponding gene body, consistent with previous reports 245

investigating replicating-independent histone turnover in budding yeast (Figure 3B) (Rufiange et 246

al. 2007; T. Kaplan et al. 2008; Dion et al. 2007; Gossett and Lieb 2012). Similarly, previous 247

studies of replication-independent exchange of the histone H3 variant, hH3.3, in mouse and 248

human cell cultures revealed high levels of turnover at promoter regions, 5’ UTRs, and the 3’ 249

ends of genes (Kraushaar et al. 2013; Jin et al. 2009; Goldberg et al. 2010). There was a 250

noticeable lack of histone turnover at constitutive heterochromatin domains, as previously 251

observed in fission yeast (Choi et al. 2005; Aygün, Mehta, and Grewal 2013). Altogether, our 252

findings with N. crassa support the general correlations between histone turnover and chromatin 253

contexts found in other models. 254

255

Histone turnover in Neurospora genes 256

To characterize histone turnover in genes, we used previously generated RNA-seq data to divide 257

all genes by expression level in wild type into quartiles (Bicocca et al. 2018), aligned them 258

relative to their putative transcriptional start and end sites, and averaged the relative signal across 259

the promoter regions and gene bodies for each quartile. We found that each expression quartile 260

possessed a distinct turnover profile (Figure 3C). Similar to observations in S. cerevisiae, D. 261

melanogaster, C. elegans, mouse, and human cells, increased levels of histone turnover 262

correlated with higher relative expression levels (8, 9, 11, 31–38). As in other model organisms, 263

higher levels of histone turnover were found over promoter regions and in the 5’ and 3’ ends of 264

genes (Rufiange et al. 2007; Dion et al. 2007; Gossett and Lieb 2012; Jin et al. 2009; Goldberg et 265

al. 2010; Mito, Henikoff, and Henikoff 2005; Deal, Henikoff, and Henikoff 2010; Ooi, Henikoff, 266

and Henikoff 2009) (Figure 3C). Notably, the interior of gene bodies exhibited relatively low 267

turnover, potentially due to SET-2-catalyzed H3K36me-mediated suppression of histone 268

exchange (Venkatesh and Workman 2015). Consistent with this possibility, previous work on 269

H3K36me in N. crassa revealed that SET-2-mediated H3K36me is mainly enriched over gene 270

11

bodies, marking most (~80%) N. crassa genes, although not those with low or no expression 271

(Bicocca et al. 2018). The level of turnover within gene bodies was lower in the top three 272

expression quartiles than the basal turnover found at genes within the lowest quartile, perhaps 273

reflecting this mechanism, which is dependent on Pol II elongation (Venkatesh and Workman 274

2015). 275

The overall shape of histone turnover profiles over genes in N. crassa appears to reflect 276

an additional conserved feature of nucleosome organization in gene bodies of eukaryotes. 277

Previous genome-wide studies uncovered nucleosome-free regions (NFRs) present immediately 278

upstream of the transcriptional start sites in yeasts (Albert et al. 2007; N. Kaplan et al. 2009; 279

Mavrich, Ioshikhes, et al. 2008; Whitehouse et al. 2007; Yuan et al. 2005), worms (Johnson et al. 280

2006; Valouev et al. 2008), flies (Mavrich, Jiang, et al. 2008), medaka (Sasaki et al. 2009), and 281

humans (Ozsolak et al. 2007; Schones et al. 2008). Expressed genes in N. crassa, represented by 282

the top three expression quartile profiles, appear to organize nucleosomes at the 5’ end of gene 283

bodies as in other model systems. There are noticeable dips in H3-3xFLAG enrichment just 284

upstream of the transcriptional start site, presumably due to the presence of NFRs (Figure 3C). 285

Overall, it appears the general associations between active transcription and histone turnover in 286

N. crassa is similar to those found in other eukaryotes. 287

288

Histone turnover in N. crassa heterochromatin mutants 289

Heterochromatin is generally thought to be compact, largely transcriptionally silent chromatin 290

that renders underlying DNA inaccessible to trans-acting factors (Allshire and Madhani 2018). 291

Studies in S. pombe have shown heterochromatin to be refractory towards replication-292

independent histone turnover relative to transcriptionally active euchromatin (Aygün, Mehta, and 293

Grewal 2013; Choi et al. 2005). Mutants deficient for heterochromatin-associated factors such as 294

the histone deacetylase (HDAC) Clr3, the histone H3 lysine 9-specific methyltransferase Clr4, or 295

the chromodomain proteins that bind this histone mark, Swi6 and Chp2, exhibit increases in 296

histone turnover at heterochromatin domains, suggesting destabilization of heterochromatin 297

structure (Aygün, Mehta, and Grewal 2013; Choi et al. 2005). Constitutive heterochromatin in N. 298

crassa sports distinct histone marks and conserved features typical of this category of chromatin. 299

Trimethylation of lysine 9 on histone H3 (H3K9me3) is catalyzed by the histone 300

methyltransferase, DIM-5 (Tamaru et al. 2003; Lewis et al. 2010). This mark is recognized by 301

12

the chromodomain protein HP1 (Freitag et al. 2004), which functions as a platform to recruit the 302

DNA methyltransferase DIM-2 to methylate associated cytosines (Honda and Selker 2008). HP1 303

also assists in directing the HDAC complex, HCHC, to heterochromatin, whose activity is 304

dependent on its catalytic subunit, HDA-1 (Honda et al. 2012, 2016). To test how the loss of 305

different heterochromatin factors may affect histone turnover in N. crassa, we built reporter 306

strains in ∆dim-2, ∆dim-5, ∆hpo, and ∆hda-1 backgrounds, and assayed for H3-3xFLAG 307

incorporation by next-generation sequencing. 308

In all mutant backgrounds tested, H3-3xFLAG patterns in euchromatin were almost 309

indistinguishable from that observed in wild type, suggesting that loss of heterochromatin 310

machinery has little effect on histone turnover in euchromatin (Figure 4A). However, there were 311

differences in heterochromatin domains. Heterochromatin domains in wild-type strains exhibited 312

relatively low levels of histone exchange, but were found to be flanked by short regions of rapid 313

turnover (Figure 4A and B). It is of note that high levels of histone exchange were also observed 314

at heterochromatin boundaries in S. pombe and D. melanogaster, and that histone turnover has 315

been previously suggested to limit heterochromatin spreading (Aygün, Mehta, and Grewal 2013; 316

Dion et al. 2007; Deal, Henikoff, and Henikoff 2010). 317

Histone turnover in heterochromatin in ∆dim-2 strains, which lack all DNA methylation, 318

appeared largely normal in that turnover remained suppressed across heterochromatin domains. 319

However, we observed a modest increase in turnover at the edges of heterochromatin domains 320

(Figure 4A and B). These results suggest that DNA methylation has little influence on histone 321

turnover in N. crassa. Loss of DIM-5, and thus H3K9me3, or of the HDAC HDA-1, led to 322

greater disruption of heterochromatin domains. In ∆dim-5 strains, we observed reduced turnover 323

at domain edges and increased turnover within the interior of heterochromatin domains (Figure 324

4A and B). Similar increased turnover within the interior of heterochromatin domains was 325

observed in ∆hda-1 strains, but unlike ∆dim-5 strains, there were concomitant increases in 326

turnover at domain edges (Figure 4A and B). Despite these increases, the general profile of 327

histone turnover appeared largely normal in these mutants, perhaps reflecting redundant 328

mechanisms to maintain heterochromatin structure. These results are similar to observations at 329

the S. pombe mating type locus, where loss of the HDAC Clr3 led to greater increases of histone 330

turnover than from loss of Clr4 and heterochromatin-associated H3K9me (Aygün, Mehta, and 331

Grewal 2013). The greatest increases in histone turnover were observed in ∆hpo strains. Similar 332

13

to the case in ∆hda-1 strains, we found increased turnover at domain edges, and histone turnover 333

was much higher overall throughout heterochromatin domains (Figure 4A and B). That loss of 334

HP1 led to greater destabilization of heterochromatin is puzzling as HP1 is dependent on 335

H3K9me3, and thus DIM-5 catalytic activity, for localization to heterochromatin (Freitag et al. 336

2004), and DIM-5 is responsible for all H3K9me3 in N. crassa (Tamaru et al. 2003). However, 337

this result is consistent with the observation that HP1 chromodomain mutants retain a low level 338

of DNA methylation, suggesting that HP1 is still recruited to heterochromatin domains despite 339

lacking the ability to bind H3K9me3 (Honda et al. 2016). 340

341

Conclusion: A simple method to profile histone turnover in N. crassa 342

We have created a strain that expresses 3xFLAG-tagged histone H3 under the control of a light-343

inducible promoter to profile histone turnover genome-wide. Inducible tagged histones have 344

been successfully used in the past in multiple organisms to profile histone turnover (Ahmad and 345

Henikoff 2002; Dion et al. 2007; Choi et al. 2005; Rufiange et al. 2007; Kraushaar et al. 2013; 346

Aygün, Mehta, and Grewal 2013). However, our reporter strain provides greater ease of use and 347

magnitude of expression of H3-3xFLAG, without compromising physiological levels of histone 348

occupancy (Figure 3A). Whereas turnover assays in yeasts, mice, and flies involved an 349

incubation period for induction and incorporation on the order of hours (Ahmad and Henikoff 350

2002; Dion et al. 2007; Choi et al. 2005; Rufiange et al. 2007; Kraushaar et al. 2013; Aygün, 351

Mehta, and Grewal 2013), our strain expresses readily detectible H3-3xFLAG expression and 352

chromatin incorporation as early as 20 minutes (Figures 1B and 3A), and induction does not 353

require a change of growth medium. Though we used blue light for induction, previous work 354

characterizing the vvd promoter has shown that white light (400-700 nm) from conventional 355

lamps are sufficient (Hurley et al. 2012). The rapid nature of induction in our strain should be 356

useful for profiling histone exchange genome-wide in any context and would be amenable to 357

profile chromatin at regions with quick transcriptional kinetics. 358

359

ACKNOWLEDGEMENTS 360

We thank Tom Stevens of the University of Oregon for the gift of anti-PGK1 antibody and Non 361

Chotewutmontri for help characterizing our light treatment. 362

363

14

FUNDING 364

This work was supported by grants to E.U.S. (NIH GM035690 and R35GM127142). V.T.B. and 365

W.K.S. were supported in part by an NIH postdoctoral fellowship (CA180468) and by an NIH 366

T32 training grant (GM007413), respectively. 367

368

CONFLICT OF INTEREST 369

The authors declare no competing financial interests. 370

371

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REFERENCES 372

Afgan, Enis, Dannon Baker, Marius van den Beek, Daniel Blankenberg, Dave Bouvier, Martin 373

Čech, John Chilton et al., 2016 The Galaxy Platform for Accessible, Reproducible and 374

Collaborative Biomedical Analyses: 2016 Update. Nucleic Acids Research 44 (W1): W3–375

10. https://doi.org/10.1093/nar/gkw343 376

Ahmad, Kami, and Steven Henikoff, 2002 The Histone Variant H3.3 Marks Active Chromatin 377

by Replication-Independent Nucleosome Assembly. Molecular Cell 9 (6): 1191–1200. 378

https://doi.org/10.1016/S1097-2765(02)00542-7 379

Albert, Istvan, Travis N. Mavrich, Lynn P. Tomsho, Ji Qi, Sara J. Zanton, Stephan C. Schuster, 380

and B. Franklin Pugh, 2007 Translational and Rotational Settings of H2A.Z Nucleosomes 381

across the Saccharomyces Cerevisiae Genome. Nature 446 (7135): 572–76. 382

https://doi.org/10.1038/nature05632 383

Allshire, Robin C., and Hiten D. Madhani, 2018 Ten Principles of Heterochromatin Formation 384

and Function. Nature Reviews Molecular Cell Biology 19 (4): 229–44. 385

https://doi.org/10.1038/nrm.2017.119 386

Aramayo, Rodolfo, and Eric U Selker, 2013 Neurospora crassa, a Model System for Epigenetics 387

Research. Cold Spring Harbor Perspectives in Biology 5 (10): a017921–a017921. 388

https://doi.org/10.1101/cshperspect.a017921 389

Aronson, Benjamin D., Keith A. Johnson, Jennifer J. Loros, and Jay C. Dunlap, 1994 Negative 390

Feedback Defining a Circadian Clock: Autoregulation of the Clock Gene Frequency. 391

Science. https://doi.org/10.1126/science.8128244 392

Aygün, Ozan, Sameet Mehta, and Shiv I S Grewal, 2013 HDAC-Mediated Suppression of 393

Histone Turnover Promotes Epigenetic Stability of Heterochromatin. Nature Structural & 394

Molecular Biology 20 (5): 547–54. https://doi.org/10.1038/nsmb.2565 395

Bicocca, Vincent T, Tereza Ormsby, Keyur K Adhvaryu, Shinji Honda, and Eric U Selker, 2018 396

ASH1-Catalyzed H3K36 Methylation Drives Gene Repression and Marks H3K27me2/3-397

Competent Chromatin. ELife 7: 1–19. https://doi.org/10.7554/eLife.41497 398

Chen, Chen Hui, and Jennifer J. Loros, 2009 Neurospora Sees the Light: Light Signaling 399

Components in a Model System. Communicative and Integrative Biology 2 (5): 448–51. 400

https://doi.org/10.4161/cib.2.5.8835 401

Choi, Eun Shik, Jin A. Shin, Hyun Soo Kim, and Yeun Kyu Jang, 2005 Dynamic Regulation of 402

16

Replication Independent Deposition of Histone H3 in Fission Yeast. Nucleic Acids 403

Research 33 (22): 7102–10. https://doi.org/10.1093/nar/gki1011 404

Chow, Cheok Man, Andrew Georgiou, Henrietta Szutorisz, Alexandra Maia e Silva, Ana Pombo, 405

Isabel Barahona, Elise Dargelos, Claudia Canzonetta, and Niall Dillon, 2005 Variant 406

Histone H3.3 Marks Promoters of Transcriptionally Active Genes During Mammalian Cell 407

Division. EMBO Reports 6 (4): 354–60. https://doi.org/10.1038/sj.embor.7400366 408

Davis, Rowland, 2000 Neurospora: Contributions of a Model Organism. Oxford University 409

Press, Oxford. https://doi.org/10.1017/S0016672301215080 410

Deal, Roger B, Jorja G Henikoff, and Steven Henikoff, 2010 Genome-Wide Kinetics of 411

Nucleosome Turnover Determined by Metabolic Labeling of Histones. Science 328 (5982): 412

1161–65. https://doi.org/10.1126/science.1186777 413

Dion, M. F., T. Kaplan, M. Kim, S. Buratowski, N. Friedman, and O. J. Rando, 2007 Dynamics 414

of Replication-Independent Histone Turnover in Budding Yeast. Science 315 (5817): 1405–415

8. https://doi.org/10.1126/science.1134053 416

E. Martegani, F. Tome, F. Trezzi, 1981 Timing of Nuclear Division Cycle in Neurospora crassa. 417

Journal of Cell Science 136: 127–36 418

Elvin, Mark, Jennifer J. Loros, Jay C. Dunlap, and Christian Heintzen, 2005 The PAS/LOV 419

Protein VIVID Supports a Rapidly Dampened Daytime Oscillator That Facilitates 420

Entrainment of the Neurospora Circadian Clock. Genes and Development 19 (21): 2593–421

2605. https://doi.org/10.1101/gad.349305 422

Freitag, Michael, Patrick C Hickey, Tamir K Khlafallah, Nick D Read, and Eric U Selker, 2004 423

HP1 Is Essential for DNA Methylation in Neurospora. Molecular Cell 13 (3): 427–34. 424

http://www.ncbi.nlm.nih.gov/pubmed/14967149 425

Galazka, Jonathan M, Andrew D Klocko, Miki Uesaka, Shinji Honda, Eric U Selker, and 426

Michael Freitag, 2016 Neurospora Chromosomes Are Organized by Blocks of Importin 427

Alpha-Dependent Heterochromatin That Are Largely Independent of H3K9me3. Genome 428

Research 26 (8): 1069–80. https://doi.org/10.1101/gr.203182.115 429

Goldberg, Aaron D., Laura A. Banaszynski, Kyung Min Noh, Peter W. Lewis, Simon J. 430

Elsaesser, Sonja Stadler, Scott Dewell et al., 2010. Distinct Factors Control Histone Variant 431

H3.3 Localization at Specific Genomic Regions. Cell 140 (5): 678–91. 432

https://doi.org/10.1016/j.cell.2010.01.003 433

17

Gossett, Andrea J., and Jason D. Lieb, 2012 In Vivo Effects of Histone H3 Depletion on 434

Nucleosome Occupancy and Position in Saccharomyces cerevisiae. PLoS Genetics 8 (6). 435

https://doi.org/10.1371/journal.pgen.1002771 436

Heintzen, Christian, Jennifer J. Loros, and Jay C. Dunlap, 2001 The PAS Protein VIVID Defines 437

a Clock-Associated Feedback Loop That Represses Light Input, Modulates Gating, and 438

Regulates Clock Resetting. Cell 104 (3): 453–64. https://doi.org/10.1016/S0092-439

8674(01)00232-X 440

Heinz, Sven, Christopher Benner, Nathanael Spann, Eric Bertolino, Yin C. Lin, Peter Laslo, 441

Jason X. Cheng, Cornelis Murre, Harinder Singh, and Christopher K. Glass, 2010 Simple 442

Combinations of Lineage-Determining Transcription Factors Prime Cis-Regulatory 443

Elements Required for Macrophage and B Cell Identities. Molecular Cell. 444

https://doi.org/10.1016/j.molcel.2010.05.004 445

Honda, Shinji, Vincent T. Bicocca, Jordan D. Gessaman, Michael R Rountree, Ayumi 446

Yokoyama, and Eun Y. Yu, 2016 Dual Chromatin Recognition by the Histone Deacetylase 447

Complex HCHC Is Required for Proper DNA Methylation in Neurospora crassa. 448

Proceedings of the National Academy of Sciences, 201621475. 449

https://doi.org/10.1073/pnas.1621475114 450

Honda, Shinji, Zachary A. Lewis, Kenji Shimada, Wolfgang Fischle, Ragna Sack, and Eric U. 451

Selker, 2012 Heterochromatin Protein 1 Forms Distinct Complexes to Direct Histone 452

Deacetylation and DNA Methylation. Nature Structural & Molecular Biology 19 (5): 471–453

77, S1. https://doi.org/10.1038/nsmb.2274 454

Honda, Shinji, and Eric U. Selker, 2008 Direct Interaction between DNA Methyltransferase 455

DIM-2 and HP1 Is Required for DNA Methylation in Neurospora crassa. Molecular and 456

Cellular Biology 28 (19): 6044–55. https://doi.org/10.1128/MCB.00823-08 457

Honda, Shinji, and Eric U. Selker, 2009 Tools for Fungal Proteomics: Multifunctional 458

Neurospora Vectors for Gene Replacement, Protein Expression and Protein Purification. 459

Genetics 182 (1): 11–23. https://doi.org/10.1534/genetics.108.098707 460

Hurley, Jennifer M., Chen Hui Chen, Jennifer J. Loros, and Jay C. Dunlap, 2012 Light-Inducible 461

System for Tunable Protein Expression in Neurospora crassa. G3: Genes, Genomes, 462

Genetics 2 (10): 1207–12. https://doi.org/10.1534/g3.112.003939 463

Jamieson, Kirsty, Elizabeth T. Wiles, Kevin J. McNaught, Simone Sidoli, Neena Leggett, 464

18

Yanchun Shao, Benjamin A. Garcia, and Eric U. Selker, 2016 Loss of HP1 Causes 465

Depletion of H3K27me3 from Facultative Heterochromatin and Gain of H3K27me2 at 466

Constitutive Heterochromatin. Genome Research 26 (1): 97–107. 467

https://doi.org/10.1101/gr.194555.115 468

Jin, Chunyuan, Chongzhi Zang, Gang Wei, Kairong Cui, Weiqun Peng, Keji Zhao, and Gary 469

Felsenfeld, 2009 H3.3/H2A.Z Double Variant-Containing Nucleosomes Mark 470

‘Nucleosome-Free Regions’ of Active Promoters and Other Regulatory Regions. Nature 471

Genetics 41 (8): 941–45. https://doi.org/10.1038/ng.409 472

Johnson, Steven M, Frederick J Tan, H. L. McCullough, Daniel P Riordan, and A. Z. Fire, 2006 473

Flexibility and Constraint in the Nucleosome Core Landscape of Caenorhabditis elegans 474

Chromatin. Genome Research 16 (12): 1505–16. https://doi.org/10.1101/gr.5560806 475

Kaplan, Noam, Irene K. Moore, Yvonne Fondufe-Mittendorf, Andrea J. Gossett, Desiree Tillo, 476

Yair Field, Emily M. LeProust et al., 2009 The DNA-Encoded Nucleosome Organization of 477

a Eukaryotic Genome. Nature 458 (7236): 362–66. https://doi.org/10.1038/nature07667 478

Kaplan, Tommy, Chih Long Liu, Judith A. Erkmann, John Holik, Michael Grunstein, Paul D. 479

Kaufman, Nir Friedman, and Oliver J. Rando, 2008 Cell Cycle- and Chaperone-Mediated 480

Regulation of H3K56ac Incorporation in Yeast. PLoS Genetics 4 (11). 481

https://doi.org/10.1371/journal.pgen.1000270 482

Kraushaar, Daniel C., Wenfei Jin, Alika Maunakea, Brian Abraham, Misook Ha, and Keji Zhao, 483

2013 Genome-Wide Incorporation Dynamics Reveal Distinct Categories of Turnover for 484

the Histone Variant H3.3. Genome Biology 14 (10). https://doi.org/10.1186/gb-2013-14-10-485

r121 486

Langmead, Ben, and Steven L. Salzberg, 2012 Fast Gapped-Read Alignment with Bowtie 2. 487

Nature Methods 9 (4): 357–59. https://doi.org/10.1038/nmeth.1923 488

Lewis, Zachary A., Keyur K. Adhvaryu, Shinji Honda, Anthony L. Shiver, Marijn Knip, Ragna 489

Sack, and Eric U. Selker, 2010 DNA Methylation and Normal Chromosome Behavior in 490

Neurospora Depend on Five Components of a Histone Methyltransferase Complex, DCDC. 491

PLoS Genetics 6 (11): e1001196. https://doi.org/10.1371/journal.pgen.1001196 492

Luger, Karolin, Mekonnen L. Dechassa, and David J. Tremethick, 2012 New Insights into 493

Nucleosome and Chromatin Structure: An Ordered State or a Disordered Affair? Nature 494

Reviews Molecular Cell Biology 13 (7): 436–47. https://doi.org/10.1038/nrm3382 495

19

Mavrich, Travis N., Ilya P. Ioshikhes, Bryan J. Venters, Cizhong Jiang, Lynn P. Tomsho, Ji Qi, 496

Stephan C. Schuster, Istvan Albert, and B. Franklin Pugh, 2008 A Barrier Nucleosome 497

Model for Statistical Positioning of Nucleosomes throughout the Yeast Genome. Genome 498

Research 18 (7): 1073–83. https://doi.org/10.1101/gr.078261.108 499

Mavrich, Travis N., Cizhong Jiang, Ilya P. Ioshikhes, Xiaoyong Li, Bryan J. Venters, Sara J. 500

Zanton, Lynn P. Tomsho et al., 2008 Nucleosome Organization in the Drosophila Genome. 501

Nature 453 (7193): 358–62. https://doi.org/10.1038/nature06929 502

Mito, Yoshiko, Jorja G. Henikoff, and Steven Henikoff 2005 Genome-Scale Profiling of Histone 503

H3.3 Replacement Patterns. Nature Genetics 37 (10): 1090–97. 504

https://doi.org/10.1038/ng1637 505

Ooi, Siew Loon, Jorja G. Henikoff, and Steven Henikoff, 2009 A Native Chromatin Purification 506

System for Epigenomic Profiling in Caenorhabditis elegans. Nucleic Acids Research 38 (4): 507

1–14. https://doi.org/10.1093/nar/gkp1090 508

Ozsolak, Fatih, Jun S. Song, X. Shirley Liu, and David E. Fisher, 2007 High-Throughput 509

Mapping of the Chromatin Structure of Human Promoters. Nature Biotechnology 25 (2): 510

244–48. https://doi.org/10.1038/nbt1279 511

Ramírez, Fidel, Devon P. Ryan, Björn Grüning, Vivek Bhardwaj, Fabian Kilpert, Andreas S. 512

Richter, Steffen Heyne, Friederike Dündar, and Thomas Manke, 2016 DeepTools2: A Next 513

Generation Web Server for Deep-Sequencing Data Analysis. Nucleic Acids Research 44 514

(W1): W160–65. https://doi.org/10.1093/nar/gkw257 515

Rufiange, Anne, Pierre Étienne Jacques, Wajid Bhat, François Robert, and Amine Nourani, 2007 516

Genome-Wide Replication-Independent Histone H3 Exchange Occurs Predominantly at 517

Promoters and Implicates H3 K56 Acetylation and Asf1. Molecular Cell 27 (3): 393–405. 518

https://doi.org/10.1016/j.molcel.2007.07.011 519

Sachs, Matthew S., Eric U. Selker, Baoyu Lin, Christopher J. Roberts, Zongli Luo, David 520

Vaught-Alexander, and Brian S. Margolin, 1997 Expression of Herpes Virus Thymidine 521

Kinase in Neurospora crassa. Nucleic Acids Research 25 (12): 2389–95. 522

https://doi.org/10.1093/nar/25.12.2389 523

Sasaki, Shin, Cecilia C. Mello, Atsuko Shimada, Yoichiro Nakatani, Shin Ichi Hashimoto, 524

Masako Ogawa, Kouji Matsushima et al., 2009 Chromatin-Associated Periodicity in 525

Genetic Variation Downstream of Transcriptional Start Sites. Science 323 (5912): 401–4. 526

20

https://doi.org/10.1126/science.1163183 527

Schones, Dustin E., Kairong Cui, Suresh Cuddapah, Tae Young Roh, Artem Barski, Zhibin 528

Wang, Gang Wei, and Keji Zhao, 2008 Dynamic Regulation of Nucleosome Positioning in 529

the Human Genome. Cell 132 (5): 887–98. https://doi.org/10.1016/j.cell.2008.02.022 530

Steensel, Bas Van, 2011 Chromatin: Constructing the Big Picture. EMBO Journal 30 (10): 531

1885–95. https://doi.org/10.1038/emboj.2011.135 532

Tamaru, Hisashi, Xing Zhang, Debra McMillen, Prim B. Singh, Jun-ichi Nakayama, Shiv I. 533

Grewal, C. David Allis, Xiaodong Cheng, and Eric U. Selker, 2003 Trimethylated Lysine 9 534

of Histone H3 Is a Mark for DNA Methylation in Neurospora crassa. Nature Genetics 34 535

(1): 75–79. https://doi.org/10.1038/ng1143 536

Thorvaldsdóttir, Helga, James T. Robinson, and Jill P. Mesirov, 2013 Integrative Genomics 537

Viewer (IGV): High-Performance Genomics Data Visualization and Exploration. Briefings 538

in Bioinformatics 14 (2): 178–92. https://doi.org/10.1093/bib/bbs017 539

Valouev, A., J. Ichikawa, T. Tonthat, J. Stuart, S. Ranade, H. Peckham, K. Zeng, et al. 2008 A 540

High-Resolution, Nucleosome Position Map of C. Elegans Reveals a Lack of Universal 541

Sequence-Dictated Positioning. Genome Research 18 (7): 1051–63. 542

https://doi.org/10.1101/gr.076463.108 543

Venkatesh, Swaminathan, and Jerry L. Workman, 2015 Histone Exchange, Chromatin Structure 544

and the Regulation of Transcription. Nature Reviews Molecular Cell Biology 16 (3): 178–545

89. https://doi.org/10.1038/nrm3941 546

Whitehouse, Iestyn, Oliver J. Rando, Jeff Delrow, and Toshio Tsukiyama, 2007 Chromatin 547

Remodelling at Promoters Suppresses Antisense Transcription. Nature 450 (7172): 1031–548

35. https://doi.org/10.1038/nature06391 549

Yuan, Guo-Cheng, Yuen-Jong Liu, Michael F. Dion, Michael D. Slack, Lani F. Wu, Steven J. 550

Altschuler, and Oliver J. Rando, 2005 Genome-Scale Identification of Nucleosome 551

Positions in S. cerevisiae. Science (New York, N.Y.) 309 (5734): 626–30. 552

https://doi.org/10.1126/science.1112178 553

554

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FIGURE LEGENDS 555

Figure 1. A simple light-inducible system for assessing replication-independent histone 556

turnover in Neurospora crassa. 557

(A) Schematic overview of the light-inducible histone H3-3xFLAG reporter strain. 558

(B) Expression time-course analysis of H3-3xFLAG induction. H3-3xFLAG was induced with a 559

two-minute pulse of blue light and H3-3xFLAG levels were examined at 20-minute intervals by 560

immunoblotting. Uninduced H3-3xFLAG levels are indicated by “DD”. Phosphoglycerate kinase 561

1 (PGK-1) levels are included for each time point as a loading control. 562

563

Figure 2. Flowchart for genome-wide profiling of replication-independent histone turnover 564

in N. crassa. 565

5 mL of Vogel’s medium N supplemented with 1.5% sucrose is inoculated with 5.0 x 106 566

conidia, and cultures were grown in complete darkness at 32˚C for 18 hours. To block DNA 567

replication, cultures were treated with 100mM hydroxyurea (HU) and then induced with light 3 568

hours later. After 30 minutes to allow for incorporation of new H3-3xFLAG into chromatin, 569

cultures were cross-linked, lysed, and the chromatin sheared. H3-3xFLAG was 570

immunoprecipitated and the associated DNA isolated and analyzed by qPCR, or prepared for 571

next-generation sequencing and sequenced. 572

573

Figure 3. Histone turnover at representative genomic regions. 574

(A) H3-3xFLAG incorporation into chromatin increases with incubation time. qPCR analysis of 575

FLAG ChIP (left) and hH3 ChIP (right) after 20 minutes or two hours of incorporation, at 576

different chromatin regions including: active genes (actin, fkr-5, and csr-1), constitutive 577

heterochromatin (Cen IIIL, 8:A6, and 8:F10), and facultative heterochromatin (Tel VIIL). 578

Control uninduced levels are indicated by “DD”. Data are presented as the mean and SD of three 579

technical replicates. 580

(B) Genome-wide profiling of histone turnover in N. crassa. Representative IGV tracks 581

displaying H3-3xFLAG enrichment after 30 minutes of incorporation from replicate ChIP-seq 582

experiments. Levels of DNA methylation (5mC), determined by bisulfite sequencing, is 583

displayed to identify constitutive heterochromatin (Aramayo and Selker 2013). 584

22

(C) Histone turnover metaplot of gene expression level. Genes were divided into expression 585

quartiles based on wild type expression levels, and H3-3xFLAG enrichment from two replicate 586

experiments (denoted by a solid and dotted line) was aligned relative to their transcriptional start 587

(TSS) and end (TES) sites, and scaled to 3 kb (number of genes in each quartile: Top 25% - 588

2433; 75-50% - 2432; 50-25% - 2431; Bottom 25% - 2433). H3-3xFLAG enrichment is 589

displayed across the aligned gene bodies as well as 1kb upstream regions for each quartile. 590

591

Figure 4. Histone turnover at heterochromatin domains in mutants affecting 592

heterochromatin. 593

(A) Representative IGV tracks displaying H3-3xFLAG enrichment in indicated heterochromatin 594

mutants. Constitutive heterochromatin is marked by DNA methylation (5mC) and highlighted in 595

yellow. 596

(B) Metaplot of histone turnover at heterochromatin domains in heterochromatin mutants. 597

Constitutive heterochromatin domains were aligned and scaled to 2.5 kb. Replicate H3-3xFLAG 598

enrichment profiles for wild-type (black) and the indicated heterochromatin mutant strains (red) 599

are displayed over the scaled heterochromatin domains and 1 kb up- and down-stream from the 600

domains. 601

3xFLAG

anti-FLAG

anti-PGK1

DD 20 min

40 min

60 min

80 min

100 min

120 min

P vvd hH3-3xFLAG his-3 +

P vvd hH3-3xFLAG his-3 +

Light

Dark

A

B20

15

kDa

50

kDa

37

3xFLAG

3xFLAG

3xFLAG

3xFLAG

3xFLAG

Fig 1

Innoculate medium (dark)

Spike with hydroxyurea (100 mM �nal)

Induce hH3-3xFLAG expression with 2 minute

light pulse

Cross-link and anti-FLAG-ChIP

qPCR or next-generation sequencing

32˚C18 hrs

Incubate 3 hours to arrest replication

Incubate 30 minutes to incorporate hH3-3xFLAG

Fig 2

10 kb

10 kb

5mC

rep 1

rep 2

genes

H3-

3xFL

AG

LG I: 935 - 1,020 kb

LG V: 4,420 - 4,590 kb

A

B

0

0. 5

1

1. 5

2

2. 5

3

3. 5

4

ac tin fkr-5 c sr-1 C en III L 8: A6 8: F10 T elV II L

Rela

tive

Enric

hmen

t (%

of I

nput

)

hH3-3xFLAG

0

1

2

3

4

5

6

7

8

ac tin fkr-5 c sr-1 C en III L 8: A6 8: F10 T elV II L

Rela

tive

Enric

hmen

t (%

of I

nput

)

Total hH3

DD

20 min

2 hrs

5mC

rep 1

rep 2

genes

H3-

3xFL

AG

Fig 3

CGene Expression Quartile

Top 25%75-50%50-25%Bottom 25%

15

45

40

35

30

25

20

-1 kb TSS TES 0.5 kbRela

tive

Enric

hmen

t (RP

KM)

Gene BodyPromoter

WT 5mC

WT

∆dim-2

∆dim-5

∆hpo

∆hda-1

genes

H3-

3xFL

AG

LG VII: 125 - 225 kbA

BWT vs . ∆ dim-2

10 kb

30

25

20

15

35

40

-1 kb edge edge 1 kb10Re

lativ

e En

richm

ent (

RPKM

)

WT

∆dim-2

WT vs . ∆ dim-5

30

25

20

15

35

40

-1 kb edge edge 1 kb10Re

lativ

e En

richm

ent (

RPKM

)

WT

∆dim-5

WT vs . ∆ hda-1

30

25

20

15

35

40

-1 kb edge edge 1 kb10Re

lativ

e En

richm

ent (

RPKM

)

WT

∆hda-1

WT vs . ∆ hpo

30

25

20

15

35

40

-1 kb edge edge 1 kb10Re

lativ

e En

richm

ent (

RPKM

)

WT

∆hpo

Fig 4