Role of the Histone Variant H2A.Z/Htz1p in TBP Recruitment, Chromatin Dynamics, and Regulated Expression of Oleate-Responsive Genes

(1)

The role of the histone variant H2A.Z/Htz1p on TBP recruitment, chromatin 1

dynamics and regulated expression at oleate-responsive genes 2

3

Yakun Wan,1 Ramsey Saleem,1 Alexander Ratushny,1 Oriol Roda,1 4

Jennifer Smith,1 Chan-Hsien Lin,1,2 Jung-Hsien Chiang,1,2 John Aitchison1* 5

1. Institute for Systems Biology, Seattle, WA 98103 6

2. Department of Computer Science and Information Engineering, 7

National Cheng Kung University, Tainan, Taiwan 8

9 10

*Corresponding author 11

Institute for Systems Biology 12 1441 N 34th St. Seattle, WA 98103 13 Phone: (206) 732-1344 14 Fax: (206) 299-6574 15 E-mail: [email protected] 16 17

Word count for Materials and Methods: 874 words 18

Word count for the Introduction, Results and Discussion: 4324 words 19

20

Running Title: Control of gene induction by Htz1p 21

MCB Accepts, published online ahead of print on 9 March 2009

at National Cheng Kung University on March 17, 2009

mcb.asm.org

(2)

Abstract 1

The histone variant H2A.Z (Htz1p) has been implicated in transcriptional 2

regulation in numerous organisms, including Saccharomyces cerevisiae. Genome-wide 3

transcriptome profiling and chromatin immunoprecipitation studies identified a role for 4

Htz1p in the rapid and robust activation of many oleate-responsive genes encoding 5

peroxisomal proteins, and in particular, POT1, POX1, FOX2 and CTA1. Swr1p, Gcn5p 6

and Chz1p dependent association of Htz1p into these promoters in their repressed states 7

appears to establish an epigenetic marker for rapid and strong expression of these highly 8

inducible promoters. Isw2p also plays a role in establishing the nucleosome state of these 9

promoters, and associates stably in the absence of Htz1p. Analysis of the nucleosome 10

dynamics and Htz1p association with these promoters suggests a complex mechanism in 11

which Htz1p-containing nucleosomes at fatty acid-responsive promoters are 12

disassembled upon initial exposure to oleic acid leading to the loss of Htz1p from the 13

promoter. These nucleosomes reassemble at later stages of gene expression. While these 14

new nucleosomes do not incorporate Htz1p, the initial presence of Htz1p appears to mark 15

the promoter for sustained gene expression and the recruitment of TBP. 16

mcb.asm.org

(3)

Introduction 1

The organization of DNA into chromatin provides cells with a key regulatory 2

mechanism for gene expression by limiting access of the genome to the transcriptional 3

machinery. The nucleosome represents a basic structural unit of chromatin and post-4

translational modifications of histones serve as signals to define active, repressed or inert 5

chromatin states. In addition, chromatin states and gene expression can be influenced by 6

the dynamics of histones and their nonallelic variants. Indeed, exchange of canonical 7

histones for histone variants appears to be a key mechanism by which the transcriptional 8

machinery overcomes the restricted access imposed by nucleosome positioning (1). Of 9

the many classes of histone variants discovered, the Z variant of H2A is perhaps the best 10

characterized. H2A.Z differs from the canonical H2A histone in both the length and 11

sequence of the C-terminus (37), and is conserved from yeast to mammals (15). Early 12

studies with H2A.Z in Tetrahymena showed that H2A.Z incorporation is linked with 13

transcriptionally active chromatin (35). The Saccharomyces cerevisiae orthologue of 14

H2A.Z is called Htz1p and is encoded by HTZ1. Although HTZ1 is not essential gene 15

under standard laboratory growth conditions, Htz1p is implicated in transcriptional 16

regulation. Global chromatin studies have revealed that Htz1p preferentially associates 17

with the two nucleosomes flanking the nucleosome free region of promoters (12, 18, 26, 18

41), and this association is inversely proportional to transcription rates (3, 18). 19

Studies of the role of Htz1p in transcriptional regulation at specific promoters, 20

such as those of GAL1 and PHO5 (1, 30) indicate that the presence of Htz1p at promoters 21

is dynamic; Htz1p is bound in their repressed states, but dissociates during the activation 22

process. Accordingly, it is proposed that nucleosomes containing Htz1p are poised to 23

undergo nucleosome displacement allowing for rapid transcriptional responses (41). 24

The yeast S. cerevisiae is an excellent model for understanding the mechanisms of 25

cellular responses to induced perturbations. Upon exposure of yeast to fatty acids, such as 26

oleate, cells respond by dramatically altering their gene expression patterns, inducing 27

genes required for peroxisomal β-oxidation and peroxisome biogenesis (32). Genetic 28

screens to identify proteins specifically required for efficient fatty acid metabolism in S. 29

cerevisiae (34) identified metabolic enzymes, proteins required for biogenesis of the 30

organelle, signaling proteins and transcriptional regulators, and chromatin modifiers. 31

mcb.asm.org

(4)

Among this latter class of proteins, this approach identified genes encoding Htz1p, RNA 1

Polymerase II, Mediator subunits and components of chromatin remodeling complexes. 2

We thus seek to understand the nature of how chromatin is regulated and remodeled in 3

response to exposure to fatty acids and the specific role Htz1p plays in these 4

regulation/remodeling processes. 5

In this report, transcriptomes of WT and htz1∆ strains were compared during 6

exposure to oleic acid. While loss of Htz1p reduced the expression of many genes, genes 7

involved in the fatty acid response were particularly sensitive. A model is proposed, in 8

which Htz1p-containing nucleosomes at fatty acid-responsive promoters are 9

disassembled upon initial exposure to oleic acid leading to the loss of Htz1p from the 10

promoter. These nucleosomes reassemble, at later stages of gene expression. While these 11

nucleosomes do not incorporate Htz1p, the initial presence of Htz1p appears to mark the 12

promoter for sustained gene expression and the recruitment of TBP. 13

mcb.asm.org

(5)

Materials and Methods 1

Strains and growth conditions. 2

All yeast strains used in this study are indicated in Table 1. Haploid strains with 3

myc-tagged genes were made by genomically tagging target genes with the sequence 4

encoding 13 copies of the c-myc epitope from pFA6a-13MYC (20) by homologous 5

recombination into BY4742 (wild type) using a previously described PCR-based 6

procedure (2) Strains were verified by PCR analysis of the tagged gene loci and Western 7

blot analysis of the fusion proteins. Examination of growth characteristics of each strain 8

suggests that the chimeras did not alter protein function. For all experiments, control 9

strains were otherwise isogenic to test strains. Strains were cultured at 30oC in the 10

following media: YPD (1% yeast extract, 2% peptone, 2% glucose), SCIM (0.17% yeast 11

nitrogen base without amino acids and ammonium sulfate (YNB-aa-as), 0.5% yeast 12

extract, 0.5% peptone, 0.079% complete supplement mixture, 0.5% ammonium sulfate) 13

containing 0.5% Tween 40 (w/v) and 0.2% (w/v) oleate. 14

15

RNA preparation and microarray analysis. 16

Yeast cultures were grown at 30oC to a density of ~1 × 107 cells/ml. Cells were 17

collected and immediately frozen in liquid nitrogen. Total RNA was isolated by hot acid 18

phenol extraction. Total RNA was treated with RNase-free DNase I and purified with a 19

Qiagen RNeasy kit. Microarray labeling and hybridization reactions were performed as 20

previously described (7). Two color microarrays, comparing RNA from experimental 21

conditions (wild-type (WT) and htz1∆ cells grown in oleate (SCIM) for 6 h) to RNA 22

from control WT cells grown in glucose-containing medium (YPD), were performed 23

using Agilent whole-genome S. cerevisiae arrays. All experiments were performed with 24

duplicate experimental and duplicate technical replicates of each condition and the Log10

25

of the average mRNA abundance ratios are reported. Differentially expressed genes were 26

identified by maximum-likelihood analysis (λ≥100) (14, 32) and significantly affected 27

genes in the mutants were identified by a change in expression of two-fold or more 28

compared to the expression in the relevant WT strains. 29

30

mcb.asm.org

(6)

For quantitative reverse transcription-PCR, total RNA was directly reverse 1

transcribed using the First Strand cDNA Synthesis Kit from Fermentas (Catalog: K1611). 2

cDNAs were treated by RNase H and diluted 1:100 for quantitative PCR. RT-PCR was 3

done using a 7900 HT Fast Real-time PCR systems and DyNAmoTM Flash SYBR Green 4

qPCR Kit (NEB, F-415L) with gene-specific oligonucleotides. mRNA levels were 5

normalized relative to ACT1 mRNA levels from three independent RT-PCR analyses. 6

Primers for RT-qPCRs are available on request. 7

8

ChIP and Real Time PCR. 9

For each chromatin immunoprecipitation (ChIP) experiment, yeast strains were 10

first grown in glucose medium (YPD) to a density of ~1 × 107 cells/ml, and then 11

transferred to oleate medium (SCIM) for indicated times. ChIP experiments were 12

performed as described by (33) with the following modifications: For HA-Htz1p ChIP, 13

cells were crossed-linked with 1% formaldehyde for 45 min at room temperature. 2 µg of 14

anti-HA antibody (12CA5) was prebound with to 50µl of pan-mouse IgG Dynabeads 15

(Dynal Biotech) and then incubated with 1mg (protein) of supernatant from the sheared 16

chromatin overnight at 4oC. TBP (Spt15p-Myc) ChIP was performed as described by 17

(33). Cells were cross-linked with 1% formaldehyde for 2 hours at room temperature. 2µl 18

of anti-Myc antibody (9E11; Abcam) was pre-bound to 50 µl of pan-mouse IgG 19

Dynabeads and then incubated with 1mg (protein) of supernatant from sheared chromatin 20

overnight at 4oC. 21

All ChIP experiments were performed in triplicate. The purified ChIP samples 22

were used in quantitative PCR (qPCR) analysis. Real-Time qPCR was performed by 23

using an iCycler instrument (ABI 7900) and DyNAmoTM Flash SYBR Green qPCR Kit. 24

The average of three independent replicates is reported as relative amplification of each 25

target of interest compared to a normalization control amplicon, within the non-promoter 26

IGRi YMR325W. Primer sequences are available on request. Occupancy level was 27

determined by dividing the relative abundance of an experimental target by the relative 28

abundance of a control target. This ratio represents the enrichment of ChIP DNA over the 29

input DNA for a specific target versus the control target. 30

31

mcb.asm.org

(7)

FACS analysis. 1

Procedures were performed as previously described (27). Fluorescence intensities 2

of individual cells were measured using a FACS Calibur flow cytometer (BD 3

Biosciences). Data analysis was performed using WinMDI 2.8 (available from 4

http://FACS.scripps.edu/). 5

6

Nucleosome scanning assay (NuSA). 7

Two hundred ml of cells at OD600 of 1.0 in either glucose or after transfer to

8

oleate-containing media for the indicated time were treated with 1% formaldehyde for 20 9

min, followed by 5 min incubation in 125 mM glycine. Cell permeablization, 10

micrococcal nuclease digestion, protein degradation and DNA purification steps were 11

performed as described in (38). DNA samples were then treated with RNase A and 12

analyzed in a 2% agarose gel to quantify nucleosomal content. The bands corresponding 13

to mononucleosomal DNA were extracted using a Qiagen gel extraction kit. Q-PCR 14

analysis on digested DNA was performed. Q-PCR primers are available on request and 15

cover the promoter regions of POT1, CTA1, POX1 and FOX2 with overlapping 16

amplicons averaging 100 bp in size. To define nucleosome occupancy, the protection 17

value of each amplicon was normalized to CEN3 values as described (6). The N+1 18

nucleosome refers to the first nucleosome downstream of the transcription start site which 19

is located at open reading frame regions. The N-1 nucleosome refers to the first 20

nucleosome upstream of the transcription start site which is located at promoter regions. 21

mcb.asm.org

(8)

Results 1

Htz1p is required for transcriptional activation of a subset of oleic acid-responsive 2

genes. 3

Transcriptome profiling was used to obtain a global understanding of how Htz1p 4

contributes to gene expression in response to external stimuli. To do so, we focused on 5

gene induction upon shift from glucose to oleic acid growth conditions. This condition 6

was chosen because we and others have previously shown that this transition leads to 7

dramatic alterations in gene expression patterns (16, 32, 33), genes involved in fatty acid 8

metabolism are significantly induced under these conditions, and because it has been 9

shown that S. cerevisiae htz1∆ strains have a specific growth defect when grown on fatty 10

acids (19, 34). In accordance with previous genome-wide analyses of oleate responses 11

(16, 32, 33), a large portion of the genome responds to the transition (Fig 1A; column 1). 12

Reflecting the non-fermentative metabolism of oleate by the coordinated activities of 13

peroxisomes and mitochondria, the most significantly enriched classes of induced genes 14

include genes linked to mitochondrial respiration and peroxisomal lipid metabolism (GO 15

terms oxidative phosphorylation, electron transport chain and aerobic respiration, 16

hypergeometric p values <10-10; components of the mitochondrial respiratory chain - 17

p~10-13; fatty acid oxidation and peroxisome organization and biogenesis related - p~10-6, 18

and the peroxisomal compartment - p~10-12). By comparison, there were many genes that 19

were relatively unresponsive in htz1∆ cells (Fig 1A, column 2; Fig 1B). This included 20

genes that were both poorly induced and genes that were poorly repressed in htz1∆ cells 21

compared to WT (Fig 1A). Among the genes induced upon oleate exposure, 292 were 22

expressed at least two-fold less in htz1∆ cells than in WT cells (Fig 1B). Interestingly, 23

these poorly induced genes were most enriched for those annotated with peroxisomal 24

functions and components; but were not enriched for annotations of mitochondrial 25

components or aspects of mitochondrial respiration (fatty acid and lipid oxidation - 26

p~4.0x10-12; and peroxisomes - p~9 x10-20) (Fig. 1C). Indeed, 26 genes (of 57 total) 27

encoding peroxisomal proteins showed significantly reduced transcription in an htz1∆ 28

background (Fig. 1C). These data suggest that Htz1p is required for the regulated 29

expression of a large number of genes upon transition from one state to another. In the 30

mcb.asm.org

(9)

case of transition to oleate, genes linked to peroxisomal fatty acid oxidation are normally 1

highly induced and their expression is the most significantly affected in the absence 2

Htz1p. 3

4

HTZ1 _{is required for normal peroxisomal beta-oxidation.}

5

The finding that normally highly induced genes linked to fatty-acid oxidation are 6

poorly expressed in htz1∆ cells is consistent with the finding that cells lacking HTZ1 7

show a specific impairment of fatty acid metabolism (19, 34). Like mutants defective in 8

peroxisomal function (e.g. pex3∆). htz1∆ cells exhibit a growth defect on fatty acid-9

containing medium (YPBO), but not on glucose (YPD) containing media (Fig 2A), nor 10

on other non-fermentable carbon sources such as glycerol (YPG) or acetate (YPA) 11

requiring mitochondrial function (34). As expected, the WT cells grew normally on 12

different carbon sources. 13

To examine the effect of Htz1p on the organelle itself, we examined peroxisomes 14

by fluorescence microscopy. WT and htz1∆ cells expressing peroxisomal thiolase Pot1p, 15

tagged by genomic integration with GFP, were incubated in oleate medium and observed 16

over a time course of induction by direct fluorescence confocal microscopy (Fig. 2B). In 17

glucose-containing medium peroxisomes were barely detectable. However, upon shift to 18

oleic acid, WT cells induced the expression and import of Pot1p-GFP as indicated by the 19

accumulation of punctate fluorescent structures (29). However, there was a dramatic 20

delay in the appearance of punctate GFP fluorescence in htz1∆ cells compared with WT 21

cells induced over the same time period. Together these data suggest that peroxisome 22

biogenesis per se is not defective in htz1∆ cells. Rather the defect in the ability to 23

metabolize oleate effectively is a result of relatively poor expression of genes required for 24

(peroxisomal) fatty acid metabolism. 25

26

Transcriptional response of POT1, POX1, FOX2 and CTA1. 27

To further examine the molecular defects associated with the loss of Htz1p, we 28

focused on 4 strongly induced peroxisomal matrix enzymes, encoded by POT1, FOX2, 29

POX1 and CTA1 (Fig. 3A, red). These genes are normally repressed on glucose, and 30

strongly induced on oleic acid (32). Quantitative RT-PCR of these mRNAs demonstrated 31

mcb.asm.org

(10)

that in the absence of Htz1p, each of these genes was repressed as in WT cells, but their 1

induction was impaired upon transition to oleate medium (Fig.3A). Interestingly, the 2

expression of each of these genes appeared to be most significantly affected at the later 3

time points after transition to oleate (compare 4 and 6 hours of induction to 0.5 and 1 4

hour of induction). These data suggest that loss of Htz1p did not dramatically alter the 5

initial response, but was important for the sustained expression of these four genes. 6

Having demonstrated a role for Htz1p in the normal regulation of POT1, FOX2, 7

POX1 and CTA1 expression in the presence of oleic acid, we next sought to determine if 8

Htz1p binds the cognate promoters of these genes using chromatin immunoprecipitation 9

(ChIP) of a strain expressing an HA-tagged version of Htz1p. Cells were grown in either 10

repressed (glucose) or activated (oleate) conditions. Htz1p-HA was immunoprecipitated 11

with anti-HA antibody and isolated DNA was analyzed by PCR. This analysis revealed 12

that Htz1p was bound to each of the four promoters (POT1, FOX2, POX1 and CTA1) in 13

their repressed states (Fig. 3B). These data are consistent with genome wide 14

characterization of levels of Htz1p association with these promoters (41). The association 15

of Htz1p with these promoters was dynamic; when cells were shifted to oleic acid 16

activating conditions, Htz1p levels on the POT1, POX1, and FOX2 promoters were 17

dramatically reduced. Dissociation from the CTA1 promoter was not observed. These 18

data suggest that loss of Htz1p from promoters is coincident with gene activation, but that 19

dissociation is not required for the induction of all genes. 20

21

Swr1p, Chz1p and Gcn5p - dependent association of Htz1p to promoters. 22

Swr1p, Chz1p and Gcn5p have been implicated in modulating Htz1p association 23

at promoter regions. Swr1p is part of the SWR1-C multisubunit protein complex, 24

necessary for Htz1p deposition at repressed promoters (24). Chz1p, was recently 25

identified as a histone chaperone that preferentially interacts with Htz1p (21) and Gcn5p 26

is the histone acetyltransferase subunit of the SAGA complex (36). To investigate 27

whether these factors affect Htz1p binding to the oleate-responsive promoters and 28

subsequent expression, the association of Htz1p with POT1, POX1, FOX2 and CTA1 29

promoters was investigated in cells lacking these proteins under conditions of repression 30

(2% glucose) and expression of these genes was monitored upon oleate induction (Fig. 31

mcb.asm.org

(11)

4). Similar to its role at the well-studied GAL1 promoter, Swr1p is required for Htz1p 1

binding to oleate responsive promoters suggesting a common role for Swr1p at disparate, 2

highly inducible promoters. Likewise, Gcn5p was required for efficient Htz1p binding. 3

This suggests that Gcn5p, which plays a role as a coactivator of transcription through 4

histone acetylation (11), controls the binding or stability of Htz1p at repressed promoters. 5

This may also be via histone acetylation. In the absence of Chz1p, Htz1p occupancy at 6

each of the four promoters was was decreased. As expected the amount of Htz1p on each 7

of these promoters in mutant strains remained low upon switch to oleate (data not 8

shown). 9

Microarray analyses in gcn5∆, swr1∆, and chz1∆ mutants support a model in 10

which initial Htz1p association with the promoter is required for subsequent full 11

induction. The expression levels of POT1, FOX2, POX1 and CTA1 were significantly 12

reduced in mutant strains compared to WT upon oleate induction. Moreover, all 26 genes 13

encoding peroxisomal proteins that showed transcriptional defects in htz1∆ cells (Fig. 14

1C) were similarly reduced in their expression at least two-fold in gcn5∆, swr1∆, and 15

chz1∆ mutants compared to WT (Fig. 4B). Together, these data suggest that factors 16

functionally associated with Htz1p, such as the chromatin remodeling complex 17

component Swr1p, histone acetyltransferase Gcn5p and chaperone Chz1p, regulate the 18

deposition or maintenance of Htz1p at repressed promoters, which in turn, facilitates 19

rapid activation of transcription. 20

21

Acetylation of Htz1p is required for efficient transcriptional induction. 22

Acetylation of Htz1p is known to occur at sites of active transcription (23). The 23

finding that Gcn5p is required for expression of oleate responsive genes suggests that 24

acetylation on Htz1p is required for the oleate response. To address this question, 25

plasmids expressing either one of two acetylation mutants of Htz1p (pCM314 (Htz1p-26

K14A) or pCM330 (Htz1p-K14R)) were introduced into htz1∆ cells and expression was 27

monitored by FACS, confocal microscopy and quantitative RT-PCR. FACS and confocal 28

microscopy demonstrated that Pot1p-GFP fluorescence in cells carrying pCM305 (WT 29

HTZ1) was stronger than that in those cells carrying empty plasmid (pRS416), or 30

acetylation mutants (pCM330, or pCM314) during a time course of oleate incubation but 31

mcb.asm.org

(12)

the peroxisomes were morphologically normal (data not shown). mRNA levels of POT1, 1

CTA1, FOX2 and POX1, determined by quantitative RT-PCR were consistent with the 2

GFP reporter analysis (Fig 5A). The K14A acetylation mutant of Htz1p showed a defect 3

in the normal induction of POT1, CTA1, FOX2 and POX1. In addition, cells expressing 4

Htz1p K14A also exhibited a growth defect on fatty-acid containing media, but not on 5

glucose containing media. This growth defect was less pronounced than the null mutant 6

of HTZ1 (data not shown). The association of Htz1p-K14R with these oleate responsive 7

promoters at two time points (0 h and 6 h), also revealed that Htz1p-K14R association 8

was diminished under glucose conditions (Fig. 5B). Significant differences in association 9

of Htz1p-K14R on these promoters were not observed during 6 h of oleate induction. 10

These data indicate that acetylation of Htz1p is required for association with oleate 11

responsive promoters during repressed conditions and the acetylation of Htz1p is not 12

required for the dissociation of Htz1p from oleate responsive promoters during oleate 13

induction (Fig. 5B). These data collectively indicate that acetylation of Htz1p is 14

important for expression of fatty acid responsive genes and normal peroxisomal matrix 15

protein assembly. 16

17

TBP is not efficiently recruited to oleate inducible promoters in the absence of 18

Htz1p. 19

We next directly analyzed in vivo binding of the transcriptional machinery to 20

repressed and activated promoters in both WT and htz1∆ strains (Fig. 6A). As expected, 21

the binding of TBP to the POT1, POX1, and CTA1 promoters increased with gene 22

expression in oleate in WT cells. The abundance of TBP did not significantly increase on 23

the FOX2 promoter following oleic acid induction but was present at higher initial levels 24

than the other three other promoters studied. Nonetheless at all four promoters in htz1∆ 25

cells, TBP binding was significantly reduced compared to WT cells. The reduced levels 26

of TBP binding were not attributable to decreased cellular levels of TBP. Western blot 27

analysis of both WT and htz1∆ cells demonstrated that TBP levels were equivalent 28

between the strains and did not significantly change during oleate induction (Fig 5B). 29

These results suggest a positive function for the Htz1p-containing nucleosomes in the 30

mcb.asm.org

(13)

recruitment of TBP to the repressed promoters during the process of transcriptional 1

activation. 2

Htz1 regulates nucleosome-promoter association during activation. 3

A nucleosome scanning assay (NuSA) was used to investigate the role of Htz1p in 4

modulating chromatin structure by measuring nucleosome occupancy and location within 5

oleate responsive promoters during activation (POT1, POX1, FOX2 and CTA1) (Fig. 7). 6

Mononucleosome-associated DNA was isolated from yeast cells before and after oleate 7

induction and quantitative real time PCR (qPCR) was used to measure dynamic 8

nucleosome occupancy during activation in WT and htz1∆ cells. The precise positions of 9

the nucleosomes were determined by qPCR corresponding to their known positions (17). 10

Overall the gross nucleosome position pattern at each of the four promoters under 11

repressed conditions was the same in WT and htz1∆ cells. The major nucleosome 12

changes were observed at position N-1 in each promoter. These nucleosomes appeared to 13

begin disassembly from each promoter at the earliest time point measured (5 min) and 14

continued through to the 30 min time point. After this initial disassembly, nucleosomes 15

were detected to have begun reassembly after 1 h of induction (Fig. 7). These 16

reassembled nucleosomes likely do not contain Htz1p. As shown in Fig. 3B, Htz1p is 17

progressively lost from these promoters during the 6 h period of induction. Notably, the 18

nucleosomes of each promoter appeared to be more protected at the later time points (6 h) 19

in htz1∆ cells compared to WT. This was most evident at the N-1 position of the 20

promoters of POX1 and CTA1 (and at the N-2 position of POX1). These data suggest that 21

upon oleate treatment the nucleosome proximal to initiation site in each promoter 22

disassembles leading to Htz1p loss and initial transcriptional activation. During 23

prolonged expression, nucleosomes reassemble, but these reassembled nucleosomes do 24

not contain Htz1p. 25

26

Interplay between Htz1p and chromatin remodeling factor Isw2p. 27

While Htz1p is proposed to contribute to nucleosome disassembly during 28

induction, the results presented above indicate that the overall chromatin structure at the 29

promoters was not extensively perturbed in htz1∆ cells. To gain insight into the potential 30

mcb.asm.org

(14)

additional mechanisms at play during the transcriptional induction, we considered 1

additional chromatin bound proteins. One such protein is Isw2p. Isw2p is an ATP-2

dependent chromatin remodeling factor that has previously been shown to be required for 3

maintenance of chromatin structure at the POT1 promoter (8, 9, 39) We therefore 4

investigated Isw2p function at the POT1, POX1, FOX2 and CTA1 promoters in WT and 5

htz1∆ cells. 6

Nucleosome protection assays in isw2∆ cells led to significant changes in the 7

nucleosome structure in all four promoters (Fig 8A). These data indicate that Isw2p plays 8

a role in chromatin structure of these four promoters and are consistent with previous 9

work on the POT1 promoter (8, 9, 39). 10

Next we used ChIP to assay the ability of Isw2p to associate with the four oleate 11

responsive promoters. Previous work has shown that in WT cells Isw2p does not stably 12

associate with these promoters, which suggests that under normal conditions, Isw2p 13

contributes to the nucleosome structure at these Htz1p-containing POT1, POX1, FOX2 14

and CTA1 promoters through transient interactions (9). Similarly, we found very low 15

levels of Isw2p association with these promoters in WT cells, in glucose and after oleate 16

induction. However, substantial amounts of Isw2p were observed in association with 17

each of these promoters in the htz1∆ cells. Isw2p remained associated with these 18

promoters during their activation, suggesting a role in establishing chromatin dynamics in 19

the absence of Htz1p (Fig. 8B). 20

mcb.asm.org

(15)

Discussion 1

2

Exposure of yeast cells to oleate results in large scale reorganization of gene 3

expression regulatory networks and provides an excellent experimental system for 4

understanding the mechanisms of gene expression at both the molecular and network 5

levels (28, 33). The resulting changes in gene expression are widespread, representing the 6

reorganization of regulatory networks governing numerous categories of gene function. 7

For example genes involved in protein translation and glycolysis are repressed, reflecting 8

the shift in growth rates and metabolism (16, 32, 33). Likewise, genes linked to 9

mitochondrial respiration and peroxisomal fatty acid metabolism is induced, reflecting 10

the cells shift to non-fermentative β-oxidation as an energy source (16, 32, 33). The 11

ability of yeast cells to adapt to this shift is dependent on the HTZ1 gene encoding the 12

histone variant Htz1p/H2A.Z (19, 32). The data presented here demonstrate that Htz1p 13

plays a critical role in this transition by contributing to the recruitment of TBP to oleate 14

responsive genes leading to rapid and robust expression of highly inducible genes. 15

Transcriptome profiling studies presented here demonstrate that expression of 16

genes that are normally highly responsive to oleate is impaired in the absence of Htz1p. 17

Because, under these conditions, many of the most strongly induced genes are required 18

for peroxisomal β-oxidation and peroxisome proliferation, lack of Htz1p renders cells 19

unable to respond efficiently to the transition and metabolize the fatty acids. 20

In order to elucidate the step-wise molecular function of Htz1p in the 21

transcriptional regulation of these genes, we generated and compared various time course 22

datasets to analyze chromatin states before and after the switch to oleic acid. We used 23

chromatin immunoprecipations to assay the dynamic association of Htz1p at the 24

promoters of four model genes (POT1, POX1, FOX2, and CTA1) encoding peroxisomal 25

matrix enzymes, the expression of which was perturbed by deletions of HTZ1. Htz1p has 26

been proposed to preferentially bind repressed promoters facilitating the rapid activation 27

of the associated genes (41). Consistent with the current models, we demonstrate that 28

Htz1p tends to be bound to these promoters in their repressed states (glucose) and 29

disassociates from these promoters once the cells are exposed to oleate; however, this 30

association and dissociation pattern occurs at levels that are promoter specific. The 31

mcb.asm.org

(16)

methods employed here did not reveal significant dissociation of Htz1p from the CTA1 1

promoter. The data suggest that Htz1p levels on the CTA1 promoter are lower (~2-fold 2

over control regions) than the other promoters examined. Thus, Htz1p does not appear to 3

dissociate from the CTA1 promoter following oleic acid induction. The mechanisms 4

underlying promoter specific effects of Htz1p and other epigenetic factors remain fertile 5

ground for future study. 6

In addition, data presented here support previous studies in both yeast and 7

mammalian cells that demonstrate that Htz1p is deposited at promoters by the chromatin 8

remodeling protein Swr1p (24, 40). Similarly, as in other transcriptional responses (23), 9

Gcn5p/Esa1p mediated acetylation at Lys14 of Htz1p is required for efficient 10

transcriptional activation. Moreover, Gcn5p/Esa1p mediated acetylation at Lys14 of 11

Htz1p is required for efficient association of Htz1p at some oleate responsive promoters. 12

The significantly decreased association of the Htz1p-K14R mutant was observed under 13

repressive conditions (i.e. glucose), and this decreased binding of Htz1p was also 14

observed in cells lacking the enzyme (Gcn5p) responsible for Htz1p acetylation. Htz1p 15

acetylation mutant cells also displayed defects in peroxisome proliferation and growth on 16

oleic acid, similar to an HTZ1 null mutant. These data demonstrate that acetylation of 17

Htz1p, mediated by Gcn5p, is required for association with oleate responsive promoters 18

during repressed conditions and for normal transcriptional induction contributed by 19

Htz1p. 20

Among the known effectors of Htz1p, Chz1p is relatively less well characterized. 21

Luk et al (21) identified a role for Chz1p as a nuclear chaperone for Htz1p though the 22

functional relationship between Chz1p and Htz1p with respect to transcriptional 23

regulation remained uncharacterized. Here, we report that Chz1p, like Swr1p and Gcn5p, 24

is also involved in the deposition of Htz1p at repressed promoters. 25

With respect to the role of Htz1p in TBP recruitment, studies of the GAL 26

promoters have drawn different conclusions. In a recent study, TBP recruitment to the 27

GAL1 promoter in htz1∆ strains was indistinguishable from that of WT cells (10). 28

However, earlier studies showed Htz1p-dependent enrichment of TBP to GAL1 and 29

GAL10 promoters during a time course of galactose induction (1). In the case of the fatty-30

acid inducible promoters tested here, absence of Htz1p led to significant reduction in the 31

mcb.asm.org

(17)

recruitment of TBP during oleate induction. We did not observe increased enrichment of 1

TBP at FOX2 promoter during oleate induction. The dynamics of TBP binding appear to 2

be promoter specific. The relative abundance of TBP at the FOX2 promoter prior to 3

induction by oleic acid (compared to later time points) suggests that activation of FOX2 4

does not require additional TBP binding and that the gene exists in a state poised for its 5

activation upon receiving the correct signals (i.e. oleic acid). Comprehensive 6

investigation of the dynamic and quantitative role of Htz1p in the recruitment of factors 7

such as mediator and TBP to different promoters throughout the genome remains for 8

future studies. 9

Our data suggest that activation of repressed genes leads to a dynamic 10

reorganization of chromatin structure. Specifically, upon oleate treatment the nucleosome 11

proximal to initiation site in each promoter disassembles. This coincides with the ejection 12

of Htz1p from the promoter. These data are in agreement with previous studies that 13

indicate nucleosome disassembly from promoters during activation provides access to the 14

transcriptional machinery (25). While Htz1p is proposed to contribute to nucleosome 15

disassembly during induction, surprisingly, the apparent rate of nucleosome disassembly 16

at the oleate responsive promoters was not dramatically different in htz1∆ cells. After 17

initial disassembly, the nucleosomes reassemble (~1 h after induction). Interestingly, 18

these new nucleosomes do not appear to contain Htz1p, but levels of transcription are 19

nonetheless higher in WT cells than in cells lacking Htz1p. These data suggest the initial 20

presence of Htz1p ensures a normal transcriptional response and provides an epigenetic 21

mark that persists after its loss, ensuring high levels of expression. Close examination of 22

the data from nucleosome protection assays suggest that, in the absence of Htz1p, 23

nucleosomes in the promoter regions of oleate responsive genes are relatively more 24

assembled, which may cause reduced expression levels at these later time points. The 25

reassembly of nucleosomes during the coincident high levels of gene expression, suggests 26

that transcriptional activity is not simply related to an overall openness of chromatin at 27

activated promoters and obstruction at repressed promoters. Rather, the precise dynamic 28

placement and specific constituents of individual nucleosomes at promoters 29

mechanistically regulates transcription by modulating access of transacting factors to 30

specific sites. Further, characterization of the dynamics of the epigenetic marks, protein 31

mcb.asm.org

(18)

components of the nucleosomes and chromatin remodeling complexes at these promoters 1

is required to delineate the mechanistic basis of the links between chromatin state and 2

transcriptional activation. 3

The observed lower expression levels in cells lacking Htz1p may also be 4

contributed by Isw2p. Isw2p is an energy-dependent chromatin remodeling factor and 5

negative regulator of gene expression (8, 9). When assayed for genome binding by ChIP-6

chip Isw2p association with the POT1, POX1, FOX2 and CTA1 promoters was not 7

detected (9). Similarly, we found no enrichment of Isw2p at these promoters in WT cells. 8

However, Isw2p bound to each promoter in the absence of Htz1p, and this association 9

persisted through the six hours of induction. Therefore, the increased association of 10

Isw2p to these four oleate responsive promoters may account for the reduced expression 11

levels in htz1∆ cells. It is also possible that in WT cells Isw2p provides a complementary 12

mechanism for chromatin structural changes independent of Htz1p. Loss of Htz1p 13

provides an opportunity for Isw2p binding that is not normally functional in Htz1p-14

containing regions of chromatin. Further studies are required to understand the global 15

relationship between Isw2p and Htz1p. 16

In mammalian cells histone variant H2A.Z can serve as a novel epigenetic marker 17

of breast cancer progression as it is associated with lymph node metastasis and decreased 18

breast cancer survival (13). In the plant Arabidopsis thaliana, histone H2A.Z is required 19

for immune resistance to the phytopathogenic bacteria Pseudomonas syringae pv. tomato 20

(22). In zebrafish, histone variant 2a z (H2afza) is essential for larval development 21

through the generation of a lethal locus with a truncation of conserved carboxy-terminal 22

residues in the protein (31). Taken together these studies implicate histone H2A.Z in a 23

number of diverse functions in different organisms. Because peroxisomes are highly 24

dynamic and responsive eukaryotic organelles whose dysfunction are linked to a host of 25

human conditions (4, 5), it is important to understand the roles of proteins like Htz1p, that 26

control aspects of chromatin structure and transcriptional responses preceding the 27

proliferation of peroxisomes and fatty acid metabolism in S. cerevisiae. 28

mcb.asm.org

(19)

Acknowledgements 1

We thank Bradley R. Cairns, Haiying Zhang and Michael Grunstein for providing 2

plasmids and strains; Jeff Ranish and members of the Aitchison laboratory for helpful 3

comments and discussion during the course of this project. This work was supported by 4

grants GM067228, GMO76547 and RR022220 from the U.S. National Institutes of 5 Health. 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

mcb.asm.org

(20)

References 1

2

1. Adam, M., F. Robert, M. Larochelle, and L. Gaudreau. 2001. H2A.Z is 3

required for global chromatin integrity and for recruitment of RNA polymerase II 4

under specific conditions. Mol Cell Biol 21:6270-9. 5

2. Aitchison, J. D., M. P. Rout, M. Marelli, G. Blobel, and R. W. Wozniak. 1995. 6

Two novel related yeast nucleoporins Nup170p and Nup157p: complementation 7

with the vertebrate homologue Nup155p and functional interactions with the yeast 8

nuclear pore-membrane protein Pom152p. J Cell Biol 131:1133-48. 9

3. Albert, I., T. N. Mavrich, L. P. Tomsho, J. Qi, S. J. Zanton, S. C. Schuster, 10

and B. F. Pugh. 2007. Translational and rotational settings of H2A.Z 11

nucleosomes across the Saccharomyces cerevisiae genome. Nature 446:572-6. 12

4. Bensinger, S. J., and P. Tontonoz. 2008. Integration of metabolism and 13

inflammation by lipid-activated nuclear receptors. Nature 454:470-7. 14

5. Berger, J., and D. E. Moller. 2002. The mechanisms of action of PPARs. Annu 15

Rev Med 53:409-35. 16

6. Biddick, R. K., G. L. Law, and E. T. Young. 2008. Adr1 and Cat8 mediate 17

coactivator recruitment and chromatin remodeling at glucose-regulated genes. 18

PLoS ONE 3:e1436. 19

7. Dudley, A. M., J. Aach, M. A. Steffen, and G. M. Church. 2002. Measuring 20

absolute expression with microarrays with a calibrated reference sample and 21

an extended signal intensity range. Proc Natl Acad Sci U S A 99:7554-9. 22

mcb.asm.org

(21)

8. Fazzio, T. G., M. E. Gelbart, and T. Tsukiyama. 2005. Two distinct 1

mechanisms of chromatin interaction by the Isw2 chromatin remodeling complex 2

in vivo. Mol Cell Biol 25:9165-74. 3

9. Gelbart, M. E., N. Bachman, J. Delrow, J. D. Boeke, and T. Tsukiyama. 2005. 4

Genome-wide identification of Isw2 chromatin-remodeling targets by localization 5

of a catalytically inactive mutant. Genes Dev 19:942-54. 6

10. Gligoris, T., G. Thireos, and D. Tzamarias. 2007. The Tup1 corepressor directs 7

Htz1 deposition at a specific promoter nucleosome marking the GAL1 gene for 8

rapid activation. Mol Cell Biol 27:4198-205. 9

11. Govind, C. K., F. Zhang, H. Qiu, K. Hofmeyer, and A. G. Hinnebusch. 2007. 10

Gcn5 promotes acetylation, eviction, and methylation of nucleosomes in 11

transcribed coding regions. Mol Cell 25:31-42. 12

12. Guillemette, B., A. R. Bataille, N. Gevry, M. Adam, M. Blanchette, F. Robert, 13

and L. Gaudreau. 2005. Variant histone H2A.Z is globally localized to the 14

promoters of inactive yeast genes and regulates nucleosome positioning. PLoS 15

Biol 3:e384. 16

13. Hua, S., C. B. Kallen, R. Dhar, M. T. Baquero, C. E. Mason, B. A. Russell, P. 17

K. Shah, J. Liu, A. Khramtsov, M. S. Tretiakova, T. N. Krausz, O. I. 18

Olopade, D. L. Rimm, and K. P. White. 2008. Genomic analysis of estrogen 19

cascade reveals histone variant H2A.Z associated with breast cancer progression. 20

Mol Syst Biol 4:188. 21

mcb.asm.org

(22)

14. Ideker, T., V. Thorsson, A. F. Siegel, and L. E. Hood. 2000. Testing for 1

differentially-expressed genes by maximum-likelihood analysis of microarray 2

data. J Comput Biol 7:805-17. 3

15. Jackson, J. D., V. T. Falciano, and M. A. Gorovsky. 1996. A likely histone 4

H2A.F/Z variant in Saccharomyces cerevisiae. Trends Biochem Sci 21:466-7. 5

16. Koerkamp, M. G., M. Rep, H. J. Bussemaker, G. P. Hardy, A. Mul, K. 6

Piekarska, C. A. Szigyarto, J. M. De Mattos, and H. F. Tabak. 2002. 7

Dissection of transient oxidative stress response in Saccharomyces cerevisiae by 8

using DNA microarrays. Mol Biol Cell 13:2783-94. 9

17. Lee, W., D. Tillo, N. Bray, R. H. Morse, R. W. Davis, T. R. Hughes, and C. 10

Nislow. 2007. A high-resolution atlas of nucleosome occupancy in yeast. Nat 11

Genet 39:1235-44. 12

18. Li, B., S. G. Pattenden, D. Lee, J. Gutierrez, J. Chen, C. Seidel, J. Gerton, 13

and J. L. Workman. 2005. Preferential occupancy of histone variant H2AZ at 14

inactive promoters influences local histone modifications and chromatin 15

remodeling. Proc Natl Acad Sci U S A 102:18385-90. 16

19. Lockshon, D., L. E. Surface, E. O. Kerr, M. Kaeberlein, and B. K. Kennedy. 17

2007. The sensitivity of yeast mutants to oleic acid implicates the peroxisome and 18

other processes in membrane function. Genetics 175:77-91. 19

20. Longtine, M. S., A. McKenzie, 3rd, D. J. Demarini, N. G. Shah, A. Wach, A. 20

Brachat, P. Philippsen, and J. R. Pringle. 1998. Additional modules for 21

versatile and economical PCR-based gene deletion and modification in 22

Saccharomyces cerevisiae. Yeast 14:953-61. 23

mcb.asm.org

(23)

21. Luk, E., N. D. Vu, K. Patteson, G. Mizuguchi, W. H. Wu, A. Ranjan, J. 1

Backus, S. Sen, M. Lewis, Y. Bai, and C. Wu. 2007. Chz1, a nuclear chaperone 2

for histone H2AZ. Mol Cell 25:357-68. 3

22. March-Diaz, R., M. Garcia-Dominguez, J. Lozano-Juste, J. Leon, F. J. 4

Florencio, and J. C. Reyes. 2008. Histone H2A.Z and homologues of 5

components of the SWR1 complex are required to control immunity in 6

Arabidopsis. Plant J 53:475-87. 7

23. Millar, C. B., F. Xu, K. Zhang, and M. Grunstein. 2006. Acetylation of H2AZ 8

Lys 14 is associated with genome-wide gene activity in yeast. Genes Dev 20:711-9

22. 10

24. Mizuguchi, G., X. Shen, J. Landry, W. H. Wu, S. Sen, and C. Wu. 2004. ATP-11

driven exchange of histone H2AZ variant catalyzed by SWR1 chromatin 12

remodeling complex. Science 303:343-8. 13

25. Petesch, S. J., and J. T. Lis. 2008. Rapid, transcription-independent loss of 14

nucleosomes over a large chromatin domain at Hsp70 loci. Cell 134:74-84. 15

26. Raisner, R. M., P. D. Hartley, M. D. Meneghini, M. Z. Bao, C. L. Liu, S. L. 16

Schreiber, O. J. Rando, and H. D. Madhani. 2005. Histone variant H2A.Z 17

marks the 5' ends of both active and inactive genes in euchromatin. Cell 123:233-18

48. 19

27. Ramsey, S. A., J. J. Smith, D. Orrell, M. Marelli, T. W. Petersen, P. de 20

Atauri, H. Bolouri, and J. D. Aitchison. 2006. Dual feedback loops in the GAL 21

regulon suppress cellular heterogeneity in yeast. Nat Genet 38:1082-7. 22

mcb.asm.org

(24)

28. Ratushny, A. V., S. A. Ramsey, O. Roda, Y. Wan, J. J. Smith, and J. D. 1

Aitchison. 2008. Control of Transcriptional Variability by Overlapping Feed-2

Forward Regulatory Motifs. Biophys J8:3715-23 3

29. Saleem, R. A., B. Knoblach, F. D. Mast, J. J. Smith, J. Boyle, C. M. Dobson, 4

R. Long-O'Donnell, R. A. Rachubinski, and J. D. Aitchison. 2008. Genome-5

wide analysis of signaling networks regulating fatty acid-induced gene expression 6

and organelle biogenesis. J Cell Biol 181:281-92. 7

30. Santisteban, M. S., T. Kalashnikova, and M. M. Smith. 2000. Histone H2A.Z 8

regulats transcription and is partially redundant with nucleosome remodeling 9

complexes. Cell 103:411-22. 10

31. Sivasubbu, S., D. Balciunas, A. E. Davidson, M. A. Pickart, S. B. Hermanson, 11

K. J. Wangensteen, D. C. Wolbrink, and S. C. Ekker. 2006. Gene-breaking 12

transposon mutagenesis reveals an essential role for histone H2afza in zebrafish 13

larval development. Mech Dev 123:513-29. 14

32. Smith, J. J., M. Marelli, R. H. Christmas, F. J. Vizeacoumar, D. J. Dilworth, 15

T. Ideker, T. Galitski, K. Dimitrov, R. A. Rachubinski, and J. D. Aitchison. 16

2002. Transcriptome profiling to identify genes involved in peroxisome assembly 17

and function. J Cell Biol 158:259-71. 18

33. Smith, J. J., S. A. Ramsey, M. Marelli, B. Marzolf, D. Hwang, R. A. Saleem, 19

R. A. Rachubinski, and J. D. Aitchison. 2007. Transcriptional responses to fatty 20

acid are coordinated by combinatorial control. Mol Syst Biol 3:115. 21

34. Smith, J. J., Y. Sydorskyy, M. Marelli, D. Hwang, H. Bolouri, R. A. 22

Rachubinski, and J. D. Aitchison. 2006. Expression and functional profiling 23

mcb.asm.org

(25)

reveal distinct gene classes involved in fatty acid metabolism. Mol Syst Biol 1

2:2006 0009. 2

35. Stargell, L. A., J. Bowen, C. A. Dadd, P. C. Dedon, M. Davis, R. G. Cook, C. 3

D. Allis, and M. A. Gorovsky. 1993. Temporal and spatial association of histone 4

H2A variant hv1 with transcriptionally competent chromatin during nuclear 5

development in Tetrahymena thermophila. Genes Dev 7:2641-51. 6

36. Sterner, D. E., and S. L. Berger. 2000. Acetylation of histones and transcription-7

related factors. Microbiol Mol Biol Rev 64:435-59. 8

37. Suto, R. K., M. J. Clarkson, D. J. Tremethick, and K. Luger. 2000. Crystal 9

structure of a nucleosome core particle containing the variant histone H2A.Z. Nat 10

Struct Biol 7:1121-4. 11

38. Whitehouse, I., O. J. Rando, J. Delrow, and T. Tsukiyama. 2007. Chromatin 12

remodelling at promoters suppresses antisense transcription. Nature 450:1031-5. 13

39. Whitehouse, I., and T. Tsukiyama. 2006. Antagonistic forces that position 14

nucleosomes in vivo. Nat Struct Mol Biol 13:633-40. 15

40. Wong, M. M., L. K. Cox, and J. C. Chrivia. 2007. The chromatin remodeling 16

protein, SRCAP, is critical for deposition of the histone variant H2A.Z at 17

promoters. J Biol Chem 282:26132-9. 18

41. Zhang, H., D. N. Roberts, and B. R. Cairns. 2005. Genome-wide dynamics of 19

Htz1, a histone H2A variant that poises repressed/basal promoters for activation 20

through histone loss. Cell 123:219-31. 21

22 23 24 25

mcb.asm.org

(26)

Figure Legends 1

2

Figure 1. Robust expression of oleate-responsive genes expression is dependent on 3

HTZ1_{. (A) Comparison of changes in mRNA levels of all yeast genes in WT (left}

4

column) and htz1∆ cells (middle column) after induction in oleate medium for 6 h. 5

Shown are the relative expression levels (Log10) of genes that were determined to be

6

significantly (λ≥100) altered in cells on oleate (compared to WT cells on glucose). 7

Relative expression levels are shown using the scale of the yellow-blue heat map (top). 8

Genes are ordered top-bottom based on relative expression in WT cells on oleate. 9

Approximately 1000 genes were significantly induced and 1000 genes were repressed 10

and changed in expression at least 2-fold. Genes that were reduced in expression were 11

significantly enriched for functions related to ribosomal biogenesis (hypergeometric 12

distribution analysis of GO terms – p ~10-50). Induced genes were enriched for oxidative 13

phosphorylation, electron transport chain and aerobic respiration (p ~ 10-10; components 14

of the mitochondrial respiratory chain (p ~ 10-13), fatty acid oxidation and peroxisome 15

organization and biogenesis related (~ p ~ 10-6), and the peroxisomal compartment (p ~ 16

10-12). For comparison the relative expression of each gene in htz1∆ cells in oleate 17

(middle column) and glucose (right column) are shown. (B) As in Panel A, but shown are 18

the relative expression levels of 292 genes significantly (λ≥100) altered in WT cells on 19

oleate and expressed at least two-fold less than their expression levels in WT cells. This 20

list is enriched for genes linked to fatty acid and lipid oxidation (p~4.0x10-12) and 21

peroxisomes (p~9 x10-20). (C) As in B, but shown are genes encoding peroxisomal 22

proteins significantly (λ≥100) altered in WT cells on oleate and expressed at least 2-fold 23

less in htz1∆ cells. 24

25

Figure 2. Deletion of HTZ1 leads to delayed peroxisome biogenesis. (A) Deletion of 26

HTZ1 impairs cell growth on oleate-containing media. Strains were grown to mid-27

logarithm phase in liquid YPD medium, and equal amounts of cells were serially diluted 28

ten-fold onto YPD and incubated at 30°C for 3 days and onto oleate-containing YPBO 29

and incubated at 30°C for 5 days. (B) Fluorescent images of WT and htz1∆ cells shown 30

are expressing the peroxisomal matrix protein Pot1p fused with GFP (Pot1p-GFP) at 31

mcb.asm.org

(27)

different time points of oleate incubation were captured on a TCS SP2 Laser Scanning 1

Spectral Confocal Microscope. 2

3

Figure 3. Htz1p dynamically dissociates from oleate responsive promoters upon 4

induction. (A) POT1, POX1, FOX2 and CTA1 mRNA levels were determined by RT-5

PCR in WT and htz1∆ strains over a time course of oleate induction. The signal obtained 6

from ACT1 mRNA was used as a loading control for normalization. Error bars represent 7

standard deviation from the mean of three independent experimental values. (B) Htz1p 8

enrichment at four promoters was determined by qPCR during oleate induction. Relative 9

enrichment values (Y axes) are the average of three independent ChIPs with qPCR 10

determination performed twice per each biological replicate. Non-promoter IGRi 11

YMR325W was used as an internal control to normalize signals of promoter enrichment. 12

In response to oleate induction, Htz1p was lost from the POT1, POX1, FOX2 and CTA1 13

promoters. 14

15

Figure 4. Swr1p, Chz1p and Gcn5p - dependent association of Htz1p to promoters. 16

(A) In vivo association of Htz1p with POT1, POX1, FOX2 and CTA1 promoters was 17

measured by ChIP in the WT, chz1∆, gcn5∆ and swr1∆ strains in 2% glucose medium. 18

ChIP was performed in glucose-containing medium. Error bars represent standard 19

deviation from the mean of three independent experimental values and two technical 20

replicates of each. (B) Comparison of changes in mRNA levels of all yeast genes in WT, 21

chz1∆, gcn5∆ and swr1∆ strains after induction in oleate medium for 6 h. As in Figure 1 22

C, genes are encoding peroxisomal proteins significantly (λ≥100) altered in WT cells on 23

oleate and expressed at least 2-fold less in htz1∆ cells. 24

25

Figure 5. Acetylation of Htz1p is required for efficient transcriptional induction. (A) 26

POT1, POX1, FOX2 and CTA1 mRNA levels were determined by RT-PCR in WT and 27

htz1∆ and Htz1p K14A mutant strains over a time course of oleate induction. The signal 28

obtained from ACT1 mRNA was used as a loading control for normalization. Error bars 29

represent standard deviation from the mean of three independent experimental values. (B) 30

Enrichment of WT Htz1p and Htz1p K14A mutant at four promoters was determined by 31

mcb.asm.org

(28)

qPCR during glucose and oleate induction for 6 hours. Relative enrichment values (Y 1

axes) are the average of three independent ChIP experiments with two technical 2

replicates of each. Non-promoter IGRi YMR325W was used as an internal control to 3

normalize signals of promoter enrichment. 4

5

Figure 6. Recruitment of TBP during oleate induction requires Htz1p. (A) The 6

association of TBP with POT1, POX1, FOX2 and CTA1 promoters was determined by 7

chromatin immunoprecipitation (ChIP) using anti-Myc antibodies, followed by gene-8

specific PCR. The relative enrichment ratio is plotted at 4 time points (0, 1, 4, 6 h) of 9

induction in oleate. ACT1 was used as an internal control to normalize signals of 10

promoter enrichment. Error bars show the standard deviation from three independent 11

experimental values with two technical replicates of each. (B) Deletion of Htz1p did not 12

affect TBP expression during oleate induction. The WT strain and HTZ1 deletion strains 13

expressing genomically integrated TBP were grown in 2% glucose overnight and then 14

transferred to oleate-containing SCIM medium at the indicated time points. Samples 15

containing equal protein were analyzed by Western blotting with anti-Myc antibody to 16

visualize TBP expression. A polyclonal antibody directed against Gsp1p was used as 17

loading control. 18

19

Figure 7. Htz1p regulates the occupancy of specific nucleosomes on POT1, POX1, 20

FOX2_{and CTA1 promoters. The NuSA assay was used to determine the nucleosome}

21

positioning and density at POT1, POX1, FOX2 and CTA1 promoters during oleate 22

induction (time of induction is indicated at left) in WT and HTZ1 deletion strains. Each 23

point represents the relative protection of each PCR amplicon, quantified by real-time 24

PCR and normalized to a centromeric control. The position of each amplicon (referenced 25

to the middle of each amplicon) within the promoter is shown on the x-axis. The 26

approximate location of nucleosome is represented by grey circle with the nucleosome 27

number referred to in the text shown on the circle. 28

29

Figure 8. Isw2p can associate with oleate responsive promoters in the absence of 30

Htz1p. (A) The NuSA assay was used to determine the nucleosome positioning and 31

mcb.asm.org

(29)

density at POT1, POX1, FOX2 and CTA1 promoters during repression (2% glucose) in 1

WT, htz1∆ and isw2∆ strains. Each point represents the relative protection of each PCR 2

amplicon, quantified by real-time PCR and normalized to a centromeric control. (B) The 3

association of Isw2p (as a C-terminal myc fusion) with POT1, POX1, FOX2 and CTA1 4

promoters was determined by chromatin immunoprecipitation (ChIP) using anti-Myc 5

antibodies, followed by gene-specific PCR. The relative enrichment ratio is plotted at 4 6

time points (0, 1, 4, 6 h) of induction in oleate. ACT1 was used as an internal control to 7

normalize signals of promoter enrichment. Error bars show the standard deviation from 8

three independent experimental values with two technical replicates of each. 9

mcb.asm.org

(30)

Table 1: Strains and plasmids used in this study 1

Strain Genotype Reference

BY4741 MATa, his3∆1, leu2∆0, met15∆0, ura3∆0 Open

BY4742 MATα, his3∆1, leu2∆0, met15∆0, ura3∆0 Open

YWY004 MATα, his3∆1, leu2∆0, met15∆0, ura3∆0, htz1::kanMX4 Open

YWY042 MATα, his3∆1, leu2∆0, met15∆0, ura3∆0, POT1-GFP::natMX This study

YWY007 MATα, his3∆1, leu2∆0, met15∆0, ura3∆0, POT1-GFP::natMX, pRS416 This study YWY005 MATα, his3∆1, leu2∆0, met15∆0, ura3∆0, htz1::kanMX4, POT1-GFP::natMX This study YWY009 MATα, his3∆1, leu2∆0, met15∆0, ura3∆0, htz1::kanMX4, POT1-GFP::natMX, pRS416 This study YWY010 MATα, his3∆1, leu2∆0, met15∆0, ura3∆0, htz1::kanMX4, POT1-GFP::natMX, pCM305 This study YWY011 MATα, his3∆1, leu2∆0, met15∆0, ura3∆0, htz1::kanMX4, POT1-GFP::natMX, pCM330 This study YWY012 MATα, his3∆1, leu2∆0, met15∆0, ura3∆0, htz1::kanMX4, POT1-GFP::natMX, pCM314 This study

YWY013 MATα, leu2∆0, ura3∆0, HA-HTZ1 Zhang haiying, 2005

YWY0165 MATα, leu2∆0, ura3∆0, HA-HTZ, chz1::kanMX4 This study

YWY0166 MATα, leu2∆0, ura3∆0, HA-HTZ, gcn5::kanMX4 This study

YWY0177 MATα, leu2∆0, ura3∆0, HA-HTZ, swr1::kanMX4 This study

YWY206 MATα, his3∆1, leu2∆0, met15∆0, ura3∆0, SPT15-13MYC::kanMX4 This study

YWY207 MATα, his3∆1, leu2∆0, met15∆0, ura3∆0, SPT15-13MYC::kanMX4, htz1::hphMX This study

Plasmid Description Reference

pRS416 CEN6-ARS4 URA3 Open

pCM314 CEN6-ARS4 URA3 HA-htz1K14A Millar CB et al, 2006

pCM330 CEN6-ARS4 URA3 HA-htz1K14R Millar CB et al, 2006

pCM305 CEN6-ARS4 URA3 HA-HTZ1 Millar CB et al, 2006

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

mcb.asm.org

(31)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

mcb.asm.org

(32)

1 2 3 4 5 6 7 8 9 10 11 12 13

mcb.asm.org

(33)

1 2 3 4 5 6 7 8

mcb.asm.org

(34)

1 2 3 4 5 6 7

mcb.asm.org

(35)

1 2 3 4 5 6

mcb.asm.org

(36)

1

mcb.asm.org

(37)

1 2 3 4 5 6 7 8 9 10 11 12 13 14

mcb.asm.org

(38)

1 2 3 4 5 6 7

mcb.asm.org