ORIGINAL PAPER

Chromatin analysis of an Arabidopsis Phytochrome A allelereveals the correlation of transcriptional repressionwith recalcitrance to histone acetylation

Gulab Rangani • Jamie L. Underwood •

Vibha Srivastava

Received: 7 May 2014 / Accepted: 22 May 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Phytochrome A (phyA) is a major photoreceptor

of red light that regulates seedling de-etiolation. The wild-

type PHYA is abundantly expressed in dark and repressed by

light through chromatin modification involving histone

hypoacetylation and enrichment of the repressive histone

mark, H3 lysine 27 trimethylation (H3K27me3). Earlier, an

Arabidopsis phyA allele, phyA-17, was reported that contains

hypermethylation in the gene body, and is repressed con-

stitutively (transcriptional repression in the dark). In this

study, chromatin analysis of phyA-17 was done to under-

stand the basis of its transcriptional repression. Specifically,

this study analyzed four different histone modifications on

phyA-17 and the wild-type PHYA (Columbia-0) in light and

dark conditions. This analysis revealed hypoacetylation of

phyA-17 chromatin in both conditions correlating with its

constitutive repression. However, relative enrichment of

H3K27me3 on phyA-17 chromatin was not detected in either

condition. Histone hypoacetylation suggested a role of his-

tone deacetylases in phyA-17 repression. Chemical inhibi-

tors of histone deacetylases, Trichostatin A and Sodium

Butyrate, induced partial de-etiolation of phyA-17 seedlings

without activating the resident phyA gene. Gene expression

analysis revealed activation of the phyA-signaling pathway

by Trichostatin A, suggesting a role of histone deacetylases

downstream in the seedling de-etiolation pathway. However,

since phyA-17 repression is not dependent on histone

deacetylases, recalcitrance to histone acetylation by histone

acetyl transferases, possibly due to hypermethylation, is

likely the basis of its hypoacetylated chromatin.

Keywords Chromatin modification � Histone

acetylation � Transcriptional repression � Trichostatin A �Sodium Butyrate � Phytochrome A

Introduction

Chromatin modification plays an important role in regu-

lating gene expression. A number of environmentally-

controlled or tissue-specific genes in plants are regulated

by acetylation and methylation of lysine residues in H3

tails among other types of histone modifications (Liu et al.

2010; Charron et al. 2009; Benhamed et al. 2006). It is not

clear how histone modification enzymes bring about

chromatin modifications precisely on their target loci. DNA

hypermethylation (cytosine methylation) correlates with

chromatin modification on a number of genomic sites in

Arabidopsis and other plant species (Mathieu et al. 2005;

Gendrel et al. 2002; Zhou 2009). DNA methylation in

eukaryotes is predominantly found in CG sites; however, in

plant genomes methylation of CHG and CHH sites

(H = C, T or A) is also found in transposable elements

(TE), repetitive sequences, and complex transgene loci

(Law and Jacobsen 2010; Martienssen et al. 2008). This

pattern of DNA hypermethylation correlates with the

enrichment of the repressive histone mark, H3 lysine 9

dimethylation (H3K9me2).

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10725-014-9942-8) contains supplementarymaterial, which is available to authorized users.

G. Rangani � J. L. Underwood � V. Srivastava (&)

Department of Crop, Soil and Environmental Sciences,

University of Arkansas, Fayetteville, AR 72701, USA

e-mail: vibhas@uark.edu

V. Srivastava

Department of Horticulture, University of Arkansas,

Fayetteville, AR 72701, USA

123

Plant Growth Regul

DOI 10.1007/s10725-014-9942-8

Euchromatic loci (e.g. protein coding genes) in Arabi-

dopsis genome display a different pattern of DNA meth-

ylation, involving CG sites within the coding region or

gene-body, and require MET1 to maintain it (Cokus et al.

2008; Lister et al. 2008). Recent studies have found the role

of gene-body methylation in regulating alternative pro-

moter activity and splicing (Lorincz et al. 2004; Maunakea

et al. 2010; Shukla et al. 2011; Zilberman et al. 2007).

However, whether it also regulates gene expression is not

clear. The body-methylated genes are generally expressed

at moderate levels, suggesting a relation between tran-

scription and DNA methylation (Zilberman et al. 2007;

Aceituno et al. 2008). Although upregulation of these

genes upon hypomethylation is not generally observed

(Lister et al. 2008; Zhang et al. 2006), their tissue-specific

or conditional upregulation cannot be ruled out. Recent

studies have linked transcriptional repression with DNA

(CG) hypermethylation in gene-body (Chawla et al. 2007;

Hohn et al. 1996; Shu et al. 2012; Iwasaki et al. 2013),

especially if it is found in the first exon (Brenet et al. 2011).

However, the mechanism of their transcriptional repression

is, so far, unknown. The importance of gene-body meth-

ylation has also been indicated by its conservation among

orthologous genes and its presence in functionally impor-

tant genes (Takuno and Gaut 2012, 2013). Previously, we

identified an Arabidopsis Phytochrome A (phyA) allele,

phyA-17, which carries transcriptional repression and gene-

body hypermethylation (Chawla et al. 2007; Rangani et al.

2012). phyA encodes a photoreceptor for seedling de-eti-

olation among other photomorphogenesis responses, it is

expressed in dark and reversibly repressed by light. phyA-

17 is tightly associated with the phyA mutant phenotype

consisting of long hypocotyls and unexpanded cotyledons

when grown in continuous Far-Red light (FRc) (740 nm).

As gene transcription is controlled by the structure of the

associated chromatin, we hypothesized that chromatin

modification via alterations in specific histone marks

underlies phyA-17 repression. The specific objectives of the

present work are to study the histone modifications asso-

ciated with phyA-17, and the role of histone modification

enzymes in bringing about the changes. We studied the

abundance of four different histone marks, two ‘expres-

sion’ marks and two ‘repression’ marks, on phyA-17

chromatin and compared it with that of the wild-type phyA

in Columbia-0 background (phyA-Col). We found deple-

tion of expression marks, acetylation of H3 lysine 9

(H3K9ac) and trimethylation of H3 lysine 4 (H3K4me3),

without enrichment of the repressive histones, dimethyla-

tion of H3 lysine 9 (H3K9me2) or trimethylation of H3

lysine 27 (H3K27me3). The decrease in H3K4me3 and

H3K9ac suggests hypoacetylation of the associated his-

tones, implicating histone deacetylases (HDA) and/or his-

tone acetyltransferases (HAT) in phyA-17 repression.

However, the role of HDA was ruled out by analyzing the

effect of HDA inhibitors, Trichostatin A (TSA) and

Sodium Butyrate, on phyA expression, invoking misregu-

lation of phyA-specific HAT. phyA is regulated by light–

dark cycle, and is activated in the dark through chromatin

modifications involving histone acetylations and H3K4me3

enrichment (Jang et al. 2011), suggesting the role of dark-

specific HAT and histone methylases. Therefore, phyA-17

repression mechanism possibly involves inhibition of

phyA-specific HAT in properly acetylating phyA chromatin

in the dark. This conclusion is also supported by the

understanding that phyA activation in dark is mediated by

active histone acetylation (Jang et al. 2011).

In addition, we found that TSA or Sodium Butyrate

treatment leads to partial seedling de-etiolation in FRc as

well as dark, indicating the activation of phyA signaling

pathway. This phenotypic reversal was observed for both

wild-type (in dark) and phyA mutant genotypes (in dark and

FRc). Gene expression analysis found activation of master

regulators of seedling de-etiolation, ELONGATED HYPO-

COTYL 5 (HY5) and LONG HYPOCOTYL IN FAR-RED

(HFR1) by TSA, suggesting the role of chromatin modifi-

cation in the regulation of these genes presumably by HDAs.

Materials and methods

Plant materials and growth conditions

Arabidopsis seedlings of Columbia-0 (Col) and phyA

mutants, L211 (phyA-211) and L17 (phyA-17) were used in

this study. For ChIP experiments, Col and L17 seeds were

plated on MS media, stratified for 2 days, and exposed to

light for inducing seed germination. The seedlings were

grown for 14 days either in continuous light or in a 16-h

light regime. Seedlings were then transferred to the dark

(t = 0) for 8 h and then to the light for 16 h to collect light

(L) sample at the end of the light treatment or kept con-

tinually in the dark since t = 0 for a total period of 24 h to

collect the dark (D) sample.

ChIP-qPCR assay

ChIP was performed using 3 g of seedlings. Chromatin was

prepared using the protocol described by W. Aufsatz at http://

www.epigenesys.eu/images/stories/protocols/pdf/20111025

150640_p12.pdf. Immunoprecipitation and DNA recovery

were carried out using the protocol modified by the Pikaard

group (http://sites.bio.indiana.edu/*pikaardlab/). Seed-

lings grown under L and D conditions were harvested and

cross-linked with 1 % formaldehyde for 15 min in a vacuum,

and nuclei were isolated using sucrose gradients. Chromatin

was sheared using a Branson Sonicator 250 using 3 mm

Plant Growth Regul

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tapered micro-tip to generate fragments of * 500 bp, and

immunoprecipitated using anti-H3K4me3 (Active Motive,

39159), anti-H3K9ac (Millipore, 07-352), anti-H3K9me2

(Millipore, 07-441), and anti-H3K27me3 (Millipore,

07-449) antibodies. Immunocomplexes were captured by

Protein A agarose beads (Millipore, 16-157), washed, and

reverse cross-linked by boiling in the presence of Chelex

resin (Bio-Rad; 142-1253). Input control (IC) DNA was

precipitated with ethanol and reverse cross-linked by boiling

in the presence of Chelex resin. Two to three biological

replicates were used in the analysis. ChIP (IP) DNA was

recovered into approximately 200 lL of TE buffer, and used

for qPCR using TaKaRa SYBR Premix ExTaq on CFX96

real-time machine (BioRad Inc.) along with IC and mock

(no-antibody control). The analysis was done using the

percentage of input (% Input) approach. The % Input values

were calculated as 100 9 2^[-(Ct of IP -Ct of IC)]. We

used Actin7, 180 bp pericentromeric repeats, FUSCA, and

LEC2 as positive or negative controls. phyA ChIP-primers

are given in Table S1.

Trichostatin A (TSA) and Sodium Butyrate treatments

Sterile seeds of Col, L17 and L211 were germinated on MS

media until the seed coat cracked. At this point, seeds were

transferred to MS media containing 5 or 10 lM TSA or 10 mM

Sodium Butyrate (Sigma Co.), and transferred to continuous FR

(FRc) light (2.5 W m-2) for 4 days. A parallel dark experiment

was done in which germinating seeds were plated on MS media

containing 1 % sucrose and 10 lM TSA or 10 mM Sodium

Butyrate, and grown in the dark for 5 days. Seedlings were then

observed and photographed under white light. Some dark-

grown seedlings were saved for RT-qPCR analysis.

Reverse transcriptase-qPCR for gene expression

analysis

For expression analysis, total RNA, extracted using RNAeasy

Plant Mini Kit (Qiagen Inc.), was converted to cDNA using

SuperScript III First stand synthesis system (Invitrogen Inc.).

Real time qPCR on cDNA pools were done using TaKaRa

SYBR Premix ExTaq on CFX96 real-time machine (Bio-Rad

Inc.). Three samples (biological replicates) were used for each

gene expression analysis against 2–3 reference genes. The

primers used for this RT-qPCR analysis are given in Table S2.

Results

Light–dark regulation of phyA-17

phyA is expressed in the dark and quickly repressed upon

exposure to the light (Canton and Quail 1999). Jang et al.

(2011) showed that this light–dark regulation is based on

reversible chromatin changes in phyA locus without any

change in DNA methylation. We analyzed light–dark reg-

ulation of the hypermethylated allele, phyA-17, in 2-week

old seedlings grown either in the dark (D) or the light

(L) and compared with that of the wild-type Col-0 allele

(phyA-Col). PHYA expression was determined by RT-qPCR

on total RNA from each sample. As expected, phyA-17 was

repressed in both L and D samples as compared to phyA-Col

(Fig. 1). Comparison of L and D samples of phyA-Col

showed 3 times repression by L, whereas that of phyA-17

showed only 2 9 repression (Fig. 1), indicating a somewhat

suppressed light-regulation of phyA-17.

Chromatin analysis

Jang et al. (2011) showed that in the dark phyA locus is

enriched with histone expression marks (e.g. H3K4me3),

whereas in the light, enrichment of repressive marks (e.g.

H3K27me3) occurs. Accordingly, phyA chromatin is

hyper-acetylated in the dark and actively hypoacetylated by

Histone Deacetylase 19 (HDA19) in response to the light.

Similarly, a number of developmentally regulated genes

undergo chromatin modifications involving changes in

H3K27me3 in conjunction with histone hypoacetylation

(Charron et al. 2009; Zhou 2009; Zhou et al. 2010). Histone

acetylation is reversible and determines the ‘permissive’

and ‘repressive’ states of the chromatin as enrichment of

acetylated histones is coupled with transcriptional activity

of the locus (Berger 2007; Servet et al. 2010; Eberharter

and Becker 2002; Ha et al. 2011). We analyzed chromatin

modification in phyA-17 and phyA-Col upon dark to light

transition in an experimental design similar to that of Jang

et al. (2011). We used 14-days old seedlings of L17 (phyA-

17) and Col (phyA-Col) grown in a 16 h light regime, and

ph

yA e

xpre

ssio

n

0

0.5

1

1.5

2

2.5

3

3.5

4

1 2 3 4LL D D

Col L17

Fig. 1 phyA expression analysis by RT-qPCR on total RNA isolated

from either light (L) or dark (D) grown Col and L17 seedlings. phyA

expression is analyzed against three reference genes (Table S2). Error

bars indicate standard deviation (n = 3)

Plant Growth Regul

123

collected samples at the end of light treatment (L) or after

24 h of the dark treatment (D). Specifically, we compared

H3K4me3 and H3K9ac enrichment in D/L samples of each

line. This analysis focused on 6 specific phyA regions

(Fig. 2a). Region 1 is upstream of promoter elements,

region 2 covers the promoter, and region 3 covers a part of

50 UTR that carries the highest RNA Pol II occupancy in

the dark (Jang et al. 2011), while the remaining regions fall

within the coding region as shown in Fig. 2a. Region 2 and

3 undergo maximum chromatin modifications in D to L

transition (Jang et al. 2011). Enrichment of H3K4me3 was

detected in all phyA regions, most prominently in region 2,

3 and 4 of D samples of Col; however, no enrichment or

only mild enrichment was observed in L17 except in region

4 (Fig. 2b). More importantly, D samples of L17 showed

much lower enrichment of H3K4me3 compared to Col in

all 6 phyA regions (Fig. 2b). Next, enrichment of H3

acetylated at lysine 9 (H3K9ac) was studied in regions 2, 3,

4, and 5. As expected, H3K9ac profile matched that of

H3K4me3 in the critical regions, 2, 3 and 4. In D samples

of Col, H3K9ac enrichment was observed in all phyA

regions; whereas D samples of L17 remained hypoacety-

lated (Fig. 2c). PHYA regions 2, 3 and 4 undergo maxi-

mum H3K9ac enrichment in L to D transition (Jang et al.

2011). Relative enrichment of H3K9ac in D sample of L17

in these regions was lower than that of Col, corresponding

with the reduced transcription rate of phyA-17. Thus,

depletion of H3K4me3 combined with H3 hypoacetylation

in the dark, an otherwise permissive environment, charac-

terizes phyA-17 repression.

Next, we explored whether depletion of the H3K4me3 and

H3K9ac is accompanied by enrichment of the repressive

marks, H3K9me2 and H3K27me3, as found in wild-type

phyA during L/D transition (Jang et al. 2011). Although,

H3K9me2 is mostly found on heterochromatic loci that are

actively methylated, it is a major histone mark that corre-

sponds with DNA hypermethylation (Bernatavichute et al.

2008; Jackson et al. 2002; Liu et al. 2010). A moderate

increase in H3K9me2 was found in region 2 and 3 in L

samples of phyA-Col (Fig. 3a). However, no significant

difference was observed between L and D samples of the

two alleles, phyA-17 and phyA-Col (Fig. 3a). Therefore,

H3K9me2 does not play a specific role in the repression

mechanism of phyA-17. Next, H3K27me3 was found to be

*2-fold enriched in L samples as compared to the D sample,

indicating the role of L in recruiting histone methylases

responsible for H3K27 trimethylation. However, the two

alleles, phyA-Col and phyA-17, generally showed similar

trends in their respective L and D samples (Fig. 3b). Further,

since D samples of L17 were not enriched with H3K27me3

when compared with D samples of Col, H3K27me3 does not

serve a specific role in phyA-17 repression.

In summary, chromatin analysis indicated that phyA-17

repression correlated with H3K4me3 depletion and H3

0123456789

2 3 4 5 6 1

Fo

ld c

han

ge

Region 2 Region 3 Region 4 Region 5

Col L17 Col L17 Col L17 Col L17

H3K9ac

(a)

(c)

ATG

5’ UTR 3’ UTRPromoter Ex 1 Ex 2 Ex 3 E

x 4

+1

0

1

2

3

4

5

6

7

8

Fo

ld c

han

ge

H3K4me3 (b)

Col L17 Col L17 Col L17 Col Col Col L17

Region 2 Region 3 Region 5 Region 6 Region 1 Region 4 L17 L17

Fig. 2 ChIP-qPCR analysis of

chromatin ‘expression’ marks,

H3K4me3 and H3K9ac.

a Position of phyA regions

(thick lines 1–6) analyzed by

ChIP-qPCR are shown below

phyA gene structure. Promoter,

transcription start site (?1), 50

and 30 untranslated region

(UTR), start codon (ATG), and

exons (ex 1–4) are indicated in

the gene structure. Thin line

between exons represents

introns. b, c Relative levels

(fold-change) of H3K4me3

(b) and H3K9ac (c) in specific

phyA regions during L/D

transition. All samples are

compared with Col L sample in

each region. Standard deviation

(n = 3) is shown as error bars

Plant Growth Regul

123

hypocaetylation without the enrichment of the repressive

H3 marks, H3K9me2 and H3K27me3. Therefore, phyA-17

repression is associated with the depletion of expression

marks and histone hypoacetylation without concurrent

enrichment of H3K27me3, the repressive histone mark that

integrates light signal in transcriptional regulation of phyA

locus.

Role of histone deacetylases

As discussed above, chromatin analysis revealed H3K9

hypoacetylation on phyA-17 correlating with its transcrip-

tional repression. Histone acetyltransferase (HAT) and

histone deacetylases (HDA) target lysine (K) residues in

H3 and H4 histones to alter the chromatin state of the

associated loci (Berger 2007; Zhang et al. 2007; Earley

et al. 2007). Repression of phyA locus in response to light

involves H3K9 hypoacetylation mediated by HDA19 (Jang

et al. 2011). Therefore, we tested whether histone deacet-

ylases (HDA) somehow target phyA-17 locus in the dark, to

lower its transcription rate. First, we tested the effect of

HDA inhibitors, Sodium Butyrate and Trichostatin A

(TSA), on seedling phenotype. Exposure to TSA (5 lM) or

Sodium Butyrate (10 mM) resulted in phenotypic reversion

in L17 (phyA-17) characterized by partially de-etiolated

seedlings in FRc (shorter hypocotyl, and partially open

cotyledons; see Fig. 4a). As expected, Col seedlings are

fully de-etiolated in FRc upon chemical treatments

(Fig. 4a). Similar effects on seedling phenotype were

observed by a higher dose of TSA (10 lM), which also

caused severe root inhibition in all seedlings (Fig. 4b).

However, these chemical inhibitors caused de-etiolation in

dark-grown seedlings (Fig. 4a, b). Accordingly, L211

(phyA-211), a null mutant, also showed phenotypic rever-

sion in FRc upon TSA treatment (Fig. 4b).

HDA19 represses light-responsive genes (e.g. photomor-

phogenesis genes) in the dark by histone deacetylation (Be-

nhamed et al. 2006); therefore, TSA treatment could activate

these genes causing seedling de-etiolation in the absence of

light signal. Further, since de-etiolation in FRc light is con-

trolled by phyA, phyA-signaling pathway is possibly acti-

vated by TSA. RT-qPCR analysis on TSA (10 lM)-treated,

dark-grown seedlings of L17 and Col-0 revealed no change in

phyA expression in either genotype (Fig. 4c), indicating no

major role of HDA genes in regulating phyA-17. These

observations were further confirmed by transferring phyA-17

in the genetic backgrounds of three hda mutations: hda9

(SALK_007123), hda15 (SALK_004027C), and hda19

(SALK_139445). We examined at least four different F2

families for phenotypic segregation among FRc-grown

seedlings. These families either segregated 3:1 ratio for short

(de-etiolated) and tall (etiolated) seedlings or showed a higher

than expected tall seedlings in the population (Data not

shown). F3 progeny of multiple double-mutant F2 plants

(derived from hda19 crosses) were also examined for phe-

notypic reversion, but no revertants were scored in F3 popu-

lations. These genetic data indicate that HDA are not involved

in phyA-17 repression, which is consistent with the observa-

tion that TSA treatment does not activate phyA-17 expression

(Fig. 4c). However, since single hda mutations do not phe-

nocopy the TSA effect, more than one HDA gene is likely

involved in regulating seedling de-etiolation pathway.

A recent study showed that dark repression of phyB-

target genes is mediated by HDA15 through its direct

association with Phytochrome Interacting Factor 3 (PIF3)

(Liu et al. 2013). However, it is not clear whether HDAs

also target the genes encoding master regulators of seedling

de-etiolation (phyA-signaling pathway), HY5, HFR1, and

LONG AFTER FAR-RED LIGHT 1 (LAF1). Previously,

Hudson et al. (2011) analyzed the effect of TSA on the

genome using microarray approach (GEO accession no.

GSE25067); however as light-grown seedlings were used

in their experiment, TSA effect on phyA-signaling cannot

be analyzed in their dataset. We analyzed the expression of

HY5, LAF1 and HFR1 genes in TSA (10 lM)-treated dark-

grown 5-day old seedlings using RT-qPCR, and found that

HY5 and HFR1 are *29 activated while LAF1 is repres-

sed (Fig. 4d). This trend of activation and repression was

consistent in all three genotypes, Col-0, L17, and L211,

(a)

(b)

00.20.40.60.8

11.21.41.61.8

Fo

ld c

han

ge

H3K9me2

Col L17 L17 L17Col Col

Region 2 Region 3 Region 5

0

1

2

3

4

5

6

7

8H3K27me3

Col L17 L17Col L17Col

Region 2 Region 3 Region 5

Fo

ld c

han

ge

Fig. 3 ChIP-qPCR analysis of chromatin ‘repressive’ marks,

H3K9me2 and H3K27me3. Relative levels (fold-change) of

H3K9me2 (a) or H3K27me3 (b) in the specific phyA regions during

L/D transition. All samples are compared with Col L sample in each

region. Standard deviation (n = 2 or 3) is shown as error bars

Plant Growth Regul

123

indicating the effect of TSA on these genes possibly

through inhibiting HDA activity.

Discussion

DNA hypermethylation spanning CG and non-CG sites often

leads to transcriptional repression. Such methylation pattern

is actively maintained by RNA directed DNA Methylation

(RdDM) pathway, and facilitates binding of repressive his-

tones such as H3K9me2 to switch the chromatin to a

‘‘repressed’’ state (Johnson et al. 2007; Matzke and Birchler

2005). This mechanism is critical for repressing transposons

and pseudogenes (Castel and Martienssen 2013; Slotkin and

Martienssen 2007), and also targets complex transgene loci

that are usually located in the euchromatic region. However,

methylation is also found in the expressed loci that are not

generally targeted by RdDM (Gehring and Henikoff 2008).

As a result, DNA methylation in protein coding genes is

predominantly confined to CG sites in the gene bodies, and

maintained by MET1 (Zilberman et al. 2007; Zhang et al.

2006). Function of the gene-body methylation is not clearly

understood; however, it is found in functionally-important or

tissue-specific genes, and genomes have evolved mecha-

nisms to maintain and conserve it (Takuno and Gaut 2012,

2013; Feng et al. 2010; Dalakouras et al. 2012), and even

restore it upon demethylation (Zubko et al. 2012). Hyper-

methylation of phyA-17 gene-body is associated with its

‘constitutive’ repression obscuring its light–dark regulation.

phyA-17 confers a strong mutant phenotype, and carries

hypermethylation in several CG sites within exon 1 (Chawla

et al. 2007; Rangani et al. 2012).

In this study, we analyzed the chromatin make up of

phyA-17, and found that it is similar to that of wild-type

phyA, except that the associated histones are hypoacety-

lated. Although, this study analyzed only a single type of

histone acetylation (H3K9ac), depletion of H3K4me3 on

phyA-17 indicates hypoacetylation of other lysine residues

within H3 or H4 histones. H3K4me3 strongly correlates

with acetylation of lysine (K) residues in both H3 and H4

histones (Ha et al. 2011; de la Paz Sanchez and Gutierrez

2009), and maps perfectly with active or open chromatin

FRc

Dark

+ TSA (10 µM)R

elat

ive

exp

ress

ion

L17

0

0.2

0.4

0.6

0.8

1

1.2

1.4

+ +

Col

PHYA

Co

l

L17

L211

Col L17 L211

HY5 HFR1 LAF1

(c)

Rel

ativ

e ex

pre

ssio

n0

0.5

1

1.5

2

2.5

3

3.5C

ol

L17

L211

Co

l

L17

L211

Col ColL17 L17

FRc Dark(a)

(b) (d)

Control

TSA (5 µM)

Sodiumbutyrate

Fig. 4 Effect of chemical inhibitors of histone deacetylases on

seedling phenotype. a Col and L17 seedlings grown in FRc light or

dark on germination media containing sodium butyrate (10 mM) or

Trichostatin A (TSA) (5 lM). Phenotypes of the respective lines in

control condition (without TSA or Sodium Butyrate) are shown

below. Note seedling de-etiolation (short seedlings and partially

expanded cotyledons) on the treatment plates regardless of light or

dark condition or genotype. b Effect of a higher dose of TSA (10 lM)

on Col, L17 and L211 (phyA null mutant) in FRc or dark. Note short

and thick seedlings with partially open cotyledons, and strong

inhibition of root growth. c Relative phyA expression in seedlings

grown in dark on germination media without TSA (-) or with 10 lM

TSA (?). Error bars indicate standard deviation (n = 3). d Expres-

sion of positive regulators of phyA-signaling, HY5, HFR1 and LAF1

in TSA treated seedlings of Col, L17 and L211. Each sample was

analyzed against untreated controls using 2–3 reference genes (see

Table S2). White bars represent untreated controls (-), and gray bars

represent TSA treated samples (?). Error bars indicate standard

deviation (n = 3)

Plant Growth Regul

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(Santos-Rosa et al. 2002) as lysine acetylation in histone

tails loosens its association with DNA. Therefore, it is not

surprising that histones associated with transcriptionally

repressed phyA-17 are hypoacetylated. However, it is

important to note that hypoacetylation occurs without

enrichment of the repressive marks, H3K9me2 and

H3K27me3, and evidently also without the recruitment of

histone deacetylases (HDA). These simple findings provide

the first glimpse of a potentially novel mechanism that

regulates of phyA-17. phyA expression is down-regulated

by light, which is a consequence of chromatin change

involving histone hypoacetylation and H3K27me3 enrich-

ment (Jang et al. 2011). phyA-17, on the other hand, remains

repressed regardless of the light or dark condition, and it

cannot be activated by hda mutations or by HDA inhibitors,

Sodium Butyrate and Trichostatin A (Riggs et al. 1977;

Yoshida et al. 1990). Therefore, constitutive repression of

phyA-17 is not maintained by histone deacetylases, but

possibly maintained through recalcitrance of phyA-17 to

histone acetyltransferases, responsible for acetylating phyA

histones in darkness. Trimethylation of H3K4 (H3K4me3),

which is mediated by histone methyltransferases, plays an

instructive role for histone acetylation. Therefore, the

working hypothesis for phyA-17 repression is that insuffi-

cient interaction of the dark-specific histone methyltrans-

ferases with phyA-17 chromatin leads to ‘constitutive’

histone hypoacetylation, eventually lowering its transcrip-

tion rate. In summary, this study suggests that gene-body

methylations could lead to hypoacetylation of the associated

chromatin, which in turn may cause transcriptional repres-

sion. However, alternative mechanisms involving feedback

effect of phyA (protein or transcript) depletion on phyA

expression cannot be overlooked.

A corollary of this study is the finding that positive regu-

lators of phyA-signaling and master-regulators of seedling de-

etiolation pathway are regulated by histone deacetylases, as

TSA-treatment induced seedling de-etiolation in FRc-grown

phyA-17 and phyA-211 mutants (Fig. 4a, b). phyA-signaling is

not fully understood; however, it involves sequestration of

COP1, a negative regulator of photomorphogenesis, to allow

expression of master regulators such as HY5, HFR1 and LAF1

(reviewed by Li et al. 2011). COP1, an E3 ubiquitin ligase,

promotes proteasome-mediated degradation of HY5, LAF1

and HFR1 in the absence of photoactive Pfr form of phyA

(PfrA) (such as in dark). However, light converts phyA-Pr to

Pfr, which is imported to the nucleus, where it is quickly

degraded by COP1. Thus, photomorphogenesis, in part, is

induced by pulling COP1 off the sites of protein degradations,

leading to the stabilization of HY5 and HFR1. We found

transcriptional activation of HY5 and HFR1 genes in TSA-

treated seedlings (dark-grown); further, since HFR1 stabilizes

LAF1 by interacting with it (and protecting it from COP1-

mediated degradation), TSA treatment effectively leads to

activation/stabilization of all three positive regulators of phyA

signaling. Therefore, TSA induced-photomorphogenesis or

seedling de-etiolation in dark, in the absence of COP1

sequestering by phyA-Pfr, suggests (a) overexpression of HY5

and HFR1 genes partially overcomes COP1-mediated inhibi-

tion of seedling de-etiolation, and (b) HY5 and HFR1 are

regulated at chromatin level through histone deacetylation.

Overall, these findings suggest that cellular machineries reg-

ulate important pathways such as seedling de-etiolation at both

protein function and chromatin levels. Chromatin modification

ensures moderate expression of these genes, which is probably

important for effective regulation of encoded proteins by

COP1 that in turn prevents seedling de-etiolation in dark.

Acknowledgments This study was funded by the Arkansas Divi-

sion of Agriculture, and Arkansas Bioscience Institute. Authors are

grateful to Arabidopsis Biological Resource Center at The Ohio State

University for providing phyA-211, hda9 (SALK_007123), hda15

(SALK_004027C), and hda19 (SALK_139445) seeds.

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