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ARNT controls the expression of epidermaldifferentiation genes through HDAC- and EGFR-dependent pathways

E. Douglas Robertson, Lynda Weir, Malgorzata Romanowska, Irene M. Leigh and Andrey A. Panteleyev*Centre for Oncology and Molecular Medicine, Division of Medical Sciences, College of Medicine, Dentistry and Nursing, Ninewells Hospital,University of Dundee, UK

*Author for correspondence (a.a.panteleyev@dundee.ac.uk)

Accepted 22 March 2012Journal of Cell Science 125, 3320–3332� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.095125

SummaryPreviously we showed that spatial and developmental modulation of ARNT (HIF1b) expression in mouse epidermis is essential formaintenance of keratinocyte differentiation, proper formation of the barrier and normal desquamation. Here, using lentiviral suppression

or induction of ARNT in TERT-immortalized (N-TERT) and HaCaT cells we assessed the nature and mechanisms of ARNTinvolvement in control of differentiation in human epidermal keratinocytes. ARNT depletion did not affect the levels of basal keratinsK5 and K14, but significantly induced expression of several key differentiation markers (an effect abolished by EGF supplementation).

Furthermore, ARNT deficiency resulted in the downregulation of amphiregulin (AREG) – the most highly expressed EGFR ligand inhuman keratinocytes – whereas upregulation of ARNT showed the opposite. In ARNT-deficient monolayer cultures and 3D epidermalequivalents, the downregulation of AREG was concurrent with a decline of EGFR and ERK1/2 phosphorylation. TSA, a potent

suppressor of HDAC activity, abolished the effects of ARNT deficiency, implying a role for HDACs in ARNT-dependent modulation ofthe AREG–EGFR pathway and downstream epidermal genes. Total HDAC activity was significantly increased in ARNT-depleted cellsand decreased with ARNT overexpression. ARNT-dependent shifts in HDAC activity were specifically attributed to significant changesin the levels of HDAC1, HDAC2 and HDAC3 proteins (but not mRNA) in both monolayer and 3D cultures. Collectively, our results

suggest that ARNT controls AREG expression and the downstream EGFR–ERK pathway in keratinocytes, at least in part, bymodulating HDAC activity. This novel regulatory pathway targeting advanced stages of epidermal differentiation might have importantimplications for skin pathology such as psoriasis, atopic dermatitis and cancer.

Key words: Amphiregulin, Differentiation, EGFR, Epidermis, Epigenetics, ERK1/2, Filaggrin, Keratinocyte

IntroductionThe aryl hydrocarbon receptor nuclear translocator (ARNT or

HIF1b) is a basic helix-loop-helix protein that belongs to a

Period–ARNT–single-minded (PAS) protein family. PAS

proteins act as heterodimeric transcription factors activated by

environmental stress with ARNT being a master dimerization

partner in this protein family. Most characterised are the

heterodimeric complexes of ARNT with aryl hydrocarbon

receptor (AhR) and hypoxia-inducible factors (HIF-aproteins), formed upon AhR or HIF-a activation by

halogenated aromatic hydrocarbons or hypoxia, respectively

(Wenger and Gassmann, 1997; Beischlag et al., 2008). ARNT is

also essential for normal development and its knockout in mice

leads to early embryonic lethality due to abnormal angiogenesis,

cardial dysfunction, defective haematopoiesis and cranial

malformations (Kozak et al., 1997; Maltepe et al., 1997).

Possible roles for ARNT acting as a homodimer independent of

HIF1a and AhR have also been suggested (Sogawa et al., 1995;

Swanson and Yang, 1999).

Given the wide-ranging role of ARNT in adaptation to

environmental stress (Gu et al., 2000), its activity is likely to

be implicated in control of adaptive responses in normal skin and

in pathogenesis of certain environmentally mediated skin

disorders. Recently, we and others showed an essential role for

epidermal ARNT in mouse skin development, keratinocyte

differentiation, barrier function and dermal vascularisation

(Takagi et al., 2003; Geng et al., 2006; Wondimu et al., 2012).

In contrast to the existing view that ARNT is ubiquitously present

in mammalian tissues, we found that its expression in mouse and

human skin is subject to significant developmental and spatial

changes. In the epidermis, ARNT expression gradually declines

through the spinous layer and is totally excluded from the

granular layer suggesting a negative regulatory link between

ARNT activity and advanced steps of keratinocyte terminal

differentiation (Geng et al., 2006). We also showed that

alterations in the structure and function of the epidermis in

K14-ARNT KO mice are associated with elevated expression of a

number of differentiation markers mapped to the epidermal

differentiation complex on mouse chromosome 3. Abnormal

epidermal differentiation in these mice was also accompanied by

deregulation of certain ligands for the epidermal growth factor

receptor (EGFR) (Geng et al., 2006).

EGFR belongs to a family of ErbB tyrosine kinase receptors.

Upon activation by one of its ligands such as amphiregulin

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(AREG), beta-cellulin (BTC), epidermal growth factor (EGF),epiregulin (EREG), epithelial mitogen (EPGN), heparin-binding

EGF-like growth factor (HBEGF), and transforming growthfactor alpha (TGFa) EGFR forms homo- or a heterodimer (withanother ErbB family member) leading to its autophosphorylation(Yarden and Schlessinger, 1987). This triggers many downstream

signalling cascades including the protein kinase C and Ras-activated ERK1/2 MAP kinase pathways (Roberts and Der, 2007;Getsios et al., 2009), with the later considered as a hallmark of

EGFR activity (Schlessinger, 2000). EGFR-mediated signaltransduction results in effects on cell proliferation, migrationand differentiation in various tissues and organs including the

skin (Yarden and Sliwkowski, 2001; Pastore et al., 2008). In theepidermis, dividing keratinocytes of the basal layer are typifiedby elevated activity of EGFR (Wang et al., 2006), whichmaintains their sustained proliferation (Hansen et al., 2000; Jost

et al., 2000). In turn, the inhibition of EGFR blocks proliferationand induces terminal differentiation of epidermal keratinocytes(Peus et al., 1997; Getsios et al., 2009) associated with induction

of differentiation markers such as K1 and K10 (Poumay andPittelkow, 1995). Furthermore, EGFR signalling is essential fornormal development of the epidermis and its appendages

(Luetteke et al., 1994; Miettinen et al., 1995; Sibilia andWagner, 1995; Hansen et al., 1997). The activation of EGFR isalso linked to pathogenesis of certain skin diseases such as

psoriasis (Nanney et al., 1986) and squamous cell carcinoma(Uribe and Gonzalez, 2011) while disruption of the EGFRpathway inhibits development of papillomas and carcinomasfrom immortalized epidermal keratinocytes (Woodworth et al.,

2000). In wound healing, EGFR plays a positive role bypromoting keratinocyte proliferation and migration (Repertingeret al., 2004).

The transition of epidermal keratinocytes through the definedstages of differentiation is associated with tightly regulatedexpressional shifts. Recently, evidence has begun to emerge

demonstrating a key role of epigenetic mechanisms (e.g. histonemodifications) in these regulations (Markova et al., 2007; Fryeet al., 2007; Sen et al., 2008; Ezhkova et al., 2009; LeBoeuf et al.,2010; Fessing et al., 2011). It was previously reported that in mouse

placenta, differentiation of trophoblast cells is controlled by ARNTthrough modulation of HDAC activity (Maltepe et al., 2005). Inaddition, class I HDAC1 and HDAC3, as well as class II HDAC4

and HDAC6, were shown to interact with HIF1a and enhanceHIF1a/ARNT-mediated transactivation (Qian et al., 2006; Kimet al., 2007). Then, it was shown that altered HDAC1 activity plays

a central role in Cyp1a1 expression driven by AhR – anotherconventional ARNT dimerization partner (Schnekenburger et al.,2007). Although these findings do not implicitly show direct

ARNT–HDAC interaction, they suggest that HDACs are mediatingdownstream effects of ARNT activity.

Here, using N-TERT and HaCaT human epidermal keratinocytecell lines with variable level of ARNT expression and 3D

epidermal equivalent models developed using these cells, weshow that ARNT deficiency positively regulates expression ofkeratinocyte differentiation markers through suppression of the

EGFR ligand AREG and concomitant downregulation of EGFRactivity and ERK1/2 phosphorylation. ARNT overexpressionshows opposite effects. This pathway is controlled through

modulation of HDAC activity, which appears to be the first stepin this novel ARNT-dependent regulatory cascade targetingadvanced stages of keratinocyte differentiation programme.

ResultsModulation of ARNT activity in N-TERT andHaCaT keratinocytesUsing transduction with lentiviral particles created from SigmaMission shRNA plasmids we developed two N-TERT

keratinocyte cell lines with stable suppression of ARNT (N-KO1 and N-KO2; Fig. 1A–D). 3D equivalents developed usingARNT-depleted keratinocytes (N-KO) showed an evident decline

in ARNT protein compared to control models (Fig. 1F).

Transduction of N-TERT keratinocytes with the lentiviral

vector pCDH1-MCS1-EF1-copGFP (al Yacoub et al., 2007)containing the full ORF fragment of ARNT cDNA resulted in asignificant increase of ARNT expression at both mRNA and

protein levels (Fig. 1C,E). This N-OXP cell line showed agradual loss of vector-retaining cells (up to 50% by day 44 ofculture; supplementary material Fig. S2). Nevertheless, 3Depidermal equivalents developed using N-OXP keratinocytes

showed at least twice the level of ARNT protein as compared tocontrol models even at the time of 3D culture completion (day 42post-transduction; Fig. 1E,G) thus confirming that gradual loss of

ARNT overexpressing cells does not impede the use of thissystem as a valid model. Of note, ARNT transduction resultednot only in a general increase of ARNT protein level in 3D

cultures but also in its ectopic appearance in the granular layer ofARNT-OXP epidermal equivalents (Fig. 1G).

In HaCaT keratinocytes, ARNT was effectively suppressed by

ARNT shRNA (H-KO1 and H-KO2 cell lines; supplementarymaterial Fig. S1A,B), or transiently upregulated with an ARNT/GFP-tagged expression vector (H-OXP1, H-OXP2; supplementary

material Fig. S1C,D).

Depletion of ARNT promotes keratinocyte differentiationWhile qPCR revealed no changes in the mRNA level of basal

keratins K5 and K14, differentiation-associated keratins K1 andK10, transglutaminase 1 (TGM1) and involucrin showed a 2.5- to3.5-fold increase in ARNT-depleted N-TERT (N-KO) cells.Transcription of late differentiation markers filaggrin and

loricrin was upregulated in these cells by 30 and 70 foldrespectively (Fig. 2A). Western blot (WB) analysis of N-KOkeratinocytes demonstrated a corresponding increase in involucrin,

TGM1 and K1/10 at the protein level (Fig. 2B, for quantificationsee supplementary material Fig. S3F). Filaggrin and loricrinproteins were not detected in N-KO cells cultured in monolayer,

even though their mRNA levels were significantly induced byARNT depletion. However, in 3D epidermal equivalents filaggrinprotein was easily detected. WB results revealed a prominent

increase of both filaggrin and K10 proteins in ARNT-depleted 3Dcultures while in ARNT-overexpressing epidermal equivalents wefound a substantial reduction of K10 and fully processed (40 kDa)filaggrin monomers (Fig. 2C, for quantification see supplementary

material Fig. S3G). Importantly, supplementation of culturemedium with EGF abolished the stimulatory effects of ARNTknockout on transcription of differentiation markers with the only

exception being K10 (Fig. 2D).

3D epidermal equivalents developed using ARNT-depleted

cells showed significant increase in the number of cornifying celllayers along with retention of nuclei (parakeratosis) as comparedto control equivalents (Fig. 2F, H&E, arrows). Additionally,

ARNT-deficient 3D culture models were characterized by areduced number of granule-containing cells and a loss of granularlayer contiguity (Fig. 2F, H&E).

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Immunohistochemistry (IHC) on paraffin sections also

revealed noticeable changes in the expression of several key

differentiation markers in N-KO epidermal equivalents (Fig. 2F).

In the control models, filaggrin was localized to the upper

suprabasal layers corresponding to the granular layer of the

normal epidermis. In N-KO models, filaggrin was increased in

the uppermost layers (Fig. 2F, Filaggrin) and also became

apparent in some immediate suprabasal cells suggesting

premature induction of its translation or processing

(supplementary material Fig. S2E, arrows). K10 was excluded

from the basal layer of the control epidermal equivalents and

showed low positivity in the uppermost layers being most

prominent in the spinous cells overlaying the basal layer (ADD

Fig. 2F, Keratin 10, control, arrows). In N-KO 3D models, the

overall level of K10 was markedly increased (confirmed by WB,

Fig. 2C) in all suprabasal layers including the uppermost

‘cornifying’ layers which were only weakly positive in controls

(Fig. 2F, Keratin 10, N-KO2).

Finally, involucrin expression in control cultures was confined

to the cytoplasm of suprabasal keratinocytes with most prominent

positivity in the granular layer (Fig. 2F, Involucrin, arrows).

ARNT-deficient 3D models showed significant increase of

involucrin positivity, which appeared even in the nuclei of

basal and spinous keratinocytes.

ARNT-overexpressing 3D cultures revealed no apparent

differences in morphology as compared to control epidermal

equivalents. At the same time, they appeared to be slightly thinner

than control models (supplementary material Fig. S2C,D), while

ARNT-deficient 3D epidermal equivalents were usually thicker

compared to controls (Fig. 2F). This effect seems to be related to a

negative correlation between the level of ARNT and the size of

keratinocytes rather than to variations in the number of cell layers

in corresponding 3D cultures.

ARNT deficiency has a negative effect upon

keratinocyte growth

As shown by direct cell counting (Neubauer hemocytometer), in

EGF-depleted medium, N-KO cells have a markedly reduced

growth rates as compared to the control. This effect of ARNT

deficiency was abolished when the medium was supplemented

Fig. 1. Modulation of ARNT level in N-TERT keratinocyte cell line and in 3D epidermal equivalents. (A) ARNT depletion in N-TERT cells by lentiviral

transduction as demonstrated by qPCR (n52). (B) ARNT depletion at protein level in ARNT-KO N-TERT cells shown by western blot (n52).

(C) Table summarising efficiency of ARNT depletion or overexpression at mRNA and protein levels in lentivirus-transduced ARNT-KO and ARNT-

overexpressing N-TERTs. (D) ARNT immunocytofluorescence in N-KO and control N-TERT keratinocytes. (E) Expression of ARNT in ARNT-OXP N-TERT

keratinocyte (N-OXP) cultures and corresponding 3D models. ARNT level (mRNA and protein) was assessed at different time points post-transduction (PT) as

detailed in supplementary material Fig. S2B. For both qPCR and WB, n52. (F) ARNT immunohistochemistry in control and ARNT-depleted 3D epidermal

equivalents (N-TERT cells). (G) ARNT immunohistochemistry in ARNT-overexpressing and control 3D epidermal equivalents.

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with EGF (Fig. 2E). The negative effect of ARNT deficiency

upon keratinocyte growth was validated by WST-1 cell

proliferation assay, which showed approximately a 25% decline

of readings (irrespective of Ca2+ concentration) in N-KO

monolayer cultures (supplementary material Fig. S3A).

Depletion of ARNT results in downregulation of amphiregulin

and suppression of EGFR activity in human keratinocytes

In line with recent findings (Stoll et al., 2010a), our data showed

that AREG is the most highly transcribed EGFR ligand in normal

(control) N-TERT keratinocytes (about 70% of GAPDH

Fig. 2. Increased expression of differentiation markers in ARNT-depleted keratinocytes. (A) Transcription of differentiation markers (qPCR) in N-KO1 cells

compared with normal N-TERT keratinocytes (n52, *P,0.05, **P,0.01; EGF-depleted medium). (B) Protein level of involucrin, K1, K10 and TGM1 (WB) in

ARNT-depleted N-KO1 cells cultured in monolyer (EGF-depleted medium). Quantification is shown in supplementary material Fig. S3F. (C) K10 and filaggrin protein

levels (WB) in 3D epidermal equivalents developed using ARNT-deficient (N-KO) and ARNT-overexpressing (N-OXP) N-TERTs. The double band of <40 kDa

corresponds to fully processed filaggrin monomer. Quantification is shown in supplementary material Fig. S3G. (D) Transcription of differentiation markers (qPCR) in

ARNT-depleted N-KO1 cells cultured in EGF-supplemented medium (n52). (E) Proliferation rates of N-KO1 and N-KO2 cells grown in EGF-depleted and EGF-

supplemented medium (direct cell counting n53, *P,0.05, **P,0.01). (F) Histology (H&E) of ARNT-deficient and control 3D epidermal equivalents (N-TERTs)

and IHC for filaggrin, K10 and involucrin. Arrows are pointing to parakeratotic nuclei (H&E) and to granular layer cells highly positive for involucrin.

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expression level). TGFa message was detected at the level of

about 10% of GAPDH while other EGFR ligands showed no or

negligible expression in control cells (Fig. 3A). Comparing the

level of AREG in N-KO cells with that of the controls revealed a

significant downregulation of AREG mRNA – more than 50% in

N-KO2 (Fig. 3B, shaded bars). Genes coding for other EGFR

ligands showed insignificant changes (supplementary material

Fig. S3C). The high dependence of AREG transcription on the

Fig. 3. EGFR pathway is suppressed in ARNT-depleted keratinocytes. (A) Transcription of EGFR ligands (qPCR) in normal N-TERT keratinocytes (n53).

(B) qPCR analysis of AREG expression in control, N-KO1 and N-KO2 cells without TSA (n55, *P,0.05) and with TSA treatment (n53). (C) WB analysis of

AREG in the culture medium conditioned by N-KO cells compared with control (n52) and the level of pEGFR (Tyr1173) in N-KO1, N-KO2 and control cells

(n52; EGF-depleted medium). (D) AREG protein positivity (IHC) in ARNT-overexpressing (N-OXP) and control 3D epidermal equivalents. (E) IHC analysis of

ARNT-depleted (N-KO) and control 3D epidermal equivalents showing staining for AREG, pEGFR, ERK1/2 and pERK1/2. Arrows denote regions of high

positivity. Arrowheads point to positive nuclei. (F) Immunocytofluorescence showed that pEGFR-specific signal (red) occurs as distinct foci within the cytoplasm.

Cells were grown in EGF-depleted or EGF-supplemented medium. (G) Blind test counting of the number of foci per cell for 100 cells (**P,0.01) in N-KO and

control N-TERT keratinocytes. (H) AREG mRNA level in control and ARNT-overexpressing HaCaT cells (n54, **P,0.01). (I) WB analysis of phosphorylated

and non-phosphorylated ERK1/2 in N-KO1, N-KO2 and control cells (n52; EGF-depleted medium).

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level of ARNT was further confirmed by a prominent increase of

AREG message in ARNT-overexpressing HaCaT keratinocytes

(Fig. 3H).

Significantly less AREG protein was secreted into the culture

medium by N-KO cells than by control keratinocytes (Fig. 3C).

Correspondingly, in ARNT-depleted epidermal equivalents,

AREG positivity (IHC) was fainter at the cell membrane and

eradicated from the nuclei as compared to controls where strong

nuclear and cell membrane positivity was observed (Fig. 3E,

AREG). Concomitantly with downregulation of AREG in

ARNT-depleted models, we found a prominent increase of

AREG positivity in ARNT-overexpressing 3D epidermal

equivalents (Fig. 3D).

EGFR phosphorylation at Tyr1173 (pEGFR) was significantly

reduced in the N-KO cells cultured in monolayer (Fig. 3C). This

was also confirmed by immunofluorescence: blind counting of

pEGFR-positive foci performed in duplicate revealed that the

number of foci per cell in N-KO keratinocytes was reduced by two

fold as compared to the controls. This result was not apparent when

the culture medium was supplemented with EGF (Fig. 3F,G). In

the control 3D epidermal equivalents, IHC showed prominent

pEGFR-positive signal at the plasma membranes of the spinous

cells and in the cytoplasm of the uppermost (‘granular’) cell layers

(Fig. 3E, pEGFR, arrows), whereas in N-KO 3D cultures pEGFR

positivity was not observed. While ARNT deficiency significantly

affected EGFR phosphorylation, it had no effect on EGFR

transcription (supplementary material Fig. S3B).

Induction of ERK1/2 phosphorylation is a classical

downstream effect of the EGFR-Ras signalling in many cell

types including epidermal keratinocytes (Roberts and Der, 2007;

Getsios et al., 2009). As shown by WB, ARNT depletion did not

affect the total level of ERK proteins but significantly reduced

ERK1/2 phosphorylation (Fig. 3I). Decrease of pERK1/2 in N-

KO cells was also confirmed by IHC in the epidermal equivalents

(Fig. 3E). In the control 3D cultures, pERK1/2 was localised to

the uppermost layers of flattening cells (same pattern as for

pEGFR, Fig. 3E, arrows) and to the nuclei of some basal

keratinocytes (Fig. 3E, arrowheads). In N-KO equivalents,

pERK1/2 positivity was lost or significantly reduced. No

apparent difference in the level of non-phosphorylated ERK1/2

proteins between control and N-KO models was observed

(Fig. 3E, ERK1/2).

Effects of ARNT depletion upon expression of downstreamtarget genes are modulated through HDAC activity

Suppression of HDAC activity using trichostatin-A (TSA), a

potent and specific inhibitor of class I and II HDACs, eradicated

the negative effects of ARNT deficiency on the level of AREG

transcription (Fig. 3B). Furthermore, TSA treatment eliminated

the stimulatory effects of ARNT-deficiency upon transcription

of keratinocyte differentiation markers as evidenced from the

qPCR analysis of TSA-treated normal and N-KO keratinocytes

when results were compared to corresponding (control and

ARNT-deficient) untreated cell cultures (Fig. 4A). At the same

time, comparison of all three manipulated cultures (normal

TSA-treated, N-KO, TSA-treated N-KO) against untreated

normal cells revealed that differentiation markers had

different responses to TSA (Fig. 4B). Transcription of

filaggrin, K10 and loricrin in N-TERT cells with a normal

level of ARNT was significantly reduced by TSA; TGM1 and

K1 mRNA showed a moderate increase while basal keratins K5

and K14 were not responsive to TSA treatment (as shown by

both methods of comparison). Combination of TSA treatment

with ARNT deficiency resulted in suppression of the stimulating

effects of ARNT knockdown for filaggrin, K10 and loricrin

mRNA while K5 and K14 again showed no response. Despite

both TSA treatment and ARNT deficiency have a positive effect

upon TGM1 and K1 transcription, combination of these two

factors resulted in abrogation of their individual stimulatory

effects.

Fig. 4. Changes in expression of differentiation markers in ARNT-depleted cells are dependent on HDAC activity. (A) Expression of differentiation

markers (qPCR) in N-KO1 cells without or with TSA treatment compared with untreated normal cells (dashed bar) or TSA-treated normal cells (white bars)

respectively (n52, EGF-depleted medium). (B) Same qPCR as in A, but results for N-KO1 cells with or without TSA treatment and for TSA-treated control

keratinocytes were plotted against untreated control cells (n52, EGF-depleted medium).

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In epidermal keratinocytes, the level of HDAC proteins and

the total HDAC activity are dependent on ARNT

An HDAC activity assay showed that in ARNT-depleted N-

TERT cells total HDAC activity was increased by 15–20%. In

ARNT-depleted HaCaT keratinocytes this increase reached 50%

(Fig. 5A,B). Correspondingly, ARNT overexpression in HaCaTs

resulted in a ,50% decrease of total HDAC activity (Fig. 5B).

To identify specific HDACs accountable for these changes we

first identified HDACs with a significant level of transcription in

control N-TERT keratinocytes (Fig. 5C). In these cells, class I

HDAC1 and HDAC3 appeared to be the most prominently

expressed histone deacetylases. Among the SIRT genes (coding

for class III HDACs), only SIRT2 showed a noticeable (but still

low) level of expression. In ARNT-depleted N-KO1 and N-KO2

cells cultured in monolayer, the mRNA level of all HDAC and

SIRT genes remained unchanged as compared to the controls

(supplementary material Fig. S3D,E). However, at the protein

level, depletion of ARNT resulted in a significant increase of

HDAC1, HDAC2 and HDAC3 (Fig. 5D). In ARNT-depleted

HaCaT cells the increase of HDAC1 and HDAC3 protein levels

was even more prominent (Fig. 5E) matching the changes in total

HDAC activity (Fig. 5A,B). Overexpression of ARNT in both N-

TERT and HaCaT keratinocytes cultured in monolayer led to a

prominent drop in HDAC1, HDAC2 and HDAC3 protein levels

(Fig. 5F,G).

Immunohistochemistry in ARNT-deficient 3D epidermal

equivalents revealed an increase in HDAC1 protein in the

keratinocyte nuclei as compared to controls (Fig. 6A). In

agreement with previous reports (Longworth and Laimins,

2006), HDAC3 protein in control 3D models was seen not only

in the nuclei but in the cytoplasm and at the plasma membrane as

well. In control cultures, the relatively low (compared to

HDAC1) nuclear HDAC3 positivity was mostly confined to the

lower cell layers whilst nuclei in the upper layers were HDAC3-

negative (Fig. 6A, HDAC3, arrowheads). In the N-KO 3D

models, high cytoplasmic positivity of HDAC3 was seen in all

cells including the uppermost layers, which were HDAC3

negative in controls (Fig. 6A, arrows). Nuclear HDAC3

staining increased in the lower layers and also appeared in

suprabasal keratinocytes (Fig. 6A). Increase of HDAC1, HDAC2

and HDAC3 protein levels in ARNT-deficient 3D cultures was

confirmed by WB (Fig. 6B). Concomitantly, in ARNT-

overexpressing 3D epidermal equivalents, the level of HDAC1

was significantly reduced (Fig. 6C).

DiscussionARNT is generally thought to be a ubiquitously expressed

protein. However, we have previously demonstrated strong

developmental and differentiation-associated regulation of

ARNT in mouse epidermis, which appeared to be essential for

epidermal development, cornification, barrier formation,

desquamation and epidermal–dermal communication (Geng

et al., 2006; Wondimu et al., 2012). Here, we elucidate a novel

ARNT-dependent mechanism that controls advanced stages of

differentiation in human epidermal keratinocytes. Given the

essential role of ARNT in the activity of HIF- and AhR-

dependent pathways implicated in adaptive response to hypoxia,

organic toxicants, ozone and UV (Wenger and Gassmann, 1997;

Sogawa et al., 1995; Afaq et al., 2009; Fritsche et al., 2007) our

studies also expose a potential mechanistic link between

environmental stress and the process of keratinocyte terminaldifferentiation in normal and diseased human skin.

According to our results, suppression of endogenous ARNT

significantly affects the process of differentiation in humankeratinocytes. Altered expression of key differentiation markersin N-KO cells is concomitant with morphological changes in

ARNT-depleted 3D epidermal equivalents i.e. increased numberof cornifying layers, diminished granular layer, and parakeratosis– all signs of deregulated differentiation.

At the same time, the epidermal markers reveal differentmodes of response to variations in the level of ARNT in vitro:basal keratins K5 and K14 showed no sensitivity at all;

suprabasal differentiation markers such as K1/10, TGM1 andinvolucrin were induced by ARNT deficiency at both mRNA(moderately) and protein levels; while filaggrin and loricrin –specific markers of the granular layer – were dramatically

induced at mRNA level (approximately 10-fold that of otherdifferentiation markers) but were not reliably detected at theprotein level in control and N-KO monolayer cultures. We

assume that monolayer culture may not provide the correctmicroenvironment for normal processing of these proteins – asituation analogous with b-casein expression in mammary

epithelial cells cultured in monolayer or in 3D conditions (Liet al., 1987). In line with this assumption, in 3D epidermalequivalents (which represent a more adequate model ofdifferentiating epidermis) filaggrin protein was easily detected

(Fig. 2C). The changes between control and ARNT-depleted or -overexpressing samples were proportional for different filaggrin-positive bands – 40 kDa, 74 kDa and 120 kDa – presumably

corresponding to filaggrin monomers, dimers and trimers (Daleet al., 1985; Sandilands et al., 2009). This fact, together withdramatic changes in filaggrin mRNA level (Fig. 2A) suggests

that ARNT controls (negatively) expression of profilaggrin generather than proteolytic processing of filaggrin protein. Ectopicappearance of ARNT positivity in the granular layer of N-OXP

3D epidermal equivalents and concomitant drop in total filaggrinprotein level in these models (Fig. 2C) further support thissuggestion.

Overall, the induction of differentiation markers in ARNT-

deficient keratinocytes and their suppression in ARNT-overexpressing 3D models (Fig. 2C) correspond to the absenceof ARNT protein in the upper layers of control 3D epidermal

equivalents (Fig. 1F; Control) and to the exclusion of this proteinfrom the granular layer of normal mouse and human epidermis(Geng et al., 2006). In general, the epidermal phenotype in

ARNT-deficient mice matches our findings in ARNT-KO 3Depidermal equivalents and monolayer cultures (profoundacceleration of differentiation in both in vivo and in vitro

systems). However, there are some discrepancies as well. In the

epidermis of ARNT-KO mouse newborns we observed somereduction of loricrin and filaggrin (Geng et al., 2006) whereasARNT-deficient 3D epidermal equivalents are characterized by

profound induction of these proteins. We believe that reductionof loricrin and filaggrin – two specific granular layer proteins – inthe ARNT-deficient mouse epidermis can be attributed to abrupt

cornification and the virtual absence of a granular layer in thesemice. Involucrin, which is normally expressed not only in thegranular but also in the spinous layer, did not show a decline in

ARNT-deficient mouse epidermis but rather is increased (Genget al., 2006) as we observed in the human system (3Dequivalents). The abrupt cornification characteristic of the

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Fig. 5. Regulation of total HDAC activity and specific HDACs in ARNT-depleted and ARNT-overexpressing keratinocytes cultured in monolayer.

(A) Total HDAC activity in nuclear lysates in N-KO1 and N-KO2 cells compared with controls (n52, **P,0.01). (B) Total HDAC activity in ARNT-depleted

and ARNT overexpressing HaCaT cells (n52, *P,0.05). (C) Expression of individual HDACs and SIRT deacetylases (qPCR) in control N-TERT cells (n52).

(D) Protein levels of HDAC1, HDAC2 and HDAC3 in N-KO1 and N-KO2 cells compared with control N-TERTs (n54). (E) Protein levels of HDAC1 and

HDAC3 in H-KO1 and H-KO2 cells compared with control HaCaTs (n52). (F) Protein levels of both HDAC1 and HDAC3 are significantly reduced in ARNT-

overexpressing HaCaT cells (H-OXP1 and H-OXP2). (G) Protein levels of HDAC1 and HDAC2 are reduced in ARNT-overexpressing N-TERT cells

(monolayer cultures).

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ARNT-null mouse epidermis is not as prominent in 3D culture

and therefore the granular layer, while abnormal, is present in this

model. This allows us to perceive the effects of ARNT depletion

upon the expression of loricrin and filaggrin. Thus, the appearing

discrepancy in expression of loricrin and filaggrin between our

previous in vivo and current in vitro results is likely attributed to

the basic developmental and structural differences between intact

epidermis (in vivo) and in vitro experimental 3D models.

Activation of the EGFR pathway by ligand binding is known

to suppress differentiation and positively affect growth in

keratinocytes (Monzon et al., 1996; Peus et al., 1997; Jost et al.,

2000; Schneider et al., 2008). Here we found that effects of ARNT

depletion upon keratinocyte differentiation (positive) and growth

(negative) are abolished by supplementing cell culture medium

with EGF thus suggesting that these effects are instigated by

downregulation of certain EGFR ligand(s). The implication of the

EGFR pathway in ARNT control over the expression of

differentiation markers is further confirmed by decreased EGFR

and ERK1/2 phosphorylation in N-KO cells and its restoration in

EGF-supplemented medium. Decrease of EGFR phosphorylation

in ARNT-depleted keratinocytes is also supported by significantly

smaller number of pEGFR-positive cytoplasmic foci, which are

presumably representing late endosomes. Previously it was shown

that EGFR endocytosis is driven by its autophosphorylation

(Waterman and Yarden, 2001). Therefore, a reduced level of

EGFR internalization (Fig. 3F) corresponds with a decline of its

phosphorylation (Fig. 3C,E) in ARNT-deficient cells.

In line with previous reports designating AREG as a principal

EGFR ligand mediating autocrine growth in epidermal

keratinocytes (Piepkorn et al., 1994; Nylander et al., 1998;

Stoll et al., 2010a) we showed that AREG is the most abundant

EGFR ligand in normal N-TERT cells. In N-KO keratinocytes

AREG declined significantly at both mRNA and protein levels

and its nuclear positivity was completely lost (Fig. 3B,C,E)

whereas other conventional EGFR ligands showed no changes

(supplementary material Fig. S3C). These findings suggest

suppression of AREG as a likely mechanistic explanation for

downregulation of EGFR pathway in ARNT-depleted cells.

Recently, it was shown that knockdown of AREG in human

epidermal keratinocytes results in reduced ERK phosphorylation,

inhibition of growth, and induction of K1/10, involucrin, loricrin

and TGM1 (Stoll et al., 2010b) – exactly the same set of changes

we observed in ARNT-depleted N-TERTs. This strongly supports

our proposition that decreased EGFR and ERK phosphorylation

and subsequent induction of differentiation markers in N-KO

cells is mediated through downregulation of AREG, but not

through modulation of EGFR expression. This view is supported

by the lack of ARNT effects upon EGFR mRNA level

(supplementary material Fig. S3B).

Loss of nuclear AREG in ARNT-depleted keratinocytes

(Fig. 3E) is of particular interest here. It has been shown

previously that the C-terminal transmembrane fragment of

AREG can be internalized and accumulated in the nucleus to

block keratinocyte differentiation (Stoll et al., 2010b). Similarly,

internalization and nuclear accumulation of the C-terminal

transmembrane fragment of another conventional EGFR ligand,

HB-EGF, leads to silencing of transcription (Nanba et al., 2003;

Toki et al., 2005). Taking these findings into account, our results

identify ARNT as an upstream positive regulator of both

membrane (EGFR activation) and nuclear functions of AREG.

The fact that induction of K10 expression in N-KO cells is not

amended by EGF supplementation suggests that, in contrast to

other differentiation markers, transcription of K10 may be

specifically regulated by nuclear C-terminal domain of AREG

Fig. 6. Level of HDAC proteins in 3D epidermal

equivalent models developed using ARNT-depleted

and ARNT-overexpressing N-TERT keratinocytes.

(A) IHC analysis of 3D epidermal equivalents showing

staining for HDAC1 and HDAC3 in control and

ARNT-depleted models. While uppermost cell layers

in control epidermal equivalents are HDAC3-negative,

in N-KO models, they are highly positive (arrows). In

control cultures, HDAC3-negative nuclei are common

(arrowheads), while they are always HDAC3-positive

in N-KO equivalents. (B) HDAC1, HDAC2 and

HDAC3 protein levels (WB) in ARNT-deficient and

control 3D models. (C) HDAC1 protein level (WB) in

ARNT-overexpressing 3D model compared

with control.

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through the EGFR-independent mechanism mentioned above

(Fig. 7).

It is increasingly apparent that HDAC activity is essential for

epidermal development and differentiation (Markova et al., 2007;

Ezhkova et al., 2009; LeBoeuf et al., 2010). ARNT controls

differentiation of trophoblast cells in the mouse placenta through

modulation of HDAC activity (Maltepe et al., 2005) and it is

very likely that similar mechanisms may control epidermal

differentiation. As shown by our results, TSA treatment abolishes

effects of ARNT deficiency upon expression of AREG and

differentiation markers thus strongly implicating HDAC activity

in ARNT control over AREG–EGFR pathway and keratinocyte

differentiation. However, we found that in normal N-TERT cells

different epidermal genes respond to TSA treatment differently and,

most interestingly, the mode of their response to TSA appeared to

match the changes in their expression in response to ARNT

depletion.

As evident from Table 1, the basal keratins K5 and K14

comprise a group of epidermal genes (Group 1), which, in

contrast to differentiation markers, are not responsive to ARNT

deficiency and also show no response to TSA. Previously, based

on the studies of differentiation in mouse trophoblast it was

suggested that HDAC inhibition phenocopies ARNT deficiency

(Maltepe et al., 2005). In normal keratinocytes, we found this to

be true for TGM1 and for K1 genes only (Group 2) which both

are induced by TSA and upregulated by ARNT deficiency.

Involucrin occupies an intermediate position between Group 1

and 2 genes as it is not responsive to TSA (as Group 1) but is

positively regulated by ARNT depletion (as Group 2 genes).

Filaggrin, K10 and loricrin genes (Group 3) are strongly induced

by ARNT depletion and are significantly suppressed by TSA

treatment in N-TERT keratinocytes. Moreover, TSA treatment of

N-KO cells not only eradicates stimulatory effect of ARNT

deficiency on Group 3 genes but significantly downregulates

their expression as compared to control. Thus, for genes of Group

3, which are negatively regulated by ARNT, HDAC activity

represents a key limiting factor. Also, there is an inverse

correlation between the changes in expression of Group 3 genes

and AREG in response to all kinds of manipulation (Table 1)

further suggesting that filaggrin, K10 and loricrin genes are

negatively regulated by ARNT through HDAC- and AREG-

mediated signalling. Interestingly, HDAC1- and HDAC2-

Fig. 7. A working model for the role of ARNT in the control of AREG–EGFR pathway and downstream differentiation markers. Our results suggest that

ARNT controls expression of differentiation markers through different mechanisms. First, ARNT regulates expression of filaggrin, loricrin and K10 (Group 3,

Table 1) through the modulation of the level of AREG. HDAC activity is essential for these effects. Nevertheless, the mechanisms by which HDAC1, HDAC2 and

HDAC3 are induced by ARNT deficiency and the mode of their interaction with ARNT to bring about regulation of downstream genes remain unclear (dashed

box 1). While being highly dependent on AREG, K10 is not responsive to EGF, suggesting an EGFR-independent regulation of K10 through the intracellular

C-terminal domain of AREG. This mechanism is hypothetical and requires further investigation (dashed box 2). Then, ARNT also has a negative effect upon

expression of involucrin, TGM-1 and K1 (Group 2, Table 1) but these genes show different response to TSA treatment compared with Group 3 and the

mechanisms of their regulation by ARNT remain to be elucidated (dashed box 3). Red arrows and T-bars show effects of experimental manipulation; blue arrows,

stimulatory activity; black T-bars, suppression.

Table 1. Effects of modulation of ARNT and HDAC activity

upon expression of epidermal differentiation markers

TSA Control cells TSA + ARNT-KO ARNT-KO

K5 < < <K14 < Q 2.06 <Involucrin < q 1.66 q 2.36TGM1 q 2.06 < q 2.86K1 q 1.56 q 1.66 q 2.56Flg Q 3.36 Q 2.06 q 30.06K10 Q 5.06 Q 33.36 q 3.76Loricrin Q 50.06 Q 100.06 q 68.46AREG q 1.56 q 1.36 Q 2.06

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knockout mice show an absence of K10 and loricrin expression in

the epidermis (LeBoeuf et al., 2010) thus supporting our

conclusion that effects of ARNT upon AREG, EGFR and

downstream differentiation markers are mediated by HDAC

activity. It was previously reported that TSA alters expression of

epidermal genes in a protein synthesis-dependent manner

(Markova et al., 2007) implying existence of an intermediate

protein regulator. Our data identify AREG as a putative

regulatory link between HDAC activity and expression of

filaggrin, loricrin and K10.

Thus, we have demonstrated that ARNT modulates

keratinocyte differentiation through HDAC- and EGFR-

dependent mechanisms. AREG appears to be the key

intermediate player linking together these two pathways. There

is evidence that PAS proteins such as AhR and HIF2a (which

both require dimerization with ARNT for their activity) control

the expression of AREG in urinal tract and cancer tissues (Choi

et al., 2006; Stiehl et al., 2011). These findings are in line with

the role of ARNT in control of AREG/EGFR pathway in the

epidermis revealed by our studies.

Importantly, while HDAC activity is essential for ARNT

effects upon AREG expression and keratinocyte differentiation,

HDACs themselves appeared to be dependent on ARNT level:

depletion of ARNT results in the prominent induction of total

HDAC activity and significant increase in HDAC1, HDAC2 and

HDAC3 protein levels. Concomitantly, upregulation of ARNT

leads to decrease of HDAC1 level (Fig. 6B,C).

All these interactions are shown on a schematic drawing

(Fig. 7) representing a putative model for ARNT control over

epidermal differentiation. This model suggests that ARNT, being

nuclear (and hence active) specifically in the lower layers of 3D

epidermal equivalents (Fig. 1F), is implicated in the deterrence

of differentiation in spinous keratinocytes. As keratinocytes

approach granular layer, ARNT is downregulated leading to an

increase in HDAC activity and suppression of AREG/EGFR

pathway thus triggering the expression of specific granular

layer proteins such as filaggrin and loricrin. This view also fits

prevalent expression of ARNT in lower layers of mouse and

human epidermis and its decline in the granular layer (Geng et al.,

2006). Our model, while instructive, still remains a subject for

further validation since the role of ARNT dimerization partners

(HIF-a proteins and AhR) in control of the ARNT-AREG-EGFR

pathway and mechanisms of ARNT control over activity of

specific HDACs are not yet clear, nor is the role of AREG in

control of K10 expression (Fig. 7, question marks). These issues

will be addressed in our future studies.

Materials and MethodsCell culture, modulation of ARNT expression and drug treatment

Human N-TERT keratinocytes immortalized with hTERT (a gift from J.

Rheinwald, Harvard Medical School, Boston, MA) were maintained in GIBCO

keratinocyte serum-free medium supplemented with BPE, Ca2+ and EGF (final

concentrations 25 mg/ml, 0.4 mM, 0.2 ng/ml, respectively) (Rheinwald and

Beckett, 1981). In experiments analysing the effect of EGF this supplement was

omitted from the media. To stably deplete ARNT in N-TERTs we used

transduction with lentiviral particles created from Sigma Mission shRNA

plasmids (SHGLY-NM_001668 and non-targeting control plasmid SHC002; seesupplementary material Table S1) as described previously (al Yacoub et al., 2007).

Successfully transduced N-TERT keratinocytes were selected for and further

maintained with 0.5 mg/ml puromycin at low Ca2+ (0.4 mM). Comparison of two

ARNT-depleted N-TERT cell lines (N-KO) with the non-targeting shRNA control

showed 60–80% decrease of ARNT mRNA (Fig. 1A,C) and 81% and 96%

decrease of ARNT protein for N-KO1 and N-KO2, respectively (Fig. 1B,C).

Immunocytochemistry demonstrated a high level of nuclear ARNT protein in

control N-TERTs and its visible decline in the nuclei of N-KO cells (Fig. 1D).

For viral overexpression of ARNT in N-TERT cells, ARNT cDNA ORF

fragment was cut out and purified from the OriGene expression vector (RG216724;

OriGene Technologies, Rockville, MD) by restriction enzyme digest with BamHIand NotI (R0136S and R0189S; NEB, Hitchin, UK). Lentiviral transfer vector

pCDH1-MCS1-EF1-copGFP (al Yacoub et al., 2007) was digested with BamHI

and NotI and the obtained ARNT fragment was ligated in. The ligation reactionwas transformed into Turbo Competent E. coli (C2984H; NEB). Resulting

ampicillin-resistant colonies were mini-prepped and checked for the correct

insertion by restriction digest and direct sequencing. These preparations (alongwith empty pCDH1-MCS1-EF1-copGFP vector for control purposes) were then

used to create lentiviral particles further utilized for transduction of N-TERT

keratinocytes (N-OXP) as described previously (al Yacoub et al., 2007). Efficiency

of transduction in N-OXP keratinocytes was monitored by FACS analysis of GFPfluorescence (supplementary material Fig. S2) using a BD Biosciences (Oxford,

UK) LSR Fortessa Flow Cytometer.

HaCaT keratinocytes (ECACC; Salisbury, UK) were transfected with ARNT

shRNA expressing vectors (Sigma; Dorset, UK) using Lipofectamine 2000 and

selected for stable transformants with puromycin. Three constructs targetingdifferent regions of the ARNT mRNA were used (supplementary material Table

S1). Cells transfected with non-targeting shRNA vector were used as a control.

The use of constructs 1 and 2 resulted in ,40 and 60% reduction of ARNT mRNA

(supplementary material Fig. S1A) and ,90% reduction of ARNT protein levelwhereas construct 3 gave only 20% reduction (supplementary material Fig. S1B).

For further studies we selected cell lines obtained using constructs 1 and 2 only (H-

KO 1 and 2). Transient over-expression of ARNT in HaCaT cells (H-OXP) usinglipofectamine-mediated transfection with an ARNT/GFP-tagged expression vector

(OriGene; Rockville, MD) resulted in ,200-5006 increase of ARNT mRNA level

(supplementary material Fig. S1C) and about 1.5- to 3.5-fold increase of total

ARNT protein (supplementary material Fig. S1D).

For suppression of total HDAC activity cells were grown in culture medium

containing 100 nM TSA for 48 hrs prior to taking lysates for protein and RNAisolation. Development of 3D epidermal equivalents using N-KO or N-OXP cell

lines resulted in formation of epidermis-like structures with significant depletion or

increase of ARNT levels, respectively, as was confirmed by IHC (Fig. 1F,G) andWB (Fig. 1E).

3D cell culture

N-TERT keratinocytes (26105) were resuspended in 400 ml PCT epidermalkeratinocyte medium (CnT-57; CELLnTEC, Bern, Switzerland) and added to

Millicell PCF 12 mm inserts with 0.4 um pore size (PIHP01250; Millipore,

Hertfordshire, UK) placed in a 60 mm Petri dish. Medium was added so that levelswere equal with that inside the insert. After formation of a confluent monolayer the

medium was replaced inside and outside the insert with 3D medium (CnT-02-

3DP1; CELLnTEC). The next day, the medium was aspirated from inside the

inserts. Inserts were left for 14 days with medium changes every three days.Membranes and attached cells were then removed from the inserts, fixed in

formalin overnight, processed through graded alcohols to xylene and embedded in

paraffin blocks. 4-mm-thick sections were cut for H&E and IHC staining using aLeica RM2135 rotary microtome.

RNA isolation and real-time PCR

RNA was isolated from cells grown to 70–80% confluency using Tri Reagent

(Sigma, Dorset, UK) according to the manufacturer’s protocol. RNA was diluted

1:10 and used in qPCR reactions with Invitrogen Superscript III Platinum SYBR

Green One-Step qRT-PCR. Reactions were run on a Bio-Rad mini-opticon real-time PCR machine as previously described (Weir et al., 2011a). Primers used are

shown in supplementary material Table S2.

Protein isolation and western blotting

Protein lysates were obtained from 70–80% confluent keratinocytes as reported

previously (Geng et al., 2006) and processed for WB (Weir et al., 2011a) usingappropriate antibodies (supplementary material Table S3).

Immunocytochemistry

Cells were cultured on 2 well permanox chamber slides (Lab-Tek; Scotts Valley,CA). For assessment of ARNT localisation cultures were fixed with acetone for

2 min at 220 C. For examination of EGFR phosphorylation, cells were fixed with

4% paraformaldehyde at RT, permeabilized with 0.1% Triton X-100 for 5 min andblocked for 30 min with 0.3% BSA in PBS blocking solution. After fixation and

blocking, cells were incubated with primary antibodies (supplementary material

Table S3) made up in either PBS or blocking solution. Washes were repeated

followed by incubation with secondary antibodies made up in PBS. Cells weremounted using ProLong Gold antifade reagent with DAPI (Invitrogen) and

examined using Zeis Axioskop2 microscope with fluorescence.

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Immunohistochemistry

Deparaffinized sections of 3D epidermal equivalents were boiled in the microwavefor 363 min in 10 mM sodium citrate (pH 6.0) for antigen unmasking, incubatedwith primary antibodies (supplementary material Table S3) at RT for 1 hr withblocking and processed further using the Vectastain Elite ABC kit (pk-6200;Vector Laboratories, Peterborough, UK). Samples were developed using the DABperoxidase kit (pk-4100, Vector Laboratories) and counterstained withHaematoxylin. Samples treated with no primary antibodies were used as anegative control.

WST-1 assay and cell counting

The water-soluble tetrazolium salt (WST-1) assay, a standard method to assess cellviability and proliferation (K301-500; BioVision; distributed by CambridgeBioSciences, Cambridge, UK), was performed as described previously (Weiret al., 2011b). To induce keratinocyte differentiation in this experiment,ARNT-deficient N-TERT cells were exposed to high Ca2+ (1.4 mM) as comparedto normal cell culture conditions (0.4 mM). For direct counting, cells were releasedwith 0.05% trypsin and counted in triplicate using a Neubauer hemocytometer.Trypan Blue was used to exclude dead cells from the live cell count.

HDAC activity assay

HDAC activity was performed using a Fluorometric HDAC activity assay kit(ab1438; Abcam, Cambridge, UK). Equal amounts of cell lysate (10–50 mg) wereused and the assay was carried out as per the manufacturer’s description. Resultswere retrieved using a SpectraMax Gemini XS fluorescence plate reader(Molecular Devices, Berkshire, UK).

AcknowledgementsThe authors are grateful to Celine Pourreyron (Skin Cancer Group,University of Dundee) for her valuable help with some of thetechniques used in this study. We also acknowledge the FlowCytometry Core Facility at the College of Medicine, Dentistry andNursing at the University of Dundee for assistance with FACSanalysis.

FundingOur work was funded by Cancer Research UK [grant number C5314/A6695].

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.095125/-/DC1

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