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. Author manuscript; available in PMC: 2009 Apr 13.
Published in final edited form as: J Invest Dermatol. 2007 Sep 20;128(3):530–541. doi: 10.1038/sj.jid.5701049

Synergistic Activation of Human Involucrin Gene Expression by Fra-1 and p300—Evidence for the Presence of a Multiprotein Complex

James F Crish 1, Richard L Eckert 1,2,3,4,5
PMCID: PMC2668529  NIHMSID: NIHMS104681  PMID: 17882273

Abstract

Involucrin is expressed in the differentiated suprabasal epidermal layers, and an AP1 transcription factor-binding site present in the involucrin promoter distal regulatory region is required for this regulation. This site binds Fra-1, but cofactor interaction at this site has not been adequately characterized. We show that Fra-1 and p300 histone acetyltransferase are present at the AP1 site, as detected by chromatin immunoprecipitation. This interaction is functional, as treating p300 expressing keratinocytes with calcium or 12-O-tetradeconylphorbol-13-acetate, results in a synergistic increase in hINV expression, and this enhanced activation can be reproduced by coexpression of Fra-1 and p300. p300 also co-precipitates with Fra-1, but protein fractionation studies suggest that this interaction requires an additional protein. Fra-1 also interacts with other proteins that interact at the AP1-5 site, including JunD, JunB, Sp1, and P/CAF. Contrary to results in some other systems, Fra-1 functions as a positive transcriptional regulator in human keratinocytes. These studies suggest that a large multiprotein complex, which includes Fra-1, p300, P/CAF, junD, junB, and Sp1 acts at the AP1-5 site to produce a synergistic increase in hINV gene expression.

INTRODUCTION

Epidermal keratinocytes undergo a programmed transition, in which undifferentiated proliferating cells are converted to non-proliferating, terminally differentiated cells (Eckert et al., 1997b). This differentiation process is tightly controlled and involves specific temporal and spatial changes in gene expression (Eckert and Welter, 1996; Eckert et al., 1997a, b). The ultimate product of keratinocyte differentiation is the corneocyte, which consists of a covalently crosslinked sheath of protein, called the cornified envelope, surrounding a network of stabilized keratin fibrils. Millions of corneocytes are assembled to construct the epidermal surface (Reichert et al., 1993; Steven and Steinert, 1994; Steinert, 1995; Steinert and Marekov, 1997).

Involucrin is a key cornified envelope precursor protein and is covalently incorporated to form the cornified envelope scaffolding via the action of type I transglutaminase (Simon and Green, 1985, 1988; Etoh et al., 1986; Yaffe et al., 1992). Involucrin expression is regulated during differentiation—the involucrin gene is not expressed in the basal epidermal layer, but expression is observed in cells occupying the upper-spinous and granular layers (Rice and Green, 1977, 1979). Involucrin is also expressed in other stratifying epithelia, and in each tissue, expression is confined to the suprabasal layers (Banks-Schlegel and Green, 1981; Crish et al., 1993). Thus, involucrin is an important model for the study of tissue-specific and differentiation-dependent gene expression. Previous studies show that a 6-kb segment of the hINV gene, including the hINV upstream regulatory region, the transcription start site, the intron, and the coding sequence, guides appropriate tissue-specific and differentiation-appropriate expression in transgenic mice (Crish et al., 1993). Targeted expression is also observed when the upstream regulatory region, nucleotides -2,473/-1, is linked to the human papillomavirus oncoproteins (Crish et al., 2000), suggesting that this region is sufficient to direct appropriate expression.

In vitro studies show that the promoter is expressed in cells derived from stratifying epithelia (Welter et al., 1995; Banks et al., 1999), is regulated by agents that modulate differentiation (Efimova et al., 1998, 2002; Efimova and Eckert, 2000), and is not active in fibroblasts (Welter et al., 1995). Deletion mapping and point mutation studies identify two regions of the upstream regulatory region, the proximal regulatory region (PRR) and the distal regulatory region (DRR), that are required for optimal expression in cultured cells (Welter et al., 1995; Banks et al., 1999). The DRR contains AP1 and Sp1 sites, separated by one nucleotide, that are required for optimal promoter activity (Welter et al., 1995). The DRR Sp1 site acts synergistically to activate transcription, in conjunction with the AP1-5 site (Banks et al., 1999). Gel mobility supershift studies indicate that junB, junD, and Fra-1 interact with the DRR AP1-5 site, and that Sp1 and Sp3 factors interact with the DRR Sp1 site (Welter et al., 1995; Banks et al., 1998; Balasubramanian et al., 2002). We have proposed that a complex is formed on the DRR that includes AP1 and Sp1 factors and other, presently unknown, coactivators (Eckert et al., 2004).

CREB binding protein and p300 acetyltransferases

CREB binding protein (CBP) and p300 are histone acetyltransferases (HATs) that are ubiquitously expressed and participate in a host of gene regulatory events (Partanen et al., 1999). An amino-acid sequence comparison of these multidomain proteins reveals numerous conserved regions, including three cysteine-histidine-rich regions (CH1, CH2, CH3), the CREB transcription factor-binding site (KIX domain), the bromodomain, the HAT domain, and the steroid receptor coactivator-1 interaction domain (Arany et al., 1994). CBP and p300 interact with basal transcription factors, including TATA-binding protein (Yuan et al., 1996) and TFIIB (Kwok et al., 1994; Yuan et al., 1996), and can complex with RNA polymerase II (Nakajima et al., 1997a, b; Cho et al., 1998; Neish et al., 1998). These interactions occur through an N- and C-terminal activation domain (AD). CBP and p300 also bind to other proteins via the CH1, CH3, KIX, and steroid receptor coactivator-1 interaction domain domains (Chan and La Thangue, 2001; Vo and Goodman, 2001). CBP and p300 can act as physical bridges by simultaneous interaction with basal transcription factors and upstream transcriptional regulators. In addition, these protein-protein interactions are influenced by the post-translational modifications, including phosphorylation (Janknecht and Nordheim, 1996; it-Si-Ali et al., 1998), sumoylation (Girdwood et al., 2003), and methylation (Chevillard-Briet et al., 2002; Girdwood et al., 2003).

These studies are designed to gain new insight regarding the composition of the gene regulatory complexes that regulate involucrin gene expression. Our studies show that p300 acts as a potent coactivator of hINV gene expression that, when overexpressed in differentiation agent-treated cells, results in a synergistic increase in hINV gene expression. We have previously shown that Fra-1 is a key regulator that binds at the hINV promoter AP1 factor DNA-binding sites (Welter et al., 1995). Our present studies indicate that Fra-1/p300 coexpression results in synergistic activation of hINV promoter activity. Moreover, chromatin immunoprecipitation (ChIP) analysis reveals that both proteins are bound to the promoter in the vicinity of the AP1-5 site. Immunoprecipitation analysis reveals that Fra-1 interacts with p300, and protein fractionation studies suggest that this interaction requires an additional unknown protein. In addition, JunD, JunB, p300, P/CAF, and Sp1 co-precipitate with Fra-1, suggesting that a large multiprotein complex regulates expression.

RESULTS

Calcium and 12-O-tetradeconylphorbol-13-acetate (TPA) are known regulators of keratinocyte differentiation. Treatment with either agent results in an increase in hINV gene expression and promoter activity (Welter et al., 1995; Banks et al., 1998; Deucher et al., 2002; Efimova et al., 2003). p300 and CBP are ubiquitously expressed acetyltransferases that participate in gene regulatory events in a range of cell types (Giordano and Avantaggiati, 1999; Chan and La Thangue, 2001; Iyer et al., 2004; Kalkhoven, 2004) by acetylating the N termini of several core histones (Giordano and Avantaggiati, 1999; Chan and La Thangue, 2001; Iyer et al., 2004). To assess the role of acetyltransferases in regulating involucrin gene expression, we examined the ability of coexpressed p300 to increase calcium- and TPA-dependent involucrin gene expression (Welter et al., 1995; Efimova et al., 1998; Agarwal et al., 1999). Keratinocytes were transfected with the involucrin promoter luciferase reporter plasmids, as shown in Figure 1a. pINV-2473 encodes the full-length involucrin promoter, whereas pINV-41 encodes 41 nucleotides that constitute the hINV minimal promoter (Welter et al., 1995). Keratinocytes were transfected with pINV-41 and pINV-2473 in the presence or absence of p300 expression vector and then treated with calcium or TPA. After 24 hours, the cells were harvested and assayed for luciferase activity. As shown in Figure 1b, pINV-41 does not respond to any of the treatments. In contrast, the activity of pINV-2473 is markedly increased by p300 or by addition of 0.3 mm calcium. Moreover, a synergistic activation is observed when cells are co-treated with p300 and calcium. As shown in Figure 1c, a similar pattern of synergistic activation is observed for cells treated with TPA + p300.

Figure 1. Calcium treatment of p300 expressing keratinocytes produces a synergistic increase in hINV promoter activity.

Figure 1

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(a) Structure of hINV luciferase reporter constructs. pINV-2473 includes the full-length hINV promoter; pINV-41 is the hINV basal promoter (TATA box only). The locations of the proximal and distal regulatory regions are indicated (PRR, DRR), as are the positions of the AP1-1 and AP1-5 sites, the C/EBP site, and the Sp1 site. The black box represents the luciferase gene and the arrow indicates the site of transcription initiation and the direction of transcription. (b and c) Calcium treatment of p300 expressing cells results in synergistic activation of involucrin gene transcription. Keratinocytes (50% confluent) were transfected with 1 μg of pINV-2473 or pINV-41 in the presence or absence of 0.5 μg of p300 expression plasmid. After 24 hours, the cultures were shifted to cell culture medium supplemented or not supplemented with 0.3 mm calcium, for an additional 24 hours. For TPA treatment, the cells were transfected as indicated above and then treated for 24 hours in the presence or absence of 50 ng TPA/ml. Cell extracts were then prepared for assay of luciferase activity. The values presented in this figure represent the averages of four separate experiments. As assessed by the Student’s t-test, for the pINV-2473 construct, the difference between the control and all other groups is significant at the 95% confidence level. The difference between the 0.3 mm calcium/TPA + p300 groups and all other groups is significant at the 95% confidence interval.

To assess the specificity of this regulation, TPA- and p300-dependent pINV-2473 activity was measured in the presence and absence of expression of the dominant-negative p300 mutant, d33, which lacks a segment of the CH3 domain (Eckner et al., 1994; Lill et al., 1997). The reduced pINV-2473 promoter activity, observed in the presence of d33, suggests that acetyltransferase activity is absolutely required for appropriate promoter activation in response to treatment with calcium or TPA (Figure 2a). As shown in Figure 2b, immunoblot with either anti-HA or anti-p300 confirms that HA-p300 and HA-d33 are expressed in the transfected keratinocytes. In addition, the anti-p300 immunoblot confirms the presence of endogenous p300.

Figure 2. TPA-dependent hINV promoter activity requires p300.

Figure 2

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(a) Keratinocytes (50% confluent) were transfected with 1 μg of pINV-2473, in the presence or absence of 0.5 μg of HA-p300 or HA-d33 expression plasmids. The final DNA concentration was adjusted to a final concentration of 2 μg with empty plasmid. At 24 hours post-transfection, the cultures were treated for 24 hours with or without 50 ng TPA/ml. Cell extracts were then prepared for assay of luciferase activity. The results are the average values derived from three separate experiments. The difference between TPA treated and p300 groups and all other groups is significant at the 95% confidence interval. (b) Keratinocytes were transfected with pINV-2473, HA-p300, and HA-d33 plasmids as outlined above. At 24 hours post-transfection the cells were harvested and total cell extracts were assayed for the presence of HA-p300 or HA-d33 by immunoblot using antibodies specific for p300 or HA epitope, followed by an appropriate secondary HRP-conjugated antibody and visualization of antibody binding using chemiluminescence reagents.

p300-dependent promoter activation via the hINV promoter DRR

Our previous studies indicate that AP1 transcription factors interact with the hINV promoter DRR AP1-5 site to increase involucrin promoter activity (Welter et al., 1995; Crish et al., 1998, 2002, 2006), and that treatment with TPA or calcium increases AP1 factor interaction at this site (Welter et al., 1995; Deucher et al., 2002). These observations suggest that the synergistic increase in promoter activity observed in TPA- or calcium-treated p300 expressing cells may be due to p300 factor synergy with AP1 factors.

To test this possibility, we assessed whether Fra-1, which binds to the AP1-5 site (Welter et al., 1995), can act with p300 to increase activity. We focused on the DRR region using a series of involucrin promoter luciferase reporter plasmids, which encode various segments of the DRR (Figure 3a). As shown in Figure 3b, activity of most of the DRR constructs is modestly increased or not regulated by Fra-1 or p300. In contrast, a marked synergistic increase is observed when both p300 and Fra-1 are present. It is noteworthy that this increase is specific for constructs in which the DRR is present, and is not observed for the minimal promoter construct—pINV-41. As noted above, the DRR encodes an AP1 site (AP1-5) that is essential for expression of the hINV transgene in mice (Crish et al., 1993, 1998, 2002). To assess the role of this site in mediating the response to p300 and Fra-1, we tested the activity of DRR(2,473/1,953)-AP1-5mm, in which the AP1-5 site is mutated (Welter et al., 1995). As shown in Figure 3c, mutation of this site results in a complete loss of the ability of p300, Fra-1, and p300 + Fra-1 to regulate activity. It is striking that mutation of the AP1-5 results in an inactivation of the p300-dependent increase in expression. This provides strong evidence that the p300-dependent synergy involves factors that bind at the AP1-5 site.

Figure 3. p300 and Fra-1 act synergistically to increase hINV promoter activity.

Figure 3

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(a) Structure of hINV luciferase reporter constructs. pINV-2473 includes the full-length hINV promoter; pINV-41 is the hINV basal promoter (TATA box only). The conventions are exactly as outlined in Figure 1. In the DRR constructs, segments of the DRR region are fused adjacent the hINV basal promoter. The DRR(-2,473/-1,953) AP1-5mm has a mutation in the AP1-5 site that results in its inactivation (Welter et al., 1995). (b and c) Keratinocytes (50% confluent) were transfected with 1 μg of the indicated DRR hINV promoter reporter plasmid in the presence of 0.5 μg of HA-p300 or FLAG-hFra-1. At 24 hours post-transfection, the cells were harvested and extracts prepared for assay of luciferase activity. The values are the mean of two separate experiments.

Evidence for interaction of p300 and Fra-1

Our previous studies demonstrate that Fra-1 binds to the hINV promoter DRR AP1-5 site, as determined by gel mobility and gel mobility supershift assay (Welter et al., 1995). To demonstrate that Fra-1 and p300 localize at the DRR region in vivo, we performed ChIP. Cells were incubated with formaldehyde to crosslink proteins to DNA, and the DNA was sheared and protein bound DNA was precipitated with anti-IgG, anti-p300, or anti-Fra-1. As shown in Figure 4, incubation with anti-p300 or anti-Fra-1 precipitates protein-associated DNA that is detected by PCR. In contrast, the anti-IgG precipitate does not produce a PCR product. This suggests that both p300 and Fra-1 are resident in the vicinity of the DRR AP1-5 element.

Figure 4. In vivo interaction of p300 and Fra-1 with the hINV promoter DRR.

Figure 4

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Normal human keratinocytes were treated with formaldehyde to crosslink intranuclear DNA-protein complexes. The nuclei were then prepared and lysed, and Fra-1 and p300 DNA complexes were immunoprecipitated using 5 μg rabbit anti-Fra-1 and 5 μg mouse monoclonal anti-p300 antibodies, respectively. Nonspecific precipitation was monitored by precipitation using normal mouse IgG (Santa Cruz, sc-2025). The DNA was released from the precipitated complex using 1% SDS/0.1 M NaHCO3 and the released product was amplified by PCR and the amplified product was detected by ethidium staining of an agarose gel. The arrows indicate migration of the product and the primers. M = markers (nucleotides) and T = PCR product observed following amplification of 1% of material used for immunoprecipitation.

To monitor direct interaction between p300 and Fra-1, we performed co-precipitation experiments. However, the available antibodies are not adequate to detect interaction given the low level of Fra-1 and p300 expression in keratinocytes. As an alternate method, we utilized an immortal epithelial cell line—HeLa-Z. We generated a HeLa-Z cell line that overexpresses FLAG-tagged human Fra-1. This cell line was generated by infection with pLNCX2-FLAG-hFra-1, a G418-selectable virus that was engineered to produce FLAG-hFra-1. A control HeLa-Z cell line was produce by infection with empty pLNCX2 followed by selection with G418. As shown in Figure 5a, the pLNCX2-hFLAG-Fra-1 cells produce substantially elevated levels of hFra-1, which can be detected using anti-Fra-1 or anti-FLAG. To determine whether FLAG-Fra-1 can interact with p300, we prepared extract from pLNCX2-FLAG-hFra-1 and pLNCX2 cells for immunoprecipitation with anti-FLAG. The top panel in Figure 5b shows an anti-FLAG immunoblot of anti-FLAG precipitated FLAG-hFra-1. This figure confirms that FLAG-hFra-1 is successfully precipitated by the antibody. We performed parallel immunoblots of the precipitated material using p300- and P/CAF-specific antibodies. The middle panel in Figure 5b shows a strong anti-p300-immunoreactive band that is specifically precipitated in FLAG-hFra-1-expressing cells. The lower panel shows a specific FLAG-hFra-1-dependent precipitation of P/CAF. These findings suggest that FLAG-hFra-1 and endogenous p300 and P/CAF are part of a multiprotein complex.

Figure 5. Evidence suggesting that p300 and Fra-1 interact.

Figure 5

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(a) Nuclear extracts were prepared from HeLa-Z (pLNCX2) and HeLa-Z (pLNCX2-FLAG-Fra-1) cell lines. Equal protein equivalents were separated by electrophoresis and immunoblotted with anti-Fra-1 or anti-FLAG. (b) FLAG-Fra-1 was precipitated from nuclear extract isolated from HeLa-Z (pLNCX2) and HeLa-Z (pLNCX2-FLAG-Fra-1) cell lines using 5 μg of mouse monoclonal anti-FLAG M2. The precipitated protein was separated by electrophoresis and immunoblotted with anti-FLAG (top panel), anti-p300 (middle panel), and anti-P/CAF (bottom panel). Each group was incubated with the appropriate HRP-conjugated secondary antibody and the signal was visualized using chemiluminescence detection reagents. The asterisks indicate nonspecific bands. (c) FLAG-hFra-1 precipitation of other regulatory proteins. Proteins were immunoprecipitated using 5 μg of mouse monoclonal anti-FLAG M2 as above and co-precipitated proteins were identified using antibodies specific for the indicated proteins.

We next assessed the ability of Fra-1 to interact with other transcription factors that are thought to be involved in regulation via the DRR. As shown in Figure 5c, immunoprecipitation of FLAG-Fra-1 results in co-precipitation of JunD, JunB, and Sp1. These findings are significant, since JunD and JunB have a role in regulating involucrin gene expression (Welter et al., 1995). Moreover, there is an Sp1 site located immediately adjacent and downstream of the AP1-5 site within the DRR, that has been shown to be functionally important (Banks et al., 1998, 1999; Crish et al., 1998). As expected, Fra-1 not only interacts with AP1 factors that specifically bind to the hINV promoter AP1 site, but also with other AP1 factors. For example, as shown in Figure 5c, it also precipitates c-jun, which does not interact at the AP1-5 site (Welter et al., 1995).

Mechanism of Fra-1/p300 interaction

The above studies, using total nuclear extracts, indicate the co-immunoprecipitation of Fra-1 and p300. However, it is not clear whether this involves a direct Fra-1/p300 interaction. To assess this, we loaded nuclear extract prepared from HeLa-Z FLAG-hFra-1 cells, on a P11 anion-exchange column, eluted the sample using a KCl step gradient, and tested the fractions for Fra-1/p300 interaction. We monitored elution of three key proteins—FLAG-hFra-1, JunB, and p300. As shown in Figure 6, FLAG-hFra-1 and junB elute in the 0.4-0.5 m KCl fractions. p300 is also present in these fractions, but also elutes at other KCl concentrations, probably reflecting its ability to interact with a wide range of proteins.

Figure 6. P11 column fractionation of nuclear extracts.

Figure 6

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Nuclear extract (NE, 2.5 mg) was prepared from HeLa-Z (pLNCX2-FLAG-hFra1) cells and chromatographed on a 3 ml P11 phosphocellulose column (Whatman) at a flow rate of 1 column volume per hour. The column, which was prepared in equilibration buffer containing 0.2 m KCl (see Materials and Methods), was sequentially eluted with 1 ml equilibration buffer containing 0.3, 0.4, 0.5, 0.85, and 1.2m KCl and 0.25 ml fractions were collected. Each fraction was then dialyzed against the equilibration buffer and an equivalent quantity of protein (15 μg) was separated by electrophoresis on a 10% (FLAG-hFra-1) or 5% (p300) polyacrylamide gel before immunoblotting. FLAG-hFra-1 was detected using peroxidase-conjugated anti-FLAG M2 mouse monoclonal antibody (Sigma F1804, 1:500), and JunB and p300 were detected using a 1:500 dilution of rabbit anti-JunB (Santa Cruz, sc-46X) and 2 μg/ml mouse monoclonal anti-p300CT (Upstate, 05-257), respectively.

To determine whether FLAG-hFra-1 and p300 interact following P11 chromatography, the 0.4 m KCl fractions were pooled, FLAG-hFra-1 was precipitated with anti-FLAG, and precipitated proteins were detected by immunoblot. As shown in Figure 7a, FLAG-hFra-1 is precipitated from extracts derived from FLAG-hFra-1 expressing cells, but not from extracts prepared from HeLa-Z cells (top panel), and the precipitated protein co-migrates with the FLAG-hFra-1 present in total nuclear extract (NE). We next determined whether junB co-precipitates with FLAG-hFra-1. As expected, JunB, which can heterodimerize with Fra-1, co-precipitates with FLAG-hFra-1 (middle panel). However, under these conditions, p300 does not co-precipitate with FLAG-hFra-1 (bottom panel). This indicates that neither Fra-1 nor junB is the direct binding partner of p300.

Figure 7. p300 interaction with FLAG-hFra-1 requires additional protein factors.

Figure 7

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Nuclear extract was prepared from HeLa-Z (pLNCX2) and HeLa-Z (pLNCX2-FLAG-hFra-1) and 2 mg was loaded onto a P11 column and eluted stepwise using the indicated level of KCl. (a) Protein (15 μg) of the 0.4 m KCl fraction was immunoprecipitated using 40 μl of a 50% slurry of anti-FLAG M2 agarose (Sigma, A2220). The immunoprecipitated (IP) proteins were then immunoblotted (IB) with peroxidase-conjugated anti-FLAG M2 mAb (Sigma, F1804), rabbit anti-JunB or mouse monoclonal anti-p300. For the JunB and p300 blots, primary antibody binding was visualized using a 1:14,000 dilution of HRP-conjugated donkey anti-rabbit IgG (Amersham, NA 934) or HRP-conjugated sheep anti-mouse IgG (Amersham, NA 931V). Samples of total nuclear extract and 0.4 M KCl fraction (15 μg) were electrophoresed in parallel for immunoblot. (b) Seventy-five micrograms of total nuclear extract, prepared from HeLa-Z (pLNCX2-FLAG-Fra-1) cells was adjusted to the indicated KCl concentration and incubated for 8 hours at 4°C before immunoprecipitation with anti-FLAG M2 agarose. The precipitated protein was electrophoresed and immunoblotted with anti-FLAG, anti-JunB, anti-JunD (Santa Cruz, sc-74x, 1:500 dilution) or anti-p300.

These findings suggest that the P11 chromatography step has removed a protein partner that is necessary for Fra-1/p300 interaction. However, it is possible that the presence of 0.4 m KCl causes p300 to dissociate. To assess the latter possibility, we adjusted the salt concentration, of total nuclear extracts, to 0.4, 0.5, 0.85, and 1.2 m KCl. We then precipitated FLAG-hFra-1 and monitored for co-precipitation of JunB, JunD, and p300. As shown in Figure 7b, all three proteins co-precipitated at all salt concentrations, suggesting that the absence of p300 co-precipitation observed in the Figure 7a is not due to salt-dependent release of p300.

Fra-1 functions as a transcriptional activator

To further assess the ability of Fra-1 to drive transcription we used a one-hybrid assay system. Keratinocytes were transfected with 0.1 μg of pFR-Luc, which encodes the Gal4 DNA response element linked upstream of luciferase, and 0.1 μg of expression plasmids encoding the Gal4 DNA binding domain (Gal4BD) fused to selected regions of Fra-1. As shown in Figure 8a, minimal promoter activity is observed in cells transfected with empty vector or with Gal4BD. In contrast, fusion of the GAL4BD to full-length Fra-1 results in a substantial increase in reporter activity. This activity is progressively reduced as the Fra-1 AD is truncated. Activity of Fra-1-Δ232-271 and Fra-1-Δ189-271 is reduced by 60 and 80%, respectively. These results suggest that the Fra-1 AD is required for activity. Colburn and co-workers have reported that the key threonine phosphorylation site in the Fra-1 AD is required for optimal transcriptional activity. To assess the role of this site, we converted T227 to glutamic acid or alanine. As shown in Figure 8b, the Fra-1 AD (residues 131-271, Fra-1-AD) is sufficient to drive transcription. However, mutation of T227 to Glu or Ala reduces activity.

Figure 8. Fra-1 as a transcriptional activator.

Figure 8

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Normal human keratinocytes were transfected with 0.1 μg of pFR-Luc in the presence of 0.1 μg of the indicated Gal4-BD-FLAG-Fra-1 protein expression constructs. At 24 hours after transfection, the cells were harvested and assayed for luciferase activity. (a) Regulation of pFR-Luc promoter activity by full-length Fra-1 and truncation mutants. Keratinocytes were transfected with 0.1 μg of pFR-Luc in the present of 0.1 μg of the indicated plasmids. After 24 hours, the cells were harvested and extracts were assayed for luciferase activity. (b) Regulation of pFR-Luc promoter activity by the Fra-1 AD and Fra-1 AD truncation mutants. Keratinocytes were transfected and harvested for assay of luciferase activity as outlined above. The data is expressed as the mean±SEM, n=4. Immunoblot analysis reveals that all of the proteins (GAL4BD-FLAG-Fra-1, and so on) were expressed at comparable levels (not shown). The results are a representative experiment from two repeats. The values are expressed as sample means±SD.

DISCUSSION

Involucrin gene expression—a role for AP1 and Sp1 transcription factors

Keratinocyte differentiating agents activate an nPKCδ, Ras, MEKK1, MEK3, p38δ/ERK1/2 signaling cascade to increase AP1 factor expression (Efimova et al., 1998, 2002, 2003; Efimova and Eckert, 2000). This leads to enhanced AP1 factor interaction at DNA-binding sites within the hINV promoter and ultimately leads to increased hINV gene expression (Eckert et al., 2004). The AP1-5 site, located in the DRR, is particularly important for this regulation (Welter et al., 1995). The absence of this site results in a marked reduction in promoter activity in cultured keratinocytes (Welter et al., 1995). Moreover, mutation of this site results in a complete loss of involucrin expression in murine transgenic models of hINV gene expression (Crish et al., 1998, 2002, 2006). These findings indicate that AP1 factors are essential for normal hINV gene expression. The AP1 transcriptional regulators comprise a family of proteins that form homo- and heterodimers to regulate transcription (Karin et al., 1997; Shaulian and Karin, 2002). Members of this family are known to be expressed in the epidermis (Welter and Eckert, 1995; Angel et al., 2001) and to have an important role in regulating epidermal keratinocyte gene expression, differentiation, and transformation (Smeyne et al., 1992; Kachinskas et al., 1994; Saez et al., 1995; Huang et al., 1996, 1997; Rutberg et al., 1996,1997; Rossi et al., 1998; Cooper et al., 2003). Although all of the AP1 family members are expressed in epidermal keratinocytes, only Fra-1, JunB and JunD bind to AP1-5 DNA binding site (Welter et al., 1995; Banks et al., 1998; Balasubramanian et al., 2002, 2005). In addition, a canonical Sp1 transcription factor-binding site is located immediately downstream and adjacent to the AP1-5 site (Banks et al., 1998). Mutation of this site results in reduced hINV expression in transgenic mouse models (Crish et al., 2006). Although Fra-1, JunB, and JunD, and Sp1 are known to bind to the DRR AP1-5/Sp1 element, very little is known about the role of coactivators in driving expression.

Involucrin gene expression—a role of HATs

HATs function to acetylate histones and other proteins, and also serve as bridge proteins during transcription complex assembly (Giordano and Avantaggiati, 1999). The acetylation catalyzed by these proteins is thought to modulate complex formation to from an open chromatin structure that is accessible for transcription (Giordano and Avantaggiati, 1999; Chan and La Thangue, 2001). As such, the p300 and P/CAF HATs have been shown to interact with a variety of transcriptional regulators (Giordano and Avantaggiati, 1999); however, the function of these proteins has not been extensively studied in epidermis or in epidermal keratinocytes. In this study, we examine the role of these proteins in regulating hINV gene expression. Our studies show that treatment of keratinocytes with differentiating agents (TPA or calcium) in the presence of overexpressed p300 results in a synergistic increase in hINV promoter activity. We further show that this activation can be completely blocked by expression of dominant-negative p300 or by mutation of the AP1-5 binding site. We anticipate that hINV gene expression is regulated by a complex and redundant set of mechanisms. Thus, it is very interesting that mutation of the AP1-5 site (elimination of transcription factor DNA interaction) or expression of dominant-negative p300 (inhibition of HAT function) is able to eliminate promoter activity. This is consistent with previous reports indicating that expression of a mutant form of P/CAF can inhibit the calcium-dependent increase in involucrin expression in HaCaT cells (Kawabata et al., 2002), and that expression of p300 or P/CAF, in immortalized keratinocyte cell lines that express reduced levels of involucrin, results in enhanced expression (Tran and Crowe, 2004). These findings strongly suggest that both AP1 factor and HAT function are absolutely required for promoter activation. Moreover, coexpression of Fra-1 with p300 results in a synergistic increase in promoter activity, indicating that these factors function together to drive transcription.

We further demonstrate, using ChIP, that Fra-1 and p300 interact at the AP1-5/Sp1 element in the DRR region—thus placing both proteins in physical proximity over this element. This finding is consistent with a previous report indicating that p300 and P/CAF interact with the hINV promoter AP1-1 and AP1-5 sites (Tran and Crowe, 2004). To study the composition and structure of this multiprotein complex, we expressed FLAG-hFra-1 in HeLa-Z cells, and then precipitated with anti-FLAG. This study revealed that endogenous p300 can co-precipitate with Fra-1, and that a second HAT protein, P/CAF, also interacts with Fra-1. These findings suggest that Fra-1 is part of a complex that may serve to increase hINV promoter activity by bringing p300 and P/CAF into proximity with the DRR element. We also assessed whether Fra-1 and p300 interact in a direct manner, since the direct interaction of p300 with fos family proteins has been reported (Bannister and Kouzarides, 1995; Bannister et al., 1995). P11 chromatography of total nuclear extract results in the loss of ability of p300 to co-precipitate with FLAG-hFra-1. These findings suggest that a Fra-1/JunB heterodimer is present, and that the Fra-1 protein present in this complex interacts with p300 via a bridging protein. The identity of this bridge protein is presently not known. It is possible that this interaction could occur via p300 or P/CAF interaction with the JunB protein present in the complex with hFra-1, as these HAT proteins have been shown to interact with jun family members (Bannister et al., 1995). However, this appears unlikely since FLAG-hFra-1 and JunB co-precipitate in the absence of p300 co-precipitation in P11 fractionated proteins.

Parallel studies indicate that JunB, JunD, and Sp1 also co-precipitate with FLAG-hFra-1, a finding that is consistent with a previous report indicating that p300 interacts with Fra-1, FosB, and junB in immortal and transformed SCC-12 keratinocytes (Tran and Crowe, 2004). Taken together, these studies suggest that a large complex, which includes junB, junD, Sp1, Fra-1, p300, and P/CAF, is likely to exist in the vicinity of the DRR AP1-5/Sp1 element. It is possible that one or more of these proteins provides the physical contact that permits Fra-1/p300 interaction—however, it does not appear to be junB. Candidates for the protein that may mediate physical interaction with p300 include Sp1, which has been reported to functionally interact with p300 (Hung et al., 2006). At least one example of a function relationship between Sp1 and p300 exists in keratinocytes—the loricrin gene is increased when keratinocytes are cotransfected with Sp1 or c-jun in the presence of p300 (Jang and Steinert, 2002), suggesting that p300 serves as a coactivator of loricrin expression (Jang and Steinert, 2002). A role for p300 is also indicated for control of keratin 14 expression, where it drives increased expression via interaction with the POU domain protein, Skin-1a (Sugihara et al., 2001).

Fra-1 as a transcriptional activator

Fra-1 has previously been thought to lack the ability to drive transcription due to the absence of a functional transactivation domain (Wisdom et al., 1992; Wisdom and Verma, 1993; Bergers et al., 1995); however, Fra-1 can also act to enhance cell responses (Mechta et al., 1997; Vallone et al., 1997; Kustikova et al., 1998; Young et al., 2002), suggesting that it is likely to have transcriptional activity. Our studies demonstrate that Fra-1 acts as a positive activator of hINV promoter activity in keratinocytes. Moreover, parallel studies indicate that the isolated Fra-1 AD can positively regulate transcription from the GAL4 promoter. These studies indicate that the hFra-1 AD is sufficient to drive gene expression in human keratinocytes. Colburn and co-workers demonstrated that mutation of threonine 227 to alanine in Fra-1 results in a loss of Fra-1 activity (Young et al., 2002). We were able to replicate this result in the present studies; however, we also observed a drop in activity for a threonine 227 to glutamic acid mutant. Thus, the charge distribution at this residue must be important for activity.

Regulation of involucrin gene expression—a role for p300 and P/CAF

Previous studies demonstrate that the hINV promoter AP1-5 and Sp1 sites, located in the distal regulatory region of the promoter, are essential for expression of the hINV transgene in murine epidermis (Crish et al., 1993, 1998, 2002, 2006). Cell culture-based studies, using normal human keratinocytes, indicate that a protein kinase C, Ras, MEKK3, MEK3 signaling cascade, acting via regulation of a p38δ-ERK1/2 complex, acts to increase the intracellular levels of AP1 and Sp1 transcription factor, which interact at the AP1-5 site and an adjacent Sp1 site. This complex includes Fra-1, junB, junD, and Sp1 (Welter et al., 1995; Banks et al., 1998, 1999). These studies strongly suggest that p300 and P/CAF participate as part of a multiprotein complex to regulate expression via interaction at the DRR AP1-5/Sp1 element; however, we cannot completely rule out the possibility that they may bind at distinct chromosomal locations. We propose that stimulation of keratinocyte differentiation results in an increase in AP1 factor (JunB, JunD, and Fra-1) levels and enhanced binding of AP1 to the DRR region. This, in turn, leads to increased recruitment of p300 and P/CAF to this region, and subsequent deacetylation of histones and other transcriptional factors ultimately leads to increased hINV gene transcription.

MATERIALS AND METHODS

Antibodies and reagents

Rabbit polyclonal antibodies specific for the transcription factors Fra-1 (sc-605X), JunB (SC-46X), JunD (sc-74X), c-Jun (sc-1694X), and Sp1 (sc-59X) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and diluted 1:500 for immunoblot. Rabbit polyclonal antibodies specific for P/CAF (Ab12188) was purchased from Abcam Inc., (Cambridge, MA) and used for immunoblot at a dilution of 1:800. The anti-p300 mouse mAb (05-257) was purchased from Upstate Cell Signaling (Lake Placid, NY), and used for immunoblot at 2 μg/ml. Mouse monoclonal anti-FLAG M2 (F3165) and anti-FLAG M2-agarose (A2220) were obtained from Sigma (Milwaukee, WI). Mouse monoclonal anti-human β-actin (Sigma, clone AC-15) was diluted 1:8,000 for immunoblot. Horseradish peroxidase-conjugated donkey anti-rabbit IgG (NA934) was from Amersham Biosciences (Piscataway, NJ) and used for immunoblot at a dilution of 1:14,000. Phorbol ester (TPA) was purchased from Sigma.

Plasmid construction

HA-p300 and HA-p300 (del 33, a dominant-negative form of p300) were generously provided by Dr Livingston (Eckner et al., 1994). The P/CAF expression plasmid was kindly provided by Dr Nakatani (Vassilev et al., 1998). Plasmids for mammalian one- and two-hybrid assays, pFR-Luc and pCMV-BD, were purchased from Stratagene (La Jolla, CA). pFR-Luc encodes the GAL4 promoter located upstream of the luciferase gene, and pCMV-BD encodes the GAL4BD expressed by the cytomegalovirus (CMV) promoter. To construct pCMV-BD-FLAG-hFra-1-AD, the complete AD, (amino acids D131-L271) from hFra-1 (GenBank no. BC016648) was amplified by PCR using hFra-1 cDNA as template to produce FLAG-hFra-1-AD in which a FLAG epitope is inserted in-phase upstream of the hFra-1 AD sequence. This product was inserted into pCMV-BD as an EcoRI/NotI fragment. Similar methods were used to produce pCMV-BD-FLAG-hFra-1-AD131-231 and pCMV-BD-FLAG-hFra-1-AD131-188. pCMV-BD-FLAG-hFra-1-AD (T227E) and pCMV-BD-FLAG-hFra-1-AD (T227A) were produced by point mutagenesis of pCMV-BD-FLAG-hFra-1-AD using the Quick Change Mutagenesis system (Stratagene). Additional plasmids, pCMV-BD-FLAG-hFra-11-271, pCMV-BD-FLAG-hFra-11-231, pCMV-BD-FLAG-hFra-11-188, which encode truncated forms of full-length FLAG-hFra-1 were produced using similar methods.

To construct the human involucrin promoter distal regulatory region luciferase reporter plasmids, pINV-41, which encodes the hINV basal promoter (Welter et al., 1995), was restricted with KpnI/PstI and HindIII and NsiI sites was inserted as part of a KpnI/PstI linker. PCR-generated segments of the hINV DRR were subcloned into this cassette as HindIII/NsiI inserts to yield pINV-DRR(-2,473/-1,953), pINV-DRR(-2,473/1,993), pINV-DRR(-2,473/-2,043), pINV-DRR(-2,473/-2,097) and pINV-DRR(-2,473/-1,953) AP1-5mm.

Transfection

Human foreskin keratinocytes were isolated and maintained as previously described (Welter et al., 1995). All experiments used keratinocytes at the third or fourth passage. For transfection, 1 × 105 keratinocytes were plated in 2 ml of keratinocyte serum-free medium per 10 cm2 dish. At 24 hours, the cells were transfected with 2 μg of expression plasmid using 6 μl of Fugene 6 reagent (Roche, Indianapolis, IN). At 24 hours post-transfection, the medium was replaced with 2 ml fresh medium or medium containing 0.3 mm calcium or 50 ng TPA/ml. Luciferase activity was monitored at 24-72 hours post-transfection, and the results were expressed as luciferase activity/μg protein. Human keratinocytes were obtained with approval of the human studies review panel and all procedures follow the Declaration of Helsinki Principles.

Retrovirus production and construction of FLAG-hFra-1 HeLa Z cells

pLNCX2-FLAG-hFra1 was constructed be inserting the human Fra-1 coding sequence, kindly provided by Dr D Chalbos (Philips et al., 1998), into pLNCX2 retrovirus (BD Biosciences, San Jose, CA). The hFra-1 cDNA was amplified as an XhoI/ClaI fragment and cloned into pGEMT (Promega, Madison, WI). The upstream primer encoded the XhoI site and a FLAG epitope, and the downstream primer encoded a ClaI site. The PCR-generated product was then cloned into pGEMT (Promega) to yield pGEMT-FLAG-hFra-1. The FLAG-Fra-1 fragment was released with XhoI/ClaI and inserted into XhoI/ClaI-restricted pLNCX2 to generate pLNCX2-FLAG-hFra-1. The structure of was confirmed by DNA sequence analysis. AmphoPack-293 retrovirus packaging cells (BD Bioscience) were cultured in DMEM supplemented with 10% fetal calf serum, 4 mm l-glutamine, 1 mm sodium pyruvate, 100 U/ml penicillin, and 100 μg/ml streptomycin. To produce virus, AmphoPack 293 cells (1 × 106 cells/50 cm2 dish) were transfected with 10 μg of pLNCX2 or pLNCX2-FLAG-hFra1 vector and the cells were maintained in 5 ml of medium (DMEM containing 10% fetal bovine serum and l-glutamine). The culture medium was aspirated at 10 hours post-transfection, replaced with 10 ml of fresh medium, and the cells were incubated for 48 hours at 37°C. The resulting virus-containing medium was used for infection of HeLa-Z cells.

For this purpose, 1 ml of this medium was mixed with 1 ml of fresh medium (DMEM containing 10% fetal bovine serum), polybrene was added to a final concentration of 5 μg/μl, and the mixture was added to 60% confluent HeLa-Z cells and incubated for 2.5 hours at 37°C with occasional mixing. An additional 8 ml of medium was subsequently added, and the cells were incubated for an additional 2 days. The cells were then selected with 0.5 mg/ml G418 (Gibco, Pittsburgh, PA) and candidate clones were isolated using cloning rings and positively identified as FLAG-hFra-1 producers by immunoblot using mouse monoclonal M2 anti-FLAG (Sigma, 01865). These clones were then expanded.

Bulk culture of FLAG-hFra-1 HeLa-Z cells and preparation of HeLa-Z nuclear extracts

Approximately 2 × 106 FLAG-Fra-1 expressing HeLa-Z cells, were grown attached in a 50 cm2 dish in 30 ml of growth medium (DMEM containing 10% fetal bovine serum and l-glutamine). The cells were harvested and transferred to suspension culture in 250 ml of Joklik media (Sigma, M0518) that was supplemented with 10% fetal bovine serum (Sigma, F- 6178) and 0.5 mg/ml G418 (Gibco, 10131-035). The culture was maintained at 37°C with stirring for 5 days. The suspension was then transferred to 3 l of G418-free medium and growth, with stirring, was continued at 37°C for an additional 14 days. The cells were then collected by centrifugation at 1,000 × g, washed twice with 0.2 mm phenylmethylsulfonyl fluoride-containing phosphate-buffered saline, and resuspended in 30 ml of ice-cold hypotonic buffer (10 mm Tris-HCl, pH 7.3, 10 mm KCl, 1.5 mm MgCl2). All subsequent steps were performed at 4°C.

Nuclear extracts were prepared using the procedure of Dignam (Dignam et al., 1983) and 2.5 mg of nuclear extract, in 5 ml, was loaded onto a 6 ml P11 phosphocellulose column (Whatman, Kent, OH) at a flow rate of 1 ml/min. The column was then equilibrated with 2 column volumes of equilibration buffer (20 mm Tris-HCl, pH 7.9, 0.2 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, 1 mm DTT, and 0.1 m KCl and 20% glycerol), and bound proteins were eluted by increasing the concentration of KCl in 0.1 m increments (Kershnar et al., 1998). The fractions were collected and stored frozen at -80°C.

Immunological analysis

Equal amounts of protein were separated by electrophoresis on 5 or 10% denaturing polyacrylamide gels, and transferred to PROTRAN BA83 nitrocellulose membrane (Schleicher and Schuell, Keene, NH). The membranes were blocked in 10 mm Tris-HCl, pH 7.2, containing 100 mm NaCl, 0.1% Tween-20, and 5% nonfat dry milk, and then incubated overnight at 4°C with primary antibody. The blot was washed and then exposed to an appropriate horseradish peroxidase-conjugated secondary antibody, and bands were visualized using a chemiluminescent detection system (Amersham Biosciences).

ChIP analysis

Human keratinocytes were grown to a final concentration of 3 × 106 cells per 150 mm plate and chemically crosslinked with formaldehyde (Fisher Scientific, Grand Island, NY) at a final concentration of 1%. After 15 minutes at 25°C, fixation was stopped by the addition of glycine to a final concentration of 0.125 m. The cells were rinsed twice with cold phosphate-buffered saline, harvested, and incubated on ice for 15 minutes in 0.3 ml of cell lysis buffer containing 5 mm Pipes-KOH, pH 8, 85 mm KCl, 0.5% NP-40, 0.5 mm phenylmethylsulfonyl fluoride, and 1 μg/ml leupeptin and 1 μg/ml aprotinin (Solomon and Varshavsky, 1985). The nuclei were collected at 6,000 × g and resuspended in 200 μl of sonication buffer containing 50 mm Tris-HCl, pH 8, 10 mm EDTA, and 1% SDS. Following incubation on ice for 10 min, the nuclei were sonicated six times for 10 seconds each on setting four with a Sonic Dismembrator-60 (Fisher Scientific) and centrifuged at 15,000 × g for 10 minutes at 4°C. The chromatin-containing supernatant was diluted five-fold with ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mm EDTA, 16.7 mm Tris-HCl, pH 8.1, 167 mm NaCl) and then precleared by addition of 60 μl of salmon sperm DNA/Protein A beads (Upstate Cell Signaling) for 30 minutes at 4°C, followed by centrifugation at 15,000 × g for 5 minutes.

Before immunoprecipitation, an aliquot of extract was set aside representing 1% of the material used for immunoprecipitation. For immunoprecipitation, the cleared supernatant was incubated overnight at 4°C with 5 μg of either anti-Fra1 (Santa Cruz, sc-605X) or anti-p300 (Upstate Cell Signaling, 05-257). The immune complex was collected by binding to 60 μl of salmon sperm DNA/protein A beads for 1 hour at 4°C with rotation. Washing and elution of immune complex were carried out as previously described (Gilmour et al., 1991). Briefly, the beads were collected by centrifugation at 3,000 × g for 5 minutes and washed sequentially with low-salt buffer (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris-HCl, pH 8.1, 150 mm NaCl), high-salt buffer (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris-HCl, pH 8.1, 500 mm NaCl), LiCl immune complex wash buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1mm EDTA, 10 mm Tris-HCl, pH 8.1, and finally in TE (10 mm Tris-HCl, pH 8, 1 mm EDTA). Crosslinks were reversed by addition of 250 μl of elution buffer (1% SDS, 0.1 M NaHCO3) and incubation for 15 minutes with rotation at 25°C, followed by heating at 65°C for 4 hours. The released DNA was then precipitated by addition of 2.5 volumes of ethanol and incubation at -20°C overnight. The hINV gene promoter sequence was detected by PCR using the promoter-specific upstream (5′-TCAGCCCTAGAATGTTGAGG) and downstream (5′-TCATGACCTCTCTGTTCC) primers to amplify nucleotides -2,282/-1,813 of the hINV promoter. The 470-bp PCR product was separated by electrophoresis on a 1% agarose gel and detected using ethidium bromide.

Co-immunoprecipitation analysis

Nuclear extract (5 μg/μl) was diluted 1:1 with Tris-buffered saline (50 mm Tris-HCl, 150 mm NaCl, pH 7.4) containing 0.2 mm phenylmethylsulfonyl fluoride and 1 μg/ml each of aprotinin, leupeptin, and pepstatin, and incubated with 40 μl of a 50% slurry of anti-FLAG M2-agarose (Sigma) for 2 hours at 4°C with gentle rotation. Immune complexes were then collected by centrifugation at 1,000 × g for 2 minutes and washed three times with modified radioimmunoprecipitation assay buffer (20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100). The immune complexes were eluted from the M2-agarose with Laemmli sample buffer, and separated by denaturing and reducing PAGE. Proteins were transferred onto Protran BA 83 membrane (Whatman) and incubated with the appropriate primary and secondary antibodies, and antibody binding was visualized using chemiluminescence detection reagents (Amersham Pharmacia, Piscataway, NJ).

ACKNOWLEDGMENTS

This work was supported by a National Institutes of Health grant (RLE) and utilized the facilities at the Skin Diseases Research Center of Northeast Ohio (NIH AR39750).

Abbreviations

AD

activation domain

CBP

CREB binding protein

ChIP

chromatin immunoprecipitation

CMV

cytomegalovirus

DRR

distal regulatory region

HAT

histone acetyltransferase

TPA

12-O-tetradeconylphorbol-13-acetate

Footnotes

CONFLICT OF INTEREST

The authors state no conflict of interest.

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