From the
Loricrin gene expression is limited to terminally
differentiating keratinocytes of stratified squamous epithelia. To
define the regulatory elements that mediate the expression of the
loricrin gene, we replaced the loricrin coding sequences from a
6.5-kilobase genomic fragment with the chloramphenicol
acetyltransferase gene and transfected this construct into cultured
mouse keratinocytes. High expression levels were observed in both
undifferentiated as well as differentiating cells. Transgenic mice
bearing a similar construct, but with
Mammalian epidermis undergoes a progressive differentiation to
form a mature stratified squamous epithelium. Four histologically
distinct cell layers can be identified in the epidermis, each layer
expressing a characteristic set of marker proteins specific for the
maturation state (Eckert, 1989). Keratinocytes of basal phenotype
express keratins K5
Loricrin, a major protein of
the cell envelope, is first found in granular cells, and along with
filaggrin, is one of the most predominant markers for late epidermal
differentiation. In situ hybridization has shown that loricrin
transcripts are localized only in the upper spinous and granular layers
of the epidermis (Mehrel et al., 1990; Hohl et al.,
1991a). In mouse epidermis the loricrin protein is first localized as
small, non-membrane bound granules known as L-granules (Steven
et al., 1990). Through an unknown mechanism, loricrin is first
released from the granular aggregates and is then deposited and
cross-linked by transglutaminases as a dense marginal band on the
inside of the plasma membrane of the corneocytes (Hohl, 1990).
Recent studies have begun to investigate the mechanisms regulating
epidermal differentiation. Calcium is thought to be a major regulator
of epidermal differentiation. A calcium gradient has been identified in
the epidermis, with the lowest calcium concentrations in basal cells,
and concentrations increasing through the spinous and granular layers
(Malmqvist et al., 1984; Menon et al., 1985). In
vitro studies have shown that keratinocytes maintain their
proliferative, basal phenotype when cultured in medium containing less
than 0.1 mM calcium. When the medium contained 0.1 mM
calcium or higher, cells were induced to differentiate, resulting in
the loss of proliferative capacity and an induction of terminal
differentiation markers (Hennings et al., 1980), suggesting
that calcium concentration could regulate the transcription of
epidermal differentiation markers in vitro. More recently
loricrin transcription has been shown to be up-regulated by calcium,
and repressed by retinoic acid in vitro (Hohl et al.,
1991b). These same factors that affect loricrin transcription in
vitro also affect the transcription of filaggrin and the
differentiation-specific keratins K1 and K10 (Kopan et al.,
1987; Roop et al., 1987; Yuspa et al., 1989; Fleckman
et al., 1989; Hohl et al., 1991b). Furthermore, the
protein kinase C pathway has been implicated in the induction of
epidermal differentiation by calcium (Dlugosz and Yuspa, 1993).
The
most studied differentiation-specific epidermal gene is HK1, one of the
first genes up-regulated during the induction of terminal
differentiation. A region in the 3`-flanking sequence of the HK1 gene
was determined to be responsible for the calcium induction of the gene
(Huff et al., 1993; Rothnagel et al., 1993). Recent
detailed molecular analysis of this 3`-element has identified the
cis-elements of the calcium response element (Lu et al.,
1994). This element contains a composite AP-1/steroid hormone binding
site and functions to modulate the differentiation-specific expression
of the HK1 gene in vitro. Since the K1 gene is one of the
first genes up-regulated during the differentiation process, we were
interested in comparing the regulation of this gene to the regulation
of a gene that was induced late in epidermal differentiation. The
loricrin gene was a good candidate since it is not transcribed until
the cells reach the late spinous and early granular cell layers. The
mouse loricrin gene has recently been cloned and characterized
(Rothnagel et al., 1994). In order to better understand the
regulation of loricrin, a gene up-regulated late in epidermal
differentiation, we have created reporter constructs using the
chloramphenicol acetyltransferase (CAT) gene and the
The construction of the
loricrin reporter constructs were carried out in two parts as follows.
A ClaI site was introduced at the stop codon by PCR
amplification using the primers 5`-CCATCGATAAGGTCACCGGGTTGC-3` and
5`-CACTTCCCTATCCTTTCT-3`. The PCR product of 1410 bp was digested with
PstI and ClaI, yielding a 170-bp fragment which was
then cloned into the 3`-flanking sequence of the loricrin gene.
Similarly a ClaI site was introduced at the translation start
codon by PCR amplification using the primers 5`-CTCTAGATTCCGTAAGGC-3`
and 5`-CCATCGATGTTGTTTAAGGAGAAG-3`. The 1470-bp product was cut with
ClaI and PstI and the resulting 830-bp product cloned
into the 5`-flanking sequences of the loricrin gene. The 3` sequences
were cloned adjacent to the 5` sequences creating a loricrin expression
vector as shown in Fig. 1 a. A CAT reporter and a
The tissues analyzed for
For DNase
I footprint analysis, all initial binding steps were carried out as
above with the exception that only 100 ng of poly(dI-dC) was added to
the reaction. After the 10-min incubation with protein, the probe was
digested for 30 s with 0.5-15 µg of DNase I (Worthington
Biochemical Corp., Freehold NJ). Digestion was stopped by the addition
of 100 µl of 10 mM EDTA, 0.1% SDS, 50 µg/ml proteinase
K, and 1.5 µg of sonicated salmon sperm, followed by a 30-min
incubation at 37 °C. Samples were extracted with phenol/chloroform
followed by ethanol precipitation. Samples were resuspended in 3 µl
of formamide, heated to 90 °C for 5 min, then loaded on a 7%
denaturing polyacrylamide gel and run at 65 watts. Gels were dried onto
Whatman 3MM paper for 1 h at 80 °C followed by autoradiography.
Since
neither the 5`- nor 3`-flanking region could enhance CAT expression
from a heterologous promoter, a CAT reporter gene was introduced into
the 6.5-kb fragment, replacing the loricrin coding sequences and
leaving both 5`- and 3`-flanking and noncoding sequences intact
(Fig. 1 A). This construct, pML(6.5)CAT, was analyzed by
transient transfection into primary murine keratinocytes. High levels
of CAT activity were observed in cells cultured under conditions that
promote differentiation (0.35 mM calcium) as well as in
undifferentiated cells maintained in low calcium (0.05 mM)
culture conditions (Fig. 1 B). The high activity of this
construct in low calcium does not correspond to the expression of the
endogenous loricrin gene, which has been shown previously to be
expressed only in differentiated keratinocytes cultured in media with
calcium levels of 0.12 mM and higher (Hohl et al.,
1991b). Significantly, the pML(6.5)CAT reporter construct was not
active in transiently transfected primary murine dermal fibroblasts
(data not shown).
From this study, we can conclude that the correct
transcription of the mouse loricrin gene in vitro and in
vivo requires the synergistic effects of elements in both the
5`-proximal promoter and a distal element(s) not present in the 6.5-kb
sequences. In addition, the elements which direct cell type specificity
of the promoter can be uncoupled from those regulatory elements
required to give differentiation-specific expression. The elements
required to give cell and tissue type specificity are contained within
a 6.5-kb genomic fragment, but this fragment lacks the elements
required to direct correct expression with respect to differentiation
state. In addition, the loricrin proximal promoter contains an
evolutionarily conserved, functional AP-1 element which is absolutely
necessary for the transcription of the loricrin gene in vitro.
Previous work has shown that transcription of the loricrin gene
in vitro can be regulated by calcium, retinoic acid, and cell
density (Hohl et al., 1991b). To gain a greater understanding
of this regulation at a molecular level, various 5` and 3` fragments
from a 6.5-kb mouse genomic loricrin construct were cloned into a
heterologous SV40 promoter CAT construct and assayed by transient
transfection into primary murine keratinocytes. These fragments were
unable to enhance significant CAT expression from the heterologous
promoter. Since previous studies on the HK1 gene suggested that both 5`
and 3` elements are necessary for efficient expression of the reporter
gene in vitro (Rothnagel et al., 1993), and work on
the HK14 gene showed the importance of distal elements in the
regulation of this gene (Leask et al., 1990), we constructed a
composite pML(6.5)CAT reporter containing both 5`- and 3`-flanking
sequences to ensure expression in the event that transcription of the
loricrin gene required synergistic effects of both 5` and 3` regulatory
elements. When this expression vector containing a CAT reporter was
transfected into primary keratinocytes and analyzed in low and high
calcium medium, we observed surprisingly high activity in cells
cultured to promote both basal and differentiated phenotypes. Previous
reports, examining endogenous loricrin expression in vitro,
showed that loricrin transcription was only activated when cells were
induced to differentiate, at calcium levels above 0.12 mM
(Hohl et al., 1991b). In addition, in situ hybridization and immunofluorescence data has previously shown
that the loricrin gene is expressed in the granular layer of the
epidermis in vivo (Mehrel et al., 1990). When a
Recent work on the human loricrin gene by Yoneda and
Steinert (1993) has shown that 9 kb of 5`-flanking sequence and 9 kb of
3`-sequence could direct tissue and differentiation specific expression
of a human transgene in transgenic mice. It should also be noted that
data from our laboratory, which will be reported elsewhere, indicates
that the distal element(s) which restricts expression of the mouse
loricrin gene to the granular layer is located within additional
sequences found in a larger 14-kb mouse genomic fragment.
In order
to localize the regulatory elements required to drive expression in
keratinocytes, a series of deletions were made to the 5`-flanking
sequences. These 5` deletions showed that deleting up to 60 bp 5` from
the initiation of transcription in the basal promoter resulted in no
reduction of activity. It was not until the remaining 60 bp of
5`-flanking sequence, including the TATA box and the first exon were
removed by deleting into the intron sequence, that a reduction in CAT
activity was observed (Fig. 3).
It was interesting to find
that the 5` promoter contained an AP-1 site which is perfectly
conserved between the human and mouse loricrin genes (Rothnagel et
al., 1994). This AP-1 site is retained in the most active 5`
deletion (p
While some data point
to AP-2 as well as other transcription factors such as Sp1 and the Pou
proteins as regulators of keratinocyte gene expression (Leask et
al., 1990, 1991; Andersen et al., 1993; Magnaldo et
al., 1993a, 1993b; Yukawa et al., 1993; Faus et
al., 1994;), and recent reports indicate the possible presence of
a keratinocyte-specific transcription factor termed basonuclin (Tseng
and Green, 1992, 1994), several lines of independent data are now
pointing to the AP-1 family of transcription factors as important
regulators of epidermal differentiation-specific gene expression. The
identification of functional AP-1 elements in genes of differentiation-
specific expression coincides well with the demonstrated expression of
AP-1 factors within the epidermis. Subpopulations of basal cells in the
epidermis have been shown to express c- jun mRNA, while the
differentiation of the epidermis is accompanied by an induction of
c-Fos and JunB expression (Wilkenson et al., 1989; Fisher
et al., 1991; Smeyne et al., 1992). These
observations suggest the differential expression of these and possibly
other AP-1 transcription factors are involved in regulating the
expression of differentiation-specific genes in the epidermis.
As
previously stated, the K1 gene is normally expressed in the
differentiated suprabasal cells of the epidermis. Like the loricrin
promoter, we have shown that a portion of the HK1 promoter can direct
tissue-specific expression of a reporter gene, yet the expression of
the HK1 promoter is promiscuous and present in a portion of basal cells
(Chung et al., 1994). This laboratory has evidence to show
that the elements that repress HK1 expression in basal cells and
restrict expression to cells above the basal layer are responsive to
retinoic acid and these elements lie some 6 kb upstream from the HK1
promoter.
While the most intuitive argument would be the additional elements
located outside the 6.5-kb clone are acting as repressors (the gene is
on by default and off in basal and spinous cells in the presence of
additional sequences), this may not necessarily be the case. Byrne and
Fuchs (1993) have published data on the expression of a
Clearly, a complex
interplay of different transcription factors are involved in the
regulation and expression of epidermal genes during both proliferation
and differentiation, and as more information is gathered, it will be
interesting to compare the tissue- and differentiation-specific
elements regulating these epidermal genes.
We thank Rachel Welborn for her help with figure
preparation, and Bo Lu for his expert advise.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-galactosidase as the
reporter gene, corroborated these in vitro findings and showed
tissue- and cell type-specific, but not differentiation-specific
expression. Deletion analysis of the promoter region determined that
sequences up to -60 base pairs from the start of transcription
could be removed without significant loss of promoter activity. Within
these proximal 60 base pairs is an evolutionarily conserved AP-1
element that is recognized by both purified c-Jun and AP-1 factors from
keratinocytes in vitro. Mutation of this AP-1 site abolished
the activity of the loricrin promoter. These studies show that elements
directing expression of the loricrin gene to the stratified squamous
epithelia are contained within a 6.5-kilobase genomic fragment, and
those elements required to restrict expression to differentiated
keratinocytes lie outside this region.
(
)
and K14 (Fuchs and Green,
1980; Woodcock-Mitchell et al., 1982). In the epidermis,
terminal differentiation begins in the basal layer, where once induced
to differentiate, keratinocytes down-regulate K5 and K14 and
up-regulate several major proteins, including keratins K1 and K10 (Roop
et al., 1983). Differentiation continues in sequential order
through the spinous and granular layers, where cells express the
differentiation-specific proteins filaggrin and loricrin (Rothnagel
et al., 1987; Mehrel et al., 1990). Keratinocyte
differentiation finally terminates with the cells loosing internal
organelles, forming a thickened intracellular cell envelope, and an
intercellular lipid multilamellar structure in the stratum corneum
prior to desquamation as corneocytes.
-galactosidase (
-gal) gene linked to 1.5 kb of 5`-flanking
sequence and 2.2 kb of 3`-flanking sequence from the mouse loricrin
gene. Regulatory elements within these sequences can direct cell- and
tissue-specific expression both in vitro and in vivo.
We show that loricrin transcription in vitro requires binding
of protein factors to an AP-1 consensus site in the loricrin proximal
promoter sequences, and that tissue-specific expression can be
uncoupled from differentiation-specific expression both in vitro and in vivo.
Plasmids, Constructs, and PCR Cloning
All
transfections employed pSVCAT (Gorman et al.,
1982) as the positive control. Fragments to be tested for enhancer
activity were cloned into pA10CAT
(Laimins et al.,
1982). The 3.4-kb 5` BamHI and the 3.1-kb 3` BamHI
fragments were cloned separately into the BamHI site of
pGEM7Z. These BamHI fragments were also cloned into the
BamHI and BglII sites of pA10CAT
.
Digestion of the clones with HindIII (from the pGEM7Z
polylinker) and PstI released 1.9-kb 5` and 2.2-kb 3`
fragments, which were cloned into the HindIII and
PstI sites of pGEM3Z. Digestion of these clones with
BamHI allowed insertion of the fragments into the
BamHI and BglII sites of pA10CAT
. These
5` BamHI and BamHI/ PstI, and the 3`
BamHI and PstI/ BamHI fragments were inserted
in both orientations relative to CAT.
-galactosidase reporter with ClaI ends (generated by PCR)
was subsequently cloned into the unique ClaI site to create
the pML(6.5)CAT vector and the pML(6.5)
-gal vector. All 5`
deletion constructs were generated utilizing unique restriction sites
in the flanking sequence and named by the size of the deletion in base
pairs (p
990, KpnI; p
1480, BclI; p
2230,
EcoRV; p
2610, NsiI).
Figure 1:
A, schematic showing the
organization of the mouse loricrin gene and the pML(6.5) lacking the
loricrin coding sequences. The 6.5-kb genomic loricrin clone contains
1.5 kb of 5`-flanking sequence, a small noncoding exon, a 1.1-kb
intron, a single coding exon, and 2.2 kb of 3`-flanking sequence
(Rothnagel et al.,1994). Using PCR site-directed
mutagenesis techniques, the entire loricrin coding region was deleted
from the 6.5-kb genomic fragment and a unique ClaI site was
introduced, leaving all other sequences intact. The ClaI
restriction site was used to introduce the reporter genes to produce
the pML(6.5)CAT and pML(6.5) -gal constructs. B,
BamHI; K, KpnI; P, PstI;
S, SacI. B, the pML(6.5)CAT reporter was
transiently transfected into primary murine keratinocytes in low (0.05
mM) and high (0.35 mM) calcium medium. Lane
1, pSV
CAT, high calcium; Lane 2,
pA10CAT
, high calcium; Lane 3, pML(6.5)CAT, low
calcium; Lane 4, pML(6.5)CAT, high calcium. C, expression of pML(6.5)
-gal in the epidermis of neonatal
transgenic mice. Note expression is present throughout the epidermis,
from the basal cell layer through to the cornified layers, but not in
the underlying dermis.
To test the
functionality of the AP-1 site in the proximal promoter, it was mutated
from TGAGTCA to TCCCGGG, creating a SmaI restriction enzyme
site using PCR primers as follows. The 5` reaction used the primer
5`-CCCCGGGATCTATTGATCAGGCACC-3` and a primer 5` to the BclI
site, 5`-AGTAAAGTCAGTAGTCAG-3`, generating a 610-bp fragment. The 3`
reaction used the primer 5`-CAATAGATCCCGGGG-3` and a primer 3` to the
EcoRV site, 5`-CTCCACCAGAGGTCTTTCC-3`, generating a product of
1290 bp. The products of these two reactions were purified and used in
a third reaction along with the two external primers to generate a
single combined product of 1900 bp. This product was purified and
digested with BclI and EcoRV and the fragment
containing the mutated AP-1 site re-cloned into the pML(6.5)CAT vector.
The resulting mutant promoter was subsequently sequenced to ensure that
spacing and all other sequences in this region were intact.
Cell Culture, Transfections, and CAT
Assays
Preparation of mouse primary keratinocyte and primary
dermal fibroblast cultures and their transfections were carried out as
described by Rothnagel et al. (1993). Keratinocytes and
fibroblasts (as controls) were transfected with 10 µg of plasmid
DNA/60-mm dish, with 20 µg of sonicated salmon sperm as a carrier,
with the exception of the 5` RACE experiment, where 50 µg of
plasmid p2230 DNA was transfected. Cells were harvested 4 days
post-transfection and the CAT assay performed as described previously
(Harper et al., 1988), without a sonication step. CAT assays
were normalized using an equal amount of protein from each extract.
Thin layer chromatography was performed using silica gel chromatogram
sheets in 95% chloroform and 5% methanol, followed by autoradiography.
Chromatograms were later quantitated using a Betascope blot analyzer
(model 603, Betagen Corp., Mountain View, CA). Experiments were
repeated at least three times and in duplicate.
5` RACE-PCR Amplification and Sequencing
Four days
post-transfection with p2230, total RNA was extracted from the
cultures using the RNAzol B method (Biotecx Laboratories, Inc.,
Houston, TX). The 5` rapid amplification of cDNA ends was carried out
utilizing the 5` RACE System (Life Technologies, Inc., Gaithersburg,
MD) according to the manufacture's instructions. One µg of
total RNA extracted from the transfected cells was used in the first
strand synthesis reaction using a primer specific to the CAT coding
region (5`-ACGAAATTCCGGATGAGC-3`). The resulting cDNA was then
C-tailed. The PCR reaction was carried out using the supplied anchor
primer and a primer specific to the loricrin intron sequence
(5`-CCATCGATGTTGTTTAAGGAGAAG-3`). The first round of amplification was
carried out using 5 µl of C-tailed DNA and 200 ng of each primer in
a total 50-µl reaction. The product of this amplification was
purified and 1 µl removed and used for an additional round of
amplification. In all cases the reaction conditions were 92 °C for
5 min; then 30 cycles of 92 °C for 30 s, 55 °C for 30 s, 72
°C for 30 s, followed by 72 °C for 5 min. A product of
approximately 100 bp was cloned into a TA cloning vector (Invitrogen,
San Diego, CA) and sequenced using Sequenase 2.0 (U. S. Biochemical
Corp., Cleveland, OH) (Fig. 4).
Figure 4:
Analysis of the transcription start site
of the p2230 deletion construct. The p
2230 deletion CAT
construct was transiently transfected into primary murine
keratinocytes, total RNA isolated, and RACE-PCR used to amplify the 5`
ends of p
2230 transcripts. The boxed ATG denotes the
start of the loricrin coding region. The intron-exon boundary is
indicated by the curved arrow. Alignment of the amplified
sequence ( underlined) with the loricrin gene showed that
transcripts were initiated from within the intron. This analysis
revealed a mRNA CAP site (-90 bp relative to the start site), a
TATA Box (-128 bp), and a CAAT box (-191 bp) present within
the intron of the mouse loricrin gene.
Generation and Identification of Transgenic
Mice
The pML(6.5) -gal construct was released from pGem7Z
by digestion with ApaI. The fragment was isolated by agarose
gel electrophoresis and purified (NA45 paper; Schleicher & Schuell,
Keene, NH). Mouse embryos were isolated (Hogan et al., 1986)
and the DNA microinjected into the pronuclei of one cell mouse embryos
obtained from female ICR (Charles River) mice mated to FVB males
(Frederick Cancer Research Facility). The embryos were then transferred
to the oviduct of pseudo-pregnant females and normal gestation allowed.
Positive transgenic mice were identified by tail DNA isolation (Hogan
et al., 1986) and PCR amplification. Positive founder
transgenics carrying the construct were detected by PCR analysis of
genomic DNA isolated from tail biopsies. Primers
5`-CCCGGGTTCGAATTATTTTTGACACCAGAC-3` and 5`-CTCACGCGTGGCAGC-3` specific
to the
-galactosidase gene were used for analysis. Expression
levels were analyzed by staining ear sections from the transgenics for
-gal activity.
Tissue samples for
sectioning were fixed for 1-2 h at 4 °C in 2%
paraformaldehyde, 0.2% glutaraldehyde, 0.01% sodium deoxycholate, 0.02%
Nonidet P-40, in 0.05 M sodium phosphate buffer, pH 7.4. The
tissue was then washed twice in phosphate-buffered saline containing 2
mM MgCl-Galactosidase Assay
at room temperature, followed by
5-bromo-4-chloro-3-indoyl
-D-galactoside staining at 30
°C in 0.3 M sodium phosphate buffer, pH 7.4, containing 43
mM MgCl
, 10 mM
K
Fe(CN)
, 10 mM
K
Fe(CN)
, 0.03% sodium deoxycholate, 0.06%
Nonidet P-40, 3.3 mg/ml 5-bromo-4-chloro-3-indoyl
-D-galactoside. After staining the tissue was washed in
phosphate-buffered saline containing 3% Me
SO and then
stored in phosphate-buffered saline until embedded in paraffin and
sectioned.
-gal activity by
chemiluminescence assay were excised from transgenic or non-transgenic
control animals, frozen in liquid nitrogen, and ground using a mortar
and pestle. The cells were lysed by three rounds of freeze/thaw,
followed by vortexing. The samples were centrifuged and the protein
extract concentrations measured using the Bradford assay. For each
tissue, 10 µg of protein extract was used in the Galacto-Light
(Tropix, Inc., Bedford, MA) reporter assay system without modification.
Samples were analyzed in a Lumat LB 9501-1 with built-in injector
(Wallac Inc., Gaithersburg, MD).
Preparation of Oligonucleotides and Probes for Binding
Assays
The 27-bp probe containing the AP-1 site from the
loricrin promoter used in the gel shift assays was generated using an
oligo synthesizer. The top strand, 5`-GATCTATAGATGAGTCAGAGCAG-3`, was
annealed to the lower strand, 5`-GATCCTGCTCTGACTCATCTATA-3`, and the
overlapping ends filled in with P-radionucleotides using
Klenow enzyme. The 200-bp probe encompassing the loricrin proximal
promoter region used in the DNase I footprinting assays was generated
by PCR using primers with either BamHI (5`) or BglII
(3`) restriction enzyme sites. After amplification, the PCR product was
cut with either BamHI or BglII and labeled at one end
with
P-radionucleotides by Klenow fill-in. All probes were
purified by native polyacrylamide gel electrophoresis prior to
analysis.
Preparation of Whole Cell Protein Extracts
Primary
murine keratinocytes were prepared and cultured as described above.
Whole cell extracts were prepared using a modified procedure of
Zimarino and Wu (1987). Frozen cell pellets were resuspended and lysed
in 1 volume of Wu buffer (10 mM HEPES, pH 7.9, 1.5 mM
MgCl , 0.1 mM EGTA, 0.5 mM
dithiothreitol, 5% glycerol) with protease inhibitors, containing 400
mM KCl and centrifuged at 14,000
g for 30 min
at 4 °C. One ml of the supernatant was added to 250 µl of
saturated (at 0 °C) ammonium sulfate, and incubated on ice for 20
min, then centrifuged 14,000
g for 10 min. The
supernatant was removed, and 400-µl aliquots were added to 600
µl of saturated ammonium sulfate, incubated on ice for 20 min, and
centrifuged at 14,000
g for 10 min. The pellet was
resuspended in 100 mM KCl/Wu buffer and dialyzed 1 h in a
microdialysis unit (Life Technologies, Inc.) versus 1 liter of
100 mM KCl/Wu buffer.
Electrophoretic Mobility Shift Assays (EMSA) and DNase I
Footprinting
All steps were carried out at room temperature
unless noted otherwise. For gel shift assays, whole cell extracts,
purified c-Jun protein (Promega, Madison, WI), and HeLa cell nuclear
extracts (Promega) were preincubated with 2.5 µg of poly(dI-dC)
(Pharmacia) and unlabeled competitor oligonucleotides for 10 min. Ten
microliters of binding mixture (2.5 mg of bovine serum albumin/ml, 8%
Ficoll, 0.1 mg of pd(N)/ml (Pharmacia)) containing 50,000
cpm of
P-labeled probe was added and binding allowed to
proceed for 10 min. For supershift assays, rabbit polyclonal anti-JunD
antibodies (Santa Cruz Biotechnology) were then added and binding
allowed to proceed for an additional 20 min. Samples were analyzed on a
6% native polyacrylamide gel run at 200 V, then dried for 45 min at 80
°C onto Whatman 3MM paper followed by autoradiography.
Analysis of the Loricrin Promoter in Cultured
Cells
The cloning and characterization of the 6.5-kb genomic
loricrin fragment which were used in these studies have been reported
elsewhere (Rothnagel et al., 1994). This fragment contains the
entire loricrin coding sequence, a 1.1-kb intron located within the 5`
noncoding region, 1.5 kb of 5`-flanking and 2.2 kb of 3`-flanking
sequences (Fig. 1 A). In an initial survey for the
putative regulatory regions involved in loricrin expression, various
subfragments of the genomic clone were introduced into the enhancerless
reporter construct pA10CAT. Individual fragments consisted
of the 5` BamHI- PstI, and
BamHI- BamHI; and the 3` BamHI- BamHI
and PstI- BamHI fragments (see
Fig. 1A). These fragments contained flanking sequences
as well as intronic and coding sequences and were cloned into
BglII and BamHI sites of pA10CAT
in both
orientations relative to CAT (see ``Materials and Methods'').
Interestingly, none of these fragments were able to appreciably enhance
CAT activity in primary mouse keratinocytes (data not shown).
Analysis of the Loricrin Promoter in Transgenic
Mice
To determine the in vivo expression pattern of the
loricrin expression vector derived from the 6.5-kb genomic fragment, a
-gal reporter gene was inserted into this fragment at the unique
ClaI site and introduced into the germline of transgenic mice
(Fig. 1 A). Four founder lines which expressed high
levels of the pML(6.5)
-gal reporter transgene were identified and
used for subsequent analysis. An examination of
-gal-stained
epidermal sections from these mice revealed strong expression of the
transgene not only in the differentiated suprabasal layers but also in
undifferentiated basal layer keratinocytes (Fig. 1 C).
This expression pattern was consistent with the in vitro data
which showed that the 6.5-kb loricrin construct was expressed in
undifferentiated keratinocytes. Expression of the
-gal transgene
was only detected in tissues normally expressing loricrin, including
skin, tongue, and forestomach. The expression in tongue and forestomach
was focal and relatively weak compared to the expression detected in
the epidermis. This may indicate that additional cis-elements located
outside the 6.5-kb fragment are required for complete expression in
these tissues. Notably,
-gal staining was limited to these
epithelial tissues and not detected in any other tissues. Expression of
the
-gal transgene did not produce any observable pathology.
Control epidermis from a non-transgenic sibling did not stain for
-gal activity. An additional tissue survey of transgene expression
was performed using a more sensitive chemiluminescence assay, found
significantly higher
-gal activity in crude protein extracts from
transgenic skin and tongue, but not in tissues that do not express
loricrin such as brain, liver, kidney, muscle, or heart (Fig. 2).
Therefore, the regulatory elements present within the 6.5-kb loricrin
construct could direct tissue-specific expression of the transgene, but
is missing those elements which direct expression to the correct cell
layer within the epidermis.
Figure 2:
Relative -gal activity in various
tissues from transgenics expressing pML(6.5)
-gal. Using a
chemiluminescence assay, the relative
-gal activity of protein
extracts from: H, heart; M, muscle; K,
kidney; L, liver; B, brain; S, skin; and
T, tongue from pML(6.5)
-gal transgenic mice were
compared to non-transgenic control mice. Only skin and tongue, which
normally express loricrin, showed a significant increase in
-gal
activity over control tissues. Values shown are an average of three
normal and three control mice, with standard deviations less than
20%.
Analysis of Deletion Constructs
In an effort to
locate important regulatory sequences involved in the expression of
loricrin in keratinocytes in vitro, a series of 5` deletions
were made to the pML(6.5)CAT construct using unique restriction sites
within the 5`-flanking region and designated by the number of bases
removed (Fig. 3). These constructs were analyzed by transient
transfection into primary murine keratinocytes cultured in both
undifferentiating (0.05 mM Ca) and
differentiating (0.35 mM Ca
) culture
conditions. This analysis revealed that deletions up to 60 bp 5` from
the transcriptional start site were well tolerated and retained very
high levels of promoter activity (p
990 and p
1480 in
Fig. 3
). In fact, CAT activity actually increased with these
deletions, indicating the possibility that some repressor or
controlling elements may be located in these deleted sequences. Not
unexpectedly, deletions that extended past the TATA box and into the
intron resulted in a substantial reduction in CAT activity.
Figure 3:
Using
convenient restriction sites, a series of 5` deletions were made to the
pML(6.5)CAT reporter ( pMLC) and analyzed by transient
transfection into primary murine keratinocytes in low ( L, 0.05
mM) and high ( H, 0.35 mM) calcium medium.
The constructs are named by the size of the deletion in base pairs. The
arrow indicates to position of the TATA box in each of the
deletions. Percent CAT activity is relative to the highest expressing
construct.
Surprisingly though, the p2230 deletion was still able to
direct significant CAT expression in the absence of the TATA box and
CAP site. Note that CAT expression was not abolished until most of the
intron was removed, suggesting that a cryptic promoter may be located
within the intron sequence. In an effort to account for this activity,
5` RACE-PCR was performed on total RNA obtained from keratinocytes
transiently transfected with the p
2230 construct. Alignment of the
PCR generated sequence with the mouse loricrin gene revealed that
transcription from the p
2230 construct was initiated from a region
within the intron that encodes a weak cryptic promoter complete with a
TATA box, CAAT box, and an mRNA CAP site (Fig. 4). Comparison of
this region with the human gene revealed similar motifs, suggesting
that these sequences may be functionally significant. However, in a
survey of the 5` ends of loricrin transcripts we were only able to
detect the native transcript (as determined by Mehrel et al. (1990)) in RNA isolated from normal mouse epidermis (data not
shown), suggesting that the weak activity of the p
2230 construct
is most likely an in vitro phenomenon of the deletion and not
relevant in vivo.
The Loricrin Promoter Contains a Functional AP-1
Element
Sequence analysis of loricrin promoter revealed a
perfect AP-1 consensus sequence (TGAGTCA) present 15 bp upstream of the
TATA box (Rothnagel et al., 1994). This sequence is conserved
in both human and mouse genes and is retained in the most active
p1480 CAT construct (see Fig. 5 A). To characterize
the AP-1 element in the loricrin promoter and to determine if the
element could interact with AP-1 transcription factors, both
electrophoretic mobility shift assays and DNase I footprint analysis
were performed. In mobility shift experiments on a 27-bp fragment
encoding the AP-1 element from the loricrin proximal promoter, purified
c-Jun (Promega) produced a specific retarded complex
(Fig. 5 B, lanes 2 and 3) that could be
inhibited by preincubation with a 100-fold molar excess of an unlabeled
oligonucleotide containing the AP-1 consensus sequence (Promega)
(Fig. 5 B, lane 4). To demonstrate the interaction of
keratinocyte factors with this region, whole cell extracts were used
and either preincubated with an excess of unlabeled AP-1 competitor or
supershifted with polyclonal antibodies to JunD. As shown in
Fig. 5C, a specific retarded complex formed with
keratinocyte extracts interacting with the 27-bp fragment ( lane
2), and formation of this complex was completely abolished using a
100-fold molar excess of AP-1 competitor ( lane 1), or
partially supershifted with JunD antibodies ( lane 3),
demonstrating that this site is capable of binding members of the AP-1
family of transcription factors present in keratinocytes. To define
precisely the sites of interaction between keratinocyte nuclear factors
and the proximal promoter, a 200-bp fragment encompassing the promoter
region and including the AP-1 site was used in DNase I footprint
analyses. The results in Fig. 6show that keratinocyte extracts
produced a complex footprint with several protected regions within the
loricrin promoter, including the same DNA region protected by purified
c-Jun protein. The identity of the other sites protected within the
loricrin promoter, and their potential functions, were not determined
in this study.
Figure 5:
A, a comparison of the promoter region of
the mouse and human loricrin genes shows the conserved AP-1 element
upstream of the TATA box. The conserved CAP site is also shown as is
the BclI site used to make the p1480 deletion. B, electrophoretic mobility shift analysis of the AP-1 element with
purified c-Jun protein showed a specific complex formed
( arrow) using 0.2 µl ( Lane 2) and 0.5 µl
( Lane 3) of protein (1 foot printing unit/µl). This
complex could be competed using a 100-fold molar excess of an unlabeled
AP-1 oligo ( Lane 4). Lane 1 is a control lane with
probe alone. C, mobility shift analysis of the AP-1 element
with keratinocyte whole cell extracts show a single complex formed with
the probe ( Lane 2). The formation of this complex could be
inhibited using a 100-fold molar excess of an unlabeled AP-1 competitor
( Lane 1). In addition, incubation with polyclonal antibodies
to JunD partially supershifted the complex ( Lane 3, open
arrow), indicating that AP-1 factors from cultured keratinocytes
interact with the probe. Lane 4 is a control lane with probe
alone.
Figure 6:
DNase
I footprint analysis of the loricrin promoter. Lane 1, no
protein; Lane 2, 5 µg of keratinocyte whole cell extract;
Lane 3, 10 µg of keratinocyte whole cell extract; Lane
4, 1 footprinting unit of c-Jun protein; Lane 5, 2
footprinting units of c-Jun protein; Lane 6, no protein.
Keratinocyte extracts protected the same region of the promoter as the
c-Jun protein. The protected region coinciding with the AP-1 element is
indicated.
Mutation of the AP-1 Site Abolishes Transcriptional
Activation
The conservation of the AP-1 element in both mouse
and human genes (Rothnagel et al., 1994) and the observation
that both purified c-Jun and keratinocyte AP-1 factors bind to it,
strongly suggests an important role for this element in the regulation
of the loricrin gene. To test the functionality of this element, the
site was mutated from TGAGTCA to TCCCGGG, leaving the spacing between
the other promoter elements and adjacent sequences intact. As can be
seen in Fig. 7, mutation of this AP-1 element essentially
abolishes the activation of the CAT reporter in primary keratinocytes.
Promoter activity was reduced 93% in proliferating keratinocytes and
85% in differentiated cells, indicating that this AP-1 element is
required for activity of the loricrin promoter in vitro.
Figure 7:
The AP-1 element is necessary for loricrin
promoter activity. The AP-1 element in the proximal promoter was
mutated from TGAGTCA to TCCCGGG in the context of the pML(6.5)CAT
vector and analyzed by transient transfection in primary murine
keratinocytes cultured in either 0.05 mM calcium ( lo)
or 0.35 mM calcium ( hi). Mutation of the element
resulted in a 93% reduction in low calcium and a 85% reduction in high
calcium medium. Relative CAT activity was calculated as expression of
the AP-1 minus promoter relative to the native promoter over an average
of three experiments. Standard deviation was less than
20%.
-gal gene was cloned into the ClaI site of the reporter,
the pML(6.5)
-gal vector was expressed in a tissue-specific
manner, however, the reporter was expressed throughout the entire
epidermis, including the basal and spinous layers, supporting what was
observed in vitro with the CAT reporter. Therefore, this
6.5-kb construct contains those elements which direct expression of
loricrin to the epidermis, but must lack the regulatory elements
necessary to confine loricrin transcription to differentiated
keratinocytes.
1480) containing 30 bp of sequence 5` to the TATA box.
This site was of great interest since we have previously determined
that the calcium responsive induction of HK-1 is directly regulated by
a composite AP-1/steroid hormone binding site located in the
3`-flanking sequence of the HK-1 gene (Lu et al., 1994). In
addition, functional AP-1 sites have been identified in other
epidermal-specific genes including keratins K8 and K18 (Tamai et
al., 1991; Oshima et al., 1990), the bovine keratin 5
gene (Casatorres et al., 1994), transglutaminase I (Liew and
Yamanishi, 1992), and in the promoters of keratinocyte-specific human
papilloma viruses types 16 and 18 (Cripe et al., 1990; Offord
and Beard, 1990; Mack and Laimins, 1991; Thierry et al.,
1992). Furthermore, the involucrin gene, another cornified envelope
protein, has been found to be activated by
12- O-tetradecanoylphorbol-13-acetate (Takahashi and Iizuka,
1993), a tumor promoter known to stimulate the activity of genes
regulated by AP-1 (Boyle et al., 1991; Angel et al.,
1987; Lee et al., 1987). Furthermore, in vitro studies have determined that co-transfection of AP-1 factors
c- fos and c- jun could also mimic
12- O-tetradecanoylphorbol-13-acetate activation of the
involucrin gene (Takahashi and Iizuka, 1993). Likewise, loricrin gene
transcription could be induced in vitro by
12- O-tetradecanoylphorbol-13-acetate treatment (Dlugosz and
Yuspa, 1993). It is interesting to speculate that the loricrin and
involucrin genes may be regulated by very similar regulatory mechanisms
since both genes have almost identical gene structures and are
components of the cornified envelope, both genes are expressed in the
differentiated layers of the epidermis, and both genes are induced by
12- O-tetradecanoylphorbol-13-acetate.
(
)
Likewise, elements specifying the
differentiation-specific expression of loricrin lie in the additional
upstream sequences present in a larger 14-kb genomic construct. Because
it is known that retinoic acid also plays a role in the suppression of
loricrin transcription in vitro (Hohl et al., 1991b),
the elements responsible for loricrin suppression in basal cells may
also be retinoic acid-responsive elements. Whether this is a direct
cis-regulation by the binding of a retinoic acid receptor to the distal
elements of the loricrin gene, an indirect trans-regulation, or a
direct interaction between the retinoic acid receptors and the AP-1
factors regulating loricrin in the epidermis remains to be determined.
-galactosidase reporter driven by a truncated form of the human K5
promoter in transgenic mice. Normally the keratin 5 gene is only
expressed in basal cells in the epidermis. When the cells are induced
to differentiate, the gene is then down-regulated. It was reported that
while 6 kb of 5`-promoter could direct basal cell-specific expression
in transgenic mice, a deletion containing only 90 bp of promoter
sequence switched differentiation specificity. This 90-bp promoter,
while largely retaining its tissue specificity, was now expressed only
in differentiated cells in the epidermis.
-gal,
-galactosidase; CAT,
chloramphenicol acetyltransferase; RACE, rapid amplification of cDNA
ends; HK1, human keratin 1; HK14, human keratin 14; kb, kilobase
pair(s); bp, base pairs(s); PCR, polymerase chain reaction.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.