Development 120, 2103-2111 (1994)
Printed in Great Britain © The Company of Biologists Limited 1994
2103
A distal tyrosinase upstream element stimulates gene expression in neuralcrest-derived melanocytes of transgenic mice: position-independent and
mosaic expression
Susan D. Porter* and Cathy J. Meyer
Department of Pathology and Laboratory Medicine, University of British Columbia, 2211 Wesbrook Mall, Vancouver, British
Columbia, V6T 2B5, Canada
*Author for correspondence
SUMMARY
We have assessed the importance of a melanocyte-specific
DNase I hypersensitive site and matrix attachment region
situated 15 kb upstream of the mouse tyrosinase gene by
analysis in transgenic mice. Transgenes containing all,
part, or none of this region linked to the tyrosinase
promoter and human tyrosinase cDNA were introduced
into genetically albino mice, and pigmentation and
transgene message levels were analyzed in the resulting
transgenic lines. The effect of the upstream region was to
enhance significantly gene expression in melanocytes, and
to provide position-independent expression of the
transgene. Two exceptions to complete position independence were seen; these lines displayed a mosaic expression
pattern in which the transgene was expressed fully in some
melanocyte clones but less so in others, resulting in transverse stripes of colours ranging from near white to dark
grey. Unexpectedly, pigmentation in the eye of all transgenic lines containing the upstream region was nonuniform, in that the neural-crest-derived melanocytes of
the choroid and anterior iris contained significantly more
pigment than those derived from the optic cup (retinal
pigment epithelium and posterior iris). Transgenes containing a small part or none of the upstream region were
expressed poorly and in a position-dependent manner; of
those lines that were visibly pigmented, expression was
equal in the neural crest and optic-cup-derived cells of the
eye. Mice with transgenes containing DNA sequences
encompassing the hypersensitive site but lacking most of
the matrix attachment region were, on average, poorer
expressors than those containing the entire upstream
region; the highest expressing line of this series, however,
had a pigmentation level similar to the lines with the full
upstream region. Thus, full transcriptional enhancement
activity may lie within the segment containing the hypersensitive site, but position-independent expression may
require the flanking matrix attachment region containing
sequences.
INTRODUCTION
sequences required for autonomous establishment of an open
chromatin structure and for insulation of the transgene from
neighbouring chromatin or regulatory elements, are included
in the construct. The first of such elements isolated was the
locus control region (LCR) of the β-globin gene cluster
(Grosveld et al., 1987), and elements with similar properties
from a number of other genes have been identified (Greaves et
al., 1989; Abe and Oshima, 1990; Bonifer et al., 1990; Greer
et al., 1990; Chamberlain et al., 1991; Aronow et al., 1992).
Studies of the tyrosinase gene regulatory elements using
transgenic mice have shown that 270 bp of the promoter are
sufficient for expression in skin and eye melanocytes
(Beermann et al., 1992) and that the expression of the
transgene, although lower than that of the endogenous gene,
roughly recapitulates its developmental timing. Position independence of expression in this and in other studies using longer
tyrosinase promoters (up to 6 kb) with the tyrosinase or heterologous cDNAs (Tanaka et al., 1990; Yokoyama et al., 1990;
Tyrosinase is the key enzyme of melanin biosynthesis. Its gene
is expressed in all pigmented melanocytes, including those of
neural crest origin (residing in the skin, choroid and internal
organs) and the optic-cup-derived melanocytes of the retinal
pigment epithelium (RPE). We are interested in defining the
elements required for the establishment of an active tyrosinase
chromosomal domain in melanocytes, to understand better the
mechanisms involved in this aspect of gene regulation, as well
as to help in the identification of trans-acting factors important
in melanocyte-specific gene expression and differentiation.
The identification of cis-acting sequences important in
higher order gene control has been approached primarily by
the functional analysis of candidate sequences in transgenes
randomly integrated into the genome of mice or cultured cells.
High level expression of the genes, which is proportional to
the number of copies integrated, is considered to indicate that
Key words: melanocytes, tyrosinase, transgenic mice, mosaic, neural
crest
2104 S. D. Porter and C. J. Meyer
Bradl et al., 1991a; Klüppel et al., 1991) was not specifically
addressed; however, visual analysis of coat color suggested
that expression levels often did not correspond to copy number.
In addition, some transgenic lines (non-founders) displayed a
mosaic expression in which melanocyte clones in the coat were
pigmented to different degrees (Bradl et al., 1991b; Takeuchi
et al., 1993), clearly a variegating type position effect. The
three analyzed transgenic lines derived from microinjection of
a yeast artificial chromosome (YAC) encompassing 235 kb of
the tyrosinase gene (80 kb of the coding region and 155 kb of
upstream sequences) showed position-independent transgene
expression at levels comparable to that of the endogenous gene
(Schedl et al., 1993), suggesting that all elements necessary for
higher order gene control are located within these limits.
A sequence at 15 kb upstream from the first exon of the
tyrosinase gene is hypersensitive to DNase I in melanocytes
and has in vitro nuclear matrix attachment activity (Porter et
al., 1991). It is located within a region removed by rearrangement from the rest of the tyrosinase gene in the variegating
mutation of the tyrosinase locus, chinchilla-mottled (cm)
(Porter et al., 1991). This autosomal, somatically stable
mutation results in light and dark grey stripes on the coat of
homozygous mice, which are accounted for by altered tyrosinase mRNA levels (a roughly 10-fold difference between the
light and darkly pigmented cells, and between the dark cm/cm
and wild-type melanocytes). The difference in gene expression
between the light and dark cells could be attributed to alternate
chromatin conformations of the tyrosinase locus. Thus, the
rearrangement of the tyrosinase gene causes aberrant
chromatin formation in some clones and reduced expression in
all clones (even those with apparently normal chromatin
structure). The −15 kb region was hypothesized to encompass
a potential regulatory element important in transcriptional
enhancement of the tyrosinase gene and/or in the establishment
of open chromatin, as it was the only DNase I hypersensitive
site within 20 kb of the promoter that was separated from the
gene in the cm mutation. That all sequences required for full
expression and open chromatin conformation were not present
in the immediate vicinity of the coding sequence was
concluded independently from the transgenic experiments
discussed above.
To address the hypothesis that this region is important in
tyrosinase gene control in melanocytes, we generated transgenic mice containing the tyrosinase promoter, human tyrosinase cDNA, and part or all of this −15 kb region. We found
that the region imparted high level, position-independent
expression in all lines except two, which showed variegating
position effects. In addition, we found the region to enhance
tyrosinase expression in neural-crest-derived melanocytes, but
not in optic-cup-derived melanocytes, indicating the existence
of distinct tyrosinase regulatory elements for these two developmental lineages. The sequence was also shown to be hypersensitive in melanoma cells, but not in neuroblastoma, liver or
brain cells.
Near confluent cultures were harvested by scraping, and nuclei were
isolated and subjected to incremental DNase I (Gibco BRL) digestions as previously described (Porter et al., 1991).
Brain and liver were obtained from BALB/c mice. The tissues were
minced, washed 3 times in ice-cold SSC/10 mM Tris-HCl pH 7.5 and
homogenized in a dounce homogenizer. Nuclei were isolated (after
passage through gauze) and DNase I digestions were carried out as
above.
For Southern analysis, DNA was digested with EcoRI, separated
by gel electrophoresis, blotted to Nytran (Schleicher and Schuell) and
probed according to the manufacturer’s directions.
Transgenic mice
All DNA constructs used to generate transgenic mice contain the
mouse tyrosinase promoter and human tyrosinase cDNA, and some
contain sequences from the −15 kb region of the mouse tyrosinase
gene. The promoter was derived from λgTYR101, and the −15 kb
region from λgTYR14 (Ruppert et al., 1988). These genomic
fragments were derived from the chinchilla allele, which contains a
point change in the coding region (Beermann et al., 1992) and exhibits
no apparent alteration in the transcript size or level (Halaban et al.,
1988). The human cDNA, which includes a polyadenylation signal,
was derived from BBTY-1 (Bouchard et al., 1989). The promoter and
cDNA in all constructs are identical and consist of a 2.5 kb EcoRISau3A segment of the immediate upstream region of the tyrosinase
gene (the Sau3A site is situated 65 bp downstream of the major transcription initiation site; Ruppert et al., 1988; Yamamoto et al., 1989)
fused through a 21 bp polylinker to the EcoRI fragment of BBTY-1.
For UPT, a 3.6 kb EcoRI-HindIII fragment encompassing the −15 kb
hypersensitive site was ligated to the EcoRI site (blunt-ended) of the
promoter, and for 3PT, a 1 kb AvaII-SstI subfragment of the 3.6 kb
fragment was blunt-end ligated to the promoter. 2PT was derived
directly from UPT by cutting the final construct at the 5′ end with SstI
(which removes all sequences from the 3.6 kb fragment except the 3′
terminal 300 bp). Constructs were digested with XhoI to release the
3′ end of the construct from the vector, and either SmaI (UPT), BamHI
(PT), or SstI (3PT and 2PT) to release the 5′ end. The DNA was gelpurified by Geneclean (Bio 101) for injection.
Fertilized mouse oocytes were obtained from superovulated
BALB/c females crossed with BALB/c males (for TgUPT5, 6 and 7)
or CD-1 males (all remaining lines), and were cultured, injected and
implanted using standard methods (Hogan et al., 1986). Positive
founders were initially identified by PCR analysis of tail DNA
samples from all mice developed to term.
MATERIALS AND METHODS
Copy number determination
Tail DNA samples from the progeny of UPT founder mice were
digested with EcoRI, and analyzed by Southern blotting using the 3.6
kb EcoRI-HindIII fragment as probe. This gives a 6.1 kb transgenespecific band and a 3.6 kb band from the endogenous sequence (corresponding to two copies). Additional bands were apparent in most
samples, presumably corresponding to partial truncations. DNA from
the other lines was digested with PstI (which cuts once in the
construct) and probed with the tyrosinase promoter sequence, which
recognizes a 12 kb endogenous fragment, a band corresponding to the
full length of each construct representing head-to-tail tandem integrations, and at least one other band representing the terminal
transgene copy and separately integrated copies.
DNA was blotted onto Nytran and hybridized with random primer
32P-labelled DNA probes. Blots were exposed to either preflashed Xray film at −70°C with intensifying screens, or to non-flashed film
without screens. The autoradiograms were quantitated using a Zeineh
hand-held scanning densitometer (AAB).
DNase I hypersensitivity analyses
Cell cultures of Neuro-2a and Cloudman S91 (clone M-3) melanoma
(both from ATCC) were propagated as recommended by the ATCC.
Quantitation of transgene expression
RNA was isolated from the dorsal skin of 7-8 day transgenic offspring
of founders crossed with BALB/c mice by the method of Chom-
Melanocyte gene expression stimulation 2105
Fig. 1. DNase I hypersensitivity of
−15 kb region in different cell
types. (A) Nuclei were prepared
from the melanoma cell line clone
M-3 (Cloudman S91), BALB/c
brain, liver and the neuroblastoma
cell line Neuro-2a, and digested
with increasing concentrations of
DNaseI. Purified DNA was
restricted with EcoRI, separated
electrophoretically, transferred to
nylon and hybridized with a probe
adjacent to the hypersensitive site
(0.9 kb EcoRI-XbaI fragment from
the 3.6 kb EcoRI-HindIII fragment
(see Fig. 2) for clone M-3; the 3′
adjacent 0.65 kb XbaI fragment for
the remaining samples). (B) DNA
blots that showed no sub-band
corresponding to the hypersensitive
site were stripped and rehybridized
with a probe from the second intron of the ribosomal protein S16 gene (Wagner and Perry, 1985) as a control for the DNase I digestion. The
sub-band observed maps upstream of the first exon. Molecular weight markers are indicated for each blot (kb).
czynski and Sacchi (1987), and 50 µg of each sample were used for
analysis by RNase protection. RNA probes used for the assays were
radiolabelled with [32P]CTP (using an RNA transcription kit, Stratagene), and corresponded to the antisense strand encompassing the 3′
terminal 275 bp (HaeIII site to terminus) of the human tyrosinase
cDNA (BBTY-1) including 65 bp of vector sequence (39 of which
are also present at the 3′ end of the transgene) and, as an internal
control, the antisense 3′ terminal 188 bp (TaqI site to terminus) of the
mouse tyrosinase cDNA (Terao et al., 1989).
Assays were carried out by standard methods (Ausubel et al., 1993),
using approximately 10,000 cts/minute of each probe simultaneously.
Products of the RNase digestions were separated by denaturing gel
electrophoresis (6% polyacrylamide/7 M urea), and autoradiograms
were prepared and quantitated as described for the copy number determination analyses. An average of the ratio of the human tyrosinase
protected fragment (transgene) to that of the mouse (endogenous) was
taken of two separate assays and normalized to line UPT3 (1 transgene
copy).
Histology
All tissue samples were fixed in 10% buffered formalin and embedded
in paraffin. 4 µm sections were stained with hematoxylin and eosin
for microscopic examination.
RESULTS
Cell type specificity of the −15 kb hypersensitive site
The −15 kb region of the tyrosinase gene was examined in a
number of cell types for hypersensitivity to DNase I. As seen
in Fig. 1, neither liver, nor brain nor the neuroblastoma cell
line Neuro-2a was hypersensitive at −15 kb. The pigmented
melanoma cell line Cloudman S91 (clone M-3) was hypersensitive at this point, as expected.
Effect of the −15 kb region on expression patterns in
transgenic mice
The constructs used to generate transgenic mice are illustrated
schematically in Fig. 2. Each transgene contains the identical
promoter fragment (2.5 kb upstream from the transcription
initiation point of the mouse tyrosinase gene) and human
tyrosinase cDNA/polyadenylation signal. The human tyrosinase cDNA was used as a reporter gene to allow the level and
uniformity of transgene expression to be determined by visual
analysis of the pigmentation pattern; quantitative comparison
of expression levels was also possible as the transgene message
was readily distinguished from that of the endogenous tyrosinase gene by RNase protection analysis. The constructs
contain the following additional sequences: (1) UPT contains
the 3.6 kb EcoRI-HindIII fragment encompassing the hypersensitive site and matrix attachment region (MAR) (Porter et
al., 1991), (2) 3PT contains the 1 kb AvaII-SstI fragment,
which includes the hypersensitive site but lacks most of the
MAR, (3) 2PT contains only the 3′ terminal 300 bp of this
fragment, and (4) PT contains none of the −15 kb region.
A representative mouse of each transgenic line generated
with the above constructs is shown in Fig. 3. Each of the
pigmented mice shown here are genotypically black (an allele
of the brown locus) and agouti. None of the mice containing
the PT transgene (Fig. 3B) have visibly pigmented coats;
however, two (TgPT2 and TgPT4) have ruby-colored eyes and
pigmented tail, ears and scrotum. One line out of four
generated with the 2PT construct (Fig. 3C), Tg2PT1, is
pigmented in all normally pigmented structures, but the level
is very low. Lines generated from 3PT (Fig. 3D) are either light
grey, non-pigmented or pigmented in a mosaic pattern. The
eyes of the two pigmented lines (Tg3PT1 and Tg3PT3) are
ruby-colored. In striking contrast, all lines generated from the
UPT construct containing the entire 3.6 kb upstream fragment
are pigmented (Fig. 3A), with colors ranging from tan
(TgUPT3) to black (TgUPT5, 6 and 8). The eyes of each line
are black. An additional founder animal that died at birth was
also pigmented. Two of the TgUPT lines (1 and 9) have a
mosaic coat pattern of stripes. Mice of line TgUPT1 are more
highly pigmented overall than those of line TgUPT9, and
although some TgUPT1 mice have a distinctive dark grey and
2106 S. D. Porter and C. J. Meyer
Fig. 2. Maps of DNA constructs used to generate transgenic mice. A
restriction map of the tyrosinase locus is shown at the top and
includes the first exon (solid box), direction and initiation of
transcription (wavy arrow) and DNase I hypersensitive sites (strong
one at −15 kb, weaker one at −100 bp). Shaded boxes above the −15
kb region indicate those restriction fragments bound to nuclear
matrices in vitro (Porter et al., 1991). Each of the transgenes
indicated below the locus include 2.5 kb of the region immediately
upstream of the first exon, the human tyrosinase cDNA (lined
boxes), and various portions of the −15 kb region. A, AvaII; E,
EcoRI; H, HindIII; S, SstI; X, XbaI.
white pattern, most have stripes of varying shades of grey. The
number of pigmented clones in line TgUPT9 mice is generally
much smaller, although this is extremely variable within the
line. In addition, the ears of some mice of this line are darkly
pigmented, whereas others have no visible ear pigmentation.
The eyes of all pigmented lines were examined histologically to assess the amount and distribution of pigment (Fig. 4).
The eyes of the pigmented TgPT and Tg2PT lines, whose
transgenes contain little or none of the upstream region, had
only a small amount of pigment in the eye, and it was distributed evenly between the choroid, iris and RPE (Fig. 4D,H).
The distribution of ocular pigment in every TgUPT line and in
the two pigmented Tg3PT lines, however, was very different.
Whereas every TgUPT line had significant (typically wildtype) levels of pigmentation in the choroid and anterior half of
the iris (which is continuous with the choroid), most lines had
no detectable pigmentation of the RPE or posterior iris (which
is derived from the retina) (Fig. 4B,F). Line TgUPT1 had an
occasional cluster of lightly pigmented cells in the RPE (0-3
clusters of 2-3 cells each, per section), and lines TgUPT6 and
8, with the highest transgene copy numbers, had a moderate
but subnormal amount of RPE and posterior iris pigmentation
(Fig. 4C,G). The eyes of the pigmented Tg3PT lines were less
pigmented than those of any TgUPT line, but the distribution
of melanin granules (only in the choroid and anterior iris) was
the same (data not shown).
The approximate level of pigmentation in the ear and
scrotum (where the majority of pigmentation results from
dermal rather than epidermal melanocytes; Billingham and
Silvers, 1960), Harderian gland (a pigmented lacrimal gland),
eye structures and coat for all transgenic lines is summarized
in Table 1. Harderian gland pigmentation was evaluated histologically, but as it is difficult to quantitate, was indicated only
Fig. 3. Representative mice of all transgenic lines, arranged in order
of increasing copy number. Pigmented mice are on a black agouti
background, and all mice are of either F1 or F2 generation.
(A) TgUPT series, from left to right, lines 3, 7, 4, 9, 1, 5, 6, and 8;
(B) TgPT series, lines 8, 5, 3, 4, 7, and 2; (C) Tg2PT series, lines 6,
1, 5, and 7, and (D) Tg3PT series, lines 5, 3, 2, and 1.
as present or absent. Pigment cells in various locations were
categorized as being derived from the optic cup (RPE and
posterior iris), or from the neural crest (probably all other
melanocytes; Billingham and Silvers, 1960). It is immediately
evident from these results that a direct correlation exists
between the presence of the −15 kb hypersensitive site in the
transgene and increased expression of the gene in neural-crestderived melanocytes. There appears to be little or no effect of
the region on optic-cup-derived pigment cell expression, as the
highest level of pigmentation seen in either the TgUPT or
Tg3PT mice (lines TgUPT6 and 8, with 17 and 19 transgene
copies, respectively) is not significantly higher than that seen
in the strongest expressing line of the TgPT series (TgPT4,
with 23 transgene copies).
Another conclusion that can be drawn from these data is that
the expression of the UPT transgene in neural-crest-derived
pigment cells in various locations is apparently proportionately
Melanocyte gene expression stimulation 2107
Fig. 4. Ocular pigmentation in
wild-type and representative
transgenic mice. Sections
showing choroid and retinal
pigment epithelium (A-D) and
iris (E-H) of a wild-type
pigmented (C/c B/B A/A)
mouse (A,E) and hemizygous
mice of lines TgUPT5 (B,F),
TgUPT8 (C,G) and TgPT4 (D,H). Also shown is a schematic
diagram of the relevent features of the mouse eye. c, choroid; a,
anterior iris; p, posterior iris; l, lens; cb, ciliary body; i, iris; r, retina.
Bar, 50 µm.
normal, whereas that of the TgPT lines is not (in which
expression in the ocular and dermal melanocytes is much higher
than that in the follicular melanocytes). Melanocytes of the only
expressing Tg2PT line are pigmented in apparently correctly proportional amounts and the Tg3PT lines, although underpigmented in the eye, have otherwise proportionate pigmentation.
Three TgUPT lines with the highest transgene copy numbers
(5, 6 and 8) were bred with strains containing different genetic
coat color determinants that act downstream of tyrosinase in
the synthesis of pigment. In each case, the transgenes behaved
as true phenotypic revertants of albinism. That is, the brown
agouti, black agouti and black non-agouti (carried out only on
line 8) phenotypes were all comparable to those of wild-type
(non-albino) animals with these genetic determinants.
Quantitative analysis of transgene expression
To address the question of position independence (copy
number dependence) of transgene expression, and to estimate
quantitatively the influence of the upstream region, RNase protection analyses were carried out on RNA from the dorsal skin
of 7-8 day offspring of founder mice using both human
(transgene) and mouse (endogenous)-specific probes (Fig. 5).
An average of the ratio of human:mouse tyrosinase RNA, normalized to line TgUPT3, was taken of two experiments and
expressed as a function of transgene copy number (Fig. 6).
The results are consistent with a visual analysis of coat color
in that expression of the transgene appears to be directly proportional to the transgene copy number for all lines except
those exhibiting a mosaic coat color pattern. Analysis of
transgene expression levels in some of the TgPT and Tg2PT
lines was attempted (including Tg2PT1, the only one with a
pigmented coat), but the level was too low to be quantitated.
The Tg3PT series was not quantitatively analyzed. The pigmentation levels of the TgPT, Tg2PT and Tg3PT series mice
show, however, that transgene expression does not correlate
with copy number (see Table 1).
A comparison of coat colors of mice from different series
can also give an indication of relative transgene expression
efficiency, and thus of the quantitative effect of the upstream
region. TgUPT3 mice have a single transgene copy which is
expressed at a level (per copy number) comparable to those of
the rest of the series (Fig. 6). Tg2PT1 homozygotes, which
have 12 transgene copies, have coat colors which are indistinguishable from those of TgUPT3 hemizygotes, implying that
the difference in gene expression of the highest expressing
Tg2PT line and the average TgUPT line is approximately 12
fold. Likewise, one can conclude that the relative expression
level of the highest expressing Tg3PT line (Tg3PT3; 2 copies)
is similar to that of the average TgUPT line, as a Tg3PT3 hemizygous mouse has a coat color intermediate between that of a
TgUPT3 (1 copy) and TgUPT7 hemizygote (2 copies, with
slightly higher than average expression).
DISCUSSION
Combined evidence from a number of transgenic mouse
studies and from a molecular analysis of the chinchilla-mottled
mutation has led to the conclusion that regulatory elements
necessary for full expression of the tyrosinase gene are present
between 5.5 and 155 kb upstream from the first exon. A
candidate region centered at −15 kb was tested in transgenic
mice and was shown to encompass an element with strong
enhancer activity in melanocytes of neural crest origin. This
element was also able to insulate the transgene from all
position effects except variegating ones.
Enhancer activity of the −15 kb region
None of the six transgenic lines generated with the PT
construct (containing the mouse tyrosinase promoter and
human tyrosinase cDNA) had pigmented coats, whereas all
eight lines containing the same DNA fused to the −15 kb region
fragment were pigmented, three at wild-type levels. Mice
transgenic for the 2PT construct (with only 300 bp of the
upstream fragment) were, like the TgPT animals, poor expressors, with only one line in four showing any pigmentation.
These results point to a strong effect of this 3.6 kb upstream
region in enhancing gene expression and/or insulating the
2108 S. D. Porter and C. J. Meyer
Table 1. Pigmentation of transgenic lines
Pigmentation
Neural crest
Transgenic line
Copy
number
Coat
(follicle)
Ear/scrotum
(dermis)
Wild-type
(non-transgenic)
UPT3
UPT7
UPT4
*UPT9
*UPT1
UPT5
UPT6
UPT8
PT8
PT5
PT3
PT41
PT7
PT21
2PT6
2PT1
2PT5
2PT7
3PT5
3PT3
3PT2
*3PT1
−
1
2
3
4
8
10
17
19
2
5
5
23
25
50
6
6
7
34
1
2
2
8
++++
++
+++
+++
−/+++
+++/+
++++
++++
++++
−
−
−
−
−
−
−
+
−
−
−
+++
−
+++/−
++++
++
+++
+++
+++
+++
++++
++++
++++
−
−
−
++
−
+
−
+
−
−
−
+++
−
+++/−
Optic cup
Choroid
Harderian
gland
RPE
Posterior
iris
++++
+++
++++
++++
++
++++
++++
++++
++++
−
−
−
++
−
+
−
+
−
−
−
++
−
++
Y
Y
Y
Y
Y
Y
Y
Y
Y
nd
nd
N
Y
nd
N
nd
Y
N
nd
nd
Y
nd
Y
++++
−
−
−
−
−/+
−
++
++
−
−
−
++
−
+
−
+
−
−
−
−
−
−
++++
−
−
−
−
−/+
−
++
++
−
−
−
++
−
+
−
+
−
−
−
−
−
−
*Indicates a mosaic coat
1Pigmentation in these lines varied to some degree between individual animals. The highest level seen is indicated.
Y, yes,; N, no; nd, not determined.
transgene from position effects. It was somewhat surprising to
find such a low level of coat pigmentation in the TgPT series
of mice, as the majority of other transgenic mice containing
the same or similar promoter fragments fused to the mouse
tyrosinase cDNA (Tanaka et al., 1990) or mini-gene
(Yokoyama et al., 1990) were visibly pigmented. Possible
reasons for this discrepancy include strain differences and/or
the substitution of human sequences for those of the mouse.
It can be difficult to differentiate poor expression due to a
lack of strong regulatory elements from that due to position
effects on randomly integrated DNA, and it appears that both
of these factors play a role in the reduced expression of the
TgPT and Tg2PT series relative to the TgUPT series. The
existence of position effects can be surmised from a comparison of phenotypes within the Tg2PT series of mice, where a
line with 6 copies (Tg2PT1) is visibly pigmented, although all
other lines, with equal or greatly increased numbers of
transgene copies, are not. Cell-type-position effects are also
likely to be prevalent in the TgPT series, as the two pigmented
lines have an aberrant distribution of pigment, in which the
eyes, ears, scrotum and Harderian gland (in one of them) are
pigmented, but the coat is not. The presence of ear pigmentation in mice with little or no coat pigmentation has been
observed in other transgenic lines containing the tyrosinase
promoter (Yokoyama et al., 1990). The fact that even the
highest expressing lines (Tg2PT1 for the coat, TgPT4 for the
eye) are significantly less pigmented than any of the UPT lines
indicates that, even in the absence of position effects, transgenes lacking the −15 kb region are expressed at a much lower
level than those containing it.
The reduction of expression of transgenes lacking most or
Fig. 5. RNase protection analysis of dorsal skin RNA from
hemizygous TgUPT transgenic mice. 50 µg of each sample were
hybridized with antisense probes from the 3′ terminus of the human
and mouse tyrosinase cDNAs and digested with RNase. The
products were run on a 6% polyacrylamide/7 M urea denaturing gel.
The probes protect at least two fragments each (approximately 185
and 180 nucleotides for the mouse; and approximately 315 and 300
nucleotides for the human), most likely representing different
transcript termination points. The two major transgene-specific
transcripts appear to encompass some or all of the vector sequence;
TgUPT3 has only the lower of these bands, which is presumably due
to the integration of this transgene as a single copy, with unique 3′
adjacent sequences. The RNA used in the last lane (−) was yeast
tRNA.
all of the −15 kb region compared to those containing the entire
region, in lines presumably not showing position effects (using
a coat color comparison of Tg2PT1 and TgUPT3, described
Melanocyte gene expression stimulation 2109
mosomal position effects (Stief et al., 1989; Phi-Van et al.,
1990; McKnight et al., 1992); it also showed transcriptional
activation activity in stably transformed cells (Stief et al.,
1989), but did not appear significantly to activate linked genes
in mice (McKnight et al., 1992). The mean level of gene
activity in transgenic mice containing immunoglobulin κ gene
constructs with an intact intronic MAR was twice as high as
those containing transgenes with no MAR (Xu et al., 1989).
As in our series, however, the highest expressing animal with
no (in our case, little) transgenic MAR sequence had an
expression level similar to the average expression of the MARcontaining mice. This suggests that, in the absence of a
negative position effect, a MAR has little transcriptional activation activity in transgenic mice.
Fig. 6. Relationship of TgUPT transgene expression to copy number.
Transgene expression was quantitated by RNase protection analysis;
each point represents an average from two assays of the ratio of the
transgene-specific fragment to that of the endogenous tyrosinase
gene for a hemizygous representative of each UPT line, normalized
to line TgUPT3 (1 copy). Transgene copy number was determined
by quantitative Southern blot. Open boxes represent mice with
mosaic pigmentation (lines TgUPT1 and 9), the values of which
were not used to plot the line of best fit.
earlier), is estimated to be approximately 12-fold. Interestingly,
this is similar to the reduction of tyrosinase transcription in the
dark cm/cm melanocytes (the tyrosinase gene chromatin
structure of which is normal), relative to that of the wild-type
(Porter et al., 1991). The deficiency in transcription in the dark
cm/cm cells may therefore be completely attributable to the lack
of adjacent −15 kb sequence, and may indicate that there are
no further upstream regulatory sequences required for skin
melanocyte transcription. There was no attempt to optimize the
transgenic constructs for high level expression (e.g., by
including introns), as an internal comparison of transgene
expression levels was the primary purpose of this work. It is
thus not meaningful to compare directly transgene expression
levels with endogenous tyrosinase gene expression, and these
experiments do not address whether there exist other tyrosinase locus elements (perhaps located within introns or immediately downstream of the coding region, i.e. within the unrearranged portion of the cm locus) that contribute to full
expression of the gene.
The phenotypes of the Tg3PT series are variable and
position dependent, so it is difficult, as it is for the other lines,
to ascertain the quantitative contribution to gene expression, if
any, of the sequences immediately 5′ of the hypersensitive site.
If one compares the average relative expression of the Tg3PT
series with that of the TgUPT series, one can conclude that
deletion of MAR-containing sequences significantly reduces
expression. However, if a comparison is made of the highest
expressing Tg3PT line with the average expressing TgUPT line
(as discussed previously), the expression level seems quite
similar. The sequences lacking in 3PT compared to UPT (i.e.,
most of the MAR) may not therefore contribute to the enhancer
activity of the fragment, but do have a significant effect on
position independence. A chicken lysozyme gene MAR has
been shown to contribute to the insulation of genes from chro-
Cell type specificity of enhancer
Ocular pigment in the pigmented TgPT and Tg2PT mice was
distributed evenly between the choroid, RPE and iris. An
equivalent expression in these structures was also seen in transgenic mice with the tyrosinase mini-gene driven by the tyrosinase promoter (Klüppel et al., 1991). Interestingly, however,
the TgUPT and Tg3PT transgenes were expressed consistently
at much higher levels in the choroid and anterior iris than in
the RPE and posterior iris. Thus, neural-crest-derived
melanocytes had enhanced expression relative to optic-cupderived melanocytes. What expression there is in the RPE,
from the UPT construct, appears to be as independent of
position as in the other melanocytes, with visible pigmentation
only in the lines with highest transgene copy numbers.
It is possible that the discrepancy in gene expression is not
a function of developmental lineage per se, but rather of
differing environments. This seems unlikely for a number of
reasons, including (1) all analyzed neural-crest-derived
melanocytes throughout the body of the transgenic mice
seemed to be pigmented in normally proportional amounts
(dermal melanocytes of the ear and scrotum, choroidal
melanocytes, Harderian gland melanocytes and hair follicle
melanocytes) and (2) the posterior layer of the iris, which is
derived from the retina, but which exists in a different environment from that of the RPE, displays the same level of pigmentation as the RPE in all lines.
There appears to little or no effect of the −15 kb region on
expression in the RPE, even when the transgenes are apparently
poised for transcription in this cell type (i.e, not subject to
position effect). It is likely, therefore, that transcription factor(s)
specific to neural crest melanocytes play an important role in
tyrosinase gene expression in these cells. A number of nuclear
proteins bind this region (data not shown), but their identity has
not yet been determined. It also seems likely that there exists
an optic-cup-specific enhancer outside this region, but difficulty
in obtaining long-term cultures of melanocytes from the RPE
has precluded preliminary investigation by molecular means.
The pigmented cells of the RPE of cm/cm mice are strongly
pigmented (Deol and Truslove, 1981), suggesting that regulatory elements for this cell type lie within the non-rearranged
portion of this mutant locus. The −15 kb region was not hypersensitive to DNase I in the neural-crest-derived neuroblastoma
cell line Neuro-2a (which has neuronal characteristics),
implying that chromatin rearrangement at this site occurs after
the separation of this lineage from that of melanocytes.
The two pigmented lines of the Tg3PT series have reduced
2110 S. D. Porter and C. J. Meyer
pigmentation in the choroid relative to their coat color, as
compared to the choroidal and coat pigmentation levels of mice
of the TgUPT series. This may mean that sequences needed for
choroid melanocyte expression are missing in this construct,
or that the 3PT transgene in these cells is affected by neighbouring sequences only in this cell type.
Position-independent expression of non-mosaic
UPT animals
Position effects can cause uniformly reduced (or enhanced)
expression of randomly integrated DNA, altered patterns of
cell-type-specific expression, or variegation. Position independence in terms of a direct correlation of expression level to
transgene copy number is a criterion generally used in defining
chromosomal domain control elements. A linear relationship
of transgene expression to copy number was found for the six
non-mosaic TgUPT mice, in dramatic contrast to the mice of
all other series (TgPT, Tg2PT and Tg3PT), which displayed
little correspondence of copy number and expression level.
Elimination of most of the MAR containing sequences in the
3PT construct appeared to result in loss of position independence. Lines Tg3PT2 and Tg3PT5 (2 and 1 transgene copy,
respectively) are not detectably pigmented, whereas line
Tg3PT3, with 2 transgene copies, is substantially so. This is
consistent with the previously mentioned studies showing that
MARs can contribute to insulation of genes against position
effects. Many sequences with no MAR activity, however, have
also been shown to contribute to position independence, and
so it remains to be determined whether MAR activity itself is
required for the effect of the UPT transgene, or whether it is
another (or perhaps non-specific) attribute of the 5′ sequences.
Variegating position effects
Two of the eight TgUPT transgenic lines have a mosaic coat
consisting of differently pigmented patches, usually transverse
stripes of variable widths. This pattern reiterates that of the
clonal developmental history of melanocytes (Mintz, 1967),
although some of the transgene-induced stripes are narrower.
The darkest color of each line is roughly equivalent to that of
mice in the same series with similar copy numbers of transgenes. This suggests that the dark clones are fully expressing
the transgenes, and that much poorer (but usually some)
expression of the transgene occurs in the light clones. This is
in contrast to position effect variegation in Drosophila, where
most variegating genes are either completely on or completely
repressed (Spofford, 1976). Mice of the TgUPT1 line generally
have more clones that are visibly pigmented than do the mice
of line TgUPT9, indicating either an earlier inactivation event
(i.e. prior to melanoblast determination) in line TgUPT9, or
increased probability of inactivation. As the mice are not
inbred, there may also be different genetic modifiers that play
a role in the modulation of gene activity. The uniformity of
transgene expression of these mice in cell types other than follicular melanocytes is difficult to assess. Mosaicism of
expression in the RPE of line TgUPT1 is discernable, but no
RPE pigmentation is detectable in line TgUPT9.
Mosaic expression of other transgenes expressed in
melanocytes (Bradl et al., 1991b; Mintz and Bradl, 1991;
Takeuchi et al., 1993) and other cell types (Sweetser et al.,
1988a,b; Katsuki et al., 1988; McGowan et al., 1989) has been
observed, and as mosaicism of expression is rarely specifically
addressed, it seems likely that it is not an unusual occurrence.
Heterogeneity of (autosomal) gene expression between cellular
clones of the same cell type can also occur naturally in vivo
(Rubin et al., 1989; Michaelson, 1993), and may be of fundamental importance in normal developmental and physiological
processes (Mintz, 1971). Little is known of the mechanisms
contributing to mosaic expression of autosomal genes or transgenes in mice. Variability of chromatin structure may play a
role, as it does in chinchilla-mottled mice and in position effect
variegation in Drosophila; mosaicism in DNA methylation has
also been found to be associated with mosaic transgene
expression (McGowan et al., 1989). Neither the TgUPT1 nor
TgUPT9 line phenotype is noticably affected by the sex of the
parent transmitting the transgene, making it unlikely to be a
result of or affected by gamete-of-origin-specific imprinting
(see Sapienza, 1990).
The mosaic pattern of pigmentation in the Tg3PT1 line is
slightly unusual in that the melanocyte clones of the head are
more often pigmented than those of the body. This may reflect a
time-dependent (the anterior region of the neural crest being
formed later than others) or environmentally affected gene activation event related to the chromosomal position of the transgene.
If the variegating effects observed with the TgUPT lines
have a similar molecular basis as those observed in
Drosophila, it is not surprising that regulatory or structural
elements (enhancers and MARs) fail to inhibit the inactivation
of the gene in some clones. That is, gene inactivation in
Drosophila can spread over 60 chromosome bands in variegating mutations (see Tartof et al., 1989), implying that normal
domain boundaries and the regulatory elements contained
within the domains do not insulate internal sequences from this
particular inactivating phenomenon. This has in fact been
shown experimentally by Kellum and Schedl (1991) who
tested the activity of chromosomal domain boundary
sequences in insulating against position effects in Drosophila.
Although nine lines transformed with the white gene flanked
by these elements fully expressed the gene, a tenth line had a
variegated expression pattern, indicating that the boundary
elements did not protect against this type of position effect.
An important advantage of studying genes affecting coat
colour is the ease with which uniformity of expression can be
addressed. Other studies using transgenic mice with putative
LCR-containing transgenes have analyzed transgene
expression in total tissue, and variability of expression between
individual cells or clones has not been a focus of attention.
Thus, variegating position effects have not been accounted for
in these experiments, and mosaic expression may have
occurred unnoticed as LCR containing transgenes can often be
expressed, per copy number, within a 2- or 3- fold range of
each other. It will be interesting to determine whether variegating position effects do occur in LCR-containing transgenic
mice, and indeed what causes these effects. Until then, one
cannot conclude with certainty that the UPT construct contains
LCR activity comparable to other described LCR like
elements. Little is known about gene regulation in
melanocytes, and the identification and characterization of this
genetic element will be of use in defining regulatory mechanisms in this cell type as well as in inducing high level,
position-independent expression of heterologous genes in
melanocytes of transgenic mice.
Melanocyte gene expression stimulation 2111
We thank Blake Gilks and Robert Kay for valuable discussions and
comments on the manuscript, Brigitte Bouchard and Beatrice Mintz
for gifts of human and mouse cDNAs, respectively, and Günter
Schütz for the tyrosinase genomic clones. This work was supported
by a B.C. Health Research Foundation grant to S.D.P.
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(Accepted 20 April 1994)