MELANOGENIC FACTORS: REGULATION OF GENE EXPRESSION
J. Vachtenheim1 and J. Duchon2
Laboratory of Molecular Biology, Institute of Chest Diseases,
Prague 8 and IInd Department of Medical Chemistry and Biochemistry,
1st Faculty of Medicine, Charles University
Melanogenesis faktory: Regulace genové exprese.
Vachtenheim J., Duchori J. Sborn. Iék., Vol. 97 (1996
No. 1, p. 41-47.
* This paper is dedicated to the 50th anniversary of IInd Department
of Medical Chemistry and Biochemistry 1st Faculty of Medicine,
Mailing address/Adresa autora: MUDr. J. Vachtenheim, CSc.,
Laboratory of Molecular Biology, Institute of Chest Diseases,
18000 Prague 8 (Czech Republic).
SUMMARY: Melanogenesis is a multistep biochemical process
resulting in the formation of melanin in pigment cells in the
skin and the eye. Three melanogenic factors, tyrosinase, TRP 1,
and TRP2 participate in the pathway. Here, the regulation of
gene expression of these melanocyte-specific markers is shortly
SOUHRN: Melanogenese je biochemický proces s mnoha stupni,
vedoucí k tvorbe melaninu v pig mentových bunkach
v kuzi a oku. Tii melanogenní faktory, tyrosinasa, TRP1
a TRP2, se úcastni této biochemické cesty.
Zde jsou kratce shrnuty poznatky o regulaci genové exprese
techto pro melanocyty specifickych markeru.
Melanocytes, highly differentiated cells producing the pigment
melanin, are derived from the neural crest and are unique in
that they possess enzymes converting tyrosine and low-molecular
intermediates into the dark polymeric pigment through a specific
biochemical pathway. For many years, tyrosinase has been considered
to be the only enzyme responsible for the production of melanin
in melanocytes. It catalyzes two initial steps in the formation
of melanin, the hydroxylation of tyrosine to L-3,4-dihydroxyphenylalanine
(L-DOPA) and the oxidation of L-DOPA to DOPAquinone. Apart from
these two well-known catalytic functions, a third catalytic activity
of tyrosinase has been shown (I). This activity, the oxidation
of dihydroxyindol (DHI), seems to be true tyrosinase activity
and could be immunoinhibited by antibodies directed against electrophoretically
purified tyrosinase (2). Recently, Kobayashi et al. have shown
that extract from fibroblasts transfected with the tyrosinase
construct was able to utilize DHI as a substrate and melan-c cells
(lacking functional tyrosinase) had no ability to oxidize DHI
(3). These results clearly demonstrate the DHI-oxidase activity
Tyrosinase is not the only enzyme that catalyzes the reactions
in the melanogenic pathway. Two other regulatory factors, namely
tyrosinase-related proteins, TRP1 and TRP2, have been described
and Their activities determined. The action of TRP1 is more distal
than TRP1- in the melanogenic pathway. TRP1 has been shown to
possess dihydroxyindol carboxilic acid (DHICA) oxidase activity
(3) but low tyrosine hydroxylase was also associated with this
protein (see references in 3). In experiments analogous to tyrosinase
expression. TRP1 expression in fibroblasts resulted in DHICA conversion
in transfected cell extracts (3). TRP2 was shown to have DOPAchrome
tautomerase (DT) activity isomerizing the rnelanogenic intermediate
DOPAchrome to DHICA (4), a relatively stable jntermediate. Similarly
the expression of TRP2 in HeLa (5) or COS 6) cells results in
DOPAchrome conversion activity in cell extracts. Thus, besides
tyrosinase. two other factors facilitate the formation of melanin
in melanocytes: TRP2 produces DHICA and is therefore responsible
for preserving carboxyl group in melanins The product of TRP2,
DHICA, serves as a substrate for TRP1. Since TRP 1 was shown to
be associated with the production of eumelanin rather than phaeomelanin
in human melanoma cell lines (7), this factor probably has the
specific role to facilitate the very distal step in the production
of black type of pigment eumelanin, i.e. the oxidation of DHICA
before the polymerization takes place (3).
TYROSINASE GENE FAMILY
In the past years, by using techniques of molecular biology, cDNA
for tyrosinase as well as for the two proteins mentioned above:
(TRP1 and TRP2) that participate in melanogenesis has been cloned.
Human tyrosinase cDNA clone encodes a protein with the predicted
molecular weight 62.6kD (8). The sequence shows several potential
N-glycosylation sites and a signal peptide characteristic for
membrane associated proteins (8,9). Another isolated tyrosinase
cDNA clone has a sequence very similar to the first clone (10).
When expressed in mouse fibroblasts which normally do not synthesize
melanin, tyosinase was capable to induce melanin pigmentation
(10). Mouse tyrosinase maps to the c albino locus on chromosome
7 (8) and its amino acid sequence is &9% homologous to the
human sequence (9). The mouse and human tyrosinase genomic structures
an also similar: the protein sequences are encoded in 5 exons
spanning about 70 kb in the genomes 11-13). Human tyrosinase is
located on 11q14-q21 (14) but a tyrosinase-related segment corresponding
only to exons 4 and 5 and adjacent noncoding sequences has been
found on 11p11.2 (12).
The TRP 1 and TRP2 map at the b (brown) and slt (slaty) locus
in the mouse, respectively (15, 16). Although initially thought
to be a tyrosinase clone the mouse TRP1 has been the first gene
isolated among the melanogenic proteins (17). Both TRP1 and TRP2
from the mouse are highly homologous to their human counterparts:
TRP1 exerts higher homology (93 %) than TRP2 (84%) (5, 18). The
degree of homology of the three individual melanogenic enzymes,
tyrosinase, TRP1 and TRP2, to each other is much lower (about
40-50%) in the mouse and in human. The chromosomal loci at which
TRP1 and TRP2 reside has also been determined. In humans, TRP1
maps at the short arm of chromosome 9 and TRP2 maps at 3q31-932.
These regions correspond to homologous regions on mouse chromosomes
3 and 14, respectively (19, 20). In summary, the three proteins
are coded at distinct genes Iocated on different chromosomes,
have different enzymatic activities in the melanogenic pathway,
share some sequence similarity and constitute a "tyrosinase
family" of proteins.
TRANSCRIPTION REGULATION OF PIGMENT
CELL-SPECIFIC GENE EXPRESSION
Tissue-specific gene expression and cell differentiation are
governed by complex interaction of specific transcription factors
interacting with DNA elements in gene promoters. In melanocytes,
the regulatory 5'-sequences of tyrosinase, TRP1 and TRP2 genes
have been isolated and analysed for the presence of positive and
negative elements crucial for pigrnent cell-specific gene expression.
All the three pigment cell-specific enzymes mentioned above are
expressed in normal melanocytes. However, some or all of these
markers are often repressed in melanoma cell lines (7, 9).
For the mouse tyrosinase gene transcription, a 270-bp sequence
flanking the transcriptional start site is sufficient to confer
melanocyte-specific transcription of the reporter gene in a human
and a mouse melanoma cell line (21). This promoter sequence was
also sufficient for expression of the tyrosinase transgene both
in skin melanocytes derived from the neural crest and pigmented
retinal cells derived from the optic cup in mice. Tyrosinase in
pigmented retinal cells was expressed from day 10,5 and in hair
folicles from day 16,5 onwards in the mouse embryo (21a); thus,
the mouse tyrosinase promoter was sufficient to provide cell-type
and developmental regulation of tyrosinase in skin melanocytes
and pigmented cells in the retina. The pattern of transgene expression
mimicked the sites of expression of the endogenous tyrosinase
(21, 21a). Similarly, in human tyrosinase promoter, only 115-bp
segment of the upstream sequence was sufficient to confer tissue-specific
transcription 122). Apart from the proximal promoter sequences
required for cell-specific transcription, a short (39bp) distal
enhancer element (at position about - 1800-bp upstream from the
transcriptionaI start) was identified in the 5'-flanking region
of the human tyrosinase gene (23). This elernent, further narrowed
to only 20-bp sequence binds, however, factors from nuclear extract
from both the melanoma and HeLa cells (24). The tyrosinase promoter
differs from the TRP1 promoter because it does not contain typical
TATA box or CCAAT box present in the tyrosinase promoter (25,
26). A 5'-flanking segment of 370-bp of the mouse TRP 1 is sufficient
to direct cell-specific expression of the reporter gene in mouse
melanoma cells (25). However, despite the differences in the TRP
1 and tyrosinase gene promoters, there is an important 1 bp element
conserved in both promoters in both mouse and human genes (25,
27). This motif, termed M-box (ATGCATGTGCT), contains a 6bp sequence
(CANNTG), a E-box known to be recognized by a family of basic-helix-loop-helix
(bHLH) transcription factors comprising proteins with either specific
or ubiquitous pattern of gene transactivation. The M-box was shown
to be a positive functional element within the tyrosinase (28)
and TRP 1 (25, 27) gene promoters. Almost identical M-box has
been detected by sequencing the human TRP2 gene promoter (29)
suggesting further the possible irnportance of this element for
melanocyte-specific gene transcription. The functional analysis
of the human TRP2 promoter sequences has been also performed (29):
a 32-bp distal sequence located -447 to -415bp upstream to the
transcriptional start was sufficient for pigment cell-specific
expression but the simultaneous presence of another more proximal
region (containing M-box) was also required (29). It should be
noted that TRP2 is the first melanocyte-specific marker that is
expressed, detecting the early migratory melanoblasts at day 10
in the mouse embryo (30).
By a detailed deletional and mutational analysis of the mouse
TRP1 promoter, both positive and negative elements were identified
(27). The stretch between -44 and +107 (containing the M-box)
was able to confer cell-specific transcription of the gene. Two
positive and one negative element correlated with protein binding
and it was surprising that one of the positive element, the M-box,
was capable to regulate positively the expression in both TRP
1-expressing and -nonexpressing cell lines (27). It was further
demonstrated that M-box bound the ubiquitous bHLH transcription
factor USF (31). In the TRP1 promoter, the initiator element (-1
to +5) that binds putative melanocyte specific factor, seems to
be important for melanocyte-specific expression. The same factor
interacts also with identical upstream sequence (31). These two
elements, however, overlap two negative regulatory elements. When
the initiator element was mutated, dramatic increase of the reporter
expression was observed and the expression was detected also in
nonpigmented cells not expressing TRP1 (31). Additional upstream
octamer binding motif acts as a positive regulator of TRP 1 expression
Recently, the gene that maps to the mi (microphthalmia) locus
of the mouse has been identified (32, 33). Many different mutated
mi alleles have been described and mice homozygous for the mutated
mi gene have defects in melanin pigmentation, small eyes and are
deaf (due to the lack of melanocytes in the inner ear) (34). In
human, mutations at the mi locus causes Waardenburg syndrome type:
2 (35) and the human homologue of mi has also been cloned (36).
Since the mi protein is a member of the bHLH-leucine-zipper protein
family of transcription factors, it has become an excellent candidate
for the melanocyte-specific transcripton factor. Indeed, it has
been shown that it can transactivate transcription from the promoter
containing the M-box (22, 24, 37). Mi protein, however, binds
the CANNTG motif (37) and might probably stimulate transcription
through the interaction with this motif at the initiator sequence
of the human tyrosinase promoter (22). Microphthalmia protein
binds to DNA as homodimer or heterodimer with factors TFEB, TFE3
or TFEC. This group of proteins constitute a novel MiT gene family
(37). The mi mutations, when analysed for their DNA recognition
and oligomerization abilities in vitro, can explain the effects
of mutations known in mi mutant mice strains (37). Microphthalmia
gene therefore seems to be crutial for the melanocyte differentiation
and function. It is expressed in cells producing melanin and,
surprisingly, in heart in high levels (32). Additionally, microphtalmia
gene seems to be a target for the repression by adenoviral Ela
oncogene which is known to repress many differentiation-specific
genes. The arteficial expression of mi gene from CMV promoter
prevents the repression of tyrosinase and TRP 1 genes seen in
Ela-expressing cells (38). Moreover, the mi protein interacts
with the retinoblastoma protein in vitro (38).
Although the precise role of microphtalmia protein and other potential
transcription factors in the transcriptional regulation of the
tyrosinase family of genes and the regulation of expression of
the mi gene itself remains to be determined, the function of melanogenic
enzymes and the regulation of expression of genes encoding these
factors are now more clear than several years ago. Besides the
three melanogenic factors other proteins such as pmel17/silver
locus gene product (39) were suggested to be structural part of
the melanosome matrix and might also prove to be important for
melanogenesis in subcellular particles - melanosomes. However,
little is known about the molecular mechanisms that operate in
the conversion of normal to malignant melanocytes and the role
of differentiation-specific genes in the process.
1. K ö r n e r A., P a w ele k J.:Mammalian tyrosinase catalyzes
three reactions in the
biosynthesis of melanin. Science 217, 1982, 1163-1165.
2. Vachtenheim J., Duchon J., Matous B., Vulterin K.: Specific
antityrosinase antibodies of tyrosinase-mediated melanogenesis.
J. Invest. Derm.
86, 1986, 573-576.
3. Kobayashi T., Urabe K., Winder A., Jiménez-Cervantes
C., Imokawa G.,
Brewington T., Solano F., García-Borrón J. C.,
Hearing V. J.:Tyrosinase related
protein 1 (TRP1) functions as a DHICA oxidase in melanin biosynthesis.
J. 13, 1994, 5818-5825.
4. Tsukamoto K., Jackson I. J., Urabe K., Montague P. M., Hearing
V. J.: A second
tyrosinase-related protein, TRP-2, is a melanogenic enzyme
tautomerase. EMBO J. 11, 1992, 519-526.
5. Yokoyama K., Suzuki H., Yasumoto K., Tomita Y., Shibahara S.:
and functional analysis of a cDNA coding for human DOPAchrome
rosinase-related protein-2. Biochim. Biophys. Acta 1217, 1994,
6. Kroumpouzos G., Urabe K., Kobayashi T., Sakai C., Hearing V.
analysis of the slaty gene product (TRP2) as dopachrome tautomerase
effect of a point mutation on its catalytic function. Biochem.
Biophys. Res. Commun.
202, 1994, 1060-1068.
7. DelMarmol V., Ito S., Jackson I., Vachtenheim J., Berr P.,
Ghanem G., Morandini
R., Wakamatsu K., Huez G.:TRP-1 expression correlates with
human pigment cells in culture. FEBS Lett. 327, 1993, 307-310.
8. K wo n B. S., Haq A. K., P o m e ra nt z S. H., H ala b
a n R.:Isolation and
sequence of a cDNA clone for human tyrosinase that maps at
the mouse c-albino
locus. Proc. Natl. Acad. Sci. USA 84, 1987, 7473-7477.
9. Müller G., R u p p e rt S., S c h m i d E., S c h u
t z G.:Functional analysis of
alternatively spliced tyrosinase gene transcripts. EMBO J.
7, 1988, 2723-2730.
10. Bouchard B., Fuller B. B., Vijayasaradhi S., Houghton A.
pigmentation in mouse fibroblasts by expression of human
tyrosinase cDNA. J. Exp.
Med. 169, 1989, 2029-2042.
11. Ruppert S., Müller G., Kwon B., Schutz G.: Multiple
transcripts of the mouse
tyrosinase gene are generated by alternative splicing. EMBO
J. 7, 1988, 2715-2722.
12.Giebel L. B., Strunk K. M., Spritz R. A.:Organization and
of the human tyrosinase gene and a truncated tyrosinase-related
Genomics 9, 1991, 435-445.
13. P o n n a z h a g a n S., H o u L., K w o n B. S .: Structural
organization of the
human tyrosinase gene and sequence analysis and characterization
of its promoter
region. J. Invest. Derm. 102, 1994, 744-748.
14. B a rt o n D. E., K w o n B. S., F r a n c k e U.:Human
tyrosinase gene mapped to
chromosome 11(q14-q21) defines second region of homology
chromosome 7. Genomics 3, 1988, 17-24.
15. Jackson I. J.: A cDNA encoding tyrosinase-related protein
maps to the brown locus
in mouse. Proc. Natl. Acad. Sci. USA 85, 1988, 4392-4396.
16. Jackson I. J., Chambers D. M., Tsukamoto K., Copeland N. G.,
Gilbert D. J.,
Jenkins N. A., Hearing V.:A second tyrosinase-related protein,
TRP-2, maps to
and is mutated at the mouse slaty locus. EMBO J. 11, 1992,
17. Shibahara S.,Tomita Y., Sakakura T., Nager C., Chaudhuri
B., Muller R.: Cloning
and expression of cDNA encoding mouse tyrosinase. Nucl.
Acid Res. 14, 1986,
18. Cohen T., Muller R. M., Tomita Y., Shibahara S.:Nucleotide
sequence of the
cDNA encoding human tyrosinase-related protein. Nucl. Acid
Res. 18, 1990, 2807-
19. A b b o t t C., J a c k s o n I. J., C a r r i t t B ,
P o v e y S.:The human homolog of
the mouse brown gene maps to the short arm of chromosome
9 and extends the
known region of homology with mouse chromosome 4. Genomics
11, 1991, 471-
20. Sturm K. A., Baker E., Sutherland G. R.: Assignment of the
protein-2 gene (TYRP2) to human chromosome 13q31-q32 by
fluorescence in situ
hybridization: extended synteny with mouse chromosome 14.
Genomics 21, 1994,
21. Kluppel M , Beermann F., Ruppert S., Schmid E., HummIer E.,
Schutz G .: The
mouse tyrosinase promoter is sufficient for expression in
melanocytes and in the
pigmented epithelium of the retina. Proc. Natl. Acad. Sci.
USA 88, 1991, 3777-3781.
21a. Beerman F., Schmid E , Schutz G.:Expression of mouse tyrosinase
embryonic development: Recapitulation of the temporal regulation
mice. PNAS 89, 1992, 2809-2813.
22. B e n tle y N. J., E i s e n T., G o d i n g C. R . : Melanocyte-specific
the human tyrosinase promoter: Activation by the microphthalmia
and role of the initiator. Mol. Cell. Biol. 14, 1994, 7996-8006.
23. S h i b a t a K., M u ra o sa Y , T o m i ta Y., Tagami
S.:ldentification of a cis-acting element that enhances the
expression of the human tyrosinase gene. J. Biol. Chem. 267,
24. Y a s u m ot o K.- I., Y o k o y a m a K., S h i b a t a
K , T o m i t a Y.,
S h i b a h a r a S.:Microphthalmia-associated transcription
factor as a regulator for
melanocyte-specific transcription of the human tyrosinase
gene. Mol. Cell. Biol. 14,
25. Jackson I. J., Chambers D. M., Budd P. S., Johnson R.: The
protein-1 gene has a structure and promoter sequence very
tyrosinase. Nucl. Acid Res. 19, 1991, 3799-3804.
26. Kikuchi H., Miura H , Yamarnoto H., Takeuchi T., Dei T., Watanabe
Characteristic sequences in the upstream region of the human
Biochem. Biophys. Acta 1009, 1989, 283-286.
27. Lowings P., Yavuzer U., Goding C. R.: Positive and negative
a melanocyte-specific promoter. Mol. Cell. Biol. 12, 1992,
28. Ganss R., S c h u t z G., B e erm an n F.:The mouse tyrosinase
modulation by positive and negative regulatory elements.
J. Biol. Chem. 269, 1994,
29. Y o k o y a m a K., Y a s u m o t o K , S u z u k I H.,
S h i b a h a r a S. : Cloning
of the human DOPAchrome tautomerase/tyrosinase-related
protein 2 gene and
identification of two regulatory regions required for its
expression. J. Biol. Chem. 269, 1994, 27080-27087.
30. Steel K. P., Davidson D. R., Jackson I. J.:TRP-YDT, a new
marker, shows that steel growth factor (c-kit ligand) is
a survival factor.
Development 115, 1992, 1111-1119.
31. Yavuzer U., Goding C. R.: Melanocyte-specific gene expression:
repression and identification a melanocyte-specific factor,
MSF. Mol. Cell. Biol. 13,
32. Hodgkinson C. A., Moore K. J., Nakayama A., Steigrimsson E.,CopeIand
J e n k i n s N. A., Ar n h e i t e r H.:Mutations at
the mouse microphthalmia
locus are associated with defects in a gene encoding a novel
helix-zipper protein. Cell 74, 1993, 395-404.
33. Hughes M. J., Lingrel J. B., Krakowsky J. M., Anderson K.
transcription factor-like gene is located at the mi locus.
J. Biol. Chem. 268, 1993,
34. Jackson I. J., Raymond S.: Manifestations of microphthalmia.
Nature Genet. 8,
35. Tassaabe hj i M., N ewton V. E., Re ad A. P.:Waardenburg syndrome
caused by mutations in the human microphthalmia (MITF) gene.
Nature Genet. 8,
36. Tachibana M., Perez-Jurado J. A., Nakayama A., Hodgkinson
C. A., Li X.,
Schneider M., Miki T., Fex J., Fra ncke U., Arnheit er H:
Cloning of MITF, the
human homoIog of the mouse microphthalmia gene and assignment
chromosome 3p14.1-p12.3. Human Mol. Genet. 3, 1994, 553-557.
37. Hemesath T. J., Steigrímsson E., McGill G., Hansen
M. J., Vaught J.,
Hodgkinson C. A.,ArnheiterH., Copeland N. G., Jenkins N.
A., Fisher D. E.:
Microphthalmia, a critical factor in melanocyte development,
defines a discrete
transcription factor family. Genes & Develop. 8, 1994,
38. Yavuzer U., Keenän E., LowingJ P., Vachtenheim J.,Currie
G., Goding C. R.:The
microphthalmia gene product interacts with the retinoblastoma
protein in vitro and
is a target for deregulation of melanocyte-specific transcription.
39. Kobaya shi T., Ur abe K., Orlow S. J., Higashi K., Imokawa
G., Kwon B. S.,
Potter B., Hearing V. J.:The Pmel 17/silver locus protein.
investigation of its melanogenic functions. J. Biol. Chem.
269, 1994, 79198-29205.