0 1992 MUNKSGAARD
Pigment Cell Research 5:155-161(1992)
A &-Acting Element Involved in Mouse vrosinase Gene Expression and Partial Purification of its Binding Protein S. PONNAZHAGAN AND BYOUNG S. KWON Department of Microbiology and Immunology, and Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana 46202 The tyrosinase gene is specifically expressed in melanocytes. Understanding the molecular basis of tissue-specific expression of the tyrosinase gene will greatly explain the mechanisms controlling pigmentation. We report a nucleotide sequence, TGATGTATTC, located -236 base pairs upstream of the transcription start site, that enhances tyrosinase gene expression in mouse melanoma cells. The sequence is referred to as the tyrosinase element-1 (TE-1). TE-1 was protected from DNase I cleavage by pigment cell nuclear extracts but was not protected by non-pigment cell nuclear extract. Partial purification of TE-1 binding protein (TEBP-1) was performed from the B16 mouse melanoma cell nuclear extract using biotin-cellulose affinity chromatography. The affinity-purified fraction exhibited binding to the DNA fragment containing TE-1, and to a synthetic oligomer representing TE-1. UV-cross-linking indicated that the size of TEBP-1 is approximately 49 kD.TE-1 also directed enhanced CAT activity in the B16 melanoma cells but not in non-pigment cells. These data indicate that TE-1 may be an enhancer element that is responsible for pigment cell specific expression of the tyrosinase gene. Key words: Qrosinase, cis-Acting element, Trans-Acting factor, Qrosinase element-1 (TEl), Tissue specific transcription
INTRODUCTION The cis-acting elements controlling transcription of eukaryotic genes typically include the core promoter elements that determine the basal level of transcription and several enhancer elements that mediate the action of trunsactingregulatory factors. The collectiveinteraction ofthese factorsdetermine the overall rate oftranscription. Tyrosinase (monophenol monoxygenase, monophenol, L-dopa: oxygen oxidoreductase, E.C.1.14.18.1) is specifically expressed in the melanocytes and is an essential enzyme in melanogenesis (Hearing and Jimenez, 1989). We and others have previously reported the isolation of cDNAforthemousetyrosinasegene(Yamamotoet al., 1987; Kwon et al., 1988;Muller e t al., 1988). We have characterized the mouse tyrosinase gene (Ruppert et al., 1988)and showed that the gene-encoding tyrosinase is situated at the genetically well characterized c-locus in mouse (Kwon e t al., 1988; Kwon et al., 1989). This was also demonstrated by the rescue of c-locus mutant albino through the introduction of functional tyrosinase minigene constructs into mice (Yamamoto et al., 1989;Beerman et al., 1990). Studies also indicated that a 2.2 kb of 5' upstream regulatory region of the tyrosinase gene appears sufficient to provide appropriate developmental regulation and tissue-specific expression ofthe tyrosinasegene (Yokoyamaet al., 1990). More recently, it has been reported that a 270-base pair region upstream of the transcriptional start site of the mouse tyrosinase gene was sufficient to induce tissue-specific
expression of the tyrosinase gene in transgenic mice (Kluppel et al., 1991).These observationsindicate that essentialand tissue specific cis-acting element(s)regulating the expression ofthe mouse tyrosinase gene should lie within a short segment adjacent to the transcription start site. In this regard, we have investigated for a potential cisacting element and tram-acting factor regulating mouse tyrosinase gene expressionusing DNA binding assays, affinity purification, and transient expression system.
MATERIALS AND METHODS Cell Culture and Nuclear Extracts B16 melanoma cells were grown in DMEM supplemented with 10%fetal calf serum. Nuclear extracts were prepared essentially following the method of Dignam e t al. (1983) using HEPES buffer. Usually, 4 to 6 mg/ml protein was obtained from lo8 cells. Promoter Fragment A 0.42-kb HindIII-XmnI fragment ofmouse tyrosinase ( - 80 to - 509 bp relative to the transcription start site) was isolated from a2-kb Hind111fragment and subcloned into Address reprint requeststo Byounp S. Kwon, Department of Microbiology and Immunology, and Walther Oncology Center, Indiana University School of Medicine, Indianapolis, I N 46202. Received April 29. 1992; accepted July 7, 1992.
156
S. Ponnazhagan et al.
Affinity Purification Biotin-cellulose affinity purification was carried out following the method of Kadonaga, et al. (1986). The tetramer probe was excised from the pBluescript vector at ClaI and EcoRI sites and end-labeled by Klenow. Thymidine Gel Shift Assay nucleotides were substituted by biotin-UTP and [a-"PIGel shift assays were performed based on the method of dATP was used to monitor the binding stability of the probe Carthew et al. (1985). DNA binding reactions were per- to cellulose. One and one-half milligrams of crude nuclear formed in a volume of 25 p1 with 1 pg of poly dI-dC, 2 pg extract from B16 melanoma was incubated in HEPES buffer BSA, and 12% glycerol in HEPES buffer (pH 7.9). Two with 10% glycerol, 0.1 mglml calf thymus DNA, and 0.1 micrograms of nuclear extract or 0.2 pg of affinity puri- mglml BSA at 30°C for 10 rnin prior t o the addition of the fied fraction was preincubated for 10 min at 30"C, and 10,000 probe; 0.5 pg of biotin-labeled tetramer probe was then cpm of end-labeled probe was added to the mixture. The added to the mixture and binding reaction continued for binding reaction was allowed to proceed for 30 min at 30"C, 30 min. Following binding, 50 pg of streptavidin was added and the bound complex separated from unbound probe on and incubated for an additional 5 min. Biotin-cellulose, 5% low ionic strength polyacrylamide gels with recirculat- pretreated with calf thymus DNA and BSA, was added ing TAE buffer. and allowed to couple with streptavidin-biotin-proteincomplex at 4°C for 90 min with gentle agitation. After this bindDNase I Protection Analysis ing, the mixture was spun briefly in microcentrifuge and The 0.42-kb promoter fragment of the mouse tyrosinase unbound protein removed from cellulose. The resin with was excised from the vector and labeled with [a-"'P]-dATP probe and protein complex was washed five times with at one end (Hind111 end) with Klenow fragment of E . coli biotin-cellulose binding buffer (12 mM HEPES, pH 7.9, 4 polymerase I. The labeled probe was ethanol precipitated, mM Tris-C1, 60 mM KC1, 1 mM EDTA, 1 mM DTT, and and further purified by DEAE membrane (Schleicher & 12% glycerol) to remove unbound protein and the bound Schuell) on an agarose gel. For each footprint reaction, protein was later eluted in 50 pl elution buffer (20 mM 50,000 cpm (
Pigment Cell Research 5:155-161(1992)
A &-Acting Element Involved in Mouse vrosinase Gene Expression and Partial Purification of its Binding Protein S. PONNAZHAGAN AND BYOUNG S. KWON Department of Microbiology and Immunology, and Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana 46202 The tyrosinase gene is specifically expressed in melanocytes. Understanding the molecular basis of tissue-specific expression of the tyrosinase gene will greatly explain the mechanisms controlling pigmentation. We report a nucleotide sequence, TGATGTATTC, located -236 base pairs upstream of the transcription start site, that enhances tyrosinase gene expression in mouse melanoma cells. The sequence is referred to as the tyrosinase element-1 (TE-1). TE-1 was protected from DNase I cleavage by pigment cell nuclear extracts but was not protected by non-pigment cell nuclear extract. Partial purification of TE-1 binding protein (TEBP-1) was performed from the B16 mouse melanoma cell nuclear extract using biotin-cellulose affinity chromatography. The affinity-purified fraction exhibited binding to the DNA fragment containing TE-1, and to a synthetic oligomer representing TE-1. UV-cross-linking indicated that the size of TEBP-1 is approximately 49 kD.TE-1 also directed enhanced CAT activity in the B16 melanoma cells but not in non-pigment cells. These data indicate that TE-1 may be an enhancer element that is responsible for pigment cell specific expression of the tyrosinase gene. Key words: Qrosinase, cis-Acting element, Trans-Acting factor, Qrosinase element-1 (TEl), Tissue specific transcription
INTRODUCTION The cis-acting elements controlling transcription of eukaryotic genes typically include the core promoter elements that determine the basal level of transcription and several enhancer elements that mediate the action of trunsactingregulatory factors. The collectiveinteraction ofthese factorsdetermine the overall rate oftranscription. Tyrosinase (monophenol monoxygenase, monophenol, L-dopa: oxygen oxidoreductase, E.C.1.14.18.1) is specifically expressed in the melanocytes and is an essential enzyme in melanogenesis (Hearing and Jimenez, 1989). We and others have previously reported the isolation of cDNAforthemousetyrosinasegene(Yamamotoet al., 1987; Kwon et al., 1988;Muller e t al., 1988). We have characterized the mouse tyrosinase gene (Ruppert et al., 1988)and showed that the gene-encoding tyrosinase is situated at the genetically well characterized c-locus in mouse (Kwon e t al., 1988; Kwon et al., 1989). This was also demonstrated by the rescue of c-locus mutant albino through the introduction of functional tyrosinase minigene constructs into mice (Yamamoto et al., 1989;Beerman et al., 1990). Studies also indicated that a 2.2 kb of 5' upstream regulatory region of the tyrosinase gene appears sufficient to provide appropriate developmental regulation and tissue-specific expression ofthe tyrosinasegene (Yokoyamaet al., 1990). More recently, it has been reported that a 270-base pair region upstream of the transcriptional start site of the mouse tyrosinase gene was sufficient to induce tissue-specific
expression of the tyrosinase gene in transgenic mice (Kluppel et al., 1991).These observationsindicate that essentialand tissue specific cis-acting element(s)regulating the expression ofthe mouse tyrosinase gene should lie within a short segment adjacent to the transcription start site. In this regard, we have investigated for a potential cisacting element and tram-acting factor regulating mouse tyrosinase gene expressionusing DNA binding assays, affinity purification, and transient expression system.
MATERIALS AND METHODS Cell Culture and Nuclear Extracts B16 melanoma cells were grown in DMEM supplemented with 10%fetal calf serum. Nuclear extracts were prepared essentially following the method of Dignam e t al. (1983) using HEPES buffer. Usually, 4 to 6 mg/ml protein was obtained from lo8 cells. Promoter Fragment A 0.42-kb HindIII-XmnI fragment ofmouse tyrosinase ( - 80 to - 509 bp relative to the transcription start site) was isolated from a2-kb Hind111fragment and subcloned into Address reprint requeststo Byounp S. Kwon, Department of Microbiology and Immunology, and Walther Oncology Center, Indiana University School of Medicine, Indianapolis, I N 46202. Received April 29. 1992; accepted July 7, 1992.
156
S. Ponnazhagan et al.
Affinity Purification Biotin-cellulose affinity purification was carried out following the method of Kadonaga, et al. (1986). The tetramer probe was excised from the pBluescript vector at ClaI and EcoRI sites and end-labeled by Klenow. Thymidine Gel Shift Assay nucleotides were substituted by biotin-UTP and [a-"PIGel shift assays were performed based on the method of dATP was used to monitor the binding stability of the probe Carthew et al. (1985). DNA binding reactions were per- to cellulose. One and one-half milligrams of crude nuclear formed in a volume of 25 p1 with 1 pg of poly dI-dC, 2 pg extract from B16 melanoma was incubated in HEPES buffer BSA, and 12% glycerol in HEPES buffer (pH 7.9). Two with 10% glycerol, 0.1 mglml calf thymus DNA, and 0.1 micrograms of nuclear extract or 0.2 pg of affinity puri- mglml BSA at 30°C for 10 rnin prior t o the addition of the fied fraction was preincubated for 10 min at 30"C, and 10,000 probe; 0.5 pg of biotin-labeled tetramer probe was then cpm of end-labeled probe was added to the mixture. The added to the mixture and binding reaction continued for binding reaction was allowed to proceed for 30 min at 30"C, 30 min. Following binding, 50 pg of streptavidin was added and the bound complex separated from unbound probe on and incubated for an additional 5 min. Biotin-cellulose, 5% low ionic strength polyacrylamide gels with recirculat- pretreated with calf thymus DNA and BSA, was added ing TAE buffer. and allowed to couple with streptavidin-biotin-proteincomplex at 4°C for 90 min with gentle agitation. After this bindDNase I Protection Analysis ing, the mixture was spun briefly in microcentrifuge and The 0.42-kb promoter fragment of the mouse tyrosinase unbound protein removed from cellulose. The resin with was excised from the vector and labeled with [a-"'P]-dATP probe and protein complex was washed five times with at one end (Hind111 end) with Klenow fragment of E . coli biotin-cellulose binding buffer (12 mM HEPES, pH 7.9, 4 polymerase I. The labeled probe was ethanol precipitated, mM Tris-C1, 60 mM KC1, 1 mM EDTA, 1 mM DTT, and and further purified by DEAE membrane (Schleicher & 12% glycerol) to remove unbound protein and the bound Schuell) on an agarose gel. For each footprint reaction, protein was later eluted in 50 pl elution buffer (20 mM 50,000 cpm (
