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Deletion subcloning reveals sophisticated cellular regulation of the transglutaminasei enzyme
52
Oct. 31/Mini-Symposium
235
2 - Gene Regulation in Normal and Diseased Skin
238 “D”SE 23O-kDa CHARACTERIZATION
BLJCLO”S
PE”PHIGOlD
ANTIGEN
GENE:
OF 5’-FLANKING REGION. Daisuke Sawamura, Takashi Sate, Atushi Kant Ken Harada, Kazuo Nomura, E Hashimoto, Katsuto Tamai, Jouni Ultto. Dept. of Dermatol. Hirosaki UniverSity and Deot. Of Dermatol. Jefferson Medical College. 230-k& bulbous pemphlqoid antigen (BPAG? the malor autoantiqen an bullous pemphlgold. To clone the 5’flanking region of mouse BPAGl gene, we screened a mouse genomic library with a human 0.3 kb CDNR and Isolated a ca. 15 kb clone. Southern hybridization allowed isolation of a 3 kb Hind III fragment which was sublect to nucleotlde sequencing. Alignment of the mo”se nucleotide sequences with human BPAGl sequences revealed that the 3.0 fragment contained 2.4 kb of 5’.flankinq region, addition to exon
t I_=.
i and exon 2. TATRAAand CCAATco”se”5”* Seq”e”C.?S, a5 well as several p”tatl”e cis-regulatory sequences wereidentified in the 5’.flanking region. The
functionality of the promoter region was demonstrated by development of promoter/CAT reporter gene constructs, followed by transfections of normal mouse keratlnocytes and PAM cells in culture. The CAT assays revealed significant promoter actlvlty in both types of cells. Also transfection study using PAM, NIH3T3, 816 melanoma, and CT26 colon carcinoma cells suggests that 1.1 kb 5’. flanking DNA contains ~1s.acting elements which confer tissue specxflcity to the expression of BP*Gl gene.
236
239 THE GENE EXPRESSIONS OF TYROSlNASE AND mOS,NASE-REIATED PROTEIN ARE ENHANCED BY DIFFERENT CIS-ACTING ELEMENTS. Ynsusht Tmta’, Koushi Shlbata’. Shlgekl Shibahara’ and Hachiro Tagamr’.
‘Department of Dermatology and ‘Department of Applied Physiology and Molecular Biology. Tohoko Llmversity School of Medicine. Sendai, and ‘Pola K & D Laborat&es. Yokohama. Japan Tyroslnase IS a rate-hmiting enzyme of melanm biosynthesis. Tyrosmaserelated protem ,TRP. hro~n-locus protem, IS mvolved in the production of black melanm rather than brown. Both tyrosmase and TRP share a slgmficant homology (about 40%) m amino acid sequence, are specifically expressed in melanacytes. are Induced su”ilar,y by several b,olog,cal factors, and are probably derived from a common ancestral gene. It IS therefore important to knoiv whether the gene expresslo”s of both tyroslnase and TRP are regulated by a similar promoter function. We found the c&acting element. located between 2.0 and 1.8 kiiobase pairs (Kbp) upstream from the transcnption initiation site of human tyrosmase gene, that enhances the transwnt ex,,ress,on of the luc~ferase reporter gene ,n melanoma cells. but not in &,a cells. We further identified the oizment WI,-spec,f~ enhanrer of 39.bp core clement between -2.0 and -1.8 i6p reOn the other Fponslbie for the expresslo” of the human tyrosinase gene. hand. the downstream regmn of TRP gene. includmg the first intro”, enhances the transient expression of the luclferase reporter gene under control of the TRP gene promoter. This enhancer-like actlvlty IS detected not only m melanoma cells but also in ~e,a cells whose TRP gene expression IS assumed to be repressed. These results indicate that the promoter function of the human tyrosmase gene is completely different from that of the human TRP gene. bhanges
237
240
DELETION SUBCLONING REVEALS SOPHISTICATED CELLULAR REGULATION OF THE TRANSGLUTAMINASEI ENZYME. S.-Y. Kim. K. Yoneda. P.M. Steincn. Skin Biology branch. NIAMS; and S.-l. Chung, Lahxalory Of Cellular Development and Oncology, NIDR. NM. U.S.A. The formation of the comified cell envelope in terminally dlfferenliating epidemus involves three different transglufaminases (TGases). the membrane-associaled TGase,. ubiqullous tissue TGase2 and procnzyme TGase3. Although infomuxion is avadable on then sequences.propenies and expresslo”, litde is known about [heir precise role(s). We have made a series of deletion constructs of the TGasel system from our full-length cDNA Following expression in bacteria and purification, rhe forms were characterized with respecl1o lheu specific actlvties and subsmatespecificides. Deleoon of rhe fust 30. 63 amino acids resulied in a ien-fold increase in specific acdvity. Their x,iviIy could bc further increased two-fold on dispase treatment. D&dons beyond residue 63 resulted in a less dramadc change, and deletions beyond IO9 residues lost activity. Similarly, deletions of rhe last 240 residues retamed activity, but deletions beyond this lost activity. Thus we can define the minimal sequences required for acdvlty. The data Indicate TGasel has been modified and enlareed durine evolution by addirion of se~oencesthat effectively brake its specific activity-and ma& proteolytic.actrvation sites’common to other TGases. Consmxrs in which the first 62.97 had been deleted showed dramatic variations in substraw specilicilies toward synthetic peptides correspondmg 10 sequences of putadve TGase subsnares. Indirect immunofluorescence experiments using a new antibody to the smallest active consrru~t defined above decorated all levels of the epldemus, with some porentn.uon in the granular layer. By Western blotting we found that boiled SDS exrracts of cultured HaCat or RHEK cells contain a series of smaller processed TGasel fomx which also possesshigh specific activities. The presence of such forms in living cells suggests that epithelial cells can regulate both rhe specific acrivi!y and substrate specificity of the TGasel system by controlled pro,eolytlc processingduring retinal differentiation.
wvi PRO’TD-ONCOGI:NL lrimi~rrs .TKANSFORMING GROWTII FACI~OK+ INIXICF,) ,NI,,RlTION OF COLLACtNASI: GENE U(PRIXiION. Ala111Mauviel, Yuc Oiu Chen. und looni Ilitro. I~ep~runcnls of Dermatology, and Iiiochcmlstr) and hloleculz~r Ihology. lefferson Medical College, I’hlladelphia. ,‘A. We have ~nvcsl~garcd the moleculn mechanisms b) which TC&fl interacts nlth pro-inflammatory rylohines, intcrleukin-I (Il.-lo and ll:,g). and (urnor nccros~s lactnr-n (TNI-u), m modulating fibroblast collagen~sc gene nprcsrion m 1 ihv. T(;I:-1%blocks the indunion of rollagenase gene e\prcs~inn induced b> the r)toLines sludied. ils dclcrmined ~1 the mRNA slead\vslnlc Ic\cl and h) lrilnbienl cell tranbfcclmns !vith 3 collaaenase gone c”“s~ruc,. Annl>sis of the c\pr&on pattern of o&ogcnes of the Jon famllj rcvuled rhar Ihe I\X’O forms of ,,:, and TNf-n promote high Incl~ of c-,un mRNA, mdependontly of the presence of XI--& By contr3\1, ‘ICI:+! has hctlc cffoct on c-1x1”npression but is il potent inducer of /on-H mRNh. in the absence or in the presence of the other cytokines. Also, 3 Ir~nsmnr clcvatinn of c-fos mRNA levels xxs noted following flhroblaat s,lmu,a,ion by an, 01 ,he grmrth fdcrors studled. Using c\prcs9nn \wIors for r--jun and Jon-8 in transient Cell Iransfections. we
Oct. 31/Mini-Symposium
235
2 - Gene Regulation in Normal and Diseased Skin
238 “D”SE 23O-kDa CHARACTERIZATION
BLJCLO”S
PE”PHIGOlD
ANTIGEN
GENE:
OF 5’-FLANKING REGION. Daisuke Sawamura, Takashi Sate, Atushi Kant Ken Harada, Kazuo Nomura, E Hashimoto, Katsuto Tamai, Jouni Ultto. Dept. of Dermatol. Hirosaki UniverSity and Deot. Of Dermatol. Jefferson Medical College. 230-k& bulbous pemphlqoid antigen (BPAG? the malor autoantiqen an bullous pemphlgold. To clone the 5’flanking region of mouse BPAGl gene, we screened a mouse genomic library with a human 0.3 kb CDNR and Isolated a ca. 15 kb clone. Southern hybridization allowed isolation of a 3 kb Hind III fragment which was sublect to nucleotlde sequencing. Alignment of the mo”se nucleotide sequences with human BPAGl sequences revealed that the 3.0 fragment contained 2.4 kb of 5’.flankinq region, addition to exon
t I_=.
i and exon 2. TATRAAand CCAATco”se”5”* Seq”e”C.?S, a5 well as several p”tatl”e cis-regulatory sequences wereidentified in the 5’.flanking region. The
functionality of the promoter region was demonstrated by development of promoter/CAT reporter gene constructs, followed by transfections of normal mouse keratlnocytes and PAM cells in culture. The CAT assays revealed significant promoter actlvlty in both types of cells. Also transfection study using PAM, NIH3T3, 816 melanoma, and CT26 colon carcinoma cells suggests that 1.1 kb 5’. flanking DNA contains ~1s.acting elements which confer tissue specxflcity to the expression of BP*Gl gene.
236
239 THE GENE EXPRESSIONS OF TYROSlNASE AND mOS,NASE-REIATED PROTEIN ARE ENHANCED BY DIFFERENT CIS-ACTING ELEMENTS. Ynsusht Tmta’, Koushi Shlbata’. Shlgekl Shibahara’ and Hachiro Tagamr’.
‘Department of Dermatology and ‘Department of Applied Physiology and Molecular Biology. Tohoko Llmversity School of Medicine. Sendai, and ‘Pola K & D Laborat&es. Yokohama. Japan Tyroslnase IS a rate-hmiting enzyme of melanm biosynthesis. Tyrosmaserelated protem ,TRP. hro~n-locus protem, IS mvolved in the production of black melanm rather than brown. Both tyrosmase and TRP share a slgmficant homology (about 40%) m amino acid sequence, are specifically expressed in melanacytes. are Induced su”ilar,y by several b,olog,cal factors, and are probably derived from a common ancestral gene. It IS therefore important to knoiv whether the gene expresslo”s of both tyroslnase and TRP are regulated by a similar promoter function. We found the c&acting element. located between 2.0 and 1.8 kiiobase pairs (Kbp) upstream from the transcnption initiation site of human tyrosmase gene, that enhances the transwnt ex,,ress,on of the luc~ferase reporter gene ,n melanoma cells. but not in &,a cells. We further identified the oizment WI,-spec,f~ enhanrer of 39.bp core clement between -2.0 and -1.8 i6p reOn the other Fponslbie for the expresslo” of the human tyrosinase gene. hand. the downstream regmn of TRP gene. includmg the first intro”, enhances the transient expression of the luclferase reporter gene under control of the TRP gene promoter. This enhancer-like actlvlty IS detected not only m melanoma cells but also in ~e,a cells whose TRP gene expression IS assumed to be repressed. These results indicate that the promoter function of the human tyrosmase gene is completely different from that of the human TRP gene. bhanges
237
240
DELETION SUBCLONING REVEALS SOPHISTICATED CELLULAR REGULATION OF THE TRANSGLUTAMINASEI ENZYME. S.-Y. Kim. K. Yoneda. P.M. Steincn. Skin Biology branch. NIAMS; and S.-l. Chung, Lahxalory Of Cellular Development and Oncology, NIDR. NM. U.S.A. The formation of the comified cell envelope in terminally dlfferenliating epidemus involves three different transglufaminases (TGases). the membrane-associaled TGase,. ubiqullous tissue TGase2 and procnzyme TGase3. Although infomuxion is avadable on then sequences.propenies and expresslo”, litde is known about [heir precise role(s). We have made a series of deletion constructs of the TGasel system from our full-length cDNA Following expression in bacteria and purification, rhe forms were characterized with respecl1o lheu specific actlvties and subsmatespecificides. Deleoon of rhe fust 30. 63 amino acids resulied in a ien-fold increase in specific acdvity. Their x,iviIy could bc further increased two-fold on dispase treatment. D&dons beyond residue 63 resulted in a less dramadc change, and deletions beyond IO9 residues lost activity. Similarly, deletions of rhe last 240 residues retamed activity, but deletions beyond this lost activity. Thus we can define the minimal sequences required for acdvlty. The data Indicate TGasel has been modified and enlareed durine evolution by addirion of se~oencesthat effectively brake its specific activity-and ma& proteolytic.actrvation sites’common to other TGases. Consmxrs in which the first 62.97 had been deleted showed dramatic variations in substraw specilicilies toward synthetic peptides correspondmg 10 sequences of putadve TGase subsnares. Indirect immunofluorescence experiments using a new antibody to the smallest active consrru~t defined above decorated all levels of the epldemus, with some porentn.uon in the granular layer. By Western blotting we found that boiled SDS exrracts of cultured HaCat or RHEK cells contain a series of smaller processed TGasel fomx which also possesshigh specific activities. The presence of such forms in living cells suggests that epithelial cells can regulate both rhe specific acrivi!y and substrate specificity of the TGasel system by controlled pro,eolytlc processingduring retinal differentiation.
wvi PRO’TD-ONCOGI:NL lrimi~rrs .TKANSFORMING GROWTII FACI~OK+ INIXICF,) ,NI,,RlTION OF COLLACtNASI: GENE U(PRIXiION. Ala111Mauviel, Yuc Oiu Chen. und looni Ilitro. I~ep~runcnls of Dermatology, and Iiiochcmlstr) and hloleculz~r Ihology. lefferson Medical College, I’hlladelphia. ,‘A. We have ~nvcsl~garcd the moleculn mechanisms b) which TC&fl interacts nlth pro-inflammatory rylohines, intcrleukin-I (Il.-lo and ll:,g). and (urnor nccros~s lactnr-n (TNI-u), m modulating fibroblast collagen~sc gene nprcsrion m 1 ihv. T(;I:-1%blocks the indunion of rollagenase gene e\prcs~inn induced b> the r)toLines sludied. ils dclcrmined ~1 the mRNA slead\vslnlc Ic\cl and h) lrilnbienl cell tranbfcclmns !vith 3 collaaenase gone c”“s~ruc,. Annl>sis of the c\pr&on pattern of o&ogcnes of the Jon famllj rcvuled rhar Ihe I\X’O forms of ,,:, and TNf-n promote high Incl~ of c-,un mRNA, mdependontly of the presence of XI--& By contr3\1, ‘ICI:+! has hctlc cffoct on c-1x1”npression but is il potent inducer of /on-H mRNh. in the absence or in the presence of the other cytokines. Also, 3 Ir~nsmnr clcvatinn of c-fos mRNA levels xxs noted following flhroblaat s,lmu,a,ion by an, 01 ,he grmrth fdcrors studled. Using c\prcs9nn \wIors for r--jun and Jon-8 in transient Cell Iransfections. we