Phylogenetic Specificity of Prolactin Gene Expression with Conservation of Pit-l Function
Harry P. Elsholtz*, Sonali Majumdar-Sonnylal, Zhiyuan Gong, and Choy L. Hew Departments of Clinical Biochemistry Biochemistry (F.X., C.L.H.) University of Toronto Toronto, Ontario, Canada M5G 1 L5 The Bantina and Best Diabetes Toronto, OEtario, Canada M5G Hospital Toronto,
for Sick Children Ontario, Canada
Centre 1 L7
(H.P.E,
(H.P.E.. ’
Fei Xiong,
S.M.-S.,
and
SM.-S.)
(F.X., Z.G., C.L.H.) M5G 1X8
of the marked species-dependent expression of PRL genes observed in mammalian and teleost lactotrophs. The dramatic divergence of S-flanking sequences of mammalian and teleost PRL genes further suggests that conservation of Pit-l function is not strictly limited by evolutionary flexibility within genomic regulatory regions. (Molecular Endocrinology 6: 515-522,1992)
In mammals, the pituitary POU homeodomain protein, Pit-l, binds to proximal and distal V-flanking sequences of the PRL gene that dictate tissue-specific expression. These DNA sequences are highly conserved among mammals but are dramatically different from PRL 5’ sequences in the teleost species, Oncorbynchus tschawytscha (chinook salmon). To analyze the molecular basis for pituitary-specific gene expression in a distantly related vertebrate, we transfected CAT reporter gene constructs containing 2.4 kilobases (kb) 5’-flanking sequence from the salmon PRL (sPRL) gene into various cell types. Expression of the sPRL gene was restricted to pituitary cells, but in rat pituitary GH4 cells levels of expression were at least QO-fold lower than those obtained with a -3 kb rat PRL (rPRL) construct. Conversely, in primary teleost pituitary cells, -2.4 kb sPRL/CAT was expressed at levels about lo-fold higher than -3 kb rPRL/CAT. To determine whether species-specific transactivation by Pit-l was sufficient to explain these species differences in PRL gene expression, we isolated a cDNA clone encoding the salmon Pit-l POU domain and constructed a
INTRODUCTION The anterior pituitary hormone PRL is uniquely versatile in its range of actions-from stimulation of lactogenesis in mammals and maternal behavior in birds to regulation of ion transport across the gill epithelia in teleost fish. Unlike its functions, which appear to have evolved extensively, the primary structure of PRL as well as the organization of the PRL gene exhibit marked conservation among vertebrates. Recent cloning of the chinook salmon (Oncorhynchus tschawytscha) PRLII gene ‘.* has revealed that in groups as divergent as mammals and fish, the PRL gene consists of five exons with the position and type of each exon/intron splice site being strictly conserved. Furthermore, in nearly all vertebrates expression of the PRL gene is restricted to the pituitary lactotrophs, suggesting that developmental mechanisms that determine cell type-specific expres-
rat Pit-l expression vector that contained salmon Pit-l POU domain sequences substituted in frame. The chimeric Pit-l encoded 14 amino acids unique to salmon. Coexpression of rat Pit-l with salmon or rat PRL/CAT in transfected HeLa cells resulted in specific and strikingly comparable levels of promoter activation. Moreover, the specificity and efficacy of the chimeric salmon/rat Pit-l was similar to wild type rat Pit-l in activating salmon and rat PRL/ CAT. Our data suggest that the structure and function of Pit-l are highly conserved among vertebrates, and that Pit-l is not the primary determinant 08%%3809/92/0515-0522503.00/O Molecular Endocrinology CopyrIght 0 1992 by The Endocme
C.L.H.)
‘Chin RA, Xiong F, Hew CL, A gene encoding chinook salmon (Oncorhynchus tschawyfscha) prolactin: evolutionary implications and possible regulatory elements. Manuscript submitted *The salmonid genome contains two PRL genes due to a tetraploid event that occurred 25-100 million yr ago. The sPRL gene used in this study represents the PRL II gene which at the nucleotide level is 95% identical to the PRL I gene within the open reading frame. Both genes are actively expressed in the salmon pituitary.
Society
515 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 25 November 2014. at 04:30 For personal use only. No other uses without permission. . All rights reserved.
MOL 516
ENDO.
1992
Vol6
sion of the gene should likewise be conserved. To date, however, the molecular basis for pituitary-specific expression of the PRL gene has not been examined in nonmammalian species. Studies on the rat PRL (rPRL) gene have demonstrated an important role for 5’-flanking sequences in directing cell type-specific expression in cultured ceil (1) and transgenic animal (2) models. The elements critical for expression in lactotroph-like cell lines or targeting to the pituitary in mice have been localized to two distinct regions-a proximal activating region immediately upstream of the TATAA box, and an enhancer region between -1.8 and -1.5 kilobases (kb) distal to the transcription start site. Both regions contain multiple binding sites for the pituitary-specific POU domain protein, Pit-l (GHF-1). Tandem multimers or even single copies of these sites can confer Pit-l-dependent transactivation in cell-free systems (3) and in transfected heterologous cell lines (4). Comparison of the proximal activating region and distal enhancer of the rat PRL gene with S-flanking sequences of human and bovine PRL (bPRL) genes has demonstrated that these regions are highly conserved among mammals: the promoter sequences that encompass the proximal Pit-l binding sites of the rPRL gene (-228 to -29) average 88% identity with sequences in the other mammalian PRL promoters. Moreover, the rPRL enhancer sequences -1739 to -1549 that contain the distal Pit-l footprints Dl to D4 (3) are 80% identical to sequences -1184 to -994 of the bPRL gene (5), and highly homologous to 5’ sequences of the human PRL gene (-1.4 to -1.2 kb) shown by DNase footprinting to contain Pit-l binding sites (6). To see whether the mechanisms that dictate tissuespecific expression of the PRL gene are conserved in nonmammalian species, clones containing up to 2.4 kb of 5’-flanking sequence of the salmon PRL II gene (sPRL II; see footnotes 1 and 2) were characterized. Comparison of highly conserved 5’-regulatory sequences from mammalian PRL genes with the sPRL 5’ region indicate a dramatic divergence of sequences between mammalian and teleost classes. In this study we examine how the phylogenetic differences in these sequences affect expression of the sPRL gene in mammalian pituitary cells, comparing expression in primary cultures of teleost pituitary cells. We also examine the role of Pit-l in activating PRL gene expression in this distantly related vertebrate species.
nonpituitary mammalian and piscine cell lines (Table 1) demonstrated that under conditions permitting high levels of expression of a control Rous sarcoma virus/ chloramphenicol acetyltransferase (RSV/CAT) construct, the sPRL gene (like the rat gene) was not expressed at detectable levels. These experiments suggested that pituitary-specific factors might be required for activation of the sPRL gene. Support for this explanation was obtained with rat pituitary GH4 cells in which mammalian PRL promoters have been shown to be strongly activated. Using CAT constructs that contained either 2.4 kb or 1.3 kb 5’ sPRL genomic sequence, expression of the teleost gene was observed in GH4 cells; however, the level of expression was at least 90fold lower than that of the rPRL gene (Fig. 1). To examine more thoroughly whether the differences
Table 1. PRL/CAT Expression and Piscine Cell Lines
in Heterologous
RSV/CAT Cell type NIH-3T3 KC RCB NRK HeLa RTH
93 58 88 22 97 84
Tissue- and Species-Specific sPRL Gene
-2.4 sPRL/CAT
0.3
Harry P. Elsholtz*, Sonali Majumdar-Sonnylal, Zhiyuan Gong, and Choy L. Hew Departments of Clinical Biochemistry Biochemistry (F.X., C.L.H.) University of Toronto Toronto, Ontario, Canada M5G 1 L5 The Bantina and Best Diabetes Toronto, OEtario, Canada M5G Hospital Toronto,
for Sick Children Ontario, Canada
Centre 1 L7
(H.P.E,
(H.P.E.. ’
Fei Xiong,
S.M.-S.,
and
SM.-S.)
(F.X., Z.G., C.L.H.) M5G 1X8
of the marked species-dependent expression of PRL genes observed in mammalian and teleost lactotrophs. The dramatic divergence of S-flanking sequences of mammalian and teleost PRL genes further suggests that conservation of Pit-l function is not strictly limited by evolutionary flexibility within genomic regulatory regions. (Molecular Endocrinology 6: 515-522,1992)
In mammals, the pituitary POU homeodomain protein, Pit-l, binds to proximal and distal V-flanking sequences of the PRL gene that dictate tissue-specific expression. These DNA sequences are highly conserved among mammals but are dramatically different from PRL 5’ sequences in the teleost species, Oncorbynchus tschawytscha (chinook salmon). To analyze the molecular basis for pituitary-specific gene expression in a distantly related vertebrate, we transfected CAT reporter gene constructs containing 2.4 kilobases (kb) 5’-flanking sequence from the salmon PRL (sPRL) gene into various cell types. Expression of the sPRL gene was restricted to pituitary cells, but in rat pituitary GH4 cells levels of expression were at least QO-fold lower than those obtained with a -3 kb rat PRL (rPRL) construct. Conversely, in primary teleost pituitary cells, -2.4 kb sPRL/CAT was expressed at levels about lo-fold higher than -3 kb rPRL/CAT. To determine whether species-specific transactivation by Pit-l was sufficient to explain these species differences in PRL gene expression, we isolated a cDNA clone encoding the salmon Pit-l POU domain and constructed a
INTRODUCTION The anterior pituitary hormone PRL is uniquely versatile in its range of actions-from stimulation of lactogenesis in mammals and maternal behavior in birds to regulation of ion transport across the gill epithelia in teleost fish. Unlike its functions, which appear to have evolved extensively, the primary structure of PRL as well as the organization of the PRL gene exhibit marked conservation among vertebrates. Recent cloning of the chinook salmon (Oncorhynchus tschawytscha) PRLII gene ‘.* has revealed that in groups as divergent as mammals and fish, the PRL gene consists of five exons with the position and type of each exon/intron splice site being strictly conserved. Furthermore, in nearly all vertebrates expression of the PRL gene is restricted to the pituitary lactotrophs, suggesting that developmental mechanisms that determine cell type-specific expres-
rat Pit-l expression vector that contained salmon Pit-l POU domain sequences substituted in frame. The chimeric Pit-l encoded 14 amino acids unique to salmon. Coexpression of rat Pit-l with salmon or rat PRL/CAT in transfected HeLa cells resulted in specific and strikingly comparable levels of promoter activation. Moreover, the specificity and efficacy of the chimeric salmon/rat Pit-l was similar to wild type rat Pit-l in activating salmon and rat PRL/ CAT. Our data suggest that the structure and function of Pit-l are highly conserved among vertebrates, and that Pit-l is not the primary determinant 08%%3809/92/0515-0522503.00/O Molecular Endocrinology CopyrIght 0 1992 by The Endocme
C.L.H.)
‘Chin RA, Xiong F, Hew CL, A gene encoding chinook salmon (Oncorhynchus tschawyfscha) prolactin: evolutionary implications and possible regulatory elements. Manuscript submitted *The salmonid genome contains two PRL genes due to a tetraploid event that occurred 25-100 million yr ago. The sPRL gene used in this study represents the PRL II gene which at the nucleotide level is 95% identical to the PRL I gene within the open reading frame. Both genes are actively expressed in the salmon pituitary.
Society
515 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 25 November 2014. at 04:30 For personal use only. No other uses without permission. . All rights reserved.
MOL 516
ENDO.
1992
Vol6
sion of the gene should likewise be conserved. To date, however, the molecular basis for pituitary-specific expression of the PRL gene has not been examined in nonmammalian species. Studies on the rat PRL (rPRL) gene have demonstrated an important role for 5’-flanking sequences in directing cell type-specific expression in cultured ceil (1) and transgenic animal (2) models. The elements critical for expression in lactotroph-like cell lines or targeting to the pituitary in mice have been localized to two distinct regions-a proximal activating region immediately upstream of the TATAA box, and an enhancer region between -1.8 and -1.5 kilobases (kb) distal to the transcription start site. Both regions contain multiple binding sites for the pituitary-specific POU domain protein, Pit-l (GHF-1). Tandem multimers or even single copies of these sites can confer Pit-l-dependent transactivation in cell-free systems (3) and in transfected heterologous cell lines (4). Comparison of the proximal activating region and distal enhancer of the rat PRL gene with S-flanking sequences of human and bovine PRL (bPRL) genes has demonstrated that these regions are highly conserved among mammals: the promoter sequences that encompass the proximal Pit-l binding sites of the rPRL gene (-228 to -29) average 88% identity with sequences in the other mammalian PRL promoters. Moreover, the rPRL enhancer sequences -1739 to -1549 that contain the distal Pit-l footprints Dl to D4 (3) are 80% identical to sequences -1184 to -994 of the bPRL gene (5), and highly homologous to 5’ sequences of the human PRL gene (-1.4 to -1.2 kb) shown by DNase footprinting to contain Pit-l binding sites (6). To see whether the mechanisms that dictate tissuespecific expression of the PRL gene are conserved in nonmammalian species, clones containing up to 2.4 kb of 5’-flanking sequence of the salmon PRL II gene (sPRL II; see footnotes 1 and 2) were characterized. Comparison of highly conserved 5’-regulatory sequences from mammalian PRL genes with the sPRL 5’ region indicate a dramatic divergence of sequences between mammalian and teleost classes. In this study we examine how the phylogenetic differences in these sequences affect expression of the sPRL gene in mammalian pituitary cells, comparing expression in primary cultures of teleost pituitary cells. We also examine the role of Pit-l in activating PRL gene expression in this distantly related vertebrate species.
nonpituitary mammalian and piscine cell lines (Table 1) demonstrated that under conditions permitting high levels of expression of a control Rous sarcoma virus/ chloramphenicol acetyltransferase (RSV/CAT) construct, the sPRL gene (like the rat gene) was not expressed at detectable levels. These experiments suggested that pituitary-specific factors might be required for activation of the sPRL gene. Support for this explanation was obtained with rat pituitary GH4 cells in which mammalian PRL promoters have been shown to be strongly activated. Using CAT constructs that contained either 2.4 kb or 1.3 kb 5’ sPRL genomic sequence, expression of the teleost gene was observed in GH4 cells; however, the level of expression was at least 90fold lower than that of the rPRL gene (Fig. 1). To examine more thoroughly whether the differences
Table 1. PRL/CAT Expression and Piscine Cell Lines
in Heterologous
RSV/CAT Cell type NIH-3T3 KC RCB NRK HeLa RTH
93 58 88 22 97 84
Tissue- and Species-Specific sPRL Gene
-2.4 sPRL/CAT
0.3
