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Molecular analysis of the glucocorticoid receptor
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362 NUCI2AR RECEPTORS FOR RErINOIC ACID IN GROWTH AND DIFFE2d~TIATION OF Mf~JSE EMBRYOCARCINOMA CELLS.
MOLECULAR ANALYSIS OF THE CLUCOCORTICOID RECEPTOR. K.R. Y a m a m o t o , L.P. F r e e d m a n , P.J. G o d o w s k i , D. P i c a r d , D.D. Sakai, M. S c h e n a Dept. o f B i o c h e m i s t r y , U C S F , S a n F r a n c i s c o .
Paul T. van der Saag, Christina E. van den Brink, Ban H.K. Defize, Marie-Jose Donath, Frank &E. Kruyt, Linda vmn dar Veer and Wiebe Kruljer. Mt~reeht Laboratory, Netherlande Institute for Developmental Biology, Utrecht, the Netherlands.
The glucocorticoid receptor is a d i r e c t signal transducer, interacting specifically with both hormone a n d g e n e to e n h a n c e or repress transcription. These events are m e d i a t e d b y r e c e p t o r b i n d i n g to two c l a s s e s o f DNA sequences: enhancement by glucocorticoid response elements (GREs), repression by "negative" GREs (nGRES). Exploiting the c a p a c i t y o f this m a m m a l i a n p r o t e i n to f u n c t i o n in y e a s t at GRE-linked y e a s t p r o m o t e r s , we screened point mutants in a r e g i o n of the receptor that includes four receptor activities. Specific amino acids were identified that are required selectively for e n h a n c e m e n t or D N A b i n d i n g . T h e w i l d type a n d m u t a n t r e c e p t o r f r a g m e n t s a r e b e i n g u s e d for f u r t h e r g e n e t i c s e l e c t i o n a n d for in vitro enhancement studies. The unliganded hormone binding region represses other receptor activities, and hormone binding relieves repression. Remarkably, receptor rearrangements, a n d f u s i o n s o f the h o r m o n e binding region to unrelated proteins, demonstrate that the unliganded hormone binding region inactivates, without strict r e g a r d to s t r u c t u r e , o t h e r a c t i v i t i e s r e s i d e n t w i t h i n the s a m e p o l y p e p t i d e c h a i n .
The recently identified rm~lear receptors for retlmoic acid (RA) belong to a supergene family of steroid and thyroid hormone receptors and can therefore be considered as ligand-controlled traDscription factors. Sofar two different retinoic acid receptors (RARs) have been identified by others, RARa and RAR$, localized on different human chromosomes. Pluripotenc EC cells can efficiently be induced by RA to differentiate in derivatives of all /tree germ layers and therefore we have studied RAR expression in mouse PI9 EC cells. Undifferentiated EC cells are expressing two transcripts constitutively and no RA~8 can be detected; upon inchlctlon of differentiation by RA, expression of RAR~ is induced within 2 hours reaching its maximum around 24 hours. In contrast, in a Pl9-derived cell line (RAC65) resistant to both the growth-inhibitory and differentiation-in@/cing activity of RA no expression of RAR~ can be induced, while an abnormal smaller RAR= transcript is constitutively present. These findings have led us to che hypothesis that the a--receptor is involved in the induction of RARE, In order to test this hypothesis we have transfected RAC65 cells with hRAR~ under the control of the SVAO early promoter. Stable cell lines were isolated which are sensitive for both the growth-inhibitory and the differentiationinducing capacity of R~ Moreover RAR~ is induced upon the addition of RA to these cell lines. Experiments with transfection of RAR~ have not given similar results. These results sofar lend support to our hypothesis that RARa is actively involved in the induction of the RAR~ gene. To further support ottr hypothesis, upstream sequences of the human RAR~ gene have been isolated to study the regulation of RARE gene expression. Moreover we have analyzed the molecular nature of the possibly truncated RAR= transcript in RAC65 cells.
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REGULATION OF DROSOPHILA RETROTRANSPO SON--EXPRESSION. A.A$ Karavanov, Ju.A. Bedkov, Ju.V, Ilyin Institute D e v e ! o p m e n t a l B i o l o g y $ USSR Academy of Sciences, Moscow; Institute of ~olecular Biology, USSR Academy of Sciences~ Moscow. The ~ s v retrotransposon of Drosophi la contains two closely spaced regi~ ons binding proteins from nuclear extracts.~One of these is an imperfect palindrome having homology with the lac-operator of E.coli the other contains reiterated sequence homologous to the octame~ that is the core of ma ny enhancers. Transient expression has shown that these regions are nega tire and positive regulators of ~yOsy expression. Methylation interference and DNA footprinting have shown the binding of specific proteins to both regions. In the negative regulator proteins are bound to palindrome and to the nearby sequence that has almomost complete homology with the element responsible for the cAMP dependence of various genes expression. This finding has very important general consequence as ~Wl~Sy element insertion is the cause of many mutations of different Drosophila genes and their suppression depends on K ~ s y activity.
CREB and cAMP Activation Marc R. Montminy, The Salk Institute, La Jolla, CA cAMP mediates the hormonal stimulation of a variety of eukaryotic genes through a conserved cAMP response element (CRE). The ultimate goal of our laboratory is to characterize the structure and dynamic regulation of nuclear factors which stimulate gene expresssion in response to this second messenger. Towards this end, we have recently purified and isolated c D N A clones for a transcription factor, CREB, which binds to the CRE and stimulates transcription of cAMP-responsive genes. The phosphorylation of CREB by cAMPdependent protein kinase is critical to its ability to transactivate genes. Using an in vitro mutagenesis approach, we are currently exploring the mechanism by which phosphorylation transforms the CREB protein into a fully active transcription factor.
SllO
362 NUCI2AR RECEPTORS FOR RErINOIC ACID IN GROWTH AND DIFFE2d~TIATION OF Mf~JSE EMBRYOCARCINOMA CELLS.
MOLECULAR ANALYSIS OF THE CLUCOCORTICOID RECEPTOR. K.R. Y a m a m o t o , L.P. F r e e d m a n , P.J. G o d o w s k i , D. P i c a r d , D.D. Sakai, M. S c h e n a Dept. o f B i o c h e m i s t r y , U C S F , S a n F r a n c i s c o .
Paul T. van der Saag, Christina E. van den Brink, Ban H.K. Defize, Marie-Jose Donath, Frank &E. Kruyt, Linda vmn dar Veer and Wiebe Kruljer. Mt~reeht Laboratory, Netherlande Institute for Developmental Biology, Utrecht, the Netherlands.
The glucocorticoid receptor is a d i r e c t signal transducer, interacting specifically with both hormone a n d g e n e to e n h a n c e or repress transcription. These events are m e d i a t e d b y r e c e p t o r b i n d i n g to two c l a s s e s o f DNA sequences: enhancement by glucocorticoid response elements (GREs), repression by "negative" GREs (nGRES). Exploiting the c a p a c i t y o f this m a m m a l i a n p r o t e i n to f u n c t i o n in y e a s t at GRE-linked y e a s t p r o m o t e r s , we screened point mutants in a r e g i o n of the receptor that includes four receptor activities. Specific amino acids were identified that are required selectively for e n h a n c e m e n t or D N A b i n d i n g . T h e w i l d type a n d m u t a n t r e c e p t o r f r a g m e n t s a r e b e i n g u s e d for f u r t h e r g e n e t i c s e l e c t i o n a n d for in vitro enhancement studies. The unliganded hormone binding region represses other receptor activities, and hormone binding relieves repression. Remarkably, receptor rearrangements, a n d f u s i o n s o f the h o r m o n e binding region to unrelated proteins, demonstrate that the unliganded hormone binding region inactivates, without strict r e g a r d to s t r u c t u r e , o t h e r a c t i v i t i e s r e s i d e n t w i t h i n the s a m e p o l y p e p t i d e c h a i n .
The recently identified rm~lear receptors for retlmoic acid (RA) belong to a supergene family of steroid and thyroid hormone receptors and can therefore be considered as ligand-controlled traDscription factors. Sofar two different retinoic acid receptors (RARs) have been identified by others, RARa and RAR$, localized on different human chromosomes. Pluripotenc EC cells can efficiently be induced by RA to differentiate in derivatives of all /tree germ layers and therefore we have studied RAR expression in mouse PI9 EC cells. Undifferentiated EC cells are expressing two transcripts constitutively and no RA~8 can be detected; upon inchlctlon of differentiation by RA, expression of RAR~ is induced within 2 hours reaching its maximum around 24 hours. In contrast, in a Pl9-derived cell line (RAC65) resistant to both the growth-inhibitory and differentiation-in@/cing activity of RA no expression of RAR~ can be induced, while an abnormal smaller RAR= transcript is constitutively present. These findings have led us to che hypothesis that the a--receptor is involved in the induction of RARE, In order to test this hypothesis we have transfected RAC65 cells with hRAR~ under the control of the SVAO early promoter. Stable cell lines were isolated which are sensitive for both the growth-inhibitory and the differentiationinducing capacity of R~ Moreover RAR~ is induced upon the addition of RA to these cell lines. Experiments with transfection of RAR~ have not given similar results. These results sofar lend support to our hypothesis that RARa is actively involved in the induction of the RAR~ gene. To further support ottr hypothesis, upstream sequences of the human RAR~ gene have been isolated to study the regulation of RARE gene expression. Moreover we have analyzed the molecular nature of the possibly truncated RAR= transcript in RAC65 cells.
363
364
REGULATION OF DROSOPHILA RETROTRANSPO SON--EXPRESSION. A.A$ Karavanov, Ju.A. Bedkov, Ju.V, Ilyin Institute D e v e ! o p m e n t a l B i o l o g y $ USSR Academy of Sciences, Moscow; Institute of ~olecular Biology, USSR Academy of Sciences~ Moscow. The ~ s v retrotransposon of Drosophi la contains two closely spaced regi~ ons binding proteins from nuclear extracts.~One of these is an imperfect palindrome having homology with the lac-operator of E.coli the other contains reiterated sequence homologous to the octame~ that is the core of ma ny enhancers. Transient expression has shown that these regions are nega tire and positive regulators of ~yOsy expression. Methylation interference and DNA footprinting have shown the binding of specific proteins to both regions. In the negative regulator proteins are bound to palindrome and to the nearby sequence that has almomost complete homology with the element responsible for the cAMP dependence of various genes expression. This finding has very important general consequence as ~Wl~Sy element insertion is the cause of many mutations of different Drosophila genes and their suppression depends on K ~ s y activity.
CREB and cAMP Activation Marc R. Montminy, The Salk Institute, La Jolla, CA cAMP mediates the hormonal stimulation of a variety of eukaryotic genes through a conserved cAMP response element (CRE). The ultimate goal of our laboratory is to characterize the structure and dynamic regulation of nuclear factors which stimulate gene expresssion in response to this second messenger. Towards this end, we have recently purified and isolated c D N A clones for a transcription factor, CREB, which binds to the CRE and stimulates transcription of cAMP-responsive genes. The phosphorylation of CREB by cAMPdependent protein kinase is critical to its ability to transactivate genes. Using an in vitro mutagenesis approach, we are currently exploring the mechanism by which phosphorylation transforms the CREB protein into a fully active transcription factor.
SllO