Specific steroid response from a nonspecific DNA element

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J. Steroid Biochem. Molec. Biol. Vol. 49, No. 4--6, pp. 251-255, 1994

Pergamon

0960-0760(94)E0044-L

Copyright© 1994ElsevierScienceLtd Printed in Great Britain. All rights reserved 0960-0760/94 $7.00 + 0.00

Symposia Specific Steroid Response f r o m a Nonspecific D N A E l e m e n t D. M. Robins,* A. Scheller and A. J. A d l e r t Department of Human Genetics, University of Michigan Medical School, Medical Science H 4708, Ann Arbor, M I 48109-0618, U . S . A .

A f u n d a m e n t a l d i l e m m a o f s t e r o i d h o r m o n e r e g u l a t i o n is h o w s p e c i f i c t r a n s c r i p t i o n is a t t a i n e d in v i v o w h e n s e v e r a l r e c e p t o r s r e c o g n i z e t h e s a m e D N A s e q u e n c e in v i t r o . W e h a v e i d e n t i f i e d a n e n h a n c e r o f t h e m o u s e s e x - l i m i t e d p r o t e i n ( S l p ) g e n e t h a t is a c t i v a t e d b y a n d r o g e n s b u t n o t b y g l u c o c o r t i c o i d s in t r a n s f e c t i o n . I n d u c t i o n r e q u i r e s a c o n s e n s u s h o r m o n e r e s p o n s e e l e m e n t ( H R E ) a n d m u l t i p l e a u x i l i a r y e l e m e n t s w i t h i n 120 b a s e p a i r s . A n d r o g e n s p e c i f i c i t y r e l i e s o n a d u a l f u n c t i o n to a u g m e n t a n d r o g e n b u t p r e v e n t g l u c o c o r t i c o i d a c t i o n f r o m a site t h a t b o t h r e c e p t o r s c a n b i n d . T h e n o n r e c e p t o r f a c t o r s a r e t h e d o m i n a n t f o r c e in t r a n s c r i p t i o n a l s p e c i f i c i t y , a l t h o u g h H R E s e q u e n c e v a r i a t i o n s c a n a f f e c t t h e s t r i n g e n c y a n d m a g n i t u d e o f h o r m o n a l r e s p o n s e . T h e effect o f H R E v a r i a t i o n s s u g g e s t s t h a t r e c e p t o r p o s i t i o n is a l t e r e d r e l a t i v e to t h e o t h e r f a c t o r s . T h u s p r o t e i n i n t e r a c t i o n s t h a t e l i c i t s p e c i f i c g e n e r e g u l a t i o n a r e e s t a b l i s h e d b y t h e a r r a y o f D N A e l e m e n t s in a complex enhancer and can be modulated by subtle sequence differences that may influence precise protein contacts.

ft. Steroid Biochem. Molec. Biol., Vol. 49, No. 4-6, pp. 251-255, 1994

INTRODUCTION

transfer, this sequence can also mediate induction by androgen, progesterone and mineralocorticoid recepSteroid hormones function in precise biological tors (AR, P R and M R ) [1]. Further, purified or recomprograms via their specific receptors, which are ligandbinant receptors can bind this sequence in vitro [3, 4]. activated transcription factors [1, 2]. T h e s e proteins Therefore, the consensus is referred to as a h o r m o n e thus link extracellular cues for reproduction, response element ( H R E ) to indicate its generality. development, and homeostasis directly to gene While there may be some overlap in sets of genes regulation. In contrast to the evolutionarily conserved regulated by these hormones, their physiological structure of the nuclear receptor family, its m e m b e r s functions are generally distinct. Requisite transcripexhibit impressive functional diversity. Intriguingly, tional specificity could be enforced by subtle sequence this diversity is not reflected in the response elements differences in response elements, or by accessory of their target genes. H o w is specific hormonal factors that selectively interact with receptors to regulation of gene expression attained when multiple determine precise gene activation. receptors recognize similar D N A binding sizes? While the H R E mediates induction in transfection Transcriptional specificity is a fundamental problem by several receptors, naturally occurring enhancers of for several transcription factor families but is especially GR-responsive genes are often comprised of multiple evident for the steroid receptors. T h e glucocorticoid imperfect consensus elements that show cooperative response element ( G R E ) consensus is a palindrome of effects with each other and with sites for various other T G T T C T half-sites separated by 3 bases. In gene factors [5, 6]. A R - d e p e n d e n t genes face a greater challenge for specific expression if D N A sites are Proceedings of the XVI Meeting of the International Study Groupfor recognized by multiple receptors, because in most Steroid Hormones, Vienna, Austria, 28 Nov.-1 Dec. 1993. target organs G R is also present and in greater abun*Correspondence to D. M. Robins. tCurrent address: Department of Oncology, Johns Hopkins dance. Some A R - d e p e n d e n t genes that express in University School of Medicine, Baltimore, MD 21205, U.S.A. prostate (C3, prostate specific antigen, and probasin) 251

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have complex response elements [7-9], further suggesting that an androgen-specific response requires more than just the H R E sequence. Since m a n y natural enhancers may be composed of multiple elements, a likely mechanism for imposing specific transcription is through differential p r o t e i n protein interactions. In this way, the context of an H R E m a y have as m u c h impact as its sequence. As an example, mutations in n o n H R E sequences of M M T V show differential response to G R , PR and AR [10]. T h e existence of hormonal specificity factors may be inferred from the p r o m o t e r - and cell t y p e - d e p e n d e n t activities of some receptors [11]. Receptor structure suggests protein interaction is a major determinant of activation, because while there is extraordinary conservation of the D N A - b i n d i n g d o m a i n s , there is little similarity in the large amino termini that encode transactivation functions and p r e s u m a b l y contact components of the transcriptional machinery [2]. ANDROGEN-SPECIFIC

RESPONSE

We have been studying the androgen regulation of mouse sex-limited protein (Slp). Slp arose from a duplicated c o m p l e m e n t c o m p o n e n t gene that acquired androgen dependence from a proviral insertion [12]. T h e transcriptional enhancer of Slp, located within the proviral long terminal repeat, contains a near-perfect H R E ( H R E - 3 ) that is necessary but not sufficient for hormonal induction [13]. Multiple interacting sites for auxiliary factors are required to augment weak AR activity. We asked whether these factors might also elicit specific response to AR from D N A sites recognized by G R and PR, by cotransfection with steroid receptors into receptor-deficient CV-1 cells [14]. T h e 120 bp enhancer fragment, C'A9, conferred almost 20-fold androgen response upon t k C A T but, remarkably, showed no response to glucocorticoid or progesterone (Fig. 1). This selectivity was not intrinsic to the receptor binding site, as two copies of H R E - 3 (2 x H R E - 3 ) before t k C A T induced strongly with each receptor. T h a t specific response of C'A9 is due to nonreceptor factors rather than to the H R E sequence is also demonstrated by the fact that it is host cell-dependent. T h a t is, C'A9 does not show selective response in T 4 7 D cells but is inducible by G R as well as by A R [15]. C'A9 thus provides a model element to address the mechanism of specificity in vitro. With the specific enhancer, we could ask why G R does not activate C'A9 from H R E - 3 . Possibly G R was unable to bind H R E - 3 in the context of C'A9, perhaps due to interference of proteins b o u n d at adjacent sites. Alternatively, G R might bind but be unable to transactivate in conjunction with factors competent for A R response. T h i s latter possibility is especially intriguing since G R cooperates promiscuously with m a n y transcription factors [6].

I

2O

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PR

G/AR

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Fig. 1. A n d r o g e n - s p e c i f i c r e s p o n s e o f t h e S l p e n h a n c e r . Rep o r t e r t k C A T p l a s m i d s w i t h e i t h e r t h e C'A9 f r a g m e n t ( H R E 3 is s h o w n as a c l o s e d d i a m o n d a n d a b i n d i n g site f o r a u b i q u i t o u s p r o t e i n as a n oval) o r t w o c o p i e s o f j u s t t h e H R E (2 × H R E - 3 ) w e r e c o t r a n s f e c t e d i n t o CV-1 cells w i t h r e c e p t o r e x p r e s s i o n v e c t o r s f o r AR, GR, P R , o r t h e c h i m e r a G / A R , w h i c h is t h e G R a m i n o t e r m i n u s f u s e d to t h e A R D N A a n d ligand binding domains. Transfection and CAT assays were p e r f o r m e d as in A d l e r e t al. [14]. F o l d i n d u c t i o n s w i t h the a p p r o p r i a t e h o r m o n e a r e t h e m e a n o f at l e a s t 3 e x p e r i m e n t s , with SEM indicated.

GR CAN BIND B U T NOT ACTIVATE THE AR-SPECIFIC

ENHANCER

T o understand G R ' s failure to activate C'A9, we first asked whether G R could inhibit AR induction, as an indication of G R ' s ability to bind H R E - 3 in the enhancer context. In fact, induction was greatly diminished when G R and AR were coexpressed (Fig. 2). T h i s was not indirect competition for other ratclimiting factors, because 2 × H R E - 3 was fully inducible in the presence of both receptors [14]. T h u s the antagonistic effect of G R was target-specific. Using m u t a n t GRs, we showed that G R D N A binding was both necessary and sufficient to block AR

A/B GR

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Fig. 2. T h e G R D N A b i n d i n g d o m a i n b l o c k s A R i n d u c t i o n of C'A9. CV-1 cells w e r e t r a n s f e c t e d w i t h C'A9, A R e x p r e s s i o n v e c t o r , a n d no, w i l d t y p e o r m u t a n t G R p l a s m i d s , as i n d i c a t e d b e l o w t h e h i s t o g r a m ( G R p l a s m i d s w e r e p r e s e n t in a 2-fold excess to AR). R e c e p t o r s a r e d i a g r a m m e d to t h e left; t h e d o t in 500Y is t h e cys to t y r m u t a t i o n . F o l d i n d u c t i o n s a r e t h e a v e r a g e o f 5 t r a n s f e c t i o n s , w i t h S E M i n d i c a t e d . See A d l e r e t al. [14 I.

Androgen-specific Gene Activation action on C'A9. T h e G R plasmid X556 encodes 150 amino acids around the zinc fingers, sufficient for nuclear localization, D N A binding and weak constitutive activity [16]. X556 was as effective as intact G R in repressing AR induction of C'A9. In contrast, G R m u t a n t 500Y, in which a zinc finger cysteine was converted to tyrosine, can localize to the nucleus but cannot bind D N A [17]. Coexpression of 500Y had no effect on C'A9's response to AR. T o confirm that G R repressed A R action by blocking A R binding to H R E - 3 , as opposed to G R binding a distinct negative G R E , we used a chimeric receptor that fused the amino terminus of G R to the D N A and steroid binding domains of AR. This allowed simultaneous testing of whether the G R domain containing the major transactivation function was sufficient for GR-specific behavior, i.e. the ability to activate 2 x H R E - 3 , but not C'A9. T h e chimeric receptor G / A R behaved essentially like G R and activated the general but not specific reporter (Fig. 1). This showed that while tethered to H R E - 3 via the A R D N A binding domain, G R ' s amino terminus could not transactivate with the AR-specific complex. Further, this suggests that the AR NH2-terminus is required for AR-specific induction of C'A9. T h a t the AR-specific complex contains factors that repress G R activity, as opposed to simply do not cooperate with G R , is suggested by two experiments. First, a constitutive G R mutant lacking the ligand binding domain strongly activates a single H R E - 3 before t k C A T , yet cannot activate this site in the context of C'A9. Further, when H R E - 3 is mutated in C'A9 and reinserted at various distances in the plasmid, G R can function, evidently due to escaping repression by juxtaposed nonreceptor factors [15]. G R ' s inhibition of AR induction m a y not occur in vivo as it does experimentally, but accentuates an underlying dilemma. G R is ubiquitous and abundant relative to AR. T h e r e f o r e for androgen-specific genes it m a y be as problematic to remain inactive in the presence of glucocorticoids as to express when androgens increase. Because A R response depends on sequences that can also function as G R E s , an efficient specificity mechanism m a y be simultaneously positive for A R and null for G R , to prevent leaky G R activation (Fig. 3). T o allow expression, accessory factors need only favor interaction of A R over GR. T h e r e f o r e the specific cohorts, and not the receptor binding site, orchestrate precise hormonal response in vivo. HRE

SEQUENCE AFFECTS INTERACTIONS

RECEPTOR

T o further characterize elements of specific hormonal response, we introduced clustered point mutations across C'A9 with P C R site-directed mutagenesis [ 15]. M a n y mutations affected but did not abolish androgen response, suggesting that the enhancer was

253

AR-specific ~

~

specificfactors eralfactors

Null interaction with GR

Fig. 3. Model for specific h o r m o n a l response. A n d r o g e n specific induction is attained by interaction of AR (directly and/or indirectly) with a variety o f accessory transcription factors. When GR binds to the c o m m o n receptor binding site (HRE), it is p r e v e n t e d f r o m transactivating by s o m e components o f the androgen-specific complex.

composed of multiple, partially redundant elements. N o mutation allowed response to G R , suggesting that while each mutation had quantitative effects on androgen response, no single mutation qualitatively altered specificity. T h e behavior of the mutants suggests that H R E - 3 mediates a hormone response whose magnitude over a broad range depends on interaction with several other elements. While H R E - 3 was nonspecific in steroid response and gained specificity from interaction with nonreceptor elements, we had not tested an influence of the precise sequence of the H R E . This was at issue since simply distancing the H R E had dramatic effects on G R activity; i.e. allowing half the induction elicited by AR compared to no response on C'A9 [15]. T h e r e fore, we replaced by mutagenesis the Sip H R E - 3 with one from M M T V that is also a good match to the consensus and responds to AR, G R and PR [18]. M T V - H R E differs from H R E - 3 at 2 bp in the imperfect half-site and 2 in the spacer (Fig. 4). T h e half-site differences are not expected to affect response, whereas a G C - r i c h spacer was shown to reduce G R induction several-fold [19]. Converting H R E - 3 to the M T V sequence nearly doubled A R response. M o r e surprisingly, G R response increased 10-fold (Fig. 4). T h u s , stringent hormonal specificity was lost in C'A9M T V , although androgen preference was retained. With other mutants, both half-site and spacer sequences were shown to contribute to specificity and/or magnitude of hormonal response. Given the striking effect on specificity of H R E sequence differences, we examined the orientation of the H R E in the enhancer, since H R E - 3 , like most natural elements, is asymmetric. W h e n we inverted H R E - 3 so that the perfect half-site was proximal to the tk promoter, androgen response was unchanged, but

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InducedCAT Activity (% C'A9 with AR) 100 200

HRE

C'A9

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i

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I

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AGAACAGGCTGTTTC MTV-HRE

AGAACAGtee tTGTaaC ee HRE-H

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Fig. 4. H R E b a s e c h a n g e s w i t h i n t h e c o n t e x t o f C'A9 a f f e c t d i f f e r e n t i a l h o r m o n e r e s p o n s e . S p e c i f i c b a s e s in H R E - 3 w e r e m u t a g e n i z e d in C'A9 to t h e s e q u e n c e s s h o w n a n d r e s p o n s e o f t h e s e p l a s m i d s to A R a n d G R w a s c o m p a r e d b y c o t r a n s f e e t i o n i n t o CV-1 cells as b e f o r e . D o t s h i g h l i g h t b a s e s d i f f e r i n g f r o m H R E - 3 in its n a t u r a l o r i e n t a t i o n . See A d l e r e t al. [15l f o r d e t a i l s .

glucocorticoid response tripled (Fig. 4). Together, the effects of the H R E mutations imply that H R E location, sequence and orientation modulate the stringency of specificity, without altering the overall preferential response of the enhancer to androgen. A c o m p o n e n t of response of the H R E mutants could be differential receptor affinities. T o correlate inducibility with binding site preference, full-length baculovirus-expressed A R [20] or G R [21] (from E. M. Wilson and G. B. T h o m p s o n , respectively) was bound to H R E - 3 D N A . T h e amount of each H R E shown in Fig. 4 required to compete 500/0 of the r e c e p t o r - H R E - 3 complex was used as an assay of relative affinity. T h e

complex of AR and H R E - 3 diminished equivalently with each sequence, indicating that AR showed little ability to distinguish among these H R E s . GR, however, b o u n d 3-fold better to the M T V - H R E than to H R E - 3 [15]. T h e affinity differences of G R corresponded qualitatively with the response of C ' A 9 - H R E mutants. However, multimerized H R E s do not confer inductions reflecting their relative affinities, since all respond strongly to both receptors. Therefore, differential affinity may be a c o m p o n e n t of activation that is aggrandized or diminished dependent on interaction with other factors. Further, androgen specificity may take advantage of receptor binding sites that are less preferred by GR.

CONCLUSION .

-

÷G

+G

+G Fig. 5. R e c e p t o r b i n d i n g s i t e s affect d i f f e r e n t i a l i n t e r a c t i o n s w i t h n o n r e c e p t o r f a c t o r s . I n t h e n a t u r a l C'A9 c o n f i g u r a t i o n (top), A R i n t e r a c t s well w i t h n e i g h b o r i n g f a c t o r s b u t G R a c t i v i t y is a b r o g r a t e d . W h e n t h e H R E is i n v e r t e d ( m i d d l e ) , G R c a n f u n c t i o n to s o m e e x t e n t . W h e n t h e H R E s e q u e n c e is a l t e r e d , b o t h r e c e p t o r s i n d u c e m o r e s t r o n g l y , b u t o n l y in t h e context of the enhancer.

T h e s e studies reveal an interplay between the precise sequence of the response element and the array of accessory sites, which may allow gene expression in vivo to vary with hormone, concentration, and cell type. Receptor affinity within the enhancer may reflect affinity to nearby factors as well as to the response element (Fig. 5). For example, inverting the H R E alters receptor contacts to D N A as well as to neighboring proteins, which has little effect on AR activity but notably enhances G R (Fig. 5, middle). Changes within the H R E increase the action of both receptors, due to higher D N A affinity or better contacts with other components (Fig. 5, bottom). For all H R E arrangements in this enhancer context, however, AR response is greater than G R , revealing that the nonreceptor factors have a dominant influence to the response element in steroid-specific induction. Protein-protein interaction may prove to be a fundamental means of specific regulation for families of transcription factors that bind similar sequences.

A n d r o g e n - s p e c i f i c G e n e Activation

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