Chromatin-associated receptors for estrogen, progesterone, and dihydrotestosterone and the induction of egg white protein synthesis in chick magnum

Cell Differentiation 2, 163-170 (1973 ). © North-H olland Publishing Company

CHROMATIN-ASSOCIATED RECEPTORS FOR ESTROGEN, PROGESTERONE, AND DIHYDROTESTOSTERONE AND T H E I N D U C T I O N O F E G G WHITE P R O T E I N S Y N T H E S I S IN CHICK M A G N U M Richard D. PALMITER, Graham H. CATLIN and Ronald F. COX Department of Biochemistry, Searle Research Laboratories, High Wycombe,Bucks., England Accepted 26 April 1973

Sex steroids bind to distinct receptor proteins located on chick oviduct chromatin, and induce the synthesis of the egg white proteins: ovalbumin, eonalburain, ovomueoid, and lysozyme. Correlating these phenomena suggests that the genes for these proteins are ~ontrolled independently of each other by more than one species of receptor which in some cases may interact to produce cooperative effects on mRNA synthesis.

Estrogen (E), progesterone (P) and testosterone (T) regulate differentiation and egg white protein synthesis in the magnum portion of the chick oviduct (Brandt et al., 1956; Oka et al., 1969a, b; O'Malley et al., 1969; Muller et al., 1970; Palmiter, 1971, 1972a). There is increasing evidence for the hypothesis that steroid hormones act by binding to specific proteins (receptors) which then migrate to the nucleus, become associated with acceptor sites on chromatin, and modify transcription such that new messenger RNA (mRNA) is synthesized and transported to the cytoplasm for translation (Hamilton, 1968; O'Malley, 1971; Spelsberg et al., 1971; Jensen et al., 1972; Baxter et al., 1972). Within the context of this hypothesis, many models could explain the induction of egg white protein synthesis, from those invoking a single receptor species and a few acceptor sites on the genome which mediate metabolic responses in a cascade fashion, to more complex ones involving multiple receptors and many acceptor sites which independently regulate the responsive genes. In this study we try to distinguish among some of the possibilities by determining a) the number of distinct receptor protein species; b) the number and specificity of the chromatin acceptor sites; and c) whether the mRNAs for 4 distinct egg white proteins are induced coordinately with different hormonal treatments.

164

Steroid receptors and the induction o f egg white proteins

RESULTS AND DISCUSSION To ascertain whether there are distinct receptors for each steroid, magnums were incubated with 3H-steroids, then chromatin-associated receptors were extracted and displayed on sucrose gradients (fig. 1); 14C-ovalbumin (3.6S) was included as an internal marker. In each case a single peak was obtained, showing the presence of only one size-class of high affinity receptor specific for each hormone. However, each peak could consist of several binding components with similar sedimentation characteristics, and the existence of other components with low steroid binding affinity or present as minor species cannot be excluded. Nevertheless, it is clear that the major receptor species are distinct. Approximate sedimentation coefficients of 4, 3.5, and 6.5S can be ascribed to the receptors for T, P, and E, respectively; similar values for the E and P receptors have been observed in the magnum from unstimulated chicks (Cox et al., 197t; Toft et al., 1972). Receptors for each of these steroids have also been demonstrated in rat uterus (Giannopoulos, 1971). The binding of T to chromatin receptors was investigated further because it has been shown in other target tissues that it is reduced to dihydrotestosterone (DHT) before binding (Gloyna et al., 1969). Likewise, in the magnum, over 85% of the steroid extracted from chromatin was identified as DHT when the steroids were separated by thin-layer chromatography, regardless of whether the tissue was incu-

['est°ster°ne 2 o,

progesterone

es trogen~

I

t~ i

t f r

i

2 o1

°o

1o

20

0

0~

0 I0 20 10 20 Fraction Fig. 1. Sedimention of T, P, and E receptors extracted from oviduct magnum chromatin. Magnum tissue (0.3-0.8 g) from chicks withdrawn for 3 weeks after 10 days primary stimulation with E (Palmiter et al., 1970; Palmiter, 1972a) was incubated for 2 hr at 37°C in Medium 199 containing radioactive steroid (3H_testosterone, 3H.progesterone, or 3H_estradiol_17~3; 2 nM, 100-110 Ci per mmole, Radiochemical Center). Nuclei were isolated (Cox et al., 1971), and chromatin extracted as before (Butterworth et al., 1971). Chromatin-associated receptors were prepared by adding 0.5 M NaC1 in 1 mM EDTA, 10 mM Tris-C1, pH 7, followed by a 15 min centrifugation at 20,000g. Aliquots of the supernatant (equivalent to 150-350 tag of chromatin DNA) were mixed with 14C-ovalbumin (Palmiter et al., 1971), layered over isokinetic sucrose gradients (Noll, 1967) in 0.5 M NaCI, and centrifuged for 16 hr at 400,000g (SW 65 rotor, Beckman). Fractions were collected and counted in 16 ml scintillator fluid (Cox et al., 1971) at an efficiency of 25% for 3H and 35% for 14C. Tritium profiles were corrected to represent the radioactivity extracted from 1 mg of chromatin DNA. Sedimentation is from left to right.

R.D. PALMITER et al.

165

bated with T or DHT. Furthermore, the same amount of steroid was bound to chromatin after 2 hr o f incubation with either hormone. Competition experiments in which equal concentrations of labeled and unlabeled competing steroid* were incubated with magnum explants showed that when the competing steroid and radioactive one were the same, the radioactivity associated with chromatin receptors was halved, indicatingthat in each case the accepter sites were saturated (table I). There was little competition by the heterologous steroids for either E or P binding, suggesting that these steroid receptors have distinct accepter sites. However, P reduced the amount of DHT receptor bound to chromatin. We cannot distinguish between P competing for the active site o f the DHT receptor, or P receptors binding in DHT accepter sites. The competition of E

Table 1 Competition between steroids for chromatin binding sites. Magnums were incubated with 2 nM labeled steroid and 2 nM unlabeled steroid as in fig. 1. Chromatin-associated receptors were extracted and isolated on sucrose gradients (Cox et al., 1971). The radioactivity under the peak was estimated and compared to that from tissue incubated with the labeled steroid only (assumed to equal 100%). Labeled steroid

Unlabeled steroid

% bound to chromatin

estradiol estradiol estradiol estradiol progesterone progesterone progesterone progesterone testosterone

none estradiol progesterone

100

testosterone testosterone

testosterone DHT DHT DHT DHT DHT

testosterone

none progesterone estradiol testosterone none testosterone

progesterone estradiol none DHT testosterone progesterone estradiol

55

90 99 100 50 1O0 86 100 51 70 64 100 49 54 77 99

*The estrogen concentration (2 nM) used in these experiments is just above the physiological range observed in laying hens (O'Grady, 1968). Competition with equal amounts of unlabeled steroid was considered more meaningful than that obtained with a large excess; in all c a s e s incubation for 2 hr was sufficient to saturate the salt-extractable nuclear receptor sites with steroid (Cox et al., 1971).

166

Steroid receptors and the induction o f egg white proteins

for T binding (table 1) m a y be due to interference with the conversion o f T to D H T since E does n o t c o m p e t e for DHT binding. The n u m b e r o f a c c e p t o r sites in the m a g n u m f r o m w i t h d r a w n chicks was estim-

Table 2 Effect t~f various hormonal treatments on magnum growth and specific protein synthesis. The h~rnlones indicated (E, 173-estradiol-benzoate; P, progesterone; and DHT, 2c~-methyldihydrotestosterone propionate) were administered to immature female chicks (weighing about 100 g) for 8 days as a primary stimulation (l°S), or for 4 days as a secondary stimulation (2°S) to chicks (300--400 g) withdrawn for several weeks after 10 days of l°S with E alone, after which time specific protein synthesis was reduced to a low level (Palmiter, 1972a). The subscripts refer to the daily dose (in milligrams) administered per chick. The rate of synthesis of specific proteins, namely ovalbumin (OV), conalbumin (CON), ovomucoid (MU) and lysozyme (LYS), was determined immunologically after pulse-labeling magnum explants in culture with a mixture of radioactive amino acids (Palmiter, 1972a); the results are expressed as the percentage of total acid-precipitable radioactivity which was immunoprecipitable with specific antibodies. Nonspecific coprecipitation was estimated by adding bovine serum albumin to the radioactive homogenates followed by anti-bovine serum albumin; the radioactivity in these precipitates (about 0.2% of total protein synthesis) has been subtracted from the experimental values. Hormonal treatment

Unstimulated

Magnum weight (mg)

10

Relative rate of protein synthesis OV CON MU (% total protein synthesis) 0

LYS

0

0

0

8 days I°S E1 P1 Tl P1T1 E1 P1 E1TI

560 11 11 17 210 1,t35

47.5 0 0 0 6.1 44.7

7.8 1.I 0 0.5 3.7 7.5

2.8 0 0 0 0.3 5.1

1.7 0 0 0 0.4 1.5

Withdrawn

70

0.3

0.6

0.2

0.2

4 days 2°S E2 P2 T2 P2 T2 E2P 2 E2T z

900 390 80 340 800 2,180

47.0 37.2 0.4 24.8 51.6 42.7

9.6 8.8 0.1 9.2 18.6 11.4

2.2 t .5 0.3 t .6 8.4 6.0

2.1 2.2 0.2 1.4 1.8 1.6

30,000

64.2

12.l

8.5

1.5

Hen

R.D. PALMITER et al.

167

ated from experiments such as those shown in fig. 1. We calculate* that there are 1070 + 116, 2320 -+ 300, and 6400 -+ 1430 (S.E.M., 4 experiments)acceptor sites for DHT, P, and E receptors, respectively, assuming that each receptor binds one steroid molecule and occupies one acceptor site. Although this tissue contains 3 responsive cell types (tubular gland cells, goblet cells which synthesize avidin (Kohler et al., 1968), and ciliated cells (Oka et al., 1969a)), these numbers probably reflect the tubular gland cell acceptor sites since they comprise the vast majority of the receptive magnum cells after withdrawal. To distinguish between the actions of these hormones, they were administered singly, or in various combinations, to immature female chicks as either a primary (l°S) or secondary stimulation (2°S) and the effects on magnum growth and specific protein synthesis were measured (table 2). The relative rates of ovalbumin, conalbumin, ovomucoid, and lysozyme synthesis expressed in table 2 probably reflect the respective concentrations of active, translatable mRNA because: a)fluorescent antibody studies have shown that all 4 proteins are localized in the tubular gland cells (Palmiter et al., 1972) and b) no differential effects of these hormones on mRNA translation rates have been observed in this system (Palmiter, 1972b, 1973). Moreover, in the case of ovalbumin, its relative rate of synthesis in tissue culture is proportional to the ovalbumin mRNA activity when polysomal RNA is assayed in a cell-free protein-synthesizing system (Means et al., 1972; Palmiter, unpublished observations). The mRNA concentrations are, in turn, a function of the rates of mRNA synthesis and degradation. Since neither P nor DHT affect the rate of mRNA degradation observed after stimulation with E alone (Palmiter, 1972a, 1973), the relative rates of egg white protein synthesis observed after different hormonal treatments reflect effects of these hormones on mRNA synthesis; however, it must be kept in mind that effects of these steroids on mRNA transcription have not, as yet, been distinguished from effects these hormones may have on mRNA maturation or transport to the cytoplasm. A salient feature of the data presented in table 2 is that no steroid alone promotes maximum stimulation of growth and the synthesis of each of the egg white proteins. While some functions require only a single steroid for maximum response (e.g. ovalbumin synthesis), others require combinations of these hormones (e.g. *Calculation: for DHT about 1.2 × 104 dpm of steroid were extracted from 1 mg of chromatin DNA. Since 5 0 - 5 6 % of the steroid was extractable with 0.5 M NaC1 and approx. 15% of the magnum cells in chicks withdrawn from E-treatment are thought to be responsive to these steroids (Palmiter, manuscript in preparation), this suggests that 1.6 X 105 dpm of DHT are bound per mg o f receptive cell DNA. Using DHT at 100 Ci per mmole and assuming 2.5 pg of DNA per cell (Sober, 1968), an estimate of the n u m b e r of DHT acceptor sites per cell is obtained from 1.6 × l 0 s dpm mg DNA

mg DNA 6 × 1023 moles X 4 X l 0 s cells X 2.2 × 1 0 1 7 d p m = 1070 moles/cell

Values for E and P aceeptor sites were similarly estimated.

168

Steroid receptors and the induction o f egg white proteins

growth and ovomucoid synthesis); in some situations inhibitory effects are also apparent (e.g. the effect of P during l°S). Hormonal interaction at some level is implied. With a combination of all three steroids, each at the appropriate dose, it is possible to approximate the physiological condition exemplified by the laying hen (Palmiter, 1972a). Since each receptor species binds predominantly one steroid (fig. 1), and there is little competition between receptors for acceptor sites (table 1), we favor the idea that the hormonal interactions occur after the receptors bind to the chromatin. By integrating the data presented, and assuming that the chromatin-associated receptors mediate the transcription of the specific mRNAs studied (either directly or indirectly via intermediate proteins or RNAs), certain features of the regulatory mechanisms involved are apparent. (i) Only E appears to activate the genes which govern the differentiation of progenitor epithelial cells into tubular gland ceils, since only hormonal combinations containing E promote growth, gland formation and significant egg white protein synthesis during l°S (table 2). Following the action of E on the genes controlling differentiation, the structural genes for the egg white proteins are activated such that either E or P can modulate their expression during 2°S (table 2; Palmiter et al., 1971b, 1972a). (ii) There may be distinct acceptor sites for both E and P receptors which independently regulate the expression of each egg white protein structural gene (or battery of genes if there is gene redundancy), since either steroid can induce all 4 egg white proteins during 2°S, yet their receptors do not compete for acceptor sites (table 1). Moreover, the additive or synergistic effects of E and P on conalbumin and ovomucoid mRNA synthesis (table 2) are most easily explained with dual (or possibly multiple) acceptor sites. The location of these acceptor sites relative to the structural genes they are presumed to regulate is, of course, not known; however, their positions relative to each other may dictate whether synergistic, additive, or no mutual effects are observed. For some genes, e.g. ovalbumin genes, synergism is apparent at low doses of E and P, but not at higher doses (Oka et al., 1969b), suggesting that processes limiting the rate of mRNA production may also be operative. (iii) The relative rates of egg white protein synthesis do not remain in constant proportion to each other with different hormonal treatments (table 2). This pattern, and the finding that conalbumin synthesis is preferentially induced by low concentrations of E (Palmiter, 1972a), suggests that the genes for these proteins are controlled independently of each other, and hence are not in the same operon or regulon. (iv) P inhibits the action of E during l°S, resulting in very little growth and poor induction of the egg white proteins. P appears to prevent normal tubular gland cell differentiation and morphogenesis (Oka et al., 1969a, b, c; Palmiter et al., 1971b); however, the mechanism of this inhibition is unknown. Our understanding of the interaction between these two hormones is complicated further by the finding that E may control the production of P receptors (Toft et al., 1972).

R.D. PALMITERet al.

169

(v) DHT is inactive alone, although there are distinct receptors and acceptor sites for this steroid (fig. 1, table 1). However, it has prominent effects on magnum growth when administered with E. This effect is mediated by increased cell division and an increased rate of protein synthesis per cell, the latter being due to an increased synthesis of protein synthetic machinery (ribosomes, mRNA, etc.) rather than an increased rate of mRNA translation (Palmiter et al., 1973). The synthesis of ovomucoid mRNA appears to be stimulated more than the other egg white protein mRNAs with the combination of E and DHT (table 2). The DHT receptors are postulated to act either in concert with certain E receptors thereby potentiating their action, or at distant sites where a gene product is made which does so. If the former possibility is correct, then each egg white protein gene may have acceptor sites for all three steroid receptors. Despite some competition between P and DHT (table 1), there is no apparent interaction between these two steroids which affects growth or specific protein synthesis (table 2). (vi) Many other cellular processes are controlled by these steroids. For instance, in addition to its effects on cell division, morphogenesis, and induction of egg white protein synthesis, E stimulates amino acid transport (Oka et al., 1969a), accumulation of tRNA (O'Malley et al., 1968), ribosome synthesis (Palmiter et al., 1970), polypeptide elongation and initiation (Palmiter, 1972b), and formation of endoplasmic reticulum (Kohler et al., 1969; Palmiter et al., 1970), thus preparing this tissue for rapid protein synthesis. This multiplicity of hormonal effects may account for the large number of acceptor sites observed per magnum cell. This correlation between steroid receptors and mRNA synthesis provides a working model for studying the regulation of the genetic apparatus in chick oviduct. Our comparison between the 3 chromatin-bound receptors, magnum growth, and the induction of 4 specific gene products suggests that following the E-mediated differentiation of the tubular gland cells, the egg white protein genes are controlled independently of each other by either E or P receptors which may in certain cases interact to produce cooperative effects on mRNA synthesis. Furthermore, the growth promoting effect of DHT appears to depend on the concomitant action of E receptors. Deciphering the organization of the acceptor sites for these receptors should contribute to our understanding the mechanism of action of these steroids.

REFERENCES Baxter, J.D., G.G. Rousseau, M. Benson, R.L. Garcea, J. Ito and G.M. Tomkins: Proc. Natl. Aead. Sci. U.S. 69, 1892-1896 (1972). Brandt, J.W.A. and A.V. Nalbandov: Poult. Sei. 35,672-700 (1956). Butterworth, P.H.W., R.F. Cox and C.J. Chesterton: Eur. J. Biochem. 23, 229-241 (1971). Cox, R.F., G.H. Catlin and N.H. Carey: Eur. J. Biochem. 22, 46-56 (1971). Giannopoulos, G.: Biochem. Biophys. Res. Commun. 44, 943-951 (1971). Gloyna, R.E. and J.D. Wilson: J. Clin. Endocr. 29, 970-977 (1969). Hamilton, T.H.: Science 161,649-661 (1968).

170

Steroid receptors and the induction o]" egg white proteins

Jensen, E.V. and E.R. De Sombre: Ann. Rev. Biochem. 4 1 , 2 0 3 - 2 3 0 (1972). Kohler, P.O., P.M. Grimley and B.W. O'Malley: Science 160, 86-87 (1968). Kohler, P.O., P.M. Grimley and B.W. O'Malley: J. Cell Biol. 40, 8-27 (1969). Means, A.R., J.P. Comstock, G.C. Rosenfeld and B.W. O'Matley: Proc. Natl. Acad. Sci. U.S. 69, 1146-1150 (1972). Muller, K.R., R.F. Cox and N.H. Carey: Biochem. J. 120, 337-344 (1970). Noll, H.: Nature 215,360-363 (1967). O'Grady, J.E.: Biochem. J. 106, 77-86 (1968). Oka, T. and R.T. Schimke: J. Cell BioL 41,816-831 (1969a). Oka, T. and R.T. Schimke: J. Cell Biol. 4 3 , 1 2 3 - 1 3 7 (1969b). Oka, T. and R.T. Schimke: Science 163, 83-85 (1969c). O'Malley, B.W., A. Aronow, A.C. Peacock and C.W. Dingman: Science 162, 567-568 (1968). O'Malley, B.W., W.L. McGuire, P.O. Kohler and S.G. Korenman: Recent Progr. Hormone Res, 25, 105-153 (1969). O'Malley, B.W.: Metabolism 20,981 988 (1971). Palmiter, R.D., A.K. Christensen and R.T. Schimke: J. Biol. Chem. 245,833--845 (1970). Palmiter, R.D.: Biochemistry 10, 4399-4403 (1971 ). Palmiter, R.D., T. Oka and R.T. Schimke: J. Biol. Chem. 246,724-737 (1971a). Palmiter, R.D. and J.T. Wrenn: J. Cell Biol. 50,598-615 (197 lb). Palmiter, R.D.: J. Biol. Chem. 247, 6450-6459 (1972a). Palmiter, R.D. : J. Biol. Chem. 247, 6770-6780 (1972b). Palmiter, R.D. and G.A. Gutman: J. Biol. Chem. 247, 6459-6461 (1972). Palmiter, R.D. and M.E. Haines: J. Biol. Chem. 248, 2107-2116 (1973). Sober, H.A.: in: Handbook of Biochemistry (Chemical Rubber Company, Ohio) p. H57 (1968). Spelsberg, T.C., A.W. Steggles and B.W. O'Malley: J. Biol. Chem. 246, 4188-4197 (197t). Toft, D.O. and B.W. O'Malley: Endocrinology 90, 1041-1045 (1972).