Proc. Natl. Acad. Sci. USA Vol. 82, pp. 3005-3009, May 1985 Medical Sciences

5-Azacytidine and sodium butyrate induce expression of aromatase in fibroblasts from carrying the henny feathering trait but not from wild-type chickens (Sebright /enzyme induction/estrogen synthesis/cytochrome P-450) MARK LESHIN Department of Internal Medicine, The University of Texas Health Science Center at Dallas, Southwestern Medical School, Dallas, TX 75235 Communicated by Jean D. Wilson, January 14, 1985

ABSTRACT Male chickens with the henny feathering trait Morgan, who concluded that it was the result of an autosomal have a female feathering pattern. In two henny-feathered (8). More recent studies have confirmed that this breeds, the Sebright bantam and the Golden Campine, the trait is due to a single autosomal gene mutation; homozygous synthesis of estrogen is increased as a consequence of increased carriers ofthe gene express full activity of aromatase in skin, activity of aromatase, a cytochrome P450 enzyme that con- and heterozygous carriers have intermediate levels ofactivity verts androgen to estrogen. The activity of the enzyme is (9). These observations suggest that the mutant gene prevents elevated in tissue slices and in cultured fibroblasts from normal suppression of aromatase in nonovarian tissues. heterozygous and homozygous birds of both breeds. In con- Understanding the nature of this mutation might provide trast, aromatase activity is very low in extraglandular tissues insight into the normal process by which tissue-specific from control chickens and is undetectable in fibroblasts cul- control of enzyme activity takes place. tured from these tissues. The current studies show that two A variety of techniques have been utilized to study the agents known to alter gene expression-5-azacytidine and mechanism of tissue-specific gene expression both in in vitro sodium butyrate-markedly induce expression of aromatase systems and in intact animals. For example, genes that are activity in Sebright and Campine fibroblasts but have no effect not expressed in specific tissues are usually more extensively on aromatase activity in fibroblasts from wild-type chickens. methylated than they are in tissues in which the gene is Induction of aromatase is specific since two other microsomal expressed, and prevention of methylation in growing cells enzymes in fibroblasts-one, a component of the can result in expression of previously dormant genes (10). aromatase enzyme complex and the other a cytochrome P-450 Addition of 5-azacytidine, an inhibitor of DNA methylation, oxidase distinct from the aromatase-are not significantly to embryonic mouse fibroblasts in culture elicits a program of affected by these agents. Further study of this unique mutation differentiation into myocytes that requires the expression of should provide insight into the mechanisms by which genes are many genes (11). Since many genes are not induced by switched to an uninducible state during differentiation. 5-azacytidine the concept has arisen that expression ofgenes may involve demethylation as one part of a multistep mecha- Aromatase is a cytochrome P-450 oxidase that converts nism. Hypomethylation of DNA may cause gene expression androgens, such as testosterone and androstenedione, to only when other unknown factors are present. Another estrogen (1). The expression of the enzyme is tissue specific pharmacological agent that alters gene expression in vitro is to some degree in all species, and this tissue specificity is butyric acid. This compound has diverse actions, a major part particularly striking in the chicken (2). In most breeds of of which may be the result of hyperacetylation of chromo- chickens aromatase is expressed only in the ovaries and somal histones (12, 13). In some systems 5-azacytidine and cannot be detected in any other tissue of the female or in any butyric acid act synergistically to induce gene expression (14, tissue of the male either during embryogenesis or later in 15). development (2, 3). In the current experiments 5-azacytidine and sodium In two breeds of chicken, the Sebright bantam and the butyrate have been utilized in cultured fibroblasts from Golden Campine, the tissue-specific restriction of aromatase Sebright and Campine chickens to investigate the abnormal is disrupted (2, 4). In these birds aromatase is expressed in differentiation pattern of aromatase. 5-Azacytidine and so- many tissues of both males and females beginning at the dium butyrate, alone and in combination, cause a profound fourth day of embryonic development (3). High enzyme increase in aromatase activity in fibroblasts cultured from levels persist in several tissues during adulthood, primarily in chickens carrying the henny feathering trait but have no the skin. This increase in aromatase activity is due to an effect on expression ofthe enzyme in cells from normal birds. increase in the activity of the terminal cytochrome P-450 These findings suggest that the underlying mutation respon- oxidase component of the aromatase enzyme complex (5). sible for the henny feathering trait does not involve DNA Fibroblasts cultured from a number of tissues of Sebright methylation or histone acetylation but does permit increased chickens express aromatase, whereas fibroblasts from wild- expression of the aromatase gene when DNA methylation type chickens do not (4). The enzyme that is expressed in and histone acetylation are altered. peripheral tissues and fibroblasts of Sebright and Campine chickens is indistinguishable by kinetic analyses from the aromatase of normal ovary (6). MATERIALS AND METHODS As a result of aromatase expression in skin, male Sebright Materials. Adult Silver Sebright bantam, White Leghorn and Campine birds develop a female feathering pattern (7) bantam, and Golden Campine chickens were obtained from known as the henny feathering trait. This trait was studied by the Halbach Farm, Waterford, WI, and fertile eggs from Silver Sebright and game bantam chickens were ob- The publication costs of this article were defrayed in part by page charge tained from David Sherrill, Arlington, TX. [1,B 3H]Testoster- payment. This article must therefore be hereby marked "advertisement" one (18.5 Ci/mmol; 1 Ci = 37 GBq) was prepared from in accordance with 18 U.S.C. §1734 solely to indicate this fact. [13,2133H]testosterone (46 Ci/mmol) (New England Nu- 3005 Downloaded by guest on September 27, 2021 3006 Medical Sciences: Leshin Proc. Natl. Acad. Sci. USA 82 (1985) clear) as described (4). Celite analytical filter was from as the free steroid by three sequential thin-layer chromatog- Fisher, and charcoal (Norit A) was from Mallinckrodt. raphy procedures utilizing the systems ethyl acetate/isooc- Dulbecco's modified Eagle's medium, medium 199, chicken tane (70:30), methylene chloride/ethyl ether (80:20), and serum, and fetal calf serum were from GIBCO. 5-Azacytidine ethyl acetate/isooctane/acetic acid (45:45:10). In some ex- and sodium butyrate were from Sigma. periments the validity of this procedure was confirmed by Culture of Fibroblasts and Preparation of Fibroblast Mem- acetylation of the final product, mixing with deoxycorticos- branes. Fibroblasts were propagated as described (4) from terone acetate, and demonstration that the ratio of 3H to 14C biopsies of skin and breast muscle taken from adult chickens did not change following recrystallization. and of skin, heart, skeletal muscle, and lung removed from Proteins were assayed by the method of Lowry et al. (17) day 18 chicken embryos. with bovine serum albumin as the standard. Culture dishes with cells grown for enzyme assay con- tained 10 ml of DMEM-199 with 5% chicken serum and 5% fetal calf serum. Additions of 5-azacytidine were made to RESULTS subconfluent monolayer cultures on day 1, and on day 2 the Addition of 5-azacytidine to actively dividing monolayer medium was removed and fresh medium containing sodium cultures of Sebright and Campine skin fibroblasts resulted in butyrate was added. Cells were in logarithmic growth phase a 6- and 17-fold stimulation of aromatase activity, respec- during the treatment period. On day 3 the fibroblast monolay- tively (Fig. 1 A and B). Similar results were observed with ers were rinsed twice with 3 ml of 50 mM Tris-HCl/50 mM fibroblasts obtained from both male and female Sebright NaCl, pH 7.4 (Tris/saline). Cells were scraped into 2 ml of chickens (results not shown). No stimulation of aromatase Tris/saline and a membrane fraction was prepared as de- was observed in control skin fibroblasts that were treated in scribed (4). the same way. Comparable degrees of stimulation were Assays. The standard assay of aromatase activity in fibro- observed in whole homogenate, whole cell, and membrane blast and ovarian membranes measured the release of 3H20 assays (results not shown). A variable toxic effect of the from [1/3-3H]testosterone during the conversion of androgen 5-azacytidine on cell growth was concentration-dependent to estrogen by a previously described modification (4) of the (Fig. 1C), but this inhibition was negligible with amounts that procedure of Thompson and Siiteri (1). produced maximal stimulation ofaromatase. In all embryonic NADPH-cytochrome c reductase was measured by a Sebright fibroblasts examined and in skin fibroblasts from modification ofthe method ofMasters et al. (16) as described adult Sebrights maximal stimulation of aromatase activity (4). Membranes were resuspended in 10 mM Tris HCl (pH was observed by utilizing 0.25 or 0.5 ,iM 5-azacytidine, 7.4) with a Dounce homogenizer. NADPH-cytochrome c whereas maximal stimulation occurred at 1 and 2.5 ,uM reductase was calculated as a function of the change in 5-azacytidine in the Campine skin fibroblasts and adult absorbance at 550 nm at room temperature. Sebright muscle fibroblasts, respectively. An effect on Steroid 21-hydroxylase was assayed by utilizing mem- aromatase activity was observed within 6 hr of exposure to branes prepared as described above and subsequently 5-azacytidine but maximal stimulation required exposure for washed and resuspended in 10 mM Tris HCl (pH 7.4). 24 hr. Following removal of 5-azacytidine from the culture Enzyme activity was assayed by measuring the formation of medium, stimulation of enzyme activity persisted for as long 11-deoxycorticosterone from progesterone. Assay mixtures as 6 days (results not shown). contained 1 ,xM [1,2,6,7-3H]progesterone, 0.1 M Tris/citrate The addition of sodium butyrate to cultures ofSebright and buffer (pH 7), 1 mM NADPH, 0.25 ,uM 11-deoxy[4-14C]- Campine skin fibroblasts resulted in a 2- and 2.5-fold stimula- corticosterone, and washed membranes (0.04-0.3 mg of tion of aromatase activity, respectively, but was without protein) in a total volume of0.2 ml. Incubations were at 25°C effect on aromatase in control fibroblasts (Fig. 2 A and B). In for 1 hr, and steroids were extracted with 1 ml of ethyl contrast to 5-azacytidine, little or no toxic effect on cell acetate. Nonradioactive 11-deoxycorticosterone (50 ,ug) was growth or protein was observed with the concentrations of added, and radioactive 11-deoxycorticosterone was purified butyrate examined (Fig. 2C). Stimulation of aromatase in-

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FIG. 1. Effect of 5-azacytidine on aromatase activity. 5-Azacytidine was added to cultures of adult skin fibroblasts 1 day after plating; on day 2 the medium was replaced with fresh medium containing no 5-azacytidine, and on day 3 membranes were prepared and assayed for aromatase activity and protein. (A) Aromatase activity, pmol hr-1 per dish. (B) Aromatase activity, pmol hr-'(mg of membrane protein)-1. (C) Membrane protein, ,ig per dish. *, Sebright fibroblasts; o, Campine fibroblasts; o, control fibroblasts. Downloaded by guest on September 27, 2021 Medical Sciences: Leshin Proc. Natl. Acad. Sci. USA 82 (1985) 3007 -c- y 3.01 Ea00

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FIG. 2. Effect of sodium butyrate on aromatase activity. Two days after plating butyrate was added to cultures of adult skin fibroblasts, and on day 3 membranes were prepared and assayed for aromatase activity and protein. (A) Aromatase activity, pmol hr-I per dish. (B) Aromatase activity, pmol hr-' (mg of membrane protein)-'. (C) Membrane protein, yg per dish. *, Sebright fibroblasts; o, Campine fibroblasts; o, control fibroblasts.

creased with increasing concentrations of butyrate between ity above the level seen with 5-azacytidine alone (results not 2.5 and 10 mM. shown). The magnitude of stimulation of aromatase in Sebright A comparison of the effect of these agents on aromatase fibroblasts sequentially exposed to 5-azacytidine and butyr- activity in fibroblasts derived from skeletal muscle of ate was greater than with either agent alone. In the experi- Sebright chickens ofdifferent ages is shown in Table 1. Basal ment shown in Fig. 3 exposure of adult Sebright skin enzyme activity is lower in adult than in embryonic fibroblasts to 0.25 ,pM 5-azacytidine resulted in an increase in fibroblasts, but following treatment with 5-azacytidine and aromatase specific activity from 4.1 to 21.8 pmol hr-' (mg of butyrate aromatase activity increases to a similar level in protein)-', and exposure to 10 mM butyrate stimulated both strains. Sequential addition of 5-azacytidine and butyr- aromatase activity to 17.3 pmol hr-1 (mg of protein)-'. The ate consistently stimulated aromatase activity in fibroblasts sequential treatment with 5-azacytidine followed by sodium from several embryonic Sebright tissues-skin, skeletal mus- butyrate resulted in an aromatase activity of 70.2 cle, heart, and lung (Table 2). Because ofhigher basal activity pmol hr-' (mg of protein)-'. Simultaneous exposure of cells in these fibroblast strains, the degree of stimulation (3- to to both 5-azacytidine and butyrate during the final day of cell 4-fold) was lower than with the adult and embryonic skeletal growth resulted in a markedly decreased enhancement of muscle strains described in Table 1. Again, no stimulation of aromatase activity (results not shown). Inclusion of either aromatase by these agents was observed in fibroblasts 5-azacytidine (0.5 and 1 ,uM) or sodium butyrate (5 and 10 derived from embryonic control skin or muscle. mM) in the aromatase assay itself had no effect (results not The specificity of the stimulatory effect of 5-azacytidine shown). In the Campine fibroblasts, addition of butyrate to and butyrate on the androgen-binding cytochrome P-450 5-azacytidine-exposed cells did not enhance aromatase activ- oxidase component of the aromatase enzyme complex was

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FIG. 3. Effect of 5-azacytidine plus butyrate on aromatase activity. One day after plating of adult Sebright skin fibroblasts 0.25 ,uM 5-azacytidine was added to some dishes, and 2 days after plating the culture medium was changed and butyrate was added. On day 3 membranes were prepared and assayed for aromatase and protein. (A) Aromatase activity, pmol hr-1 per dish. (B) Aromatase activity, pmol hr-' (mg of membrane protein)-1. o, Plus 5-azacytidine; *, no 5-azacytidine. Downloaded by guest on September 27, 2021 3008 Medical Sciences: Leshin Proc. Natl. Acad. Sci. USA 82 (1985)

Table 1. Effect of 5-azacytidine and butyrate on aromatase Table 3. Effect of 5-azacytidine and butyrate on aromatase, activity in fibroblasts grown from adult and embryonic steroid 21-hydroxylase, and NADPH-cytochrome P-450 Sebright skeletal muscle reductase activities in fibroblast membranes Aromatase, pmol hr'1 NADPH- (mg of protein)-' Steroid cytochrome 5-Azacytidine 21-hy- P-450 Muscle No additions and butyrate droxylase, reductase, Aromatase, pmol hr-'- nmol min-1 Adult 0.5 71 Embryonic 3.7 78 Source of pmol hr-' (mg (mg of (mg of fibroblast of protein)-' protein)-' protein)-' 5-Azacytidine (2.5 ,uM) and butyrate (10 mM) were added accord- membranes - + - + - + ing to the standard protocol. On day 3 fibroblasts were prepared and assayed for aromatase. Adult Sebright skin 21 127 2.0 2.7 7.4 8.4 Day 18 embry- examined by measuring two other enzymes-cytochrome onic Se- c(P-450) reductase (the other component of the aromatase bright heart 20 82 4.2 7.0 11.3 10.4 enzyme complex) and steroid 21-hydroxylase (another Adult Cam- cytochrome P-450 oxidase that utilizes a steroid substrate) pine skin 0.2 3.4 4.6 6.0 11.0 12.8 (Table 3). Whereas aromatase activity was again stimulated Adult control by 5-azacytidine and butyrate in fibroblasts from adult Sebright skin 7.1 and Campine skin and from embryonic Sebright heart, minimal <0.03 <0.03 1.8 2.8 9.1 changes were observed in steroid 21-hydroxylase and 5-Azacytidine (0.25 jM for Sebright and control fibroblasts and 1.0 cytochrome P-450 reductase activities. In fibroblasts from ,uM for Campine fibroblasts) and butyrate (10 mM) were added control chickens these latter two enzymes were also not according to the standard protocol. On day 3 fibroblast membranes significantly affected by 5-azacytidine and butyrate treatment. were prepared and assayed for aromatase, steroid 21-hydroxylase, and NADPH-cytochrome P450 reductase activities. -, No addition; +, 5-azacytidine and butyrate added. DISCUSSION

The regulation of aromatase activity in the tissues of the may provide insight into the process by which selective chicken constitutes a striking example of the genetic control restriction of enzyme activity normally prevents expression of enzyme differentiation. In the normal male chicken the in tissues other than ovary. enzyme is not measurable in any tissue, beginning as early as To explore the nature of this mutation, the effects on the fourth day of embryogenesis and continuing through the aromatase activity of two agents known to alter gene expres- adult life of the bird. In the normal female, during the same sion were examined in fibroblasts cultured from normal lifespan, the enzyme is expressed only in the ovary (2, 3). chickens and from chickens carrying the henny feathering Sebright bantam and Campine chickens ofboth sexes have trait. The protocol utilized was originally used to demonstrate an autosomal mutation termed the henny feathering trait (9) induction of 3adrenergic receptors in HeLa cells and in- that causes expression of the aromatase enzyme in many volves the sequential addition of 5-azacytidine followed by tissues from at least the fourth day of embryogenesis (3) and sodium butyrate (14). The primary effect of 5-azacytidine is that causes affected males to develop a female feathering inhibition of DNA methylation (10), whereas, among other pattern (7). The mutation does not completely prevent effects, sodium butyrate has the ability to inhibit deacetyla- suppression of aromatase activity since with advancing tion of histones (13). However, both compounds have mul- development enzyme activity in these birds does decline to tiple actions and alter the expression of many but not all genes low levels in tissues other than ovary, skin, and skin both in vivo and in vitro (10, 13). appendages (2, 3). Elucidation of the nature of the abnormal In these studies it has been demonstrated that (i) aromatase activity can be enhanced only in fibroblast strains from control ofenzyme expression in the henny-feathered chicken animals carrying the henny trait, (it) the maximal level of induced activity is similar in fibroblasts derived from diverse Table 2. Effect of 5-azacytidine and butyrate on aromatase tissues and is independent of the basal level, (iii) the degree activity in fibroblasts from day 18 embryonic of induction (over baseline) but not the absolute level of chicken tissues activity is greater in fibroblasts derived from tissues of Aromatase, pmol hr- mature chickens than in fibroblasts derived from embryonic tissues, and (iv) the induction is relatively specific in that the Source of (mg of protein)-' other cytochrome P-450 enzyme tested was not influenced by fibroblast 5-Azacytidine these agents. Similar results were obtained in two breeds of membranes No additions and butyrate chicken carrying the henny trait (Sebright bantam and Golden Sebright embryos Campine). Skin 74 210 The observation that 5-azacytidine and butyrate are in- Muscle 58 168 effective in inducing aromatase activity in the control Heart 20 82 fibroblasts indicates that the normal aromatase gene in Lung 84 229 extraovarian cells is in a state that renders it impervious to the Control embryos effects of these agents. The mutation that gives rise to the Skin <0.03 <0.03 henny feathering trait has two consequences. First, it impairs Muscle <0.03 <0.03 the mechanism that normally restricts expression of the gene in extraovarian tissues. the mutation renders the Fibroblasts were propagated and cultured from day 10 Sebright Second, and control chicken embryos. 5-Azacytidine (0.25 uM) was added 1 gene susceptible to regulation by agents that inhibit DNA day after plating, and butyrate (10 mM) was added 2 days after plating methylation and histone deacetylation, processes that do not by the standard protocol. On day 3 membranes were prepared and influence gene expression in the normal chicken. It seems aromatase activity was assayed. reasonable to speculate that the mutation impairs some Downloaded by guest on September 27, 2021 Medical Sciences: Leshin Proc. Natl. Acad. Sci. USA 82 (1985) 3009

primitive control mechanism that is fundamental to tissue- compared with wild-type chickens should help reveal these specific enzyme differentiation. The precise nature of the mechanisms. mutation is unclear. Possibilities include a gene duplication, a chromosomal rearrangement, or an unrecognized mutation The able technical assistance of Kenneth R. Luckay is gratefully of the structural gene for the enzyme that interferes with the acknowledged. This work was supported by Grant AM03892 from normal suppression mechanism. the National Institutes of Health. The finding that 5-azacytidine and butyrate cause a similar 1. Thompson, E. A., Jr., & Siiteri, P. K. (1974) J. Biol. Chem. maximal degree of enhancement of aromatase activity in 249, 5364-5372. Sebright fibroblasts derived from multiple tissues and from 2. George, F. W. & Wilson, J. D. (1980) J. Clin. Invest. 66, birds of different ages may explain two previously puzzling 57-65. aspects of estrogen physiology. The first concerns the ob- 3. George, F. W. & Wilson, J. D. (1982) Endocrinology 110, servation that enzyme activity declines with age in all 1203-1207. extraovarian tissues of the so that in the 4. Leshin, M., Baron, J., George, F. W. & Wilson, J. D. (1981) J. adult animal aromatase activity is high only in skin and skin Biol. Chem. 256, 4341-4344. appendages (3). Since the enzyme is stimulated by 5- 5. Leshin, M., George, F. W. & Wilson, J. D. (1981) Trans. azacytidine to a similar degree in fibroblasts from different Assoc. Am. Physicians 94, 97-105. 6. Leshin, M., Noble, J. F., George, F. W. & Wilson, J. D. tissues of the Sebright, it is possible that DNA methylation (1983) J. Steroid Biochem. 18, 33-39. plays an important role in this secondary suppression mecha- 7. George, F. W., Noble, J. F. & Wilson, J. D. (1981) Science nism. The second issue to which these findings may pertain 213, 557-559. involves the regulation of extraovarian estrogen production 8. Morgan, T. H. (1920) Endocrinology 4, 381-385. in the mammal. Tissue-specific restriction of aromatase 9. Somes, R. G., Jr., George, F. W., Baron, J., Noble, J. F. & activity to the mammalian ovary and placenta is never as Wilson, J. D. (1984) J. Hered. 75, 99-102. absolute as ovarian restriction in the chicken. Thus, in 10. Doerfler, W. (1983) Annu. Rev. Biochem. 52, 93-124. mammals of both sexes (including the human), aromatase is 11. Constantinides, P. G., Jones, P. A. & Gevers, W. (1977) detectable in small amounts in many extraovarian tissues, Nature (London) 267, 364-366. including adipose tissue, hair follicle, and skin (and in 12. Prasad, K. N. (1980) Life Sci. 27, 1351-1358. fibroblasts derived from normal skin) (18, 19). Furthermore, 13. Kruh, J. (1982) Mol. Cell. Biochem. 42, 65-82. in the rabbit aromatase is transiently expressed in brain 14. Jahangeer, S., Elliott, R. M. & Henneberry, R. C. (1982) this may play a critical role in Biochem. Biophys. Res. Commun. 108, 1434-1440. during embryogenesis; activity 15. Ginder, G. D., Whitters, M. J. & Pohlman, J. K. (1984) Proc. brain development (20). Such temporary expression of gene Natl. Acad. Sci. USA 81, 3954-3958. activity in the embryonic rabbit brain as well as the persistent 16. Masters, B. S. S., Williams, C. H., Jr., & Kamin, H. (1967) expression in adipose tissue, skin, and skin appendages ofall Methods Enzymol. 10, 565-573. mammals may also involve processes susceptible to phar- 17. Lowry, 0. H., Rosebrough, N. J., Fair, A. L. & Randall, macological manipulation by 5-azacytidine or butyrate (or R. J. (1951) J. Biol. Chem. 193, 265-275. both). A further understanding of the mechanism underlying 18. Schweikert, H. U., Milewich, L. & Wilson, J. D. (1975) J. the sensitivity of aromatase to 5-azacytidine and butyrate in Clin. Endocrinol. Metab. 40, 413-417. Sebright and Campine chickens may help elucidate the 19. Milewich, L., George, F. W. & Wilson, J. D. (1977) Endocri- mechanisms for tissue-specific restriction of gene expres- nology 100, 187-196. sion. Knowledge of the structure of the aromatase gene and 20. George, F. W., Tobleman, W. T., Milewich, L. & Wilson, its flanking regions in Sebright and Campine chickens as J. D. (1978) Endocrinology 102, 86-91. Downloaded by guest on September 27, 2021