But Not from Wild-Type Chickens

But Not from Wild-Type Chickens

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 chickens carrying the henny feathering trait but not from wild-type chickens (Sebright bantam/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 mutation (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 chicken 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 Poultry 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).

View Full Text

Details

  • File Type
    pdf
  • Upload Time
    -
  • Content Languages
    English
  • Upload User
    Anonymous/Not logged-in
  • File Pages
    5 Page
  • File Size
    -

Download

Channel Download Status
Express Download Enable

Copyright

We respect the copyrights and intellectual property rights of all users. All uploaded documents are either original works of the uploader or authorized works of the rightful owners.

  • Not to be reproduced or distributed without explicit permission.
  • Not used for commercial purposes outside of approved use cases.
  • Not used to infringe on the rights of the original creators.
  • If you believe any content infringes your copyright, please contact us immediately.

Support

For help with questions, suggestions, or problems, please contact us