Two Distinct Mechanisms Elicit Androgen-Dependent Expression of the Mouse Sex-Limited Protein Gene

Stefanie A. Nelson and Diane M. Robins

Department of Human Genetics University of Michigan Medical School Ann Arbor, Michigan 48109-0618


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The mouse sex-limited protein (Slp) gene is expressed in liver and kidney of adult males and is testosterone-inducible in females, indicative of androgen dependence. Analysis of mRNA levels and chromatin configuration reveals that this androgen regulation is achieved by distinct means in the two tissues. In the liver, Slp expression requires pituitary function, and specifically, as shown by others, a pulsatile pattern of GH secretion that is itself determined by androgen. After hypophysectomy, Slp synthesis cannot be reestablished in liver by testosterone, although mRNA decline can be slowed. In contrast, in the kidney Slp mRNA is directly induced by androgen in hypophysectomized mice. In vivo footprinting was used to examine the role of the Slp enhancer, which directs androgen-specific transcription in transfection and contains a factor-binding site, FPIV, whose protection in vivo has been correlated with Slp expression. In kidney, FPIV was protected in intact males and hypophysectomized mice supplemented with testosterone, but not in females or untreated hypophysectomized mice, corroborating FPIV’s association with androgen-driven transcription. Surprisingly, protection of FPIV also occurred in liver of hypophysectomized males treated with testosterone, despite the lack of Slp expression. Thus androgen directly affects the Slp enhancer in kidney, where steroid is sufficient for gene activation, as well as in liver, where chromatin remodeling occurs in response to androgen, although GH is clearly required for expression. This may indicate that both GH and androgen signal transduction pathways target the Slp enhancer to elicit precise gene regulation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Precise control of gene expression in vivo often involves the interplay of multiple regulatory elements and the integration of stimuli from distinct signaling pathways. Interaction of the effects of signaling molecules, such as the steroid and peptide hormones with tissue-specific transcription factors, can provide the means by which an extracellular stimulus induces gene expression in one cell type but not in another (1, 2, 3). Additional levels of control can be established by cross-talk of multiple signal transduction pathways (4, 5, 6). While the role of individual factors can be defined in in vitro systems, these complex regulatory interactions are best examined in vivo, yet few well characterized systems are accessible to such analysis.

The mouse sex-limited protein (Slp) gene allows examination of the impact of multiple signaling mechanisms on androgen-dependent gene expression. Slp arose as a duplication of the complement component C4 gene (7), and the two remain highly homologous in their coding and 5'-flanking regions. An androgen-responsive enhancer within the long terminal repeat of a provirus that inserted 2 kb upstream has apparently imposed androgen dependence on the adjacent Slp gene (8). DNase I-hypersensitive sites map to this enhancer in chromatin and are responsive to androgen in Slp-expressing tissues, which are primarily adult male liver and kidney (9). In vitro, a 120- bp fragment of the enhancer, which contains a consensus glucocorticoid or hormone response element (HRE) and several crucial nonreceptor protein-binding sites, confers androgen-specific response on a linked reporter gene (10). Despite the strong androgen dependence of the enhancer, however, there is evidence that expression of the endogenous Slp gene is influenced by additional signaling systems, particularly that of GH (11, 12).

Androgen-dependent expression of several sex-specific hepatic genes in rodents, such as members of the cytochrome P450 and the mouse urinary protein gene families (13, 14, 15, 16), requires interaction between the steroid hormone testosterone and the hypothalamic-pituitary axis. In rodents, release of GH occurs in episodic bursts every 3–4 h in males, but is more constantly released into female serum, with no pronounced trough periods (17, 18). The trough period, rather than absolute GH level, appears critical for male-specific liver gene expression, as mice carrying constitutive GH transgenes fail to express such markers (16). Furthermore, GH-depleted animals supplemented with testosterone fail to reinitiate male-specific expression (11, 12, 19). Instead, administration of GH to GH-deficient (or female) mice, in a pulsatile, male-like pattern, induces expression of several normally male-restricted genes.

In rodents, kidney also displays sexually distinct patterns of gene regulation (20). Transcription of kidney androgen-regulated protein (KAP), ß-glucuronidase, and ornithine decarboxylase, among others, is induced by androgens in the proximal convoluted tubules (21). In contrast to liver, sexually dimorphic gene expression in kidney is less strictly dependent on GH, with testosterone capable of activating transcription in GH-deficient animals (22, 23). Further, comparison of P450 genes that express in both liver and kidney has shown that whereas pulsatile GH secretion is required for sex-specific hepatic expression, it is not required for sexually dimorphic expression of these same genes in kidney (14, 24).

It has been known for some time that a pituitary factor is required to maintain Slp serum protein levels (which mostly reflect liver synthesis) (11). As seen for other sexually dimorphic liver genes in rodents, GH itself was shown to be the necessary factor for Slp mRNA synthesis (12). In GH-deficient animals, Slp is not expressed in liver (and serum Slp levels likewise fall) and is unresponsive to doses of testosterone sufficient to induce expression in intact animals. Moreover, administration of GH in a male-like, pulsatile pattern induces hepatic expression of Slp in testicular feminization (tfm) mutant mice that lack a functional androgen receptor.

The role of GH in liver led us to compare regulation of Slp in kidney, in order to determine the relative influence of GH and androgen on Slp expression in these sites at a molecular level. First, we localized Slp synthesis within the kidney by in situ hybridization. RNase mapping revealed that while the pituitary is required for Slp expression in liver, testosterone is capable of inducing expression in kidney, even in the absence of GH. The effects of hypophysectomy and testosterone replacement on factor binding to the Slp enhancer were examined by DNase I in vivo footprinting. FPIV, a site whose occupancy correlates with regulation of Slp (25), is unoccupied in both liver and kidney after hypophysectomy and subsequent loss of endogenous testosterone, as expected. Interestingly, testosterone induces protection of the FPIV site in hypophysectomized mice in both tissues. This suggests that a chromatin remodeling event occurs in both sites directly in response to androgen, and that this event may be sufficient for subsequent Slp expression in kidney, but is clearly not so in liver. However, the GH signal that is ultimately necessary for liver expression may be transmitted through DNA-regulatory elements that overlap those used in more classically direct androgen response.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Localization of Slp Expression in Kidney
Many androgen-regulated genes in rodent kidney, such as KAP, ornithine decarboxylase, and S-adenosylmethionine decarboxylase (26, 27, 28), show localized expression to the epithelial cells of the proximal convoluted tubules. To determine whether Slp was synthesized in a subpopulation of kidney cells, in contrast to liver where most hepatocytes express Slp, we performed RNA in situ hybridizations using a Slp riboprobe that in RNase protection assays detects both Slp and C4 mRNAs (see below). Frozen kidney sections used were from regions where the renal pelvis was visible so as to enable comparison of gene expression in cortex and medulla. Comparison of sections showed a much stronger signal in male than in female kidney, with probe hybridization concentrated in the outer, cortical region (Fig. 1Go, a and b). Comparison of brightfield and darkfield images of male kidney sections at higher magnification (Fig. 1Go, c and d) revealed that expression of Slp in male kidney was excluded from glomeruli but present abundantly in surrounding regions. This distribution pattern suggests Slp may be synthesized in the proximal convoluted tubules, as are other androgen-regulated kidney proteins (27, 28).



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Figure 1. Localization of Slp Expression in Mouse Kidney by in Situ Hybridization

The Slp/C4 riboprobe was hybridized to 7 µm frozen kidney sections as described in Materials and Methods. Panels a, b, and d are darkfield images of antisense riboprobe hybidization to male kidney sections, at 20x, 40x, and 80x magnifications, respectively. Panel c, brightfield image of male kidney, in same visual field as darkfield image in panel d. Panels e and f, Hybridization of Slp/C4 riboprobe to female kidney sections, at 40x and 80x magnification. Panels g and h, Hybridization of sense strand riboprobe to male kidney sections, 40x and 80x magnification. Sections are oriented with cortex to the right. Cortex (co) and medulla (m) are indicated in panel a. Arrowsin panels c and d indicate glomeruli, which show little specific hybridization relative to surrounding tubules. Exposure time for emulsion autoradiography was 13 days for all sections shown.

 
A significant difference in signal intensity was seen in female kidney (Fig. 1Go, e and f), in accord with low Slp expression in this site as well as in liver relative to males (29). The section shown is from a different plane than the male and consists largely of cortex with little renal pelvis visible, which accounts for the more uniform signal distribution. Female sections exhibited greater signal than those with the sense riboprobe (Fig. 1Go, g and h). Most female hybridization is probably to C4 mRNA, which is also detected with this probe and is present in equivalent levels in males and females in kidney, as in liver (29). Preliminary attempts to distinguish C4 from Slp transcripts with gene-specific oligonucleotide probes were not successful. Nevertheless, the comparison of male to female hybridization intensity localizes Slp expression to the kidney cortex, and exclusion from glomeruli suggests synthesis is likely to occur in the proximal convoluted tubules.

Tissue-Specific Differences in the Pituitary Requirement for Slp Expression
Slp mRNA levels in liver and Slp protein levels in serum require GH (11, 12), but such regulation has not been examined in kidney. Further, androgen appears to act more directly in kidney, as suggested by more rapid induction of Slp mRNA than in liver after testosterone administration to females, with maximum levels attained in days rather than weeks (29). Slp mRNA also declines more rapidly in kidney upon castration, with expression only a few percent of intact male levels by 2 weeks, while substantial Slp is detected in liver as long as 3 months later (29).

To compare dependence of Slp on pituitary factors and/or androgen in liver vs. kidney, expression was assayed after hypophysectomy (glucocorticoids and thyroid hormone were replaced in the diet). The Slp riboprobe used in RNase protection was transcribed from the region of greatest sequence divergence from C4 (+2229–2428). After digestion with RNase, a 200-nucleotide fragment is protected by Slp mRNA, whereas hybridization to C4 generates fragments of 85 and 95 nucleotides. A mouse ß-actin riboprobe served to control internally for RNA quantification. To assess completeness of hypophysectomy, we initially measured IGF-I as a marker of GH action by both RNase protection and in serum of individual mice. The few males that expressed Slp in liver 2 weeks after surgery, or females that expressed after an additional 2 weeks of testosterone treatment, also failed to show appropriately reduced IGF-I levels, consistent with residual pituitary function. Thus, to ensure the absence of GH, these studies used only tissues from animals that did not express Slp in liver after 2 weeks or longer of hypophysectomy, with or without testosterone treatment.

Two weeks after hypophysectomy, hepatic Slp expression declined to less than 1% of intact male levels in both male and female mice, as measured by PhosphorImager analysis of RNase protection assays (Fig. 2Go). Intact females exhibited approximately 2% of the intact male expression. The decrease in hypo-physectomized males was greater and more rapid than that after castration (29). Intramuscular injections of testosterone propionate for 2 weeks, beginning 2 weeks after hypophysectomy, failed to induce Slp expression in the liver of either sex, with expression remaining at less than 1% that of intact males (Fig. 2Go). This hormone treatment was sufficient, however, to induce Slp expression in liver of females with intact pituitaries. Hepatic C4 levels were not affected by hypophysectomy or testosterone treatment and therefore served as convenient controls for quantification of RNA. Liver actin levels assayed by RNase protection also remained constant (data not shown). These results confirm that a pituitary factor(s), most likely GH (12), is required for expression of Slp in liver, and that testosterone alone cannot restore Slp synthesis in the absence of this factor.



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Figure 2. Expression of Slp in Liver Is Dependent on GH

RNA was isolated from liver of control, hypophysectomized (Hx), and testosterone-treated hypophysectomized (Hx + T) male and female mice. Twenty micrograms of total RNA were used in RNase protection assays as in Materials and Methods. Bands representing RNase-digested products of the riboprobe hybridized to Slp and C4 transcripts are indicated at left. Numbers in boxes indicate duration of testosterone treatment (T) and number of days after hypophysectomy (Hx) that the animals were killed. (-) indicates no treatment. For males treated with testosterone for 4 days and killed 11 days after hypophysectomy, treatment with testosterone began on day 7 after surgery. For hypophysectomized males and females treated with testosterone for 14 days, treatment began 14 days after hypophysectomy.

 
Hepatic Slp expression was also examined to see whether any androgen effect could be demonstrated in hypophysectomized males before loss of detectable mRNA. One week after surgery, Slp mRNA was less than 50% of intact male levels; by 11 days, Slp expression had decreased to approximately 1% of male levels (Fig. 2Go). When testosterone was administered 1 week after hypophysectomy for 4 days, the decline in Slp expression was slowed, but the level at 11 days was still much lower than at 7 days. The decelerated loss of Slp mRNA produced by androgen at this intermediate time point could be due to transcriptional or posttranscriptional mechanisms. In liver of intact animals, the difference between expression of Slp in males and lack of expression in females is at the level of transcription (12, 30). Because the steroid produces no increase in Slp in liver after 2 weeks of hypophys-ectomy, in contrast to administration of pulsatile GH (12), pituitary factors are implicated in this transcriptional regulation. The modest effect of androgen only demonstrable on detectable steady-state mRNA may thus be due to mRNA stabilization (see Discussion).

Slp levels were next examined in kidney of hypo-physectomized animals to reveal GH influence on Slp expression in this tissue (Fig. 3Go). As before, only RNA from animals whose livers did not express Slp (plus or minus testosterone) was used to ensure absence of pituitary function. Because kidney C4 levels appeared more variable after hypophysectomy, Slp expression was normalized to actin. As in liver, Slp levels in male kidney had fallen considerably 2 weeks after hypo-physectomy, to 5% of intact levels. In contrast to liver, however, significant Slp induction occurred from this low level with 2 weeks of androgen administration. Expression in individual hypophysectomized mice treated with testosterone is shown in Fig. 3Go; this and additional data quantitated by PhosphorImager analysis showed that androgen induction of Slp averaged 4-fold in males and 6-fold in females (n = 6 for each). Similar induction was observed whether testosterone was administered by injection or by implantation of time-release pellets (not shown). Androgen-induced Slp in kidney of hypophysectomized males reached about 20% of the intact male levels. The same regimen in females brought Slp to more than one-third the level of an intact female similarly treated, which attains one-third the Slp level of a normal male. Thus, in contrast to liver, significant Slp expression was reestablished by androgen in kidney in the absence of GH. This illustrates that in some, but not all, sites of synthesis, a component of Slp transcription appears to be directly androgen-responsive.



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Figure 3. Slp Expression Can Be Induced by Testosterone in Kidney of Hypophysectomized Animals

RNase protection assays were performed on 25 µg total RNA isolated from kidney of control, hypophysectomized (Hx), and Hx male and female mice treated with testosterone (Hx + T). Slp- and C4-protected fragments are indicated at the left. Numbers in boxes indicate days of treatment, as in liver. Hybridization to an actin riboprobe using 0.25 µg RNA, performed for control of quantification, is shown at the bottom.

 
In Vivo Footprinting Reveals Remodeling of Enhancer Chromatin by Testosterone in GH-Deficient Animals
In transfection, a 120-bp enhancer from 2 kb upstream of Slp’s start site is sufficient to confer androgen inducibility on a linked reporter gene (10). In mice, this enhancer also displays responsiveness to androgens, as the encompassing chromatin is hypersensitive to DNase I in male liver and kidney and in the tissues of females treated with androgen (9). High resolution in vivo footprinting has linked a nonreceptor protein-binding site in the enhancer, FPIV, to the hormonal, developmental, and tissue-specific regulation of Slp (25). In particular, FPIV protection correlates with the presence of testosterone and Slp expression as it occurs in females after androgen treatment and is lost upon castration of males. We used in vivo footprinting to ask whether the enhancer would reflect androgen action in the absence of GH. Liver and kidney nuclei from intact, hypophysectomized, and hypophysectomized mice treated with testosterone were isolated and treated with varying amounts of DNase I, and purified DNA was used as template for ligation-mediated PCR. Nested primers were chosen to specifically amplify a 160-bp region of the Slp enhancer, extending from the site known as FPIII downstream through HRE-3, with FPIV centrally located (25).

In vivo footprinting of kidney nuclei (Fig. 4Go) revealed noticeable changes in protection patterns at the FPIV protein-binding site. The FPIV site in male kidney was more protected than in female kidney (apparent by comparing this region within a set of lanes to flanking hypersensitive regions). This was corroborated by densitometric scanning of autoradiograms and comparison of the indicated band in FPIV to a central band in HRE-2 that does not vary with hormonal treatment (see Table 1Go). Hypophysectomy and concomitant loss of gonadal steroids resulted in loss of FPIV protection in male kidney, with the site becoming hypersensitive to a similar degree as seen in females (Fig. 4Go). The pattern of nuclease cleavage of FPIV in female kidney was not affected by hypophysectomy. Administration of testosterone to hypophysectomized animals resulted in gain of protection at FPIV in kidney chromatin from both sexes, equivalent to that seen in intact males. Thus, in kidney, occupancy of FPIV correlated with the presence of testosterone and expression of Slp, regardless of the presence or absence of GH.



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Figure 4. FPIV Protection in DNase I in Vivo Footprinting in Kidney Correlates with Slp Expression

Nuclei were isolated from kidney of control, hypophysectomized, and hypophysectomized male and female mice treated with testosterone, treated with increasing amounts of DNase I (shown as increasing concentrations from left to right), DNA was isolated and 3 µg subjected to LMPCR. From left to right, for both males and females are control, 2-week hypophysectomized (Hx), and 2-week hypophysectomized animals treated with testosterone for an additional 2 weeks (Hx + T). Hypersensitive marker bands in the FPIV region and in HRE-2 are noted by (*) on the left. The open box marks a region of the enhancer showing increased hypersensitivity in male liver.

 

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Table 1. Protection of FPIV Relative to HRE-2

 
In liver of intact animals, the FPIV site is strongly protected in males compared with females (Fig. 5Go). Upon hypophysectomy, hypersensitivity at the FPIV site increased approximately 2-fold in male liver, similar to what was seen in kidney (Table 1Go). In females, hypophysectomy did not affect hypersensitivity of the FPIV site. Surprisingly, treatment of hypophysectomized males with testosterone reestablished protection at FPIV, in three independent chromatin preparations, to nearly the same extent as in the intact male (Fig. 5Go). This was unexpected, as occupation of the FPIV site had previously been correlated with expression of Slp, which was not achieved by androgen in liver of hypophysectomized mice. In contrast to males, testosterone treatment of hypophysectomized females did not lead to significant protection of FPIV (see Discussion). Although the Slp enhancer was incapable of driving transcription in liver in the absence of GH, these data illustrate that alterations in chromatin structure in this region can be induced by testosterone alone.



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Figure 5. In Vivo Footprinting of Liver Chromatin Reveals Occupancy of FPIV Induced by Androgen in the Absence of Slp Expression

Nuclei from male (left) and female (right) control, Hx, and Hx + T mice were isolated, and DNA was analyzed by LMPCR as described in Fig. 4Go. Hypersensitive marker bands in the FPIV region and in HRE-2 are noted by (*) on the left. The open box marks a region of the enhancer showing increased hypersensitivity in male liver.

 
Aside from changes at FPIV, no other differences in protection pattern were caused by hypophysectomy. In liver, a region of the enhancer just 5' to FPIV was consistently more hypersensitive in males than in females, but this was not significantly altered by hypophysectomy. Additionally, no changes in protection were noted over the HREs, despite the decrease in endogenous androgens due to loss of pituitary gonadotropins. This is consistent with experiments performed in our laboratory and others that show receptor binding is not easily detected using this in vivo footprinting methodology (25, 31). In contrast, the marked alteration of FPIV protection provides an interesting example of regulated factor occupancy dissociated from gene expression.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Comparison of Slp expression and chromatin configuration in liver and kidney reveals striking tissue-specific differences in the means by which androgen regulation of this gene is achieved. As first demonstrated by Georgatsou et al. (12), Slp expression in liver is dependent on pulsatile GH secretion and cannot be reinduced by testosterone. However, we show here that Slp RNA synthesis in the kidney is directly responsive to androgen. Even in hypophysectomized animals, testosterone induces Slp expression severalfold in males and in females. In addition, while testosterone may not be sufficient to elicit hepatic Slp synthesis in the absence of GH, the steroid directly affects chromatin structure around the androgen-responsive enhancer. This study provides evidence for multiple pathways influencing tissue-specific expression of Slp, ultimately resulting in male-specific expression in both liver and kidney.

Given two distinct modes by which to elicit male-specific expression, we asked whether the androgen-responsive Slp enhancer was involved in the GH-mediated as well as the androgen-mediated regulatory pathway. This seemed plausible because we have previously shown that, within 10 kb of Slp 5'-flank, DNase I-hypersensitive sites correlating with expression are prominent only in the region of the enhancer and the promoter itself (9). Furthermore, the enhancer, originating from a provirus, is linked to all Slp alleles that are male-specific in expression but not to those that are hormone-independent (8). The hormone-independent alleles confirm that the sex limitation of Slp is exerted primarily at the level of transcription. These genes are natural recombinants between the homologous C4 and Slp, in which the 5'-flank of C4 drives the Slp coding region (30). Females with these genes express male levels of Slp, implying that sex-limited expression is not a function of the mRNA sequence but of the Slp upstream flank. This further argues that the enhancer encompasses crucial cis-acting elements for sex-limited expression, regardless of the precise trans-acting signals responsible.

Protection of the FPIV site in the enhancer by in vivo footprinting was used as an indicator of enhancer activity because this site was previously correlated with hormonal, developmental, and tissue-specific regulation of Slp (25). Hypophysectomy and the concomitant loss of gonadal steroids resulted in loss of FPIV protection in male chromatin in both liver and kidney, demonstrating the need for testosterone and/or pituitary factors to maintain factor binding. In kidney, treatment of hypophysectomized animals with testosterone was sufficient to induce Slp transcription and also reestablish protection of FPIV, demonstrating that for this tissue, the enhancer is likely to mediate GH-independent regulation. Intriguingly, although hepatic Slp expression is dependent on GH, in liver of GH-deficient males, androgen remodels enhancer chromatin and allows factor binding at FPIV. Thus the Slp gene in liver senses testosterone, which, while clearly not sufficient, may still play some role in expression.

Kidney Slp synthesis is apparently more directly responsive to testosterone, as are other male-specific kidney-expressed genes, such as RP-2, ß-glucuronidase, and cytochrome P450 4A (14, 15, 22, 23). Additionally, although GH deficiency interferes with hepatic expression of several cytochrome P450 enzymes, appropriate sexually dimorphic expression of these enzymes is seen in kidney of the same animals (14). Whereas testosterone induces Slp in kidney, maximal expression is not attained in hypophysectomized animals, implying a need for pituitary factors for full induction. Cell type-specific differences within kidney may account for the lack of full induction, as other genes respond differentially to androgen in subsets of proximal tubule cells. Ornithine decarboxylase responds to testosterone only in the S1/S2 cells of the outer cortex (26, 27). Similarly, KAP is regulated by thyroid hormone in the juxtamedullary S3 cells of the proximal tubules but can be induced in the S1/S2 segment by testosterone in the absence of thyroid hormone (32, 33). If Slp is under similar cell-specific, multihormonal control, only a subpopulation of cells may respond to androgen in the absence of GH, leading to intermediate levels of mRNA.

Our results suggest that the Slp gene is also sensitive to androgen in liver, although GH is clearly requisite for expression. Pulsatile injections of GH induce Slp in tfm (testicular feminization) mice, which lack functional androgen receptor, demonstrating that androgen is not essential for transcription (12). However, in vivo footprints of the Slp enhancer show sensitivity to this steroid in liver, despite the lack of gene activation. This indicates a direct effect of androgen in liver at the level of the gene, specifically in directing chromatin structure. In addition, testosterone treatment of mice 1 week after hypophysectomy slows the decline of Slp mRNA levels, due either to stabilization of mRNA or of a preexisting, active transcription complex. Thus, even in intact animals, there are likely to be direct and/or indirect effects of androgen on male-specific hepatic gene expression that are not necessarily mediated by pulsatile GH secretion.

The FPIV site shows striking changes in in vivo footprinting and is necessary for androgen inducibility in transfection (34). In different cells distinct proteins bind FPIV (25) that in kidney may be sufficient for transcription of Slp. A major role of testosterone, via the androgen receptor, may be to alter chromatin to provide access to the FPIV site. Steroid receptors often perturb chromatin structure of hormonally regulated genes (31, 35, 36, 37, 38). However, accessibility of DNA-regulatory elements does not necessarily imply transcriptional activity; assembly of specific factors into an active transcription complex, involving additional DNA sites, may be required for expression. As accessory factors may themselves be regulated, their absence may explain why steroid-induced alterations in chromatin structure, such as those induced by androgen at FPIV in liver, can be observed even in the absence of gene expression (2, 39, 40, 41). Perhaps in liver a different factor than in kidney binds this region in the presence of GH, the factor itself may be modified, or factors bound to additional DNA elements beyond this enhancer may be required. Modification of preexisting factors may be particularly relevant. Pulsatile GH secretion leads to a liver STAT 5-related factor being phosphorylated, which is necessary for its DNA binding and transcriptional activity (42). Similarly, phosphorylation of the cAMP response element-binding protein by protein kinase A increases its affinity for DNA, thus stimulating transcription from low affinity cAMP response elements (43). In liver, androgen may promote binding of the necessary factor(s) at FPIV in hypophysectomized males, but pulsatile GH may be required to modify these factors to allow transcription or to induce recruitment of coactivators.

That liver FPIV protection is induced by androgen in hypophysectomized males but not females is also intriguing. The hypophysectomized female is never exposed to pulsatile GH, which may be necessary to imprint liver genes and allow subsequent testosterone-induced factor binding. Intact females treated with testosterone secrete male-like GH pulses, thus allowing FPIV binding and Slp expression. An interplay of GH and androgen may also account for Slp’s recalcitrance to induction by testosterone before sexual maturation. Manipulation of gonadal steroids before puberty does not fully convert GH-secretory patterns, although subsequent postpubertal release is altered (17, 18, 44). If Slp requires male-specific GH pulsatility for liver expression, the inability of testosterone to elicit this pattern in immature mice may preclude induction before puberty.

In addition to experiments described here, genetic evidence supports multiple distinct modes of male-specific Slp expression. The inbred FM mouse strain carries a mutation of an as yet unidentified trans-acting gene that permits Slp expression in females, but still only after puberty (45). It is unlikely that this is due to a difference in a major hormonal regulatory pathway, as FM mice seem otherwise normal. Androgen is not required for expression as FM animals carrying the tfm mutation express Slp. Analysis of this new regulatory phenotype may shed further light on tissue-specific differences in androgen and GH regulation of Slp.

In summary, these studies of gene expression and chromatin structure provide evidence for two distinct mechanisms by which androgen dependence of Slp is achieved. In kidney, Slp can be induced directly by androgen whereas in liver, pulsatile GH secretion, established by androgen action on the pituitary, is necessary for expression. Despite the inability of androgen alone to induce expression in liver, the steroid promotes enhancer chromatin remodeling that resembles that of expressing tissues. However, in the absence of GH, this alteration in chromatin architecture is uncoupled from transcription of the gene. Both androgen and GH may influence Slp expression to varying extents in both tissues, under different circumstances, and these effects may be integrated, at least in part, at a single complex enhancer.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Animals and Treatment
BALB/c and hypophysectomized (at 7 weeks of age) BALB/c mice were obtained from Charles River Laboratories (Wilmington, MA). Drinking water of hypophysectomized animals was supplemented with 5% sucrose, and they were maintained on AIN-76A, a diet containing 25 mg thyroid powder/kg (46). Testosterone propionate (0.1 ml of 25 mg/ml in sesame oil) was administered by intraperitoneal injection every other day for 2 weeks, beginning 2 weeks after surgery. In some cases, 25 mg, 21-day release testosterone propionate pellets were implanted subcutaneously 2 weeks after surgery. After animals were killed, tissues were frozen in liquid nitrogen and stored at -70 C. All research was conducted in accord with NIH Guidelines for the Care and Use of Experimental Animals.

In Situ Hybridization
Kidneys were embedded in OCT tissue embedding medium and quick frozen in isopentane and liquid nitrogen. Sections (7 µm) were cut in a cryostat and retained when the renal pelvis became visible. Sections were placed on poly-L-lysine-coated slides, fixed in 4% formaldehyde in PBS for 1 h at room temperature, and rinsed three times in 2 x NaCl-sodium citrate (SSC). Sections were then incubated in 1 µg/ml proteinase K for 10 min at 37 C, and washed for 1 min in diethyl pyrocarbonate-treated-H2O. Slides were incubated for 10 min with mixing at room temperature in 0.1 M triethanolamine, pH 8.0, 0.25% acetic anhydride. Slides were washed for 5 min in 2 x SSC, dehydrated in ethanols (50%–100%), and air dried.

Riboprobes for in situ hybridization were prepared as for RNase protection assays (see below), except with [35S]UTP. Sense probes were transcribed from the same plasmid, but using the Sp6 promoter. After synthesis, free label was removed with a 1 ml G-50 Sephadex column.

Riboprobes were diluted in hybridization buffer (75% formamide, 10% dextran sulfate, 3 x SSC, 50 mM sodium phosphate, pH 7.4, 1 x Denhardt’s and 0.1 mg/ml tRNA) to give 1.5 x 106 cpm per 100 µl. One hundred microliters of probe in buffer were placed on each section, a coverslip was sealed over the section with rubber cement, and slides were incubated overnight at 54 C in a humidity chamber.

After hybridization, coverslips were removed and slides washed twice for 5 min in 2 x SSC at room temperature. Sections were incubated in 200 µg/ml RNase A in 10 mM Tris-Cl, pH 8.0, 0.5 M NaCl, at 37 C for 60 min. Slides were then washed in 2 x SSC for 5 min, 1 x SSC for 5 min, and 0.5 x SSC for 60 min at 62 C. After a final 0.5 x SSC wash for 5 min at room temperature, slides were dehydrated in ethanols, air dried, and dipped in Kodak NTB-2 emulsion diluted 1:1 with dH2O plus 1 mg/ml Dreft. Sections were exposed at 4 C for 14 days. Slides were developed and then stained with cresyl violet, dehydrated in ethanols and xylenes, and coverslipped.

RNA Preparation
Tissues were homogenized using a Brinkmann polytron (Westbury, NY) in RNasol solution (47), consisting of 20 ml solution D (.025 M sodium citrate, pH 7.0, 0.5% sarkosyl, 4 M guanidinium thiocyanate, 0.1 M ß-mercaptoethanol), 20 ml phenol, and 2 ml 2 M sodium acetate, at 2 ml per 100 mg tissue. Homogenates were extracted with 0.1 vol sevag (chloroform-isoamyl alcohol, 24:1) and centrifuged, and the aqueous phase was precipitated with an equal volume of isopropanol. RNA was resuspended in solution D, reprecipitated with isopropanol, resuspended in H2O, and stored at -70 C. Kidney samples were treated with RQ1 DNase (Promega, Madison, WI) to remove contaminating DNA.

RNase Protection Assays
Synthesis of the Slp riboprobe and RNase protection were performed as described (29). Actin riboprobes were transcribed from pTRI-ß-actin plasmid (Ambion, Austin, TX) using the T7 promoter. All probes were gel-purified. Hybridizations were in a volume of 30 µl in 80% formamide, 40 mM 1, 4-piperazinediethanesulfonic acid, pH 6.4, 0.4 M NaCl, 1 mM EDTA. Total RNA (25 µg) from kidney, 20 µg from liver, or 0.25 µg RNA of either tissue for actin analysis were hybridized to 1.5 x 105 cpm of probe overnight at 45 C. Hybridizations with Slp probe were digested with 40 µg/ml RNase A plus 700 U/ml RNase T1 for 45 min at 30 C. Hybridizations with actin probe were digested with 50 µg/ml RNase A plus 1000 U/ml RNase T1 for 45 min at 37 C. After RNase treatment, samples were extracted with phenol and ethanol precipitated. Pellets were resuspended in 80% formamide, 0.1% SDS loading buffer, heat-denatured, and run on 8% acrylamide/8 M urea sequencing gels. After drying, gels were scanned and data were quantified on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

In Vivo Footprinting
Nuclear isolation, DNA preparation, and ligation-mediated PCR (LMPCR) were performed as described by Scarlett and Robins (25). Frozen tissue (5 g) was ground in a mortar and pestle, washed with PBS at 4 C, resuspended in 15 ml (liver) or 10 ml (kidney) buffer H (10 mM Tris, pH 7.5, 15 mM NaCl, 60 mM KCl, 1 mM EDTA, 0.2% NP40, 5 mM MgCl2, 5% sucrose), and homogenized in a Dounce on ice with a tight pestle. Homogenates were centrifuged 10 min in a clinical centrifuge, and the pellet was resuspended in 5 ml buffer W (10 mM Tris, pH 7.5, 15 mM NaCl, 60 mM KCl, 5 mM MgCl2) and centrifuged at 200 x g at 0 C. The pellet was resuspended in 2 ml buffer W, and nucleic acid concentrations were adjusted to 0.75 µg/ml for liver and 0.5 µg/ml for kidney. MgCl2 to 1 mM and CaCl2 to 0.5 mM were added to aliquotted nuclei. DNase I (DPFF grade, Worthington Biochemicals, Freehold, NJ) was added at 0–16 µg/ml for 10 min on ice. One hundred microliters of stop buffer A (100 mM EDTA, 4 mg/ml proteinase K) were added for 15 sec at 50 C, followed with 400 µl 2.5% SDS. After 2 h at 50 C, an additional 200 µg proteinase K were added and samples incubated overnight. DNA was extracted with phenol/sevag and dialyzed against several changes of TE (10 mM Tris, pH 7.5, 1 mM EDTA) at 4 C.

LMPCR followed the procedure of Mueller and Wold (48) using the primers below. The 25'mer and 11'mer primers were used to ligate the common primer to DNA for amplification of footprinted regions. Primer 1 (P1) (from -1779 to -1799 5' to the Slp start site) was used for the first LMPCR extension and P2 for exponential amplification. P3 was used in the labeling step and corresponds to -1855 to -1888 relative to the Slp start. Primer sequences are:

11'mer: 5'-GAATTCAGATC-3'

25'mer: 5'-GCGGTGACCCGGGAGATCTGAATTC-3'

P1: 5'-GGTTCTGGGAATTGAGCTTGG-3'

P2: 5'-AATTTAAGTTTGACCCCCATAGACC-3'

P3: 5'-GTTTGACCCCCATAGACCAAGTTAGAGC-3'

DNA (3 µg) was denatured at 95 C for 5 min, and primer P1 was annealed for 30 min at 60 C. MgCl2, dithiothreitol, and deoxynucleoside triphosphates were added, primer P1 was extended with Sequenase for 5 min at 47 C, and the reaction was terminated at 70 C for 10 min. Linker oligonucleotides were added for ligation overnight at 16 C. PCR was for 16 cycles (1.5 min at 94 C, 3 min at 65 C, 3 min at 72 C) with the addition of 5 sec to step 3 with each additional cycle. After amplification, reactions were incubated 10 min at 72 C. DNA was labeled with 2.5 pmol {gamma}-[32P]-kinased primer P3 for five rounds of PCR (1.5 min at 94 C, 3 min at 68 C, 3 min at 72 C). Sodium acetate was added to 0.3 M and EDTA to 1 mM. DNA was extracted with phenol, precipitated with ethanol, and resuspended in formamide loading buffer for electrophoresis on 8% sequencing gels.

Autoradiograms were scanned using a UC630 (Umax Data Systems, Freemont, CA) and analyzed using Adobe Photoshop (Adobe Systems, Mountain View, CA) and NIH Image software (NIH, Bethesda, MD). Areas under FPIV peaks were compared with areas of HRE-2 peaks that did not vary between males and females and were not affected by hormonal treatments. Three independent chromatin preparations for liver and two for kidney were analyzed.


    ACKNOWLEDGMENTS
 
We thank Kent Christensen and Kay Brabec of the Morphology Core of the NIH-NICHD Center for the Study of Reproduction (P30 HD-18258) at the University of Michigan (Ann Arbor, MI) for guidance in histology and in situ hybridization. Cameron Scarlett generously gave time, reagents, and advice on in vivo footprinting while in this laboratory. We thank Jessica Schwartz and Sally Camper for helpful comments on GH regulation and hypophysectomized mice and Ken Zaret for comments on the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Diane M. Robins, Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan 48109-0618.

This work was funded by a grant from the NIH (GM-31546). S.N. was supported in part by the University of Michigan Cancer Biology Training Grant.

Received for publication October 17, 1996. Revision received January 10, 1997. Accepted for publication January 23, 1997.


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