Department of Human Genetics University of Michigan Medical School Ann Arbor, Michigan 48109-0618
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ABSTRACT |
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INTRODUCTION |
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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 34 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.
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RESULTS |
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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 (+22292428). 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. 2). 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. 2
). 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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Slp levels were next examined in kidney of
hypo-physectomized animals to reveal GH influence on Slp
expression in this tissue (Fig. 3). 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. 3
; 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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In vivo footprinting of kidney nuclei (Fig. 4) 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 1
).
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. 4
). 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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DISCUSSION |
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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 Slps 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.
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MATERIALS AND METHODS |
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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 Denhardts 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 016
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 -[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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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