5'-Heterogeneity of Glucocorticoid Receptor Messenger RNA Is Tissue Specific: Differential Regulation of Variant Transcripts by Early-Life Events

J. A. McCormick, V. Lyons, M. D. Jacobson, J. Noble, J. Diorio, M. Nyirenda, S. Weaver, W. Ester, J. L. W. Yau, M. J. Meaney, J. R. Seckl and K. E. Chapman

Molecular Endocrinology (J.A.M., V.L., J.N., M.N., W.E., J.L.W.Y., J.R.S., K.E.C.) University of Edinburgh Molecular Medicine Centre Western General Hospital Edinburgh, EH4 2XU, United Kingdom
Millennium Pharmaceuticals, Inc. (M.D.J.) Cambridge, Massachusetts 02139
Developmental Neuroendocrinology Laboratory (S.W., M.J.M.) Douglas Research Center Departments of Psychiatry, Neurology, and Neurosurgery McGill University Montreal, H4H IR3, Canada


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Glucocorticoid receptor (GR) gene expression is regulated in a complex tissue-specific manner, notably by early-life environmental events that program tissue GR levels. We have identified and characterized several new rat GR mRNAs. All encode a common protein, but differ in their 5'-leader sequences as a consequence of alternate splicing of, potentially, 11 different exon 1 sequences. Most are located in a 3-kb CpG island, upstream of exon 2, that exhibits substantial promoter activity in transfected cells. Ribonuclease (RNase) protection analysis demonstrated significant levels of six alternate exons 1 in vivo in rat, with differences between liver, hippocampus, and thymus reflecting tissue-specific differences in promoter activity. Two of the alternate exons 1 (exons 16 and 110) were expressed in all tissues examined, together present in 77–87% of total GR mRNA. The remaining GR transcripts contained tissue-specific alternate first exons. Importantly, tissue-specific first exon usage was altered by perinatal environmental manipulations. Postnatal handling, which permanently increases GR in the hippocampus, causing attenuation of stress responses, selectively elevated GR mRNA containing the hippocampus-specific exon 17. Prenatal glucocorticoid exposure, which increases hepatic GR expression and produces adult hyperglycemia, decreased the proportion of hepatic GR mRNA containing the predomin-ant exon 110, suggesting an increase in a minor exon 1 variant. Such tissue specificity of promoter usage allows differential GR regulation and programming.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Glucocorticoids maintain homeostasis after stress and play key roles in differentiation, nervous system function, and intermediary metabolism (1). The principal receptor for glucocorticoids, the type II or glucocorticoid receptor (GR), a member of the nuclear receptor family (reviewed in Ref. 2), is essential for life ex utero (3). Although GRs are expressed in almost all cells, the level of expression and receptor regulation vary considerably between tissues, and even within a tissue [e.g. hippocampus (4)]. The importance of maintaining an appropriate level of expression of GR for the functional effects of glucocorticoids has been demonstrated in vitro (5) and in transgenic mice a reduction of 30–50% in tissue levels of GR results in major neuroendocrine, metabolic, and immunological abnormalities (6, 7). The level of expression of GR is thus critical for the correct level of function of a cell. In some tissues, GRs are regulated by glucocorticoids themselves, but again the regulation is highly tissue specific, with GR down-regulated by glucocorticoids in some tissues, but unaltered or even induced in others (8, 9, 10). This regulation occurs chiefly at the level of transcription (9, 11). GR gene transcription therefore must be tightly regulated with appropriately high expression for the function of any particular cell.

Much evidence suggests that GR gene transcription is, in part, permanently determined or programmed by perinatal events, again in a cell-specific manner. Thus, animals exposed to short periods of infantile stimulation (handling) have, as adults, permanently elevated GR expression selectively in hippocampal neurons (12). The hippocampus is a site of glucocorticoid feedback inhibition upon the hypothalamic-pituitary-adrenal (HPA) axis, and adult rats handled as neonates are therefore more sensitive to glucocorticoid-negative feedback with decreased HPA responsivity to stress throughout life (12, 13). In contrast, prenatal treatment of rats with the synthetic glucocorticoid dexamethasone permanently reduces GR mRNA in the hippocampus (14), but increases GR mRNA in the liver (15). These animals have permanently elevated levels of plasma corticosterone, fasting hyperglycemia (attributable to elevated levels in liver of the glucocorticoid-inducible enzyme, phosphoenolpyruvate carboxykinase, the rate limiting step in gluconeogenesis), hyperinsulinemia (15), and hypertension (14). A key question therefore, is how can GR mRNA levels be regulated in a cell-specific and even opposite manner during adult life and particularly by prenatal manipulations?

Surprisingly little is known of the mechanisms that control GR gene transcription. The GR gene spans more than 80 kb and contains 8 coding exons (exons 2 to 9) (16, 17). The human (16, 18), mouse (17, 19), and rat (20) (M. D. Jacobson and K. R. Yamamoto, unpublished data) GR gene promoter regions have been cloned and partially characterized. A single promoter has been described for the human GR gene (16, 18). In mouse, expression of the GR gene is controlled by at least 3 promoters, resulting in GR transcripts with different 5'-untranslated exons designated exons 1A (restricted to T cell lines), 1B, and 1C (the latter is homologous to the exon 1 present in the human GR cDNA) (17), and very recent evidence suggests the existence of 2 more (21). It has been suggested that rat GR mRNA might also exhibit 5'-heterogeneity (20). Little is known about GR promoter usage in tissues in vivo. Here we demonstrate tissue-specific 5'-heterogeneity of rat GR mRNA and present compelling evidence for early-life environmental programming of specific GR gene promoters in the hippocampus.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
DNA Sequence of the Rat GR Gene Flanking the 5'-End of Exon 2
We have determined the sequence of 4600 bp of the rat GR gene flanking the 5'- end of exon 2 (Fig. 1Go). The majority of this region corresponds to a CpG island (68% CG, with a CG/GC ratio of >=0.8 between -1620 and -4520 relative to the translation start at +1, within exon 2) and contains the exon 1 sequence present in the published rat GR cDNA sequence (22) (-3269 to -3322; here designated exon 16). The sequence is highly conserved when compared with the mouse GR gene (19) (91% identity throughout the whole region), including the mouse exons 1B and 1C (17). The rat sequence shows moderate conservation with the corresponding human GR gene sequence (18, 23) (~70% identity over the CpG island; nucleotides -1600 to -4220, but only 40% identity over the region between -50 and -1600), including exon 1 present in the human GR cDNA (24).



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Figure 1. Sequence of 5'-DNA Flanking Exon 2 of the Rat GR Gene

Numbering is with respect to the translation start, at +1. Shaded regions indicate exon 1 sequences found in 5'-RACE clones amplified from hippocampal GR mRNA. In the case of exon 16 (which was not represented among the RACE clones) the shaded nucleotides are those present in the cDNA sequence (22 ). The start of exon 2 is at -13. GC boxes referred to in the text are underlined; a putative NGFI-A site and a sequence identical to a footprinted region in the human GR gene which binds AP2 (referred to in the text) are boxed.

 
5'-RACE (Rapid Amplification of cDNA Ends)-PCR Identifies at Least 11 Alternative 5'-Leader Sequences in GR mRNA
To investigate 5'-heterogeneity in rat GR mRNA, we carried out 5'-RACE-PCR on total RNA isolated from rat hippocampus or thymus. Eight separate 5'-RACE reactions were carried out on the products of 5 different dC-tailed cDNA reactions from hippocampal RNA; one 5'-RACE reaction was carried out on dC-tailed cDNA produced from thymus RNA. Subcloned products were sequenced with a primer complementary to exon 2 of GR. A total of 54 independent 5'-RACE products were obtained from hippocampal RNA, deriving from independent cDNAs generated in the initial reverse transcription reactions (the 5'-ends of the cDNAs terminate at different positions in exon 1, indicating they are unlikely to be generated by PCR from the same initial tailed cDNA). In addition, four independent 5'-RACE products were obtained from thymus RNA. The cDNA products fall into 10 classes based on the sequences immediately upstream of exon 2 (Table 1Go). The majority of clones (31) from rat hippocampal GR mRNA contained exon 110 (corresponding to exon 1C of the mouse GR gene and the exon 1 sequence present in the human GR cDNA sequence). In addition, we found 7 novel exon 1 sequences present in rat hippocampal GR mRNA, exons 13, 14, 15, 17, 18, 19, and 111 (Table 1Go); exons 15 and 111 are likely to correspond to the recently described mouse exons 1D and 1E (21), respectively. Of the minor exon 1 species, exons 17 and 111 represented the major variants, present in 8 (exon 17) and 5 (exon 111) of the independent clones. Exons 14, 15, and 19 were present in, respectively, 3, 2, and 3 of the independent 5'-RACE clones. Two of the classes (13 and 18) were represented each by a single independent clone. No clones were found corresponding to the exon 1 sequence present in the published rat GR cDNA sequence (22) (but see below). Of the 4 independent clones produced from 5'-RACE PCR of thymus RNA, 2 contained exon 110. In addition, a further 2 novel variants of rat GR mRNA were identified in thymus RNA, containing exons 11 and 12 (Table 1Go). Exon 11 is likely to correspond to mouse exon 1A (76% identity) (25), reportedly specific to T lymphocytes (17).


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Table 1. 5'-RACE Reveals at Least 11 Alternative Exons 1 in GR mRNA

 
Sequence comparisons located 8 of the alternate exons 1 to the CpG island upstream of exon 2 (Table 1Go; summarized in Fig. 2Go). Each of the exons 1 that mapped to this region was flanked, at the 3' end, by the conserved GT dinucleotide splice donor site (Table 1Go). Sequences corresponding to exons 11, 12, and 13 were not found within the sequenced region, and Southern blot hybridization showed they were not located within {lambda}208 (see Materials and Methods for details of {lambda}208), indicating that they lie at least 15 kb 5' of exon 2 (data not shown).



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Figure 2. Relative Positions of Alternative Exons 1 of the Rat GR Gene

Shown are the relative positions of exons 14, 15, 16, 17, 18, 19, 110, and 111, all located within the CpG island 5' of exon 2. Exon 11 is located at least 15 kb 5' of exon 2. Note, the size of exon 2 is not to scale.

 
Exon 110 Is Present in the Majority of GR mRNA Transcripts in Hippocampus, Liver, and Thymus, but Some Other Promoters Are Tissue Specific
To investigate the relative abundance of each of the variant GR mRNA transcripts, RNase protection analysis was carried out with cRNA probes generated using 5'-RACE clones as templates. Each probe was complementary to a specific exon 1, and also to 186 nucleotides of exon 2 (common to all GR mRNA transcripts), thus giving 2 protected products; a fragment of 186 nucleotides representing GR transcripts containing exon 2 but lacking the target exon 1 sequence, and a larger fragment complementary to transcripts containing both exons 1 and 2. The sum of both fragments equates to total GR mRNA, allowing the amount of exon 1-exon 2 containing mRNA to be calculated as a percentage of total GR mRNA transcripts, after correction for differences in specific activity. In hippocampus, liver, and thymus, as well as heart, kidney, lung, and testis, transcripts containing exon 110 predominate (Fig. 3AGo, Table 2Go, and data not shown). Interestingly, although exons 15 and 17 were both present in hippocampal GR mRNA (together accounting for ~17% of GR mRNA in hippocampus), in liver and thymus they were below the limit of detection of the RNase protection assay (i.e. <1% of the total) (Table 2Go). Furthermore, exon 111-containing transcripts were relatively more abundant in hippocampus than in liver RNA but were below the limit of detection of the assay in thymus (Table 2Go). Exon 11, identified in a 5'-RACE clone from thymus, was detectable only in thymus RNA by RNase protection analysis (Fig. 3BGo and Table 2Go). Exon 16 was not detected among the products of 5'-RACE PCR on hippocampal RNA, possibly as a consequence of the hybridization of the 5'-RACE UAP-anchor primer [which contains a G(GGIIG)3 sequence] to a C8 sequence close to the 3'-end of exon 16; 5'-RACE products likely to contain only very short amounts of exon 1 sequence were not analyzed. However, exon 16 mRNA transcripts were expressed in all three tissues, representing around 10–20% of total GR mRNA (Table 2Go). Levels of exon 14-- and exon 12-containing GR mRNA were below the limit of detection of the RNase protection assay (Table 2Go).



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Figure 3. RNase Protection Analysis of the Abundance of Alternative Exon 1-Containing GR mRNAs in Rat Hippocampus, Liver, and Thymus

RNase protection assays were carried out on 50 µg total RNA from adult male rat hippocampus (H), liver (L), or thymus (T). Lanes marked Y contained yeast RNA. Lanes marked + contained undigested probe; M, markers. A, RNase protection of exon 110-containing GR mRNA. Arrowheads indicate the positions of the 186-nucleotide fragment protected by GR mRNA transcripts containing exon 2 but not exon 110 and the 306-nucleotide fragment protected by transcripts containing exon 110 and exon 2. The lane containing thymus RNA was from an adjacent gel run in parallel under identical conditions. B, RNase protection of exon 11-containing GR mRNA. Arrowheads mark the 186-nucleotide fragment protected by GR mRNA transcripts containing exon 2 but not exon 11 and the 228-nucleotide fragment containing exon 11 and exon 2. Note that, for quantitation, the 215-nucleotide band (which also contains exon 11 spliced to exon 2) was included.

 

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Table 2. Relative Abundance of Alternative Exon 1-Containing GR mRNA in Rat Hippocampus, Liver, and Thymus

 
Distribution of Alternate Exon 1-Containing GR mRNA Transcripts within the Hippocampus
Using in situ mRNA hybridization, we mapped the distribution of transcripts containing the major alternate first exons of the GR gene expressed in the hippocampus. A similar sized cRNA probe complementary to the 5'-end of the common exon 2 hybridized in a pattern equivalent to that documented for GR mRNA in previous studies (26) (Fig. 4AGo). Using cRNA probes specific to the major alternate first exons employed in the hippocampus, we found that the predominant exon 110-containing transcript was distributed very similarly to total GR mRNA, with high expression in the dentate gyrus and CA1 and lower expression in CA3 and CA4 (Fig. 4EGo). In contrast, GR mRNA transcripts containing exons 15, 17, or 111 showed a more homogeneous distribution, although in each case expression was highest in the dentate gyrus and CA1 region of hippocampus (Fig. 4FGo, C, and G, respectively).



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Figure 4. In Situ Hybridization Analysis of the Distribution of GR mRNA within Hippocampus

In situ mRNA hybridization was carried out on rat hippocampus using exon 1-specific cRNA probes. Distribution of GR mRNA containing (A) exon 2 (total GR mRNA) and alternative exons 1 as follows: (C) exon 17; (E) exon 110; (F) exon 15; and (G) exon 111. Representative sense controls are shown in (B) exon 2 (sense control) and (D) exon 17 (sense control). Arrows indicate dentate gyrus (DG), CA1, and CA3. Exposure times were 5 days (A, B, and G), 6 days (C, D, and E), and 12 days (F).

 
Promoter 17 Activity Is Highest in Central Nervous System (CNS)-Derived Cells
To investigate whether the alternate exons 1 are associated with promoter activity, regions of the rat GR gene were joined, within each of the alternate exons 1, directly to a luciferase reporter gene (Fig. 5AGo). Luciferase activity therefore arises from chimeric RNA transcripts encoding part of an alternate exon 1 of the GR gene at the 5'-end and represents the activity of the promoter that directs transcription through that individual exon 1. Although transcription may additionally originate from alternate promoters present on the same genomic DNA fragment, these transcripts will not be transcriptional fusions to luciferase and will not, therefore, result in luciferase activity (there is no splice acceptor site upstream of the luciferase gene in these constructs). To measure promoter activity of the whole CpG island, including exons 14-111, plasmid P2 was constructed in which the GR gene is joined to luciferase within exon 2 (just before the translation start) (Fig. 5AGo). In P2, RNA initiating at any of the transcription start sites will be spliced, from the donor site at the 3'-end of the respective exon 1 onto the acceptor site at the 5'-end of the common exon 2; luciferase reporter activity therefore reflects the total promoter activity of the DNA fragment inserted into P2.



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Figure 5. Transfection Analysis of Promoter Activity Associated with the Alternate Exons 1 of the Rat GR Gene

A, Diagrammatic representation of constructs used in transfection analysis. Restriction fragments containing regions of the rat GR gene were fused, within specific exons, to the luciferase reporter gene in a modified pGL3-Basic vector. P2(rev) contains the identical fragment to P2, in the reverse orientation with respect to luciferase. {blacktriangleup} indicates the splice acceptor site in the intron 5' of exon 2. B, Promoter activity of regions of the GR gene in three cell lines, HepG2 hepatoma cells, C6 glioma cells, and B103 neuroblastoma cells. Activity of P2 (spanning the whole CpG island, fused to luciferase within exon 2) was nominally set to 100% for each cell line, and activity of the other constructs was expressed relative to this value. Values represent means ± SEM.

 
Promoter activity was assayed in transiently transfected HepG2 (human hepatoma), C6 (rat glioma), and B103 (rat neuroblastoma) cells. P2 had the highest promoter activity in all three cell lines examined, whereas the same fragment in the reverse orientation with respect to luciferase had no significant activity (Fig. 5BGo). Activity of all constructs was similar in all three cell lines with the exception of P17. P17 was, apart from P2, the most active construct in B103 and C6 cells but was relatively less active in HepG2 cells (Fig. 5BGo). Promoter activity of P110 was high and P111 activity low in all three cell lines (Fig. 5BGo).

Differential Regulation of Variant GR mRNA Transcripts by Early-Life Events
Neonatal handling causes marked and permanent increases in GR mRNA expression in hippocampus (27). Strikingly, neonatal handling induced expression of GR mRNA containing the hippocampus-specific exon 17 by 2.5- to 3-fold selectively across all hippocampal subfields (Fig. 6AGo), whereas expression of exon 17-containing GR mRNA in cortex, where neonatal handling has no effect on expression of GR, was unchanged by the manipulation (Fig. 6AGo). In contrast, the level of expression and distribution of the major exon 110, and the other hippocampus-specific exons 15- and 111-containing GR mRNAs were unaffected by neonatal handling (Fig. 6BGo and data not shown). To start to examine whether this effect was confined to the hippocampus, we examined rats exposed prenatally to dexamethasone (dexamethasone administered during week 3 of gestation). This manipulation selectively and permanently increases hepatic GR mRNA levels by 25% (15). In these animals RNase protection assays show a significant decrease in the relative amount of exon 110-containing GR mRNA in the liver in the dexamethasone-treated group (73 ± 3%; n = 10) compared with controls (82 ± 2%; n = 9, P < 0.05) (an increase in one of the minor GR mRNA species would reduce the level of exon 110-containing RNA as a percentage of the total). These data suggest that prenatal dexamethasone treatment induces one of the minor mRNA variants. However, RNase protection assays demonstrated that the level of exon 16-containing GR mRNA was unchanged in livers from adult rats treated with dexamethasone prenatally (9 ± 1%; n = 4), compared with controls (9 ± 1%; n = 5). Exon 11, 15, 17, and 111-containing GR mRNAs remained very low or were undetectable, suggesting that an as yet unidentified exon 1-containing GR mRNA is induced in these animals.



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Figure 6. Exon 17-containing GR mRNA Is Induced in Hippocampus by Neonatal Handling

In situ mRNA hybridization analysis of GR mRNA containing exon 17 (A) or exon 110 (B) within the dentate gyrus (DG), the CA1 and CA3 pyramidal cell fields of the hippocampus and the cortex (CTX). Expression was measured in handled animals (hatched columns) and nonhandled animals (black columns) and is expressed as the number of grains over an area equivalent to a CA1 neuron. Values represent mean ± SEM; n = 5. *, P < 0.05.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The organization of the 5'-end of the rat GR gene is complex. Here we show the gene encodes at least 11 alternate first exons, some of which are tissue-specific, and one of which is differentially and permanently induced by early-life manipulation. None of the alternate exons 1 is predicted to alter the amino acid sequence of the GR itself; there is an in-frame stop codon present immediately 5' to the translation initiation site in exon 2, common to all the mRNA variants. Of the 10 alternate exons 1 we identified by 5'-RACE, 4 correspond to alternative exons 1 previously identified in mouse, exons 11, 15, 19, and 110 (17, 21). Rat exon 11 lies at least 15 kb upstream of exon 2 (it is not present on {lambda}208 containing 15 kb of DNA 5' of exon 2) and is probably in a similar position to the corresponding mouse exon. All the other alternate exons 1 identified here are novel. Sequence analysis of DNA flanking the 5'-end of exon 2 revealed that most of the novel exons 1 lie within a CpG island highly conserved between rat and mouse. The 3-kb CG-rich region, therefore, contains at least 8 alternate exons 1 (including exon 16, present in the published rat GR cDNA sequence), at least 5 of which are conserved in the mouse. The CpG island is also conserved in the human GR gene, indicating that the use of alternate exons 1 in GR gene expression may also occur in humans.

At least 6 of the alternate exons 1 are present in vivo in rat GR mRNA. In all adult rat tissues examined, GR mRNA containing exon 110 predominated, accounting for at least half of total GR transcripts. Exon 16 was also present ubiquitously, in a substantial minority of total GR mRNA. All other alternate exons 1 were, to varying extents, expressed in a tissue-specific manner. Exon 11 was well represented in GR mRNA in thymus, but was absent from hippocampus and liver. Preliminary data suggest that exon 11 is not restricted to a specific subset of cells in thymus, but is expressed similarly in thymocytes and thymic epithelium (A. Dammermann, C. Blackburn, and K.E. Chapman, unpublished observations). Hippocampal RNA contained significant levels of the minor exon 15-, 17-, and 111-containing GR mRNA variants that were expressed at either low or undetectable levels in liver and thymus. Five other exon 1 variants (12, 13, 14, 18, and 19) are unlikely to be of significance as they were poorly represented in the 5'-RACE PCR or were undetectable by RNase protection assays. It is unlikely that any further GR mRNA variants are present at significant levels in hippocampus as the sum of the exon 1 variants examined was close to 100% of total mRNA. Interestingly, exon 110-, 16-, and 111-containing transcripts accounted for only 90% of the GR mRNA in liver, suggesting that additional novel exon 1 sequences may be present.

In transient transfection experiments, a construct encoding the whole CpG island of the GR gene, including 8 of the alternate exons 1 and the splice acceptor site within the intron 5' of exon 2, fused to a luciferase reporter gene within exon 2 (P2), exhibited substantial promoter activity in all cell lines tested. This activity results from transcripts originating at any point within the CpG island that are spliced from an appropriate donor site onto the splice acceptor site 5' to exon 2, and represents the sum of the activity of individual promoters on the genomic DNA fragment. Promoter activity was also associated with particular regions of the CpG island, where the fusion to luciferase was made within specific exon 1 sequences. In these cases, no splice acceptor site is available within the luciferase gene, and a transcriptional fusion is generated between the specific exon 1 and the luciferase reporter; luciferase activity therefore reflects transcription through the specific exon 1. Relative activity of these constructs in different cell types was similar with one notable exception, P17 (see below). The low activity of P111, compared with the shorter constructs or to P2, probably results from promoter competition by the stronger promoters directing transcription of exon 16 and exon 110, neither of which will generate productive RNA transcripts encoding luciferase, due to the lack of a splice acceptor site. Interestingly, P17, fused to luciferase within exon 17, had the highest activity of any single promoter construct (P2 activity reflecting activity of the whole region) in B103 and C6 cells, both CNS derived. The activity of this construct was low in hepatic cells, in which P16 and P110 had the highest activity. In vivo, GR mRNA transcripts containing exon 17 were present at significant levels in hippocampus, but absent from liver, suggesting that factors present in cells of CNS origin are responsible for transcription initiation at the promoter upstream of exon 17 in rat hippocampus.

Neonatal handling induces an increase of approximately 50% in total GR mRNA levels in all subfields of the hippocampus, but not in cortex (27). Only the 17 variant GR mRNA was induced in the hippocampus by handling, with a 2- to 3-fold increase, also across all fields of the hippocampus. RNase protection assays, carried out on RNA extracted from the whole hippocampus (which will include glia and interneurons, as well as pyramidal cells and the granule cells of the dentate gyrus) showed that exon 17-containing GR mRNA is normally present in approximately 10% of total GR mRNA in hippocampus. The observed induction of 17 may not appear sufficient to account for the overall increase of approximately 50% in steady state GR mRNA levels after handling (27). However, the heterogeneity of the hippocampus as a whole may have resulted in a dilution of exon 17-containing GR mRNA if it is expressed predominantly in the pyramidal cell layers of the hippocampus and granule cells of the dentate gyrus, thereby lowering the estimate of the amount of exon 17-containing GR mRNA present in these hippocampal neuronal layers obtained by RNase protection assays of whole hippocampus. Indeed, we have previously noted a similar discrepancy between the magnitude of change in mRNA encoding the type I inositol 1,4,5-triphosphate receptor during human pregnancy measured by RNase protection assays and in situ mRNA hybridization (28). Although we cannot exclude the possibility that an additional minor variant of GR mRNA is induced by neonatal handling, none of the other main variant GR transcripts were altered by handling. These data suggest that neonatal handling programs increased hippocampal GR via increased transcription from a novel promoter, 17, active predominantly in CNS-derived cells. A similar permanent induction of a minor promoter of the GR gene appears likely in the liver after prenatal dexamethasone exposure. Within the overall increase in GR mRNA in liver of prenatally treated rats, the proportion containing the predominant exon 110 fell, although we were unable to identify a specific transcript induced. Nevertheless, the clear implication is that early life programming events selectively alter otherwise minor tissue-specific GR gene transcripts, whereas the major and ubiquitous promoters are unaffected, thus programming GR levels for the lifetime of the animal in a tissue-specific manner. Conversely, it is possible that manipulations that decrease GR levels may decrease the levels of the minor GR mRNA variants.

5HT appears crucial in mediating the effects of neonatal handling upon GR expression in hippocampus (29, 30), with subsequent effects upon HPA axis activity (31, 32). The transcription factors NGFI-A and AP2 have been implicated in the induction of GR in the hippocampus after handling or with 5HT (33). Intriguingly, a sequence in the human GR gene that binds AP2 in vitro (34) is completely conserved in the rat GR gene (at -2718). Additionally, within the CpG island, the GR gene contains 16 GC boxes (GGGCGG), which form the core consensus Sp1 site (35) and which may also bind NGFI-A; indeed, there is a sequence exactly matching the consensus binding site for the family of zinc finger proteins that includes NGFI-A (36) immediately upstream of exon 17. We speculate that the increases in AP2 and NGFI-A induced by neonatal handling cause increased transcription from a promoter adjacent to exon 17, leading to increased total GR mRNA.

It remains possible that transcription may originate at a common promoter further upstream, resulting in a common exon 0, which is then spliced upstream of the alternate exons 1. We consider this to be extremely unlikely for the following reasons. First, sequence analysis of 58 independent 5'-RACE clones neither provided evidence for a common 5'-leader sequence nor revealed any lack of colinearity with the genomic sequence. Second, the predominant rat exon 110 is homologous to exon 1 of the human GR gene (16, 18) for which a transcription start site has been mapped. A number of transcription start sites exist (typically for a TATA-less CG-rich promoter), but all are located within the region corresponding to rat exon 110, and all appear to extend to the same 3'-splice site (16, 18). Similarly, sequencing of our 5'-RACE clones and RNase protection analysis (to map transcription start sites) suggests that a number of transcription starts exist at least for exon 110 and probably for other exons 1 also (J. A. McCormick, V. Lyons, and K. E. Chapman, unpublished observations). Indeed, the 5'-end of the longest of our 5'-RACE clones containing exon 110 corresponds exactly to one of the transcription starts mapped for human GR mRNA (18) (Fig. 1Go). Finally, our transfection data suggest that promoter activity is associated with the 5'-flanking regions of specific exons 1. Thus, it is most probable that alternate exon 1 usage results from transcription initiation at a number of predominant transcription start sites within the CpG island, associated with promoter activity. CpG islands are frequently associated with multiple transcription initiation sites, often spread over a distance of up to 1 kb, resulting in transcripts with differing exons 1, all of which, however, are spliced at the same 3'-splice donor site onto exon 2 (e.g. Refs. 37, 38). Multiple 5'-ends are not usually associated with alternate splice donor sites, giving rise to discrete alternate exons 1, as we have observed for the rat GR gene. It is possible that, as the CpG island in the GR gene is very large, transcription initiates at a large number of initiation sites spread over the entire 3-kb region. The probability of splice donor sites occurring within such a large region is high, and the splicing machinery associated with the RNA polymerase complex may simply splice from the first appropriate splice donor site that occurs to the splice acceptor site before exon 2. This hypothesis is supported by the sequence of mouse exon 1E (corresponding to exon 111) which, at the 5'-end, includes a portion of exon 1C (corresponding to exon 110) as well as the intervening genomic DNA (21). Possibly, transcription originated too far 3' within exon 110 to utilize the exon 110 splice donor site; splicing then occurred at the next available splice donor site, 3' of exon 111. Certain sites within the CpG island will be favored for transcription initiation, and this will probably vary in a tissue-specific manner. Indeed, we see the highest number of variant exons 1 in hippocampus, a tissue exhibiting a high degree of complexity. This hypothesis predicts that more alternative exons 1 may exist in the CpG island if more splice donor sites are predicted, and we have preliminary evidence that this is the case (V. Lyons and K. E. Chapman, unpublished observations).

The use of multiple and tissue-specific promoters provides a flexible mechanism for distinct tissue-specific regulation of individual promoters by hormonal signals and has been described for other members of the steroid receptor family (39, 40, 41). GRs are widely expressed in virtually all cell types, although expression levels and functions vary considerably. The complex organization of the 5'-end of the GR gene may reflect this need for diverse tissue-specific regulation. We speculate that exon 110 is constitutively expressed in all tissues, providing a basal or minimal constitutive level of GR gene transcription (e.g. Ref. 8). The existence of tissue-specific promoters (e.g. 11 in thymus and 17 in hippocampus) permits differential regulation of GR in specific cell types and may explain the opposite regulatory effects of glucocorticoid hormones on the levels of GR in T lymphocytes and hippocampus (10, 42). In addition, the presence of several minor promoters clustered together may permit regulation of one or more by signal transduction pathways, resulting in moderate, but biologically significant, changes in total GR mRNA in a specific cell type and thus, ultimately, the glucocorticoid signal on the target genes. Our data illustrate the complexity of transcriptional regulation of GR and provide a basis to understand tissue-specific effects of early-life programming.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Experimental Animals
Animals were maintained under controlled lighting (lights on 0700 to 1900 h) and temperature (22 C) with water and food available ad libitum. Tissues for RNA isolation and in situ hybridization were from adult (3–8 months) male Wistar rats (Charles River UK Ltd, Margate, Kent, UK). Animals treated in utero with dexamethasone were male offspring (8 months old) of female Wistar rats administered dexamethasone (100 µg/kg per day) during week 3 of pregnancy, as previously described (15). For neonatal handling, Long-Evans rats (Charles River Canada, St. Constant, Québec, Canada) were used as previously described (30). Handling was carried out daily for 2 weeks. Male animals were selected at random from a total of five litters and were used when they reached adult age (3–5 months).

All studies involving animals described herein were approved of by the UK Home Office and were performed in strict accordance with the UK Home Office Animals (Scientific Procedures) Act, 1986.

Isolation of RNA
Total RNA was isolated using the guanidinium isothiocyanate method (43). Integrity was verified by electrophoresis on formaldehyde-agarose gels.

5'-RACE PCR
5'-RACE PCR was performed using a commercial kit (Life Technologies , Gaithersburg, MD) according to the manufacturer’s instructions. First-strand synthesis of GR cDNA was carried out at 42 C for 30 min on 5 µg total RNA using 8 U/µl SuperScript II RT and 100 nM primer GSP1 (5'-AAGGGATGCTGTATTCA-3') in a 25 µl reaction containing 20 mM Tris HCl (pH 8.4), 50 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 400 µM deoxynucleoside triphosphates (dNTPs). After RNase H treatment, cDNA was added to a 24 µl reaction containing 20 mM Tris HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 200 µM dCTP, and 0.4 U/µl terminal deoxynucleotidyl transferase. dC-tailed cDNA (5 µl) was used in a PCR reaction with 400 nM anchor primer (5'-CUACUACUACUAGGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3'), 440 nM primer GSP2 (5'-ACTCCAAATCCTTCAAGAGGTCA-3'), 20 mM Tris HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 200 µM dNTPs, and 2.5 U Taq DNA polymerase (Promega Corp., Madison, WI), with 35 cycles of PCR amplification (96 C, 45 sec; 45 C, 40 sec; 72 C, 1.5 min), followed by 10 min, 72 C. A nested PCR was carried out on the products of the first PCR reaction, under the same conditions with the following primers: UAP (5'-CUACUACUACUAGGCCACGCGTCGACTAGTAC-3') and GSP3 (5'-TTGGAATCT-GCCTGAGAAGC-3'). PCR products were cloned into pGEM-T or pGEM-T-easy (Promega Corp.) and sequenced using GSP3.

Subcloning and Sequence Analysis of the Rat GR Promoter
{lambda}208 contains exon 2 and approximately 15 kb of the rat GR gene flanking the 5'-end of exon 2 (M. D. Jacobson, unpublished data). The sequence between -4600 and +500 (the translation start close to the 5'-end of exon 2 is designated +1) was determined from restriction fragments subcloned from {lambda}208 on both strands using the Sequenase II system (Amersham International, Buckinghamshire, UK) or the Thermo Sequenase 33P-radiolabeled terminator cycle sequencing system (Amersham International). Sequence analysis, including identification of putative transcription factor-binding sites was carried out using computer software available at the UK MRC Human Genome Mapping Project Resource Centre.

Accession Number
The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank, and DDBJ Nucleotide Sequence Databases under the accession number AJ271870.

Transfection Analysis Of GR Promoter Activity
Plasmids that fused the rat GR gene to a luciferase reporter gene were constructed from appropriate restriction fragments ligated into pGL3-Basic (Promega Corp.) containing a modified polylinker as follows: P2, a HindIII/SspI fragment encoding rat GR from -4572 to -9 (the ATG translation start is designated +1); P2(rev), the same fragment in the reverse orientation with respect to luciferase; P16, a HindIII/PstI fragment encoding -4572 to -3336; P17, a HindIII/BglI fragment encoding -4572 to -2931; P110, a HindIII/HincII fragment encoding -4572 to -2318, and P111, a HindIII/PstI fragment encoding -4572 to -1767. Plasmid DNAs used for transfections were purified by CsCl density gradient centrifugation.

HepG2 (human hepatoma), C6 (rat glioma), and B103 (rat neuroblastoma) cells were maintained in DMEM supplemented with 10% (vol/vol) FBS, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Twenty four hours prior to transfection, HepG2 and C6 cells were seeded at 5–7 x 105 cells per 60-mm dish and B103 cells at 2 x 105 cells per 60-mm dish. Cells were transfected using the calcium phosphate procedure (44) with 1 µg modified pGL3-Basic or an equimolar amount of GR promoter-luciferase plasmid (plasmids varied markedly in size), 1 µg of the ß-galactosidase expression plasmid pCH110 (Pharmacia Biotech, Piscataway, NJ), and carrier pGEM-3 (Promega Corp.) to a total of 10 µg. Forty eight hours after transfection, cells were lysed and luciferase activity determined as previously described (44). ß-Galactosidase activity was determined using the Tropix Galacto-Light kit (Cambridge Bioscience, Cambridge, UK), and luciferase activity/ß-galactosidase activity was calculated. Transfections were carried out in triplicate; each experiment was repeated at least twice and two independently prepared plasmid DNAs were used for each promoter construct.

RNase Protection Assays
With the exception of exon 16 (see below), exon 1-specific cRNA probes were synthesized from corresponding 5'-RACE subclones, linearized, and transcribed with either T7 or SP6 phage polymerase, as appropriate, in the presence of either [{alpha}-32P]-UTP or [{alpha}-32P]-GTP (3000 Ci/mmol; Amersham International). The template used to synthesize an exon 16-specific cRNA probe was made by subcloning into pGEM-T-easy an RT-PCR product generated from total rat liver RNA using GSP3 (complementary to exon 2) and 5'-primer (5'-ACC- TGGCGGCACGCGA-3').

RNase protection assays were carried out using a HybSpeed RPA kit (Ambion, Inc., Austin, TX). Hybridization conditions were optimized in preliminary experiments using synthetic RNA templates. Total RNA (50 µg) was coprecipitated with 5–10 x 105 cpm cRNA probe, resuspended in 20 µl hybridization buffer (supplied with the kit) at 95 C, and incubated at 68 C for 1 h. Reactions were incubated with RNase A/T1 (1:25 dilution) for 30 min, 37 C, and RNA products were separated on a 4% polyacrylamide gel containing 7 M urea and visualized using autoradiography or a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). Data were analyzed using Student’s t test. Significance was set at P < 0.05.

In Situ mRNA Hybridizations
[35S]UTP-labeled RNA probes were synthesized as previously described (26). After DNase I treatment, unincorporated nucleotides were removed by passage over a Sephadex G-50 Nick column (Pharmacia Biotech, St Albans, UK). Exon 15-, 17-, 111-, and exon 2-specific templates were generated by PCR carried out on subclones of {lambda}208 using the following oligonucleotides tagged with sequences encoding either a T3 promoter (to make sense RNA) or a T7 promoter (to make cRNA or antisense RNA): 15, 5'-primer (5'-TATTAACCCTCACTAAAGGGTAAGAGGAGGGCGGACT-3'), 3'-primer (5'-TTAAT-ACGACTCACTATAGGGCCAGCGCGCTCACACT-3'); 17, 5'-primer (5'-CATTAACC-CTCACTAAAGGGC-ACCGTTTCCGTGCAT-3'), 3'-primer (5'-TTAATACGACT-CAC-TATAGGGCAGCGTGTGCCGACCT-3'); 111, 5'-primer (5'-TATTAACCCTCACTAAA-GGGAGCGGCGTCTGGACC-3'), 3'-primer (5'-TTAATACGACTCACTATAGGGCTA-GCGCTCAAGTTGTC-3') and exon 2, 5'-primer (5'-ATTAACCCTCACTAAAGGGCC-AATGGACTCCAAAGAA-3') and 3'-primer (5'-ATAATACGACTCACTATAGGGAA-TCTGCCTGAGAAGC-3'). The template used to synthesize exon 110-specific cRNA was generated by PCR from an exon 110 5'-RACE clone using UAP and 3'-primer (5'-ATAATACGACTCACTATAGGGCTTTGGAGTCCA- TTGGCA-3').

In situ-hybridization histochemistry was carried out as previously described (26, 45). Silver grains were counted under bright-field illumination using an image analysis system (MCID, Research Imaging, St. Catherine’s, Ontario, Canada). Results were analyzed blind and background, counted over adjacent areas of neuropil, was subtracted. Data were analyzed using Student’s t test. Significance was set at P < 0.05.


    ACKNOWLEDGMENTS
 
We are very grateful to Professor K. R. Yamamoto for providing materials, information, and helpful advice on this work, and to Professor D. Schubert for B103 cells. We thank members of the Molecular Endocrinology group and Drs. C. J. Kenyon, B. R. Walker, and M. C. Holmes, in particular, for many discussions on this work and for helpful comments on the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Karen E. Chapman, Molecular Endocrinology, University of Edinburgh, Molecular Medicine Centre, Western General Hospital, Crewe Road, Edinburgh, EH4 2XU, UK.

This work was supported by a Wellcome Trust programme grant and Senior Clinical Research Fellowship (J.R.S). J.A.M. is supported by a studentship from the Medical Research Council.

Received for publication September 14, 1999. Revision received November 29, 1999. Accepted for publication January 5, 2000.


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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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