Estrogen-Dependent Transcriptional Activation and Vitellogenin Gene Memory

Robert S. Edinger1, Elizabeth Mambo and Marilyn I. Evans

Department of Biochemistry School of Medicine Robert C. Byrd Health Sciences Center West Virginia University Morgantown, West Virginia 26506-9142


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The concept of hepatic memory suggests that a gene responds more rapidly to a second exposure of an inducer than it does during the initial activation. To determine how soon estrogen-dependent DNA/protein interactions occur during the primary response, in vivo dimethylsulfate footprinting was carried out using genomic DNA amplified by ligation-mediated PCR. When estrogen was added to disrupted cells from a hormone-naive liver, changes within and around the estrogen response elements occurred within seconds, indicating a direct and rapid effect on this estrogen-responsive promoter that had never before been activated. Because this effect was so rapid relative to the delayed onset of mRNA accumulation during the primary response, run-on transcription assays were used to determine the transcription profiles for four of the yolk protein genes during the primary and secondary responses to estrogen. As with the accumulation of mRNA, the onset of transcription was delayed for all of these genes after a primary exposure to estrogen. Interestingly, after the secondary exposure to estrogen, the vitellogenin I, vitellogenin II, and very low density apolipoprotein II genes displayed a more rapid onset of transcription, whereas the primary and secondary profiles of apolipoprotein B transcription in response to estrogen were identical. Because the apoB gene is constitutively expressed in the absence of estrogen, and the vitellogenins are quiescent before the administration of the hormone, hepatic memory most likely represents a relatively stable event in the transition to an active state of a gene that is committed for tissue-specific expression.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Activation of gene expression by the steroid/thyroid hormone receptor superfamily proceeds by mechanisms that depend on recognition sites, promoter context, cell type, and developmental stage (1, 2). Strategies for gene activation include nucleosome disruption, recruitment of chromatin remodeling proteins, release of repressors, and recruitment of coactivators that result in an active chromatin configuration (3, 4). Glucocorticoid-responsive genes provide the best documented examples of the conversion of chromatin from a closed to an open configuration during activation by steroid hormones. For example, at the mouse mammary tumor virus (MMTV) promoter, glucocorticoid administration results in a generalized sensitivity to deoxyribonuclease I (DNase I) within 7 min, and DNase I-hypersensitive sites appear in 20 min, coincident with activation of transcription (5). The rat tyrosine aminotransferase gene promoter also displays DNase I-hypersensitive sites after 20 min of glucocorticoid treatment, and the appearance of these sites coincides with a rapid increase in the rate of transcription (6). These hormone-induced hypersensitive sites disappear after hormone withdrawal.

In the chicken liver, after the initial activation of the egg yolk protein genes by estrogen, subsequent responses to estrogen occur more rapidly. Consequently, the concept of hepatic memory is derived from the observation that after the administration of estrogen to young birds or roosters, there is a delay before the onset of rapid accumulation of yolk protein mRNA. If the initial response is allowed to decay to baseline, subsequent administration of estrogen results in an earlier onset of rapid accumulation of vitellogenin I (VTGI), vitellogenin II (VTGII), vitellogenin III (VTGIII), and very low density apolipoprotein II (apoVLDLII) mRNAs (7, 8, 9, 10). Thus, the hepatocytes appear to remember the prior exposure to estrogen, even though the level of estrogen receptor mRNA, as well as the kinetics of accumulation and number of estrogen receptors tightly bound in the nuclei are identical after the primary and secondary injections of estradiol (11).

The yolk protein genes are committed to tissue-specific expression early in development (10, 12), and a bird given a single injection of estrogen at 15 days of embryogenesis can retain memory for up to 6 months after hatching (13). Accordingly, memory is marked in such a way that it persists through several rounds of cell division. This persistence led to the examination of estrogen-dependent alterations in the chromatin structure of these genes with respect to memory. The appearance of DNase I-hypersensitive sites in the 5'-flanking region of the VTGII gene accompanies activation by estrogen, a subset of which remains after hormone withdrawal (14). However, these markers disappear within 7 weeks, long before the memory decays. The demethylation observed within the estrogen receptor-binding site after estrogen treatment persists longer than the DNase I sensitivity. Nevertheless, by 25 weeks after hatching, this site is remethylated in some of the birds that still retain memory. Conversely, in birds given very low doses of estradiol, memory can be achieved in the absence of demethylation at this site (13). The conclusion drawn from these studies is that neither the appearance of DNase I-hypersensitive sites nor specific demethylation within the estrogen response element (ERE) can be related to memory.

Subsequently, mapping of the promoter regions of the VTGII and apoVLDLII genes by in vivo dimethylsulfate (DMS) footprinting revealed 20 estrogen-dependent changes in the promoter of the VTGII gene (15) and 36 such changes for apoVLDLII (16). These footprinted regions in the apoVLDLII promoter mark functional elements as shown by transient transfection assays using both deletion analysis (17) and site-directed mutagenesis (18, 19). These data provide specific targets for investigating the timing of individual estrogen-dependent changes in DNA/protein interactions and whether hepatic memory is characterized by an earlier appearance of these events.

Even though an altered chromatin structure model for hepatic memory is attractive, it seems equally likely that other mechanisms could account for the shortened lag including changes in synthesis, processing, export, or stability of the RNA. The rates of transcription of both the VTGII and apoVLDLII genes increase in response to estrogen (20). In this manuscript, changes in the rates of transcription are described for four of the yolk protein genes during the lag period in both primary and secondary stimulated birds.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In vivo DMS Footprinting of EREs Reveals Rapid Estrogen-Dependent Changes
Previous footprinting across the apoVLDLII promoter demonstrated estrogen-dependent changes in the accessibility of guanosine residues to DMS in both the perfect AGGTCAnnnTGACCT proximal (-164 through -178; ERE1) and the 4-bp mismatch GGGGCTnnnTGACCC (mismatches underlined) distal (-208 through -222; ERE2) EREs (16). To determine whether the rapidity of appearance of these changes might be related to hepatic memory, the footprinting assays were repeated at various times after the administration of estrogen. Although the rapid accumulation of apo-VLDLII mRNA begins after 4 or 2 h of estrogen stimulation for the primary and secondary response, respectively (9, 21), estrogen-dependent changes in reactivity to DMS occurred within 5 min after an injection of estradiol into the thigh muscle of young birds (data not shown).

Because this reaction took place faster than it was possible to dissect the birds, liver from a hormone-naive bird was dispersed as described in Materials and Methods and divided into three portions. These crude cell suspensions were untreated (vehicle only), or treated with estradiol for 30 or 60 sec, before the addition of DMS. Under the conditions used, DMS methylates guanosines and, to a lesser extent, adenosines. Each residue may become protected from or more exposed to the methylating reagent depending on alterations in DNA/protein interactions and/or changes in DNA conformation.

The footprinting results from these reactions are shown in Fig. 1Go. The organization and lower strand sequence of the footprinted regions across the apo-VLDLII promoter are shown in Fig. 1AGo. The footprint of the lower strand of ERE1 is shown at the top of Fig. 1BGo (stippled box), in which the adenosine at position -164 and the guanosines at positions -165, -166, and -170 were protected from methylation by DMS within 30 sec of the administration of estradiol. Protection also occurred rapidly within the vitellogenin gene-binding protein (VBP; Refs. 22 and 23) site that is 3' of ERE1 at guanosines -149, -150, -155, and -161 (Fig. 1BGo, white box). The in vitro footprint of VBP overlaps ERE1 at positions -164 and -165. The guanosines in ERE2 were protected at -208, -209, and -210, and enhanced reactivity was observed at -216 and -218, also within 30 sec (Fig. 1CGo, hatched box). The specificity of these changes is demonstrated by constant signals at guanosine residues outside the protein-binding regions (e.g. G-249 at the top of panel C). The appearance of these altered DNA/protein interactions occurs very early with respect to the onset of rapid accumulation of apoVLDLII mRNA, which is delayed for 4 h, raising the question of whether the lag and memory are encoded pre- or posttranscriptionally.



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Figure 1. Rapid Estrogen-Dependent Altered DMS Accessibility within the EREs of the ApoVLDLII Promoter

In vivo footprinting of the lower strand was determined by ligation-mediated PCR as described in Materials and Methods. The data in this figure represent results in dispersed cells from a freshly dissected hormone-naive liver. A, Organization of the footprinted region of the apoVLDLII promoter. The lower strand sequence of the footprinted region is shown. Guanosines that are protected from reacting with DMS are indicated by open circles whereas those with increased DMS reactivity are marked by closed circles. The positions of VBP (-146 through -165), ERE1 (-164 through -178), and ERE2 (-208 through -222) are shown as white, stippled, and hatched boxes, respectively. B, The region of the apoVLDLII promoter containing ERE1 and VBP sites. CTAG represents the dideoxy sequence of the footprinted regions. Lane 0 received DMSO 30 sec before the addition of DMS; 30 and 60 sec represent the time between treatment with estradiol dissolved in DMSO and the addition of DMS to the cells. The numbers between the lanes represent the positions of the nucleotides with altered reactivity to DMS. The boxes to the right mark the positions of ERE1 (stippled) and VBP (white). C, The distal apoVLDLII promoter containing ERE2. The lanes are labeled as in B. The numbers to the right of the lanes represent the positions of the nucleotides with altered reactivity to DMS. The box between the lanes marks the position of ERE2 (hatched). Although the sequence in this region of the gel is difficult to visualize, all of the bands that appear in the sample represent guanosine residues.

 
A Delayed Increase in the Rate of Transcription Results in Delayed mRNA Accumulation
The yolk protein genes VTGI, VTGII, VTGIII, and apoVLDLII display a lag after the administration of estrogen before the rapid accumulation of mRNA. The basis for this delay is unknown. The lag during the primary response varies from 4 h for apoVLDLII to 20 h for VTGI. After the administration of estradiol to hormone-naive birds, both the rate of transcription of the apoVLDLII gene and the accumulated apoVLDLII mRNA increased 2- to 3-fold during the first 2 h of this primary response (Fig. 2Go, A and B). Between 3 and 4 h the rate of transcription increased rapidly (Fig. 2CGo, circles), as did the accumulation of apoVLDLII mRNA (Fig. 2DGo, circles). These data indicate that the delay in the accumulation of mRNA reflects a delay in the initiation of transcription of the apoVLDLII gene.



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Figure 2. Delayed ApoVLDLII Transcription Results in Delayed mRNA Accumulation

Run-on transcription and RNA analysis were carried out as described in Materials and Methods. The full-length genomic clone was divided into the 5' (bp 6–1011), middle (bp 996-2020), and 3' (bp 2005–3031) regions to allow for differential probing across the gene in the run-on assay. In preliminary experiments, no differences were observed in the intensity of hybridization to these three probes at any of the time points examined. Hence the three probes were combined to maximize the signal, thereby increasing the sensitivity of the assay. A, A representative transcription assay showing hybridization to the antisense and sense strands of the apoVLDII gene and hybridization to the ß-actin antisense cDNA sequence. The lack of consistent hybridization to the sense probe suggests that the noncoding strand is not transcribed. The sporadic sense strand signals that are observed reflect a variable background inherent in the assay. B, Signals from a representative Northern blot showing hybridization to the same antisense probes used in panel A. C, ApoVLDLII data from four transcription assays normalized to the ß-actin signals. The primary (circles) and secondary (squares) responses are shown. The bars indicate the range of the average data for two birds. D, The apoVLDLII mRNA data from four Northern blots, normalized to the ß-actin signals. The primary (circles) and secondary (squares) responses are shown. The bars represent the range of the average data for two birds.

 
Birds given a secondary injection of the hormone at 6 weeks of age exhibited a 10-fold increase in the rate of apoVLDLII transcription within 1 h (Fig. 2CGo, squares), compared with the 3 h required to achieve this level after a primary estrogen stimulation (Fig. 2CGo, circles). Similarly, the accumulation of apo-VLDLII mRNA at 1 h after secondary stimulation (Fig. 2DGo, squares) was equivalent to the primary signal at 3 h (Fig. 2DGo, circles). Thus, an earlier onset of transcription resulted in the earlier accumulation of apoVLDLII mRNA after the secondary administration of estradiol.

Regulation of the VTGII Gene
Expression of the VTGII gene in response to estradiol occurs more slowly than expression of the apoVLDLII gene. After a primary injection of estradiol, the rate of transcription of the VTGII gene increased 3-fold by 6 h and continued to increase gradually to a 10-fold increase over baseline within 10 h (Fig. 3AGo, circles). These data indicate that not only does VTGII have a longer lag than apoVLDLII, taking 3 times as long as apoVLDLII to achieve a 3-fold increase, but the subsequent change in the rate of transcription is not nearly so rapid for VTGII as was observed for apoVLDLII. While apoVLDII transcription increased from 3-fold to 10-fold over baseline between 2 and 3 h after estradiol treatment (Fig. 2CGo, circles), 4 h were required, i.e. from 6–10 h after hormone treatment, to increase the rate of VTGII transcription from 3-fold to 10-fold over baseline (Fig. 3AGo, circles). The accumulation of VTGII mRNA increased approximately 5-fold during this 10-h period (Fig. 3BGo, circles). Consequently, the slow accumulation of VTGII mRNA during the primary response is due to a delayed initiation coupled with a slow rise in the rate of transcription.



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Figure 3. VTGII Transcription and mRNA Accumulation

A, VTGII data from four transcription assays normalized to the ß-actin signals. The primary (circles) and secondary (squares) responses are shown. The bars represent the range of the average data for two birds. B, The VTGII mRNA data from four Northern blots, normalized to the ß-actin signals. The primary (circles) and secondary (squares) responses are shown. The bars represent the range of the average data for two birds.

 
The secondary response of the VTGII gene to estradiol occurred much earlier. The rate of VTGII transcription after a secondary exposure to estradiol increased 20-fold within 3 h (Fig. 3AGo, squares), twice the level achieved in the first 10 h after primary stimulation (Fig. 3BGo, circles). The secondary accumulation of VTGII mRNA at 5 h (Fig. 3BGo, squares) was equivalent to the 10-h signal during the primary response (Fig. 3BGo, circles). Thus, the secondary response of the VTGII gene not only displayed an earlier onset, but the rate of transcription increased more rapidly than it did during the primary response to estradiol.

Transcriptional Regulation of the VTGI Gene
The VTGI gene is the slowest of the egg yolk protein genes to begin to accumulate RNA in response to estradiol. This is the first description of the transcriptional response. As shown in Fig. 4Go, after a primary injection of estradiol there was a 15-h delay before a rapid rise in the rate of transcription of the VTGI gene. Over the next 7 h, from 15–22 h after estradiol, the rate of transcription increased 10-fold (Fig. 4AGo, circles). VTGI mRNA increased even more slowly, with only a 2-fold increase over background detected after 22 h (Fig. 4BGo, circles). When the birds were given a secondary injection of estradiol, the rate of VTGI transcription at 8 h was increased approximately 5-fold (Fig. 4AGo, squares), equivalent to that observed at 16 h during the primary response (Fig. 4AGo, circles). VTGI mRNA increased 2-fold over baseline between 20 and 22 h after the primary administration of estradiol (Fig. 4BGo, circles). During the secondary response, an equivalent 2-fold increase was achieved by 10 h (Fig. 4BGo, squares).



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Figure 4. VTGI Displays the Slowest Response to Estrogen

A, VTGI data from four transcription assays normalized to the ß-actin signals. The primary (circles) and secondary (squares) responses are shown. The bars represent the range of the average data for two birds. B, The VTGI mRNA data from four Northern blots, normalized to the ß actin signals. The primary (circles) and secondary (squares) responses are shown. The bars represent the range of the average data for two birds.

 
Estrogen Increases Transcription of the Apolipoprotein B Gene
Constitutive expression of the apolipoprotein B (apoB) gene occurs in chickens of both sexes. During periods of egg laying, the level of apoB mRNA is increased approximately 6-fold in the liver of hens (24). Because synthesis of apoVLDLII and the three VTG mRNAs is initiated from quiescent genes in response to estrogen, it was anticipated that the mode of regulation of apoB by estrogen may differ from that of the other yolk protein genes. The rate of transcription of the apoB gene was measured after primary and secondary injections of estradiol, and the results are shown in Fig. 5Go. The rate of transcription increased slowly during the first 2 h after injection of the hormone, reaching a rate of 2- to 3-fold over background during this period. During the next hour there was a rapid rise, which resulted in a 6-fold increase over baseline in the rate of transcription of the apoB gene, indicating that the previously reported increase in apoB mRNA is the result of an increase in the rate of transcription.



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Figure 5. Transcription of the ApoB Gene Suggests Hepatic Memory Reflects Initial Activation Regardless of Inducer

ApoB data from four transcription assays normalized to the ß-actin signals. The primary (circles) and secondary (squares) responses to estrogen are shown. The bars represent the range of the average data for two birds.

 
It is striking that even though the response of the apoB gene to estrogen showed a lag followed by a transition to a rapid rate of transcription, no evidence of hepatic memory accompanied this activation. The response to the secondary administration of estradiol (Fig. 5Go, squares) was the same as that observed after the primary injection (Fig. 5Go, circles). The apoB gene is initially expressed during differentiation of the liver (25); subsequently the expression of this gene is turned up by estrogen, not on. Consequently, hepatic memory may be established during the original activation event, the result of either induction by estrogen or a developmental program.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The experiments reported in this manuscript were designed to examine the basis of hepatic memory. Because memory persists through several rounds of cell division, we, and others (13, 14), reasoned that memory might be marked by estrogen-dependent changes in chromatin structure. Due to these previous studies, which demonstrated that none of the identified estrogen-dependent changes in the chromatin persist long enough to account for memory, we examined the promoter of the apoVLDLII gene for the rapidity of appearance of changes in response to estradiol. We anticipated that changes across the promoter would occur in an ordered fashion over the 4- or 2-h lag period preceding the primary and secondary response, respectively. Surprisingly, direct and rapid effects of estrogen within the ERE1, ERE2, and VBP sites of the apoVLDLII promoter were observed by in vivo DMS footprinting when dispersed cells from a hormone-naive liver were treated with estradiol (Fig. 1Go). The rapid alterations at the VBP site are of particular interest because this demonstrates that the delay in transcriptional response is neither a consequence of impeded accessibility to non-estrogen receptor proteins nor the delayed appearance of VBP. These changes in DMS reactivity occurred within seconds and preceded the onset of rapid accumulation of apoVLDLII mRNA by 4 h. The results of run-on transcription assays demonstrated that the delay in mRNA accumulation is the result of delayed initiation of transcription (Fig. 2Go).

These data establish that chromatin alterations precede, and are not a consequence of, transcription from the apoVLDLII promoter. This is the first report to demonstrate this phenomenon using the native estrogen receptor, although previous studies using altered receptors showed that it was possible to induce estrogen-dependent chromatin structural changes in the absence of transcription (26, 27). There are several candidate proteins that could be involved in this estrogen-dependent response, particularly, those chromatin-remodeling proteins that have been shown to interact with the estrogen receptor. Examples include the SWI2/SNF2 subunit of the SWI/SNF complex (28, 29), the heterochromatin protein 1-interacting protein TIF1{alpha} (30, 31), and the nucleosome assembly factor SPT6 (32, 33).

The concept that chromatin remodeling precedes transcription suggests that induction by estrogen takes place in two steps. The first involves altering the chromatin from a closed to an open configuration. The second step involves assembly of the transcription complex with the particular combination of coactivators that allow rapid loading of polymerases at each estrogen-responsive promoter. An example of this mode of regulation takes place at the MMTV promoter where glucocorticoids alter chromatin structure and induce gene expression (34), presumably through the ability of the activated glucocorticoid receptor to recruit the nucleosome-disrupting SWI/SNF complex to the promoter (35).

The events that occur during the lag between the immediate changes observed and the initiation of transcription remain unknown. What could account for such a delayed response, and why is this lag different for each yolk protein promoter? In the frog, protein synthesis is not required for transcription of the vitellogenin genes (36). In vitro DNase footprinting experiments have shown that all of the DNA-binding proteins that interact with the apoVLDLII promoter are present in both hormone-naive and estrogen-stimulated liver (17). Although a requirement for protein synthesis has not been reported in the chicken, there are numerous proteins involved in transcriptional activation that do not bind DNA, but act through protein/protein interactions, some of which may be candidates for induction by estrogen. In addition to the proteins that direct chromatin remodeling mentioned above, numerous other potential intermediary factors can bind to the estrogen receptor. Examples of such proteins that interact with the receptor in a ligand-dependent manner include GRIP1 (glucocorticoid receptor interacting protein 1) (37), steroid receptor coactivator 1 (SRC-1) (38), RIP140 (receptor interacting protein, 140 kDa) (39), CBP (CREB-binding protein) (40), and TRIP1 (thyroid hormone receptor interacting protein 1)/SUG1 (suppressor of Gal 1) (41, 42). Of special interest is CBP, which not only has intrinsic histone acetyltransferase activity (43, 44), but associates with two other histone acetyltransferase proteins, PCAF (P300/CBP associated factor) (45) and ACTR (activator of retinoid receptors) (46), raising the possibility that powerful and specific histone acetylation may accompany estrogen-dependent transcription. These and other regulatory cofactors, conceivably induced in response to estrogen, may interact in unique combinations to activate specific promoters. The presence of the various lag periods before expression of the yolk protein genes provides an opportunity to investigate the similarities and differences in the sequence of events that precedes the initiation of transcription among these closely related estrogen-responsive genes.

Finally, our data indicate that memory may not be exclusive to the estrogenic response. The delay before an increase in transcription of the apoB gene after the administration of estrogen is identical whether the birds were hormone naive or previously treated with estrogen and withdrawn (Fig. 5Go). While it is possible that the regulation of the apoB gene is different from the rest of the members of this closely related family, we prefer the explanation that the apoB locus already has memory, which was established when constitutive expression began in the hormone-naive embryo. Memory tells the gene that it has been expressed previously, regardless of the inducer, and reflects a relatively stable event that occurs during the transition from a gene committed for expression to an active gene.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Animals and Hormone Treatment
White Leghorn chickens were obtained from Truslow Farms (Chester, MD). All birds were killed by cervical dislocation at 4–6 weeks of age. For the footprinting experiments hormone-naive birds were killed and dissected, and 0.5 g liver was dispersed in 4 ml Waymouths medium (GIBCO BRL, Gaithersburg, MD) using a Polytron PT 10/35 with a PTA-10TSM generator (Brinkmann Instruments, Westbury, NY) at setting 2 for 15 sec. This crude cell suspension was divided into three portions and incubated with 10 nM 17ß-estradiol for 0, 30, or 60 sec before the addition of 40 µl DMS/g tissue. The estradiol was dissolved in dimethylsulfoxide (DMSO). This vehicle was added to the control cells (0 estradiol) 30 sec before the addition of DMS. These mixtures were incubated on ice for 3 min before the isolation of nuclei and purification of DNA (47). For run-on transcription and RNA analysis, birds were injected ip with 20 mg 17ß-estradiol dissolved in propylene glycol per kg body weight at 6 weeks of age for the primary response, and at 2 and 6 weeks of age for the secondary response. The 6-week-old birds were killed after estradiol administration at the times indicated in the figures. All experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

Oligonucleotides and Plasmids
The nested primers used to visualize the lower strand of the promoter region of the apoVLDLII gene were:

P1 (+60 through +46) 5'-GAGCAGAGAGTGCAG-3'

P2 (+35 through +10) 5'-TCCTGCTTTCTCTCTCCCAGGCTGAA-3'

P3 (-88 through -111) 5'-GAAGCAAATTCTGCTCAGCAGGATG-3'

The sequencing primer (-62 through -82) 5'-GCTTTGTTTCCCCATTTGCTC-3' was offset from P3 by the length of the common linker. Plasmids used to derive sense and antisense SP6 transcripts (Riboprobe Systems, Promega, Madison, WI) contained chicken cDNA for apoB (24), VTGII (48), VTGI (10), ß-actin (a gift from Bruce Paterson), or genomic DNA for apoVLDLII (49).

In Vivo DMS Footprinting
Methylated genomic DNA was cleaved with piperidine (50). Ligation-mediated PCR was performed as described by Mueller and Wold (51) and modified by McPherson et al. (52). Briefly, 1 µg of the cleaved DNA was denatured at 95 C for 5 min, and the first strand synthesis gene-specific primer 1 was annealed for 30 min at 50 C. Oligonucleotide P1 was extended at 37 C for 10 min using 0.17 U/µl Sequenase DNA polymerase version 1.0 (USB, Cleveland, OH) in Sequenase buffer [40 mM Tris, pH 7.5; 5 mM MgCl2; 20 mM dithiothreitol; 0.1 mM each of deoxyribonucleoside triphosphate (NTP)]. After the first strand synthesis, the Sequenase was inactivated by heating for 10 min at 68 C. Equimolar concentrations of the common linkers (51) were heated at 95 C for 5 min and allowed to cool to room temperature. The ligation was performed with 0.18 U/µl of T4 DNA ligase (New England BioLabs, Beverly, MA). After ligation, the DNA ligase was inactivated at 68 C for 10 min. The ligated DNA was ethanol precipitated, washed, and resuspended in distilled water. Amplification of the ligated products was performed with 0.1 U/µl of Taq DNA polymerase (Perkin-Elmer, Norwalk, CT) in 1x Taq buffer: (65 mM Tris, pH 8.8; 10 mM ß-mercaptoethanol; 16.5 mM (NH4)2SO4; 1.5 mM MgCl2; 240 µM MnCl2; 400 µM each of dNTP; 100 µg/ml BSA; 10 pmol each of oligonucleotides P2 and common linker 1). Amplification was carried out for 25 cycles (1 min at 94 C; 2 min at 52 C; 2 min at 74 C). To 15 µl of the 100-µl amplification reaction, 5 µl of labeling mixture [0.5 µl of 10x Taq buffer; 1 µl of (10 mM MgCl2 + 8 mM MnCl2); 1.5 µl of 5 mM dNTP mixture; 2 pmol P3, 5' end labeled with a 2:1 molar excess of [{gamma}32P]ATP; and 1 U Taq DNA polymerase] was added. Labeling was carried out for seven cycles (1 min at 94 C; 2 min at 66 C; 2 min at 74 C). After labeling, the DNA was precipitated in 95% ethanol in the presence of 25 µg/ml carrier tRNA. The DNA was washed in 70% ethanol, lyophilized, and resuspended in 12 µl of DNA sequencing loading buffer. One third of the labeled DNA was separated on a 6% polyacrylamide/7 M urea sequencing gel. The dried gel was exposed to x-ray film for autoradiograghy.

Run-on Transcription
Nuclei were isolated (53), stored at -70 C in 100-µl aliquots containing 4 x 107 nuclei, and thawed 5 min on ice for each experiment. 32P-labeled RNA was synthesized (54, 55), and the labeled RNA was isolated (56) using Nick columns (Pharmacia Biotech, Piscataway, NJ). Nytran (Schleicher & Schuell, Keene, NH) filters containing 250 µg of each antisense and sense RNA transcript, UV cross-linked at 254 nm and 240 KJ (Stratalinker 1800, Stratagene, La Jolla, CA) then dried 1 h under vacuum at 80 C, were prehybridized in 50% formamide, 250 mM Na phosphate, pH 7.2, 1 mM EDTA, 250 mM NaCl, 100 µg/ml single-stranded DNA, 40 µg/ml tRNA, and 7% SDS. Hybridization in the same solution containing 20 x 106 dpm of the [32P]RNA was performed at 55 C for 48–72 h. The filters were washed at room temperature 4 x 5 min in 2x saline-sodium citrate (SSC) and at 65 C 3 x 20 min in 25 mM Na phosphate, pH 7.2, 1 mM EDTA, and 1% SDS. The hybridization signals were visualized using the PhosphorImager and quantitated using ImageQuant version 4.1 (Molecular Dynamics, Sunnyvale, CA).

Northern Analysis
Total RNA was prepared from 1 g of liver as described (57). Ten micrograms of total RNA were separated on a 1.2% agarose, 0.66 M formaldehyde gel. The RNA was transferred to Nytran membranes in 10x SSC by capillary action (58). The filters were UV cross-linked and dried. Prehybridization and hybridization were done in 50% formamide, 5x Denharts (50x is 1% Ficoll 400, 1% polyvinylpyrrolidone, and 1% BSA, fraction V), 5x SSC, 100 µg/ml salmon sperm DNA, and 1% SDS. Sp6-derived specific antisense 32P-labeled transcripts were added at a concentration of 1 x 106 dpm/ml in fresh hybridization buffer and hybridized at 58 C for 16 h. The filters were washed 2 x 20 min in 2x SSC, 1% SDS at room temperature, then 2 x 20 min in 0.1x SSC, 1% SDS at 68 C. The filters were imaged and quantitated as stated above.


    ACKNOWLEDGMENTS
 
We thank Drs. Ken Zaret and Lisa Salati for helpful discussions, and Drs. Jeanine Strobl, John Durham, and Diane Robins for critical comments on the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Marilyn Evans Ph.D., Associate Professor of Biochemistry, School of Medicine, Box 9142, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, West Virginia 26506-9142.

This work was supported by NIH Grant HD-26339 (to M.I.E.). R.S.E. is the recipient of a Grant-in-Aid of Research from Sigma Xi, the Scientific Research Society.

1 Present address: Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213. Back

Received for publication August 21, 1997. Revision received September 19, 1997. Accepted for publication September 22, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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