(Received for publication, May 19, 1995; and in revised form, July 19, 1995)
From the
The rat angiotensin II type 1a receptor (AT1a-R) gene is expressed in a cell-specific manner. We demonstrated that the negative regulatory element (NRE) between -489 and -331 is active in PC12 cells (Murasawa, S., Matsubara, H., Urakami, M., and Inada, M. (1993) J. Biol. Chem. 268, 26996-27003). Gel retardation assays confirmed that PC12 cells have a trans-acting factor bound to the NRE. By means of a DNase I footprint assay we identified the core of the NRE as an (A + T)-rich sequence (TAATCTTTTATTTTA) located at nucleotides -456 to -442. Oligonucleotides corresponding to the NRE core sequence bound to nuclear protein. Site-directed mutagenesis at nucleotides -451 to -448 eliminated the specific protein/DNA binding and restored expression of the AT1a-R in transient transfection assays (2.7-fold increase). The NRE did not negatively affect the thymidine kinase promoter. No homology was found with known NREs, suggesting that this is a novel NRE. Southwestern blotting revealed a 53-kDa, specific binding protein in PC12 cells and the rat brain, but not in the liver, spleen, adrenal gland, and kidney. These findings demonstrate that the NRE of the rat AT1a-R is an (A + T)-rich sequence located at nucleotides -456 to -442 and the 53-kDa protein is a specific binding protein, and suggest that this protein may be a trans-acting factor which determines the neuron-specific down-regulation of the AT1a-R gene.
Angiotensin II has multiple physiological effects in the
cardiovascular, endocrine, and nervous systems that are initiated by
binding to specific receptors located on the plasma
membrane(1) . Two major subtypes (type 1 and type 2) of
angiotensin II receptors have been revealed by their differential
affinity for nonpeptide drugs(2) . Angiotensin II type 1a
receptor (AT1a-R) ()cDNAs have been cloned from rat vascular
smooth muscle cells(3) , bovine adrenal zona glomerular
cells(4) , and rat kidney(5) . Angiotensin II mRNA is
expressed in a variety of cells and tissues including vascular smooth
muscle cells, liver, kidney, and spleen, while the mRNA abundance is
low in other tissues such as heart, brain, thymus, and testis. AT1a-R
gene expression is regulated in an ontogenic manner(6) . Thus,
the rat AT1a-R gene is cell-specifically and developmentally regulated.
We characterized one negative and three positive cis-regulatory elements in the 5`-flanking region of the rat AT1a-R gene(7) . The negative cis-regulatory element (NRE) was located between -489 and -331 and inhibited the promoter activity of the 590-bp 5`-flanking region by a factor of 10. The trans-acting factor that binds to the element was present in PC12, not in vascular smooth muscle and glial cells. This suggested that the trans-acting factor is a major determinant which regulates the expression of the rat AT1a-R gene in PC12 cells. However, the NRE was located within 159 nucleotides (nt) from -489 to -331 and the core sequence has not been mapped in detail.
Here, we identified the core sequence to clarify the negative cis-regulation, using the gel retardation assay and the DNase I footprint analysis. We showed that the core sequence is (A + T)-rich (TAATCTTTTATTTTA, nt -456 to -442). Southwestern blotting revealed that a nuclear protein of about 53 kDa bound to the NRE in PC12 cells and the rat brain, but not in vascular smooth muscle cells, glial cells, kidney, spleen, adrenal gland, and liver.
The 159-bp NRE fragment (nt -489 to
-331) obtained by XhoI and HindIII digestion
was labeled with [-
P]dCTP using Klenow
fragment (Takara Shuzo, Kyoto, Japan) and purified as
reported(8) . Oligonucleotides corresponding to the NRE
(5`-TAATCTTTTATTTTA-3`), mutation 1 (5`-TAATATTTTCTTTTA-3`), and
mutation 2 (5`-TAATCGGGGATTTTA-3`) were synthesized, labeled with
[
-
P]ATP using T4 polynucleotide kinase
(Takara Shuzo, Kyoto), and annealed to make double-strand DNA. Nuclear
extracts were incubated for 15 min on ice in a 30-µl reaction
mixture containing 12 mM Hepes, pH 7.9, 60 mM KCl,
0.1 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, 12% glycerol, and 2 µg of
double-stranded poly(dI-dC) in the presence or absence of excess
competitor DNA. A radiolabeled DNA probe was added (0.1
0.5 ng;
15,000 cpm), and the incubation was continued for 30 min at room
temperature. Thereafter, the mixture was loaded on a 6% polyacrylamide
gel in 1
TBE (90 mM Tris-HCl, pH 8.0, 89 mM boric acid, 2 mM EDTA), and electrophoresed at 140 V for
3 h followed by autoradiography.
Figure 2: DNase I footprint analyses of the NRE fragment. The NRE fragment (nt -489 to -331) was labeled at the XhoI site (A) and the BssHII site (B) as the sense probes, and at the NotI site (C) as an antisense probe. These probes were incubated in the presence or absence of nuclear extract from PC12 cells, and partially digested with DNase I as described under ``Materials Methods.'' Five µg of nuclear extract was used in panel A and increasing amounts of nuclear extract were used in panel B and C. G + A lane indicates the sequence ladder by the Maxam-Gilbert reactions. The protected portion is shown in the left side of panels.
A mutation corresponding to NRE mutation 2 was created by PCR overlap extension mutagenesis(10) . Briefly, two DNA fragments having overlapping ends were first amplified using two sets of primers (A and B, C and D), from a AT1a 980-Bluescript construct into which a 1242-bp EcoRI-SacI fragment containing the 980-bp 5`-flanking region (-980 to +262) had been subcloned(7) . Primers A and D were T3 and T7 primers for pBluescriptKS(-), respectively. Primers B and C had the following sequences: B, 5`-TTCAGAGCCGTAAAATACCAGATTA-3` (antisense: nt -432 to -456); C, 5`-TATTTTACGGCTCTGAA-3` (sense: nt -448 to -432). Primer B contained an oligo-NRE mutation 2 (Fig. 5A) at the 3` end, and primers B and C were designed to overlap at the 5` end (underlined). The two respective PCR products were mixed and amplified again with primers A and D. The resultant PCR product was subcloned into the pGEM-T vector (Promega, Madison, WI), and sequenced using T7 and T3 primers to confirm the mutated NRE and other DNA sequences. The PCR product was further subcloned into the 5` end of the CAT gene.
Figure 5:
Nucleotide sequences of NRE mutations and
functional analyses of NRE in the promoter activity. A,
nucleotide sequences of NRE mutations 1 (-452 and -447) and
2 (-451 to -448) are shown in comparison with wild NRE
sequences. B, the NRE fragment (nt -489 to -331)
was deleted from 980 bp of 5`-flanking region and fused to the CAT
reporter gene as described under ``Materials and Methods.'' A
mutation corresponding to NRE mutation 2 was designed by means of PCR
overlap extension mutagenesis. These CAT fusion genes (15 µg) were
co-transfected with 5 µg of -galactosidase genes into PC12
cells. The CAT activity levels were normalized with
-galactosidase
activities and protein contents, and expressed as relative values to
those of the promoterless CAT construct. For quantitative comparison,
the relative value of the wild AT1a 980-CAT construct in PC12 cells is
assigned a value of 1.0. The mean activities and the standard error of
the mean from six separate assays are presented. Analysis of variance
and the Dunnet's test were used for multigroup comparisons. *, p < 0.01 versus value of wild CAT construct in
PC12 cells.
To construct a NRE-thymidine kinase (TK)-CAT fusion gene, the NRE fragment (nt -489 to -331), obtained by digestion with XhoI and BamHI, was blunt-ended at XhoI site by Klenow. The TK-CAT construct (a gift from Dr. Y. Mori, Harvard Medical School) was cut with HindIII and BamHI in the upstream linker site and HindIII site was blunt-ended. The NRE fragment was subcloned between blunt-end and BamHI sites of the TK-CAT construct.
Fig. 1shows the result of gel retardation analyses using 159 bp of the NRE fragment (nt -489 to -331) as a probe. The NRE fragment formed five retarded bands (arrows A, B, C, D, and E in Fig. 1) upon incubation with cellular nuclear extract from PC12 cells. Although these bands differed in mobility, the addition of a 100-fold molar excess of the same unlabeled NRE fragment completely competed with the slowest band (arrow A). Other retarded bands (arrows C and D) also competed with the unlabeled NRE fragment, whereas the inhibition was not complete even with a 100-fold molar excess of the competitor. The arrow B and E bands were not inhibited with an excess of the competitor. The retarded band corresponding to arrow A was not detected in glial cells, A10 cells, and vascular smooth muscle cells. No specific band complex was observed when the nuclear extract from glial cells, A10 cells, or vascular smooth muscle cells were incubated with the NRE fragment.
Figure 1:
Gel retardation analysis of the NRE
fragment. The NRE fragment (nt -489 to -331) was labeled
and used in gel retardation analyses with nuclear extract (2 µg)
from PC12, glial cells, and A10 cells and primary cultured vascular
smooth muscle cells of rat aorta (VSMC). The unlabeled NRE
fragment at a 20 to 100
molar excess was the
competitor.
An oligonucleotide (oligo-NRE) was designed to encompass the protected 15-bp sequences. With oligo-NRE as the labeled probe, the nuclear extract from PC12 cells produced a single strong band that was eliminated with an excess of the same oligo, but not by an unrelated oligo (oligo I) encompassing from nt -489 to -457 of the more upstream NRE region. The nuclear extract from glial cells did not form a retarded band. The oligonucleotides known as the NRE for the human major histocompatibility complex class I (CCAAAATTATCTGAAAAAGGTTATTAAAA) (18) or the rat prolactin (TATAATTTTATA) (19) did not compete with the DNA-binding protein (Fig. 3A). Thus, oligo-NRE contains a binding site for a sequence-specific DNA-binding protein.
Figure 3:
Gel retardation analyses of the oligo-NRE. A, the oligo-NRE (nt -456 to -442) was labeled and
used in gel retardation analyses with nuclear extract (2 µg) from
PC12 cells. Glia refers to gel retardation using nuclear extract (2
µg) from glial cells. Oligo I, II, and III show gel retardation
when oligonucleotides encompassing the more upstream region (oligo I,
nt -489 to -457) and the NRE sequences for the human major
histocompatibility complex class I gene (oligo II,
CCAAAATTATCTGAAAAAGGTTATTAAAA) (18) and the rat prolactin gene
(oligo III, TATAATTTTATA) (19) were used as competitors (molar
ratio, 100). B, the NRE fragment (nt -489 to
-331) and the oligo-NRE (nt -456 to -442) were used
as the probes and the competitors, respectively, and incubated with
nuclear extract (2 µg) from PC12 cells. Arrows indicate
the retarded products specifically bound to the
probe.
Gel retardation analyses were performed using the promoter NRE fragment as a probe, to determine whether or not the retarded band could be competed by the oligo-NRE. Excess oligo-NRE interfered with the protein-NRE binding and reduced the amount of a slowest retarded product (Fig. 3B). These findings suggest that the slowest retarded band (arrow A in Fig. 1) is due to the interaction between nuclear protein and oligo-NRE sequence.
Figure 4: Gel retardation analyses of the oligo-NRE mutations. A, the NRE mutation 1 (-452 and -447) was labeled and competed with the unlabeled NRE mutation 1. B, the NRE mutation 2 (-451 to -448) was labeled and reacted with nuclear extract. C, the wild oligo-NRE was the probe and competed with wild oligo-NRE or unlabeled NRE mutation 2. Nuclear extract (2 µg) was prepared from PC12 cells. The arrow indicates the retarded band. Sequences for the wild oligo-NRE and the mutations are shown in Fig. 5.
Figure 6:
Southwestern blots of nuclear protein
prepared from PC12 cells and rat brain. Nuclear extracts (50 µg of
protein per lane) were separated by SDS-PAGE, blotted onto a
nitrocellulose membrane, then detected with the P-labeled
NRE fragment (159 bp, nt -489 to -331), oligo-NRE (nt
-456 to -442,) or oligo-NRE mutation 2 (Fig. 5)
probes. The unlabeled NRE and wild oligo-NRE were used as competitors.
The arrow indicates the 53-kDa protein specifically bound to
the probe. Films were exposed for 7 (PC12 cells) or 30 days (rat brain)
at -80 °C with intensifying screens. Size markers are
indicated in kDa on the left side of
gels.
Figure 7:
Effect
of the NRE on a heterologous promoter. PC12 cells were transiently
transfected with fusion genes containing the thymidine kinase (TK) promoter placed 5` to the CAT gene without NRE (top), with the NRE (solid bar) placed upstream of
the TK promoter (middle). The CAT activity levels were
normalized with -galactosidase activities and protein contents,
and expressed as relative values to those of the promoterless CAT
construct (bottom). For quantitative comparison, the relative
value of the promoterless CAT construct is assigned a value of 1.0. Arrows indicate the transcription initiation sites. The
experiments were separately repeated five times and the results were
shown in the mean ± S.E.
We demonstrated that three positive cis-regulatory elements and one strong NRE are present in the 5`-flanking region of the rat AT1a-R gene, and suggested that this NRE is one of major determinants that regulate the level of gene expression in PC12 cells (7) . Here, we discovered and mapped the core sequence of the NRE (5`-TAATCTTTTATTTTA-3`) between -456 and -442. This element reduced AT1a-R gene expression by a factor of 2.7 in transient transfection assays using PC12 cells. Site-directed mutagenesis in this core sequence affected the specific DNA-protein interaction and eliminated the suppression of AT1a-R gene expression in the transient transfection assays. These data demonstrated that specific protein-DNA interaction at this sequence down-regulates the gene expression in PC12 cells.
Although NREs have been detected in a variety of genes(13) , the molecular mechanisms by which they exert their effects remain obscure. While the NREs can exhibit enhancer-like qualities and function on heterologous promoters in a distance and orientation-independent fashion(14) , they often reduce rather than abolish the activity of heterologous promoters and demonstrate a preference for a specific promoter(15, 16) . The NRE in the rat AT1a-R gene, when transferred to the TK promoter, had no significant effect on the transcriptional activity, suggesting that the NRE in the AT1a-R gene works more effectively in conjunction with the native AT1a-R gene promoter than with the heterologous promoter.
Recently, many cis-acting, negative transcriptional elements in mammals have been identified(13) , some of which include (A + T)-rich sequences. These include human Ig heavy chain (AATATTTT)(17) , human major histocompatibility complex class I (CCAAAATTATCTGAAAAAGGTTATTAAAA)(18) , rat prolactin (AAATAAA, TATAATTTTATA)(19) , and mouse Ig (ATTAATTTAT)(20) . However, the (A + T)-rich NRE sequence identified in the rat AT1a-R gene did not match these (A + T)-rich sequences and was not competed with the NRE oligos for the human major histocompatibility complex class I and rat prolactin (Fig. 3A), indicating that this NRE is a novel negative element.
The rat AT1a-R gene has a subtype gene, AT1b-R, which has very high homology (96%) in the coding sequence(21, 22, 23, 24) . Although the receptor-mediated second signal was similar to that in the AT1a-R, the profile for the tissue distribution between the AT1a-R and AT1b-R mRNAs was quite distinct. The AT1a-R is the dominant form expressed in the liver, kidney, vasculature, lung, ovary, testis, and heart, whereas the AT1b-R is expressed in greater quantities in the adrenal gland, anterior pituitary, and uterus(21, 22, 23) . Very recently, Guo and Inagami (25) have sequenced the rat AT1b-R promoter region, in which the homology between both subtypes was low and the (A + T)-rich NRE sequence observed in the AT1a-R gene was not detected. Since the abundant expression of the AT1b-R gene is restricted in a few organs compared with that of the AT1a-R gene, a much stronger NRE may be present and regulate a tissue-specific expression. In the 5`-flanking region of the human AT1a-R gene, Guo et al.(26) and Takayanagi et al.(27) found that the cis-regulatory region inhibits the gene transcription between -881 to -642, and -962 to -114, respectively. However, the (A + T)-rich NRE sequence observed in the rat AT1a-R gene was not located in these regions, and a similar element (AAATTTATTTTA) was present more upstream. This study demonstrated that the trans-acting protein bound to the NRE of the rat AT1a-R gene is present in PC12, but not in vascular smooth muscle cells and glial cells. Thus, whether the (A + T)-rich sequence in the human AT1a-R gene can function as a NRE depends on a suitable cell model containing an abundant amount of the specific trans-acting protein.
Southwestern blots suggested the involvement of a 53-kDa protein in the AT1a-R promoter function in PC12 cells. We showed in a previous study (7) that the expression of the AT1-R is very low in PC12 cells: the mRNA level was detectable only by the reverse transcriptase-PCR method, not by the Northern blot, and the AT1-R protein was not quantified by the binding assay. Sumners et al.(28) have reported using the neonatal rat brain, that glial cells predominantly express AT1-R, whereas neurons contain a small amount of AT1-R. We also found that the promoter region of the rat AT1a-R gene contains a positive cis-regulatory element active only in glial and PC12 cells (7) , suggesting that the regulatory mechanism of rat AT1a-R gene expression may differ between the central and peripheral tissues. The finding that a nuclear protein bound to the NRE in the brain, but not in vascular smooth muscle cells, glial cells, the spleen, kidney, adrenal gland, and liver, also supports this contention. The expression of the AT1a-R gene is also inhibited in the adrenal gland and the pituitary gland, in which the AT1b-R subtype is predominant. The 53-kDa protein was not detected in the adrenal gland, suggesting that other regulatory mechanisms to suppress the AT1a-R gene transcription may function in these tissues. Since the NRE has only a 2-3-fold effect in inhibiting the AT1a-R promoter activity and no effect on the heterologous promoter, the inhibitory action of the 53-kDa protein may not be sufficient to determine the neuron-specific down-regulation or tissue-specific regulation of the AT1a-R gene. Recent evidence demonstrates that the blood pressure is effectively reduced in the AT1a-R knock-out mice(29) . The cloning of a gene encoding the 53-kDa protein bound to the NRE may help the isolation of a novel nuclear protein that moderately reduces blood pressure by inhibiting the expression of the AT1a-R gene.