(Received for publication, June 23, 1995; and in revised form, August 3, 1995)
From the
We have utilized a genetic selection system in yeast to identify novel estrogen-responsive genes within the human genome and to define the sequences in the BRCA-1 gene responsible for its estrogen responsiveness. This approach led to the identification of a new subclass within the Alu family of DNA repeats which have diverged from known Alu sequences and have acquired the ability to function as estrogen receptor-dependent enhancers. Importantly, these new elements confer receptor-dependent estrogen responsiveness to a heterologous promoter when assayed in mammalian cells. This transcriptional activity can be attenuated by the addition of either of three different classes of estrogen receptor antagonists, indicating that these elements function as classical estrogen receptor-dependent enhancers. Furthermore, this enhancer activity is restricted to a specific subset of DNA repeats because consensus Alu elements of four major subfamilies do not respond to the estrogen receptor. Previously, most Alu sequences have been considered to be functionally inert. However, this work provides strong evidence that a significant subset can confer estrogen responsiveness upon a promoter within which they are located. Clearly, Alu sequences must now be considered as important contributors to the regulation of gene transcription in estrogen receptor-containing cells.
The steroid hormone estrogen is a key intracellular modulator of
the processes involved in establishment and maintenance of female
reproductive function(1) . In addition, its actions play an
important role in maintaining female cardiovascular tone and regulating
bone cell differentiation(2) . In pathological states, the
mitogenic activity of estrogen facilitates progression of breast
cancers (3) and is implicated in the abnormalities of uterine
function observed in endometriosis and possibly uterine
fibroids(4) . These actions of estrogen all appear to be
mediated through specific high affinity receptors located within target
cell nuclei(1) . Molecular cloning has revealed that the
estrogen receptor (ER) ()is a member of a superfamily of
receptors which mediate the nuclear actions of the sex steroids,
retinoic acid, vitamin D
, and thyroid hormones(5) .
In the absence of hormone, ER resides in a latent form in target cell
nuclei associated within a large macromolecular complex comprising heat
shock protein 90 (hsp90), hsp70, p59, and other proteins(6) .
Upon ligand binding, the receptor undergoes a dramatic conformational
change(7, 8) , initiating a cascade of events leading
ultimately to the association of an ER dimer with specific estrogen
response elements (EREs) within the regulatory regions of target genes (9) . The mechanism by which the bound receptor modulates gene
transcription is unknown.
Because of its diverse biological
functions and the implied complexity of its targets, there has been a
keen interest in defining the genes which are regulated by estrogen. To
date, a relatively small number of genes have been identified in humans
which are modulated directly by the estrogen receptor. These include
the genes for pS-2, the progesterone receptor, oxytocin,
c-fos, and -globulin(10) .
However, it was the dissection of the Xenopus laevis vitellogenin A2 promoter which led to the definition of a
consensus ERE, the palindromic sequence GGTCAcagTGACC, which confers
estrogen responsiveness to a heterologous promoter(11) . To
date, only a single estrogen-responsive gene within human genomic DNA
has been shown to contain this consensus sequence(12) . In
fact, recent studies have indicated that sequences distinct from the
vitellogenin ERE, which function only in specific cell and promoter
contexts, may be more important in facilitating ER
function(13, 14) . Given the complexity of the
biological events that occur in response to estrogen, it is likely that
a large number of genes which are directly regulated by this hormone
remain to be identified. As a consequence of the need to define the
targets of ER action in mammalian cells, we have developed a genetic
screen in Saccharomyces cerevisiae to identify novel
estrogen-responsive genes within human genomic DNA.
Previously our laboratory has shown that ER can function as a
hormone-dependent transcription factor in yeast and used this
observation to develop a functional screen based on nutritional
prototrophy to identify novel estrogen response elements from a library
of random oligonucleotides(13) . The fact that these elements
identified in yeast function analogously in mammalian cells indicated
that this approach could be used to identify fragments from human
genomic DNA which permitted ER action. To accomplish this, we
constructed a library of genomic fragments from Sau3A-digested
MCF-7 cell genomic DNA. Size-fractionated DNA was cloned into the
promoter of an enhancerless GAL1-HIS3 fusion contained within the
pBM2389 vector (Fig. 1)(13, 20) . A library of
over 150,000 recombinants with an average insert length of 300 bp was
obtained. Following amplification in Escherichia coli, the
plasmid library was transformed into a yeast strain, YPH500, containing
the plasmid pRS415-hER expressing the wild-type human ER under control
of the copper-inducible CUP-1 promoter. Transformed yeast were then
plated on minimal media plates containing 17-estradiol and uracil,
but lacking histidine. In addition, we included aminotriazole, a
competitive inhibitor of the HIS3 gene product, to enhance the
stringency of the screen and eliminate background growth due to
leakiness of the GAL1 promoter. Using this approach, we identified
greater than 500 colonies which grew in the absence of added histidine
and contained putative enhancers within the cloned DNA. Because it was
likely that this primary screen would identify ER-independent
enhancers, those which operate in response to endogenous yeast
transcription factors, we performed a secondary screen to determine
estrogen dependence of the selected clones. For this purpose, the
colonies obtained from the primary screen were assayed for their
ability to grow on minimal media plates containing either no histidine,
histidine alone, or histidine + 17
-estradiol. A vector
containing the consensus vitellogenin A2 ERE within pBM2389 was used as
a positive control for this assay (indicated by squares in Fig. 1). The results of one of these secondary screens are shown
in Fig. 1. Those clones which were determined to be
estrogen-responsive are indicated (circles in Fig. 1).
We identified 16 clones which contained sequences from MCF-7 cell
genomic DNA which permitted activation of transcription by
17
-estradiol-activated ER.
Figure 1:
Identification of estrogen
receptor-dependent enhancers using a genetic selection protocol in Saccharomyces cerevisiae. A genomic library constructed within
pBM2389 was transformed into yeast allowing selection of fragments that
were conditionally responsive to stimulation with 17-estradiol.
Clones that would not grow in the absence of hormone (indicated by the circles) were selected for analysis in mammalian transfection
experiments. pBM2389 containing the vitellogenin A2 consensus ERE was
used as a control and is indicated by the area boxed in by the
square.
Before proceeding with a detailed
analysis of these estrogen-dependent clones, we wanted to determine
whether or not they would function as estrogen-responsive enhancers in
mammalian cells. To this end, plasmids were isolated from yeast, and
their inserts were cloned directly into the BamHI site of
pBL-TK-Luc which contains the ERE-negative thymidine kinase promoter
fused to the firefly luciferase gene(18) . The ER-dependent
enhancer activity within each plasmid was then assayed in HepG2 cells
following co-transfection of an ER expression vector (pRST7ER) and
subsequent analysis of luciferase activity following administration of
17-estradiol. As a positive control, we assayed the ER-dependent
enhancer activity of a single copy of the consensus vitellogenin ERE
inserted into the identical site within pBL-TK-Luc (TK-ERE-Luc). The
results of this analysis, shown in Fig. 2A, indicate
that TK-ERE-Luc exhibited a significant basal transcriptional activity
which was induced 3-fold in the presence of added 17
-estradiol.
Although greater inductions are obtained using multimerized copies of
this ERE (data not shown and (13) ), we felt that only a
comparison of the activity of a single-copy ERE to novel enhancers was
relevant in this instance. This analysis revealed that most of the
sequences identified in yeast permitted significant ER-dependent
enhancer activity when compared to TK-ERE-Luc. Notably,
estrogen-responsive fragment-2 (ERF-2), ERF-3, ERF-9, ERF-10, ERF-15,
ERF-16, and ERF-17 all had induction values greater than the TK-ERE-Luc
control (Fig. 2A). Interestingly, several clones which
function in yeast do not appear to function in HepG2 cells. It is
possible that these may represent estrogen-responsive sequences which
operate only in cooperation with additional yeast factors or that their
activity is not manifest in the cell and promoter background used for
these initial studies. For the purposes of this study, however, we
limited our analyses to those clones which functioned as ER-dependent
enhancers in HepG2 cells.
Figure 2:
Analysis of novel estrogen-responsive
enhancers in mammalian cells. A, 16 estrogen-responsive
enhancers, identified in yeast, were subcloned into pBL-TK-Luc and
tested for 17-estradiol-dependent transcriptional activity in
HepG2 cells which had been co-transfected with an ER expression
plasmid(13) . The activity manifest by each clone in the
absence of hormone (solid bars) or in the presence of 100
nM 17
-estradiol (open bars) are indicated. A
plasmid containing the vitellogenin A2 ERE (TK-ERE-Luc) and an
enhancerless control (TK-Luc) were also included as controls. B, the transcriptional response of the clone ERF-3 was
accessed in the presence of expressed ER over a range of
17
-estradiol concentrations as indicated. C, effect of ER
antagonists on 17
-estradiol-mediated induction of ERF-3. The
ER-dependent enhancer activity of ERF-3 was assayed in the presence of
100 nM 17
-estradiol alone (Vehicle) or in the
presence of 17
-estradiol and a 10-fold molar excess of each of
three ER antagonists as indicated. D, dissection of ERF-3. The
insert within the ERF-3 clone was sequenced and is shown. The DNA
sequence which is related to the known Alu consensus sequences
is indicated by bold letters. Potential EREs and half-sites
related to the consensus ERE sequence are underlined. The Alu-related region within ERF-3 (ERF-3a) and the
additional divergent sequence (ERF-3b) were cloned
independently into the TK-Luc vector and assayed for transcriptional
activity as above.
In order to demonstrate that the
estrogen-induced enhancer activity of the isolated genomic clones
represented a classical activity mediated through ER, we performed a
detailed analysis of the most active clone, ERF-3. When ERF-3 activity
was compared with that of the TK-ERE-Luc, it was confirmed that this
clone exhibited significantly more transcriptional activity than did
the consensus ERE. However, the kinetics of the
17-estradiol-mediated induction on both clones were similar
(EC
10
M) (Fig. 2B). It has recently been shown that ER can
activate transcription independent of DNA binding by associating with
c-Jun at AP-1 responsive promoters(14) . This activity,
although ER-mediated, can be distinguished from more direct actions of
ER in that the former is not inhibited, but is in fact induced by
steroid receptor antagonists. To distinguish between these two
mechanisms of ER action, we tested the ability of a series of
mechanistically different ER antagonists to modulate the
transcriptional activity of both the TK-ERE-Luc and the ERF-3
constructs (Fig. 2C). When tested alone,
17
-estradiol, but not keoxifene, ICI182,780, or nafoxidine
promoted ER-dependent activation of both promoters (data not shown).
Importantly, we were able to show that each of the antagonists examined
were capable of inhibiting the 17
-estradiol-mediated activation of
ERE-TK and ERF-3 in a similar manner. Cumulatively, these results
indicate that the ERF-3 clone contains a bona fide estradiol-responsive enhancer whose transcriptional activity is
regulated by a classical ER-mediated signal transduction pathway. More
importantly, however, these results validate the general approach used
to detect novel estrogen-responsive enhancers.
Given that ERF-3 was potentially derived from a novel estrogen-responsive gene, it was sequenced to determine if it contained any similarities to known EREs (Fig. 2D). We were surprised to find that most of the sequence (178 bp out of 253 bp total) corresponded to the Alu family of repetitive DNA (represented in bold letters). The remaining 75 bp do not appear to be related to any known Alu sequence. We are unsure whether or not these sequences are contiguous within the intact genome; however, it is possible that they are and represent a genetically altered Alu. In view of the fact that we used Sau3A to fragment genomic DNA in the construction of our library, it is suspect that the divergence from Alu consensus occurs close to a Sau3A restriction site. Nevertheless, within the sequence which was homologous to Alu DNA, we identified an imperfect ERE (5`-GGTCAnnnTGGTC-3`) which diverged from the vitellogenin A2 ERE by 2 base pairs in the right arm of the palindrome (underlined in Fig. 2D) and a half-site (5`-TGACC-3`) located 9 bp downstream of the imperfect ERE. Additionally, two half-sites separated by 22 bp were found in the non-Alu sequence (underlined in Fig. 2D). Because both the Alu-related and nonrelated sequences within ERF-3 contained potential EREs, we cloned them separately into pBL-TK-Luc. In this way we were able to determine that each in fact functioned independently as ER-dependent enhancers (Fig. 2D). These data suggest that the imperfect ERE within the Alu sequence (ERF-3a) functions as a genuine estrogen-inducible enhancer. The activity of the composite clone (ERF-3) seems to represent synergism between the two independent estrogen-responsive sequences and may be functioning in a manner similar to the vitellogenin B2 ERE where two imperfect palindromes cooperate to yield a functionally important element(21) . Unlike our case, however, the individual B2-derived palindromes do not demonstrate independent activity. The surprising finding that an independent functional ERE was located wholly within a highly repetitive Alu DNA sequence defines a role for this repetitive DNA heretofore unrecognized.
It is unlikely that all Alu repeats function as ER-dependent enhancers due to their abundance.
We were interested therefore in determining what specifically within
the ERF-3-derived Alu was responsible for ER responsiveness.
Fortuitously, we got the answer when two independent projects
converged. We have determined that the newly identified BRCA-1 (breast
cancer susceptibility gene) gene is positively regulated by
17-estradiol in a series of cultured breast cancer cells. (
)Because of the potential link between estrogens and breast
cancer, we wished to determine the molecular basis of the observed
17
-estradiol-induced up-regulation. Specifically, we were
interested in mapping the sequences within the BRCA-1 locus responsible
for this activity. To this end we constructed a library in the pBM2389
vector of DNA fragments derived from a P1 clone (p1141) which contained
a large portion of the 5` end of the BRCA-1 locus(15) . This
library was transformed into YPH500, and estrogen-dependent colonies
were isolated as described previously. In this manner, we identified
one clone (BCER-1) which functioned as an ER-dependent enhancer in
transfected mammalian cells. Sequencing of this clone revealed that it
was in fact an Alu sequence which displayed 100% sequence
identity in the region corresponding to the ERE-related imperfect
palindrome within ERF-3. This Alu repeat was subcloned into
pBL-TK-Luc and tested in HepG2 cells. The results shown in Fig. 3A indicate that this Alu element
functioned as an ER-dependent enhancer manifesting 75% of the activity
of the TK-ERE-Luc control when assayed in the presence of
17
-estradiol. As observed previously, with ERF-3, all three
distinct classes of ER antagonists can effectively inhibit
17
-estradiol-induced activation of the BCER-1-derived element when
present in a 10-fold molar excess (Fig. 3B). Thus, we
have independently identified another Alu sequence from a
different source of genomic DNA which can function as an independent
enhancer in the presence of estradiol-activated ER. The Alu family of repeat sequences can no longer be considered to be
functionally inert, but in fact can function as efficient ER-dependent
enhancers, the efficacy of which is influenced by promoter context.
Figure 3:
An Alu element within the BRCA-1
locus confers estrogen responsiveness to a heterologous promoter when
assayed in mammalian cells. A, the transcriptional activity of
the Alu element (BCER-1) within the BRCA-1 locus was accessed
in HepG2 cells following co-transfection with an expression vector for
ER. Two individual clones from the same Alu element are shown.
Elements were tested in the presence and absence of 17-estradiol
as indicated. B, effect of ER antagonists on
17
-estradiol-mediated activation of BCER-1. Experimental setup is
as described previously in Fig. 2.
The Alu family of DNA repeats represents approximately 5%
of the total mass of the human genome (500,000 copies)(22) ; as
such, it is unlikely that all Alu sequences function as
ER-dependent enhancers. However, the two estrogen-responsive enhancers
which we have identified within ERF-3 and BCER-1 contain specific base
changes which distinguish them from Alu sequences which are
more abundantly distributed throughout the genome. The sequence
comparisons between the Alu elements identified in our screens
and the consensus sequences of major subclasses of Alu repeats
are shown in Fig. 4A. These consensus Alu elements are approximately 282 bp in length, of which 49 bp are
shown (22) . The PS (primate-specific) consensus sequence,
which is likely to represent several of the oldest Alu subfamilies, describes the majority of Alu members(22, 23) . The AS (anthropoid-specific)
subfamily, which evolved next, can be identified by a diagnostic
mutation indicated by the dashes representing a deletion of an A and G
residue. The latest classes to evolve were the CS (catterhine-specific)
and HS (human-specific), which are identified by specific mutations
represented by nucleotide substitutions. The older PS subclass is
distinguished from the later CS and HS subclasses by an additional
mutation in the first half-site of the ERE palindrome which converts
the sequence GGCCA (PS and AS) to AGCCG (CS and HS). Thus, by this
nomenclature, ERF-3 belongs to the AS subclass whereas BCER-1 is a
member of the PS subfamily of Alu repeats. Potential
estrogen-responsive sequences are underlined from which a consensus Alu ERE was derived
(5`-GGTCAnnnTGGTC(n)TGACC-3`). More
importantly, however, ERF-3 and BCER-1 differ from the consensus
sequences of the 4 major classes of Alu repeats by a T residue
which exists in the left arm of the imperfect palindrome within ERF-3
and BCER-1 instead of a C residue which is found in all the major
classes of Alu repeats. Additionally, the same base change
appears again in ERF-3; however, because of an additional deletion (AG, Fig. 4A), it is deemed to be a member of
the AS Alu family. It is not clear whether this activating
base change was present before amplification of several independently
evolved subfamilies or whether these represent independent mutations
occurring in subfamily members after integration. However, this change
is quite common in several of the older
subfamilies(23, 24) , and it seems likely that some
form of selection for this base change must have occurred otherwise
parallel subgroups of several major subfamilies would be needed to
explain its abundance.
Figure 4:
Estrogen receptor-dependent enhancer
activity is not an inherent property of all Alu sequences. A,
partial sequence (in the antisense orientation) from four main
subfamilies of Alu are shown along with the corresponding
sequence of ERF-3a and BCER-1. Dots represent homologous
sequence, dashes correspond to deletions. Nucleotide
substitutions are indicated. Underlined sequences correspond
to putative EREs from which a consensus Alu ERE was derived. B, the transcriptional activity of the four Alu subfamily consensus sequences were accessed in HepG2 cells in the
presence of co-transfected ER. Each subfamily consensus was subcloned
into pBL-TK-Luc and tested for ER-dependent enhancer activity in the
presence or absence of 17-estradiol as indicated. The methodology
and data presentation were performed as described in Fig. 2.
Having determined that the Alu sequences contained within ERF-3 and BCER-1 differed from the consensus Alu sequence by either one (PS and AS), two (CS), or three bases (HS) within the 5` half-site within the putative ERE sequence, it was important to show that the consensus Alu sequences would not function as ER-dependent enhancers. Consequently, we cloned the individual Alu consensus sequences into TK-Luc and examined their ability to confer estrogen responsiveness upon a heterologous promoter in HepG2 cells in the presence of expressed ER (Fig. 4B). The results shown indicate that neither consensus permits ER-dependent enhancer activity. We conclude, therefore, that the Alu sequences within ERF-3 and BCER-1, by acquiring specific point mutations, have evolved from the AS and PS family of repeats enabling them to function as ER-dependent transcriptional enhancers. In addition, the more recently made CS and HS Alu sequences are less likely to give rise to ER-dependent enhancers due to the acquisition of additional mutations within the 5` half-site of the putative ERE sequence.
Although no
specific function for Alu DNA repeats have been determined,
their physical insertion into the coding regions of NF-1 (25) and factor IX (26) can disrupt gene function and
lead to neurofibromatosis and hemophilia, respectively. In addition, Alu repeats have also been implicated as possible sources of
protein variability(27) . These events, however, are rare and
do not reflect an intrinsic function of Alu repeats. Our
evidence, which indicates that a subclass of Alu sequences
possesses ER-dependent enhancer activity suggests that the presence of
an Alu element in a gene can, in addition to altering the
architecture of the gene, confer upon it estrogen responsiveness.
Although our screens have looked specifically for estrogen-induced
enhancers, we believe due to the sequence similarity of most hormone
response elements that there may exist other classes of Alu elements which posses enhancer sequences which would respond to
other nuclear hormone receptors. Specifically, mutations within the
putative Alu ERE could give rise to vitamin D or retinoic acid
receptor-dependent enhancers which can operate through direct repeats
of the GGTCA sequence(28, 29) . It is important to
note that both the ERF-3 and BCER-1 Alu sequences are from the
oldest subfamilies of Alu sequences and therefore are likely
fixed in the human genome. Only the most recent members representing
the HS and CS subclass are considered competent to move about the
genome by retroposition(22) . Therefore, estrogen-inducible
enhancers represented by Alu sequences of the class we have
identified are not likely to move around the genome except by
recombination. Interestingly, Alu-mediated recombination
within the low density lipoprotein receptor gene resulting in a form of
familial hypercholesterolemia has been found(30) . Other events
of recombination leading to insulin-dependent diabetes (31) and
adenosine deaminase negative severe combined immunodeficiency syndrome (32) have also been reported. We have performed searches of the
available sequences in the GenBank and have found a number
of perfect matches to this class of Alu elements within the
regulatory regions of known genes. One of the most interesting is
within the promoter of the hepatic lipase gene (GenBank
accession number M35426), a suspected target of estrogen
action(33) . The finding that Alu sequences can
function as ER-dependent enhancers will surely impact the way we think
about what is often considered functionally inactive DNA.