(Received for publication, May 18, 1995; and in revised form, August 9, 1995)
From the
Steroid hormone receptors regulate mouse mammary tumor virus (MMTV) gene expression by binding to hormone response DNA elements present in the long terminal repeat. Tissue-specific expression of MMTV is unlikely to be regulated by steroid hormone-receptor complex alone, and mammary cell-specific factors might play a role in the hormone-induced transcriptional activation. In this report we have investigated the function of a novel cis-acting element designated Kil (-204 to -188) which is located adjacent to the distal glucocorticoid response element, in steroid hormone-induced transcription of MMTV. Electrophoretic mobility shift assays indicate that cellular factors bind to the Kil element, and dexamethasone stimulation results in alterations in the binding pattern of proteins in this region. By transient transfection assays using wild type and deletion mutants of the Kil element, we show that this novel cis-acting element is necessary for hormone-induced transcription of MMTV and functions in mammary tumor cells but not in NIH/3T3 cells. Mutagenesis of the Kil sequence suggests that the entire Kil element functioning as one unit is necessary for hormone-induced transcription of MMTV. When placed in the context of heterologous promoters, neither Kil element nor glucocorticoid response element is able to induce significant hormone-induced transcription of MMTV. The presence of both the DNA elements in tandem results in optimal induction of transcription in the presence of steroid hormones. Our results also indicate that the Kil element functions in human breast carcinoma cell lines such as T47D and MCF-7. These results suggest that Kil element in combination with distal glucocorticoid response element functions as a mammary cell-specific enhancer to regulate MMTV transcription.
Steroid hormone receptors are cytoplasmic proteins, which, upon
binding to their ligands, translocate into the nucleus and bind to DNA
elements in a sequence-specific manner and modulate the transcription
of cellular genes(1) . Transcription from the long terminal
repeat (LTR) ()of mouse mammary tumor virus (MMTV) has been
used extensively to understand the regulation of gene expression by
steroid hormones. Glucocorticoids, progesterones, and androgens induce
the transcription of MMTV (2, 3, 4) upon
binding of their respective receptors to the hormone response elements.
These elements are located between -202 and -59 bases
upstream of the transcription initiation site in the MMTV promoter,
which can be divided into the proximal and distal glucocorticoid
response elements (GREs). The GRE consists of four repeats of the
hexanucleotide 5`-TGTTCT-3` to which the steroid hormone receptors were
shown to bind and exert their effects on transcription. The proximal
glucocorticoid responsive element (GRE) contains three repeats of the
hexanucleotide, while the distal GRE (-188 to -170)
contains only one of the hexanucleotide.
Apart from steroid
hormone-receptor complex, which is a major determinant of MMTV
transcription, cellular factors have been implicated in determining
this tissue specificity(5, 6, 7) . Growing
evidence suggests that there exists functional interaction between
hormone receptor molecules and other cellular transcription factors. A
direct physical interaction of glucocorticoid receptor (GR) with
transcription factors such as the p65 subunit of NF-B(8) ,
Ets related factor(9) , octamer transcription factor
1(10) , Spi-1(11) , and SWI1 proteins (12) have
been shown to modulate glucocorticoid receptor function. Transcription
factors such as NF-1 (13) and Oct-1 (14, 15) which bind close to proximal GRE sites seem
to modulate the MMTV promoter transcription. The glucocorticoid
receptor bound to the distal GRE site (-186 to -170) of the
MMTV LTR was found to cooperate with other binding sites to which
transcription factors such as NF-1, octamer transcription factor
(Oct-1), SP1, and CACCC box binding factor (15, 16, 17) were shown to bind. This
synergism between the GRE and other cis-regulatory elements
has been found to be dependent on the nature of the cells used,
suggesting that different nuclear factors present in different cell
types contribute to the tissue-specific expression of MMTV. In this
respect, it is interesting to note that a majority of MMTV proviruses
that cause T-cell lymphomas have alterations in the sequence near the
distal
GRE(18, 19, 20, 21, 22) ,
and these deletions or alterations seem to confer increased levels of
LTR-directed transcription even in the absence of glucocorticoid
hormones. It has been suggested that these deletions might result in an
MMTV that allows preferential viral expression in T-cells (23) in the absence of hormonal stimulation. In addition, MMTV
provirus DNA isolated from a kidney adenocarcinoma also contains
alterations in this region(24) . Thus, an alteration in the U3
region (-350 to -186) of the viral LTR appears to alter the
target cell specificity suggesting that this region might contain
sequence elements for binding of mammary cell-specific factors.
In this report, we describe the identification of a novel cis-acting regulatory sequence within the MMTV promoter which is subject to frequent alterations in mutant MMTV proviral DNAs found in T-cell and kidney tumors. This sequence, designated as Kil, is located between -204 and -188 of the LTR, adjacent to the distal GRE. Electrophoretic mobility shift assays of the probes derived from this region indicate that cellular factors bind to this region and that the binding is modulated by the action of steroid hormones. Functional analysis by transient transfection assays in mouse and human cell lines indicates that this sequence is essential for hormone-dependent transcriptional activation of MMTV promoter. Hence, the distal GRE, in combination with the Kil site, function as an inducible enhancer.
The following is the sequence of various mutants of the Kil region in MMTV promoter confirmed by nucleotide sequencing.
CCCAAATTTATTCAAAT
Wt-Kil
GGGCCCTTTATTCAAAT
M1-Kil
CCCAAATTTATTGATCA
M2-Kil
GGGCCCTTTATTGATCA
M1.2-Kil
CCTGATCATATTGATCA
Mu-Kil
The following is the sequence of oligonucleotides cloned at the HindIII and XbaI sites of E1b-CAT vector so that these can be end labeled by Klenow enzyme and used in the mobility shift assay experiments.
3` AGGGTCCCAAATTTATTCAAATACAGATC 5`
Kil sequence
3` ACAATGTTTGACAAGAATTTTGCGAGATC 5`
GRE sequence
5` AGGGTCCCAAATTTATTCAAATTACCAATGTTTGACAAGAATTTTGCGAGATC 3`
Kil-GRE sequence
Figure 1: A, schematic representation of the mouse mammary tumor virus long terminal repeat sequences. Three proximal GRE (-120 to -89) and the distal glucocorticoid response elements (-186 to -170) are indicated. The binding sites for NF-1 and Oct-1 sites near the TATA box are shown. The region which is subject to frequent changes in variant MMTV proviruses associated with T-cell and kidney tumors is shown by arrows. The Kil sequence (-204 to -188) and distal GRE sequences are shown below. B, electrophoretic mobility shift assay of Kil, GRE, and Kil-GRE probes with whole cell extracts from GR cells, uninduced or induced by dexamethasone. Lane 1 indicates the labeled Kil probe alone; lane 2 represents the Kil probe incubated with whole cell extract derived from dexamethasone-induced GR cells, while lane 3 shows uninduced GR cells. Lane 4 represents GRE probe alone, while lanes 5 and 6 represent GRE probe incubated with dexamethasone-induced and uninduced extracts. Lane 7 represents free Kil-GRE probe, while lanes 8 and 9 show Kil-GRE probe incubated with dexamethasone-induced and uninduced whole cell extracts, respectively. Note the altered slower mobility of complex B (induced extract) compared to complex C (uninduced extract).
Figure 2: A, schematic representation of the wild type MMTV-CAT and Kil deletion mutant used in the transactivation experiments. The wild type plasmid (Wt) contains MMTV LTR sequences from -421 to +126 base pairs linked to the CAT gene. In the mutant MMTV-CAT, the Kil sequences (-204 to -188) are deleted. B, deletion of Kil sequence affects the dexamethasone-stimulated transcription of MMTV in mouse cells. GR cells were transfected with wild type and mutant (Kil-deleted) MMTV-CAT, and the CAT activity was assayed 36 h after transfection. Wt represents the wild type MMTV and Mt the deletion mutant (-204 to -188). GR is the mouse mammary carcinoma cell line.(-) and (+) represent without and with dexamethasone treatment. C, dexamethasone is unable to induce MMTV-CAT transcription in NIH/3T3 cells. NIH/3T3 cells were transfected with wild type and mutant (Kil-deleted) MMTV-CAT, and the CAT activity was assayed 36 h after transfection. Wt represents the wild type MMTV and Mt the deletion mutant (-204 to -188).(-) and (+) represent without and with dexamethasone treatment.
To examine whether the observed transcriptional activation is tissue-specific, we transfected wild type and mutant vectors into NIH/3T3 cells and studied the induction of CAT activity in the presence and absence of dexamethasone. Results presented in Fig. 2C show that dexamethasone treatment of NIH/3T3 cells fails to induce MMTV transcription beyond the basal levels (Fig. 2C, lanes 2 and 3). When the Kil sequence was deleted, considerable reduction in the basal levels of CAT activity was observed (Fig. 2C, lanes 4 and 5) which did not increase with hormone treatment. These results suggest that dexamethasone might induce or associate with cellular factors in GR cells but not in NIH/3T3 cells. Absence of hormone induction in NIH/3T3 cells can be attributed to the absence or low levels of steroid receptors in this cell line or it could be due to the absence of specific cellular factor(s) that are induced by dexamethasone. It is possible that dexamethasone-inducible factors are expressed in mammary cell lines but not in fibroblasts.
Figure 3:
A, steroid hormone-induced MMTV
transcription is dependent on the progesterone receptor content in the
human breast carcinoma cell line T47D. Transient transfection and CAT
assays were performed after transfecting the wild type and mutant
MMTV-CAT constructs into the T47D cell line. D and P represent the cells treated with dexamethasone and progesterone,
respectively. Mt represents the mutant MMTV-CAT in which the
Kil sequences are deleted. CAT activity in cells transfected with
-galactosidase expression vector alone was taken as one unit and
the fold activity was calculated for comparison. B, steroid
hormones induce MMTV transcription in MCF-7 cells. Wild type and Kil
deletion mutants of MMTV-CAT plasmids were transfected into MCF-7 cells
and induced with dexamethasone (D) and progesterone (P) and assayed for CAT activity.
To test if a similar kind of synergism exists between Kil binding proteins and the GR, we have used MCF-7 cells which express significant levels of all the steroid receptors. Wild type and mutant MMTV-CAT constructs were transfected into MCF-7 cells, and the ability of progesterone and dexamethasone to induce MMTV transcription was assayed. In the absence of hormone stimulation, low levels of CAT activity was observed (Fig. 3A, lane 2) and treatment of cells with dexamethasone or progesterone had a strong stimulatory effect on CAT activity (Fig. 3A, lanes 3 and 4). This activity was totally abolished in mutant MMTV-CAT constructs which lacked the Kil sequences (Fig. 3B, lanes 5, 6, and 7). Our results using progesterone and dexamethasone suggest that these hormones activate MMTV transcription and that Kil sequences are essential for this activity in human cell lines and are dependent on the steroid receptor content in the cells(38) . The results indicate that proteins binding to Kil sequences modulate the steroid receptor function and regulate MMTV transcription. It is interesting to note that the basal activity which was detectable in mouse cells, such as GR and NIH/3T3 cells with mutant MMTV-CAT (Kil deleted), could not be detected easily in human breast carcinoma cells.
Figure 4: A, mutations in the Kil sequence affects the steroid hormone-induced transcription of MMTV in human adenocarcinoma cell line MCF-7 cells. After introducing the mutations in the Kil sequence, the mutants were subcloned at HindIII and XbaI sites of MMTV-CAT and used in the transient transfection experiments in MCF-7 cells. Wt represents the unaltered Kil sequence. Mt represents the Kil deletion (-204 to -188) mutant. M1 represents the mutations in the 5` part of the Kil sequence in which the GGGTTT sequence is changed to CCCGGG. M2 indicates the mutant in which the (Oct-1 like) sequence AGTTTA is mutated to ACTAGT. M1.2 represents the presence of mutations in both the 5` part of the Kil sequence and the Oct-1-like sequence (a combination of both M1 and M2 mutants). Mu represents the presence of an unrelated sequence in place of Kil. Transient transfections were performed in the presence of dexamethasone and progesterone along with the control. B, schematic representation of the fold increase in the CAT activity of various mutants represented in A. The fold increase in CAT activity was calculated in comparison to the untransfected MCF-7 cells as a control which is equivalent to 1. The first set represents the basal activity of various constructs without any hormone treatment. Lanes 2-7 indicate the wild type MMTV-CAT, M1, M2, M1.2, and Mu, respectively. Different hormones used for induction are indicated at the bottom of each set. The fold activity is the average of three independent experiments.
Figure 5: A, schematic representation of the E1b TATA-CAT vector in which the distal GRE and the Kil sequences are cloned at the HindIII and XbaI sites. CAT represents the E1b promoter driving the expression of CAT. Kil-GRE-CAT represents E1b CAT containing both Kil and GRE cloned upstream of E1b CAT at HindIII and XbaI sites. Kil-CAT represents E1b CAT containing only Kil sequences in E1b CAT whereas GRE-CAT indicate only GRE sequences cloned upstream of E1b-CAT at HindIII and XbaI sites. B, synergistic effect of the Kil and GRE sites of the MMTV promoter on the activation of a heterologous promoter E1b-CAT. 10 µg of each construct was transfected into GR cells and later on were subjected to hormone induction in the absence (lane 2) or presence of dexamethasone alone (lane 3) or serum alone (lanes 4, 6, 8, and 10) or dexamethasone and serum (lanes 5, 7, 9, and 11) and harvested after dexamethasone induction. D represents dexamethasone and S represents serum, SD represents serum plus dexamethasone.
In this communication, we have identified a novel cis-acting element in the MMTV promoter (5`-GGGTTTAAATAAGTTTA-3`) positioned at -204 to -188, adjacent to the distal glucocorticoid response element (GRE). This sequence, designated as the Kil element, is necessary for hormone-induced transcription of MMTV. Mobility shift assays show that the Kil element is a novel binding site for multiple nuclear factors and is distinct from the GRE site. In addition, our results show that distal GRE in combination with Kil functions as a hormone-inducible enhancer of MMTV transcription in mouse and human cell lines. Comparison of wild type MMTV LTR sequences with variant proviral MMTV LTR isolated from mouse kidney carcinoma indicate that sequences that encompass the Kil site are frequently altered in this region(24) . Frequent alterations or deletion of the Kil sequence was also observed in GR T-cell leukemia-42 and in several T-cell lymphomas that develop in GR mice(18, 19, 20, 21, 22, 35) . While the significance of these alterations is at present unclear, this change in the sequence appears to allow the expression of MMTV promoter-regulated genes in tumors in the absence of steroid hormone stimulation(19) . Our results show that this deleted region contains a cis-acting element which functions in cooperation with distal GRE and confers hormone responsiveness to the MMTV promoter in mammary cell lines.
Purified glucocorticoid receptor has been shown to bind to distal GRE and does not bind to the Kil sequence(33, 34) . DNase footprint experiments by other workers with nuclear extracts show protection of the area from -206 to -170 which consists of the Kil sequence and the distal GRE. To understand the DNA-protein interactions in this region, we have performed mobility shift experiments with whole cell extracts made from unstimulated and dexamethasone-stimulated GR cells using oligonucleotides spanning this region. Our results indicate that an oligonucleotide which contains both the distal GRE and Kil motifs forms a novel protein-DNA complex whose mobility is altered upon dexamethasone stimulation. It is possible that dexamethasone stimulation results in the association of GR with pre-existing or newly synthesized proteins and bind to Kil-GRE. Some of the proteins might be bridging between glucocorticoid receptor occupying the GRE and Kil binding proteins binding to Kil sequences. It is possible that the Kil element is responsible for binding of more than two proteins. This is supported by our mutational data which indicate that the Kil element consists of at least two or three binding sites. The sequence of the 5` end is not similar to any known DNA binding site and is therefore most likely occupied by a novel enhancer binding protein. The central binding site is A/T-rich and is similar to the recognition elements for myocyte-enhancer binding factor 2 (MEF2) or related serum response factor or a closely related protein(39, 40) , but expressed in mammary tissue. The 3` sequence resembles the binding site for Oct-1. These results suggest that multiple proteins might bind to the Kil element and regulate MMTV transcription. Steroid hormone stimulation results in alterations in the binding activity of any of these proteins, and the association of GR with these proteins might result in a multiprotein complex that exerts a strong regulatory influence on induced transcription of MMTV. The identity of the components of this multiprotein complex will provide further information on the protein-protein interactions between the GR bound to the distal GRE and Kil binding proteins.
When the GRE probe was incubated with whole cell extracts, one complex was observed, as the glucocorticoid receptors in the extract bind to the GRE sequence with high affinity. When the Kil site was used as a probe, we could not detect any complex formation, indicating that GR does not bind to the Kil site by itself. This is consistent with an earlier observation that purified GR does not bind to the Kil motif (33, 34) . However, it is interesting to note that some of the nuclear factors that bind to Kil can be competed away with unlabeled GRE (data not shown) suggesting that GR can bind to the Kil site, most likely in association with other nuclear factors. In fact, one such protein, SWI3, has previously been shown to interact with glucocorticoid receptors through its DNA binding domain(12) . This observation provides the possibility that glucocorticoid receptors can interact with unrelated DNA sequences in association with other DNA-binding factors. It is possible that dexamethasone stimulation results in hyperphosphorylation of the glucocorticoid receptors which in turn associate with a different set of transcription factors and bind to the Kil element. Support for this hypothesis comes from the observation that glucocorticoid receptors are hyperphosphorylated in hormone-treated cells(41) .
To understand if alterations in the binding activity near the Kil-GRE has a functional significance, we have systematically analyzed the function of the Kil sequences in hormone-induced transcription of MMTV. Linker scanning mutations carried out by Cato et al.(4) across the length of the MMTV promoter indicated that the region surrounding the distal GRE is important for glucocorticoid and androgen response in T47D cells. When part of this sequence, 5`-GGGTTTAAA-3`, was converted into 5`-CCCGGGAAA-3`, these mutations decreased the glucocorticoid and androgen but not the progesterone response. In addition, earlier studies had indicated that the 5` part of the Kil motif, 5`-TTTAAA-3`, is the binding site for a factor(s) present in MCF-7 cells(42) . In view of these observations and based on our preliminary results, we created mutations in the Kil sequence and performed transient transfections in MCF-7 cells. Our data suggest that the Kil binding site might be composed of at least two or three binding sites (Oct-1 and MEF2). It is possible that the Kil element might be responsible for binding of multiple factors as a complex. The 5` part might serve as a binding site for an unidentified protein. The middle portion of the Kil sequence (TTTAAATAAG) is similar to the MEF2 site (CTTTAAATAA) present in muscle gene promoters(39) . In addition, a TA-rich binding site, TATAAATA (distinct from the classical TATA box), and a TA-rich binding protein (TARP) has been shown to activate muscle and brain creatine kinase promoters(43) . It is possible that MEF2 or MEF2-like proteins or related serum response factors (40) present in mammary epithelium bind to Kil sequences. We are in the process of testing whether any of these proteins bind to Kil sequences and also interact with the steroid receptors. Mutations in the octamer sequence also drastically affect hormone-stimulated transcription, suggesting that the sequence integrity of the entire Kil element is essential for basal and hormone-stimulated transcriptional activity of the MMTV promoter. It is possible that more than two proteins might be binding to the Kil element and function as a multiprotein complex as observed with the Ban2 enhancer(44) .
The function of the Kil element is consistent with a model of Enhanson organization(45) : a tissue-specific regulatory unit consisting of many regulatory elements to create a unique function. An enhanson contains multiple levels of organization that permits a high degree of transcriptional regulation through a combinatorial binding with a limited set of transcription factors. The octamer family of transcription factors has been shown to be necessary for promoter and enhancer activity of a variety of genes and in tissue-specific expression of cellular genes. We propose that the octamer-like motif present as part of the Kil sequence near the distal GRE may be involved in tissue-specific and hormone-induced expression of MMTV in combination with other Kil-binding proteins. We propose that a combination of GRE with the Kil element acts as an inducible enhancer and functions synergistically with proximal promoter for tissue-specific expression of MMTV.
Taken together, our results suggest the presence of a novel regulatory element mediating transcriptional activity of MMTV promoter by steroid hormone receptors. Since MMTV induces mammary tumors by activating wnt genes and that this transcriptional activation is achieved through the enhancer element(s) in the LTR of the integrated proviruses(46, 47) , we speculate that the Kil enhancer element might contribute significantly to the activation of the wnt genes during the induction of mouse mammary tumors. Further analysis of the Kil binding proteins should provide valuable insights into our understanding of tissue-specific expression of MMTV.