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Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom
Received 8 November 2004/ Returned for modification 2 December 2004/ Accepted 21 January 2005
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Oct-1 is a ubiquitously expressed POU domain transcription factor that regulates transcription via an octamer element [ATGC(A/T)AAT] found in the promoters and enhancers of a diverse range of genes (23, 29, 57, 62). The POU domain is a conserved bipartite DNA-binding domain consisting of two subdomains, a POU-specific domain and a homedomain, separated by a flexible linker. Oct-1 regulates the expression of both ubiquitously expressed and cell type-specific genes. The ability of Oct-1 to differentially regulate genes can be explained by its combinatorial interactions with other transcriptional regulators on individual promoters. Such regulators can be promoter-selective coactivators, such as HCF, Bob-1, VP-16, and the SNAP complex, or DNA-binding transcription factors, such as GR, androgen receptor, and STAT5 (10, 11, 23, 35, 47, 48).
While Runx2 has largely been regarded as a bone-specific transcription factor, it is also expressed in mammary epithelial cells (3, 4, 25, 45, 53, 54). Oct-1 is also expressed in mammary epithelial cells and has been implicated in the regulation of the mammary gland-specific gene ß-casein (21, 69, 70). The ß-casein gene is an established paradigm for the study of mammary gland-specific gene expression (2, 9, 13, 14, 19, 34, 49, 63, 64). ß-Casein is a milk protein whose expression is induced by hormones during lactation. Three essential regulatory elements have been identified in the promoter of the ß-casein gene (2, 9, 13, 19, 34, 49, 63, 66). Two of these elements, termed block A and block B, have been well characterized and shown to mediate transcriptional activation via STAT5 and GR (9, 13, 19, 34, 59, 63, 66). In contrast, less is known about the molecular mechanism by which the third essential element, block C, contributes to ß-casein expression. Block C recruits a nuclear protein complex in mammary epithelial cells, the formation of which is dependent upon an octamer consensus sequence which recruits Oct-1 (49, 52, 66, 69, 70).
Here we show that block C is actually a composite element consisting of a consensus Runx-binding site adjacent to an octamer sequence. We demonstrate that Runx2 is required for the activation of ß-casein transcription via the Runx-binding site and that Runx2 and Oct-1 form a novel complex on the Runx/octamer element. Analysis of the complex revealed autoinhibitory domains for DNA binding in both the N-terminal and the C-terminal regions of Runx2. Oct-1 stimulates the recruitment of Runx2 to the ß-casein promoter by interacting with the C-terminal region of Runx2. A model is proposed in which Oct-1 stimulates Runx2 recruitment by relieving the autoinhibitory function of the Runx2 C-terminal region.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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EMSAs.
Oligonucleotides were radiolabeled with [
-32P]dCTP by using the Klenow fragment according to standard protocols (50). The following oligonucleotide sequences were used to investigate the binding of Runx2 and Oct-1 to the ß-casein promoter: Runx, 5'-CTAGAACTGACCGCAGCTGGCCGT-3' (7); Runxß-casein, 5'-CTAGTTACAAACCACAAAATTA-3'; MutRunxß-casein, 5'-CTAGTTACAAAGGAGAAAATTA-3'; octamer, 5'-GATCCTTAATAATTTGCATACCCTCA-3'; and Runx/octamer+10 bp, 5'-CATGACAAACCACAATGACTTACGAAATTAGCATGTCA-3'. The sequences of the other oligonucleotides used in electrophoretic mobility shift assays (EMSAs) are denoted in the figures. EMSAs were performed as previously described (25).
Plasmid constructs. The ß-casein promoter was amplified from genomic DNA derived from HC11 cells by nested PCR with the following oligonucleotides. First-round amplification primers were mouse ß-casein P1 (5'-ATCTTTGATGTTTCCATATC-3') and mouse ß-casein P2 (5'-CTTTAGTGGAGGACAAGAGA-3'), and second-round amplification primers were mouse ß-casein P3 (5'-GATCGAGCTCTGCTTCATAACTGAGGTTAAAG-3') and mouse ß-casein P4 (5'-GATCAAGCTTCTGAAAGGATGATGTCCTATC-3') (66). The resulting 360-bp fragment was ligated to the SacI and HindIII sites of the pGL3-Basic firefly luciferase reporter vector (Promega). The Runx-binding site in the ß-casein promoter was specifically mutated by using a QuikChange mutagenesis kit (Stratagene) according to the manufacturer's protocol with the oligonucleotides mousePMTOP (5'-GAGTATCTTACAAAGGAGAAAATTAGCATGTC-3') and mousePMBOT (5'-GACATGCTAATTTTCTCCTTTGTAAGATACTC-3'). The octamer-binding site in the ß-casein promoter was specifically mutated by using oligonucleotides ß-caseinOctMut P1 (5'-CAAACCACAAAATTCGCCGGTCATTAAGTG-3') and ß-caseinOctMut P2 (5'-CACTTAATGACCGGCGAATTTTGTGGTTTG-3').
The pRunx2i expression vector was previously described (25). Runx2 and AML-1/ETO were expressed by using pCMV-OSF2 and pCMVAML-1/ETO, respectively (16, 38). Oct-1 and POU were expressed by using pCDNA3-HA-Oct-1 and pCDNA3-HA-POU, respectively (55). The Runx2 deletions were created by PCR with pRK-Flag-Cbfa1 as a template. Details of the oligonucleotides used can be provided on request (1). The PCR products were ligated to the EcoRI and XbaI sites of pRK5. The integrity of the plasmid sequences was confirmed by sequencing.
Protein production. Wild-type and truncated human Oct-1-glutathione S-transferase (GST) fusion proteins were created by PCR with pCDNA3-HA-Oct-1 as a template. The products were ligated to the XhoI and XbaI sites of pGEX-KG. Proteins were expressed by using a TNT kit with SP6 polymerase according to the manufacturer's instructions (Promega). GST protein purification and pull-down assays were performed as described previously (56). The Runt domain was expressed in Escherichia coli as a GST fusion protein from plasmid pGEX-AML-1Runt and purified as described previously (7, 67a).
Cell cultures and transfections. HC11 and UMR106 cells were maintained as previously described (25). HC11 cell lines were transfected in either 6- or 24-well plates by using Lipofectamine 2000 transfection reagent (Invitrogen) and a total of 2 or 0.8 µg of DNA, respectively, as previously described (25). Transfection of HC11 cells that were cultured in the presence of lactogenic hormones was performed as follows. HC11 cells (1.5 x 105) were placed in a 24-well dish. After 16 h, RPMI medium containing 10% fetal bovine serum (FBS), gentamicin (50 µg/ml), bovine insulin (5 µg/ml), prolactin (5 µg/ml), and dexamethasone (0.1 µM) was added to the cells. After 24 h, the cells were transfected with a total of 800 ng of DNA containing 360 ng of the reporter plasmid being investigated and 40 ng of pRLSV40. At 5 h after transfection, the transfection mixture was removed and replaced with RPMI medium containing 10% FBS, gentamicin (50 µg/ml), bovine insulin (5 µg/ml), prolactin (5 µg/ml), and dexamethasone (0.1 µM). At 54 h after transfection, the cells were lysed, and luciferase activity was determined by using a dual-luciferase reporter assay (Promega).
Transfection of HC11 cells with pRunx2i was performed as follows. HC11 cells (3 x 105) were placed in a 24-well dish and cultured with RPMI medium containing 10% FBS, gentamicin (50 µg/ml), bovine insulin (5 µg/ml), prolactin (5 µg/ml), and dexamethasone (0.1 µM). After 24 h, the cells were transfected with a total of 800 ng of either pSUPER or pRunx2i (8, 25). At 5 h after transfection, the transfection mixture was removed and replaced with RPMI medium containing 10% FBS, gentamicin (50 µg/ml), bovine insulin (5 µg/ml), prolactin (5 µg/ml), and dexamethasone (0.1 µM). At 48 h after transfection, the cells were transfected for a second time with a total of 1 µg of DNA. Each transfection mixture contained 360 ng of the reporter plasmid being investigated, 40 ng of pRLSV40, and 600 ng of either pSUPER or pRunx2i. The cells were lysed at 48 h after the second transfection, and luciferase activity was determined.
All transfections were performed in triplicate, and data are presented as means and standard deviations (SDs). Values are relative to the luciferase activity of pGL3-Basic.
To obtain nuclear extracts from cells that had been transfected with either pSUPER or pRunx2i, the first transfection of the HC11 cells was the same as that described above. At 48 h after transfection, the cells were transfected for a second time with a total of 1 µg of DNA. Each transfection mixture contained 400 ng of pGFP and 600 ng of either pSUPER or pRunx2i. At 24 h after the second transfection, the cells were sorted by fluorescence-activated cell sorting; viable green fluorescent protein-expressing cells were collected and used to prepare nuclear extracts.
ChIP assays. Chromatin immunoprecipitation (ChIP) assays were performed by using a ChIP assay kit (Upstate Biotechnology) according to the manufacturer's protocol, except that formaldehyde cross-linking was performed for 30 min. Complexes were immunoprecipitated with 0.8 µg of anti-Runx2 antibody (Oncogene), anti-Oct-1 antibody (Santa Cruz), or anti-Flag antibody (Sigma). The sections of the ß-casein promoter containing the Runx- and octamer-binding sites were detected by PCR with the primers mouse ß-casein P5 (5'-TAGAATTTCTTGGGAAAGAC-3') and mouse ß-casein P2 (see above).
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-, and 
-caseins from a wide range of species (Fig. 1B).
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To determine which Runx factor(s) binds to the ß-casein promoter, nuclear extracts were incubated with the Runx-binding site in the presence of antibodies specific for individual Runx factors (Fig. 1D). Nuclear extracts produced two bands when analyzed by EMSAs. In the presence of anti-Runx2 antibodies, the lower of the two bands was abolished and a supershifted complex was formed (Fig. 1D, lane 3). The Runx1- and Runx3-specific antibodies did not result in a specific supershift The data demonstrate that endogenously expressed Runx2 is able to bind to the Runx-binding site in the ß-casein promoter.
To establish whether endogenous Runx2 is required for the activity of the ß-casein promoter, cells were transfected with either the wild-type ß-casein reporter or the Runx-binding site mutant reporter in the presence of pRunx2i, which expresses Runx2-specific small interfering RNA. When cotransfected with pRunx2i, the activity of the wild-type reporter plasmid was reduced by more than 50% (Fig. 1E). In contrast, pRunx2i had no effect on the activity of the Runx-binding site mutant reporter plasmid. The amount of Runx2 was significantly reduced in cells transfected with pRunx2i, as determined by supershift analysis with Runx2-specific antibodies (Fig. 1F). Taken together, the data demonstrate that both the intact Runx-binding site and Runx2 are required for full transcriptional activation of the ß-casein promoter.
Runx2 and Oct-1 form a complex on the Runx/octamer element. Having established that block C is a composite Runx/octamer element and that Runx2 is required for the activation of the ß-casein promoter, we next sought to determine whether a Runx2/Oct-1 complex forms on the ß-casein promoter. The Runx/octamer element was incubated with nuclear extracts in the presence or absence of competitor DNA (Fig. 2). In the absence of competitor DNA, three complexes were observed: C1, C2, and C3 (Fig. 2B, lane 1). Complexes C1 and C2 were abolished in the presence of unlabeled octamer DNA, indicating that these complexes contain an octamer-binding protein (Fig. 2B, lane 2). When the Runx sequence was used as a competitor, complexes C1 and C3 were abolished, indicating that both of these complexes contain a Runx protein (Fig. 2B, lane 3). All three complexes were absent in the presence of unlabeled Runx/octamer DNA (Fig. 2B, lane 4). This specific pattern of competition indicates that complex C2 contains an octamer-binding protein, C3 contains a Runx factor, and C1 contains both an octamer-binding protein and a Runx factor.
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To confirm that the endogenous ß-casein promoter is bound by endogenous Runx2 and Oct-1, ChIP assays were performed with Runx2- and Oct-1-specific antibodies. The ß-casein promoter could be amplified from immunoprecipitated complexes obtained with either anti-Runx2 or anti-Oct-1 antibodies but not with irrelevant anti-Flag antibodies (Fig. 2D). Runx2 and Oct-1 are therefore bound to the endogenous ß-casein promoter.
Overexpression of Runx2 and Oct-1 resulted in the activation of the ß-casein promoter to a greater extent than did that of either protein alone (Fig. 2E). The magnitude of activation was dependent upon both Runx and octamer sequences, since mutation of either sequence significantly inhibited promoter activity. Importantly, Oct-1 was still able to activate the promoter when the octamer sequence was intact and the Runx sequence was mutated, but maximum activity was reduced by more than 50%. In contrast, Runx2 was unable to activate the promoter when the Runx sequence was intact and the octamer sequence was mutated. Runx2 therefore requires an intact octamer element to contribute to the activity of the ß-casein promoter. Taken together, these findings suggest that Oct-1 can bind to and activate the promoter in the absence of Runx2 but that it cooperates with Runx2 to achieve maximum activation of the promoter. Runx2 therefore boosts the level of ß-casein promoter activity in an Oct-1-dependent manner.
Oct-1 stimulates the binding of Runx2 to the Runx/octamer element. The Runx2/Oct-1 complex was investigated further by using in vitro-translated Runx2 and Oct-1 in EMSAs (Fig. 3). Both proteins formed distinct complexes when incubated individually with the Runx/octamer element (Fig. 3A, lanes 2 and 3). In contrast, when both proteins were incubated together, they preferentially formed a distinct ternary complex (Runx2/Oct-1) in the presence of excess Runx/octamer DNA (Fig. 3A, lane 4). The formation of this ternary complex was dependent on the presence of both the Runx and the octamer sequences, since it was completely abolished when either sequence was mutated (Fig. 3A, lanes 8 and 12). This experiment also indicated that Oct-1 stimulates the recruitment of Runx2 to DNA. We therefore performed a titration experiment in which increasing amounts of Oct-1 were incubated with constant amounts of Runx2 and the Runx/octamer element (Fig. 3B). The amount of the ternary complex increased with increasing amounts of Oct-1 to a level greater than that observed with Runx2 alone (Fig. 3B). Hence, Oct-1 stimulates Runx2 recruitment to the Runx/octamer element. Increasing amounts of Runx2 also resulted in increasing amounts of the ternary complex (Fig. 4A). However, in this experiment, the amount of the ternary complex was only slightly larger than that observed with Oct-1 alone, demonstrating that Runx2 has only a minor effect on the binding of Oct-1 (Fig. 4A, compare lanes 2 and 7).
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Runx2 contains N-terminal and C-terminal autoinhibitory domains for DNA binding. To establish whether Oct-1 stimulates the recruitment of Runx2 via a direct interaction, we initially deleted the residues C terminal to the Runt domain and tested the ability of Oct-1 to stimulate the recruitment of the truncated Runx2 protein (Fig. 5A and B). Deletion of the C-terminal region to residue 292 did not prevent the efficient formation of a ternary complex with Oct-1. However, this truncated protein had a significantly increased ability to bind autonomously to DNA and formed a binary complex as efficiently as the ternary complex (Fig. 5B, lanes 6 and 7). In contrast, a smaller deletion to residue 462 did not affect the ability of Runx2 to bind autonomously to DNA; it was still able to form a stimulated Runx2/Oct-1 complex. These findings suggested that the C-terminal region of Runx2 contains an autoinhibitory domain for DNA binding between amino acids 292 and 462. More precise mapping of the autoinhibitory region established that truncation to residue 424 resulted in an observable increase in autonomous DNA binding, which was further enhanced upon deletion of additional C-terminal residues to amino acid 240 (Fig. 5C, even-numbered lanes). In contrast to their differential abilities to bind autonomously to DNA, all deletion proteins were able to form a ternary complex with Oct-1 as efficiently as wild-type Runx2 (Fig. 5C, odd-numbered lanes). Thus, the C-terminal region of Runx2 inhibits its DNA-binding function and is required for Oct-1-dependent recruitment to the Runx/octamer element.
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Taken together, these observations demonstrate that the C-terminal region of Runx2 is required for its Oct-1-dependent recruitment to the Runx/octamer element and that the major autoinhibitory domain for DNA binding resides in this region.
The C-terminal region of Runx2 interacts with Oct-1. To investigate whether Runx2 and Oct-1 interact, coimmunoprecipitation experiments were performed. We expressed the proteins in HeLa cells by using plasmids expressing Runx2 and HA-Oct-1. Runx2 could be coimmunoprecipitated with the anti-HA antibody, suggesting that Runx2 and Oct-1 interact directly (Fig. 7A). Further analysis of this interaction with GST pull-down assays demonstrated that wild-type Runx2 interacted with GST-Oct-1 (Fig. 7B). The interaction diminished progressively when GST-Oct-1 was incubated with a series of C-terminally truncated Runx2 proteins. Deletion of residues from the C terminus to amino acid 462 significantly reduced binding to Oct-1, and the interaction was almost completely abolished when the deletion in Runx2 was extended to amino acid 343 (C-343). Taken together, the data demonstrate that Runx2 interacts with Oct-1 predominantly via the C-terminal region of Runx2. While we could not define a discrete Oct-1 interaction domain, at least part of it resides within the same region as the autoinhibitory domain for DNA binding.
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Model of Runx2/Oct-1 complex formation. The crystal structures of the POU domain/DNA complex and the Runt domain/DNA complex were used to model the POU domain and the Runt domain bound to the ß-casein Runx/octamer element (Fig. 8). The model clearly shows that the two domains can bind to the adjacent sequences without contacting each other. The structure of the C terminus has not been resolved, and it is depicted as interacting with the Runt domain to block DNA binding. We propose that the inhibition of DNA binding is relieved by interaction of the Runx2 C-terminal region with the POU domain of DNA-bound Oct-1 (Fig. 8).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Regulation of ß-casein gene expression by the Runx2/Oct-1 complex. It is well established that the primary mechanism of activation of the ß-casein gene is via STAT5 and GR but it is also clear that the Runx2/Oct-1 complex is required (2, 9, 13, 19, 34, 49, 63, 64). We have not yet investigated the regulation of the Runx2/Oct-1 complex by signal transduction pathways but it is possible that both the formation and activity of the complex are regulated throughout the pregnancy cycle. Indeed, the activity of both proteins can be regulated by phosphorylation, which may be important in the context of ß-casein transcription (17, 20). Another potential role for the Runx2/Oct-1 complex is to act as a "landing pad" for other transcription factors. For example, both proteins have been shown to interact with GR, suggesting that the Runx2/Oct-1 complex may recruit GR (42, 48). Oct-1 was recently shown to interact with STAT5; if this interaction occurs on the ß-casein promoter, then the Runx2/Oct1 complex may be important in maintaining promoter architecture during activation by interacting with STAT5 bound at blocks A and B (35). Runx2 is also a nuclear matrix-binding protein and may serve to recruit the ß-casein promoter to transcriptionally active nuclear subdomains via its interaction with Oct-1 on the Runx/octamer element (reviewed in reference 58).
The Runx2/Oct-1 complex is reminiscent of the Runx1/Ets-1 complex in that both proteins in these complexes bind to adjacent sites, and the formation of the complex is stimulated by relieving autoinhibition of DNA binding as a result of their interaction (18, 22, 26, 31). In the Runx1/Ets-1 complex, the two proteins reciprocally stimulate their DNA binding by interacting via their respective autoinhibitory domains, resulting in truly cooperative binding. The Runx2/Oct-1 complex differs slightly, in that Runx2 binding is stimulated by Oct-1, but Oct-1 binding is only slightly enhanced by the presence of Runx2. The importance of the juxtaposition of the two sites also suggests that there is little flexibility in the protein-protein interaction between the C-terminal region of Runx2 and the POU domain. In addition, while the model depicted indicates that the two DNA-binding domains do not interact directly, it is possible that the DNA is deformed and that the Runt and POU domains are in contact when bound to the DNA (Fig. 8).
Autoinhibition of Runx2 DNA binding. Autoinhibition of DNA binding was not previously reported for Runx2, although it has been observed that a GST fusion protein containing the isolated Runt domain has a greater affinity for DNA than GST-Runx2 (15). The intramolecular mechanism of autoinhibition is likely to be similar to that of Runx1, as the domains map to similar regions (18, 22, 26, 31).
Regions located both N terminal and C terminal to the Runt domain have the capacity to autoinhibit DNA binding, although the C-terminal region possesses the major autoinhibitory function. The N-terminal autoinhibitory domain is located within the first 40 amino acids of Runx2. Deletion of the first 40 amino acids of Runx1 also resulted in an increase in DNA binding (22). Both proteins contain a block of 19 amino acids that is highly conserved and is therefore likely to contain the N-terminal autoinhibitory region. This region is also conserved in Runx3, suggesting that N-terminally mediated autoinhibition of DNA binding is a feature of all three mammalian Runt domain transcription factors. The major autoinhibitory region in the C-terminal region of Runx2 mapped to between residues 396 and 462. This region contains the well-defined nuclear matrix targeting signal, and it will be important to determine whether inhibition of DNA binding and association with the nuclear matrix are related or distinct activities (67).
The finding that Runx2 contains an autoinhibitory domain for DNA binding is also likely to be an important feature of its function in osteoblasts. Runx2 DNA-binding activity is stimulated in response to activation by the mitogen-activated protein kinase and protein kinase C pathways (17). Upon stimulation of these pathways, the C-terminal PST region of Runx2 becomes phosphorylated; this region encompasses the autoinhibitory domain, and we hypothesize that phophorylation stimulates DNA binding by relieving autoinhibition mediated by the C-terminal region.
In summary, we have shown that Runx2 forms a novel complex with Oct-1 on the highly conserved Runx2/Oct-1 element and contributes to the expression of the ß-casein gene. This is the first evidence that Runx2 is involved in the regulation of a mammary gland-specific gene. Given the importance of Runx transcription factors in tissue development, it is possible that Runx2 has a more extensive role in the mammary gland. It will therefore be important to address the role of Runx2 in vivo by generating mice in which the Runx2 gene has been specifically deleted in the mammary gland.
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ACKNOWLEDGMENTS |
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This work was funded by a Wellcome Trust research career development fellowship (to P.S.) and a BBSRC studentship (to C.K.I.).
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FOOTNOTES |
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REFERENCES |
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