Identification of a novel transcriptional repressor element located in the first intron of the human BRCA1 gene.

Loss or lowered expression of BRCA1 in non-familial breast cancer has been shown in several recent studies. Understanding how BRCA1 expression is regulated should provide new insights into the role of BRCA1 in sporadic breast cancer. We have recently identified a critical 18-base pair (bp) DNA element within the minimal BRCA1 promoter whereupon the formation of a specific protein-DNA complex and transcription of BRCA1 is dependent. We now report a non tissue-specific transcriptional repressor activity, located more than 500 bp into the first intron of BRCA1. Progressive deletions from the 3'-end of intron 1 and reporter gene assays localized the repressor activity to an 83-bp region. Electrophoretic mobility shift assays with this 83 bp DNA and various sub-fragments of it showed binding of nuclear proteins to a 36 bp BstNI-BseRI fragment. Functional transcriptional repression by this 36 bp DNA could be conferred on a heterologous thymidine kinase promoter. Analysis of multiple reporter gene constructs containing the BRCA1 genomic region driving transcription in both directions suggests that the putative negative regulatory element functions to block transcription only in the BRCA1 direction, although the promoter is shared by the divergently transcribed NBR2 gene.

Characterization of a repressor element and a juxtaposed tissue-restricted activator element located on the distal neu gene promoter.

The proto-oncogene neu (HER2 or c-erbB2) is overexpressed with or without gene amplification in 20-30% of breast cancers. In patients, neu amplification or overexpression in breast and ovarian cancer correlates with poor prognosis and tumor resistance to chemotherapy. neu-induced transformation can be reversed by the suppression of neu gene transcription. To further understand how neu gene transcription is regulated and to identify a possible transcriptional repressor(s) of neu, we identified a negative regulatory element known previously to be located within a 1-kilobase (kb) DNA fragment of an unknown sequence, upstream of the proximal neu gene promoter. One of several DNA fragments subcloned from this region suppressed transcriptional activity of the proximal neu gene promoter. Sequencing of the 1-kb fragment confirmed the location of the repressor element to be between an AluI and a RsaI sites, around 1.4 kb upstream to the translation start site. Various deletions were introduced into the AluI-RsaI fragment and subcloned into both the native neu promoter and a heterologous thymidine kinase promoter. Subsequent transfections and reporter gene assays in cell lines of various tissues of origin confirmed and narrowed the repressor activity to a 120-base pair NlaIV-MslI fragment located between -1385 and -1266. Importantly, specific protein binding activity to this element could be detected with nuclear extracts isolated from these cell lines. In contrast, a 28-base pair MslI-RsaI fragment (-1265 to -1238), located immediately 3' of the putative repressor element, was found to form protein-DNA complexes with only nuclear extracts isolated from a colon carcinoma cell line. This specific protein binding activity correlated with a previously unknown transcriptional stimulatory activity only in this cell line.

Transcription of BRCA1 is dependent on the formation of a specific protein-DNA complex on the minimal BRCA1 Bi-directional promoter.

BRCA1 is the first tumor suppressor gene linked to hereditary breast and ovarian cancers. Its involvement in sporadic breast cancer, however, remains unclear. Recent studies showed that a loss or lowered expression of BRCA1 is not uncommon in nonfamilial breast cancers. In addition, there have been cases of inherited BRCA1-linked breast cancer with as yet unidentified mutation. Misregulation of BRCA1 at the transcription level is a possible mechanism for loss of BRCA1 expression. To understand transcriptional regulation of the BRCA1 gene, we cloned and examined the BRCA1 promoter, by both functional reporter gene analyses and protein-DNA complex formation electrophorectic mobility shift assays. A bi-directional promoter could be located within a 229-base pair (bp) intergenic region between BRCA1 and its neighboring gene, NBR2. Deletion analyses further delineated a minimal 56-bp EcoRI-HaeIII fragment, which could drive transcription in the NBR2 gene direction 2-4-fold higher than in the BRCA1 direction in all cell lines tested. Furthermore, transcriptional activity in the BRCA1 direction was undetectable in the muscle cell line C2C12, whereas activity in the NBR2 direction was maintained. These results were consistent with the expression pattern of the respective genes. A specific protein-DNA complex was detected when nuclear extracts from HeLa cells and Caco2, a colon cell line, were incubated with the 56-bp minimal promoter. This protein binding activity was further localized to an 18-bp fragment and might involve a tissue-specific factor, because binding was not detected in the C2C12 cell line. The correlation of the detection of this protein-DNA complex only in those cell lines that expressed the chloramphenicol acetyltransferase reporter gene in the BRCA1 direction suggests a significant positive role of this complex in the transcription of the BRCA1 gene.

Association of the TLX-2 homeodomain and 14-3-3eta signaling proteins.

Homeodomain proteins play important roles in various developmental processes, and their functions are modulated by polypeptide cofactors. Here we report that both in vitro and in vivo, 14-3-3eta is associated with the TLX-2 homeodomain transcription factor that is required for mouse embryogenesis. Expression of 14-3-3eta shifts the predominant localization of TLX-2 in COS cells from the cytoplasm to the nucleus. Tlx-2 and 14-3-3eta are expressed in the developing peripheral nervous system with spatially and temporally overlapping patterns, and they are also coexpressed in PC12 cells. Increased expression of either gene by transfection considerably inhibited nerve growth factor-induced neurite outgrowth of PC12 cells, and cotransfection of both genes led to a synergistic effect of suppression. These findings define 14-3-3eta as a functional modulator of the TLX-2 homeodomain transcription factor and suggest that the in vivo function of TLX-2 in neural differentiation is likely regulated by signaling mediated by 14-3-3eta.

c-myc reverses neu-induced transformed morphology by transcriptional repression.

Amplification or overexpression or both of either the c-myc or the human neu (C-erbB-2) gene are common events in many primary human tumors. Coamplification or overexpression or both of both genes have been reported in some breast cancers. The possibility of cooperation between the c-myc and the normal rat neu (c-neu) genes in transforming cells was examined. Surprisingly, the expression of c-myc in B104-1-1 cells, and activated rat neu oncogene (neu*)-transformed NIH 3T3 line, resulted in morphologic reversion. This reversion was found to be a consequence of a transcription-repressive action of c-myc on the neu gene via a 140-bp fragment on the neu gene promoter. The effective concentration of a positive factor(s) interacting with this fragment seemed to be lowered by the expression of c-myc. Our findings lend support to arguments concerning the long-suspected function of c-myc as a transcriptional modulator. They also imply that an oncogene such as c-myc, or possibly the rapidly explored class that encodes transcription factors, under certain conditions may act to reverse a transformed phenotype that is induced by another oncogene instead of contributing positively towards the transformation process. Therefore, the activity of an oncogene may depend on the environment in which it is expressed. In addition, we may have identified the neu gene as a cellular target gene of negative regulation by c-myc.

Multiple cis- and trans-acting elements involved in regulation of the neu gene.

A 2.4-kb rat neu genomic DNA fragment that hybridized to the 5'-most coding sequence of the rat neu cDNA was cloned. S1 nuclease mapping identified multiple transcriptional initiation sites. DNA sequence analysis revealed that this fragment contained 64 bp of the first intron, 81 bp of the first exon, and the upstream noncoding sequence of the neu gene. The sequence immediately upstream of the translation start site was G + C rich (greater than 75%) and contained a consensus CCAAT sequence despite the absence of a TATA box. An Sp1-binding site was found, in addition to various sequence motifs common to the promoters of the human neu gene (erbB2), the epidermal growth factor receptor gene, and the simian virus 40 enhancer. A 2.2-kb EcoRI-Narl fragment containing sequences upstream from the 3'-most transcriptional start site was fused to the bacterial chloramphenicol acetyltransferase reporter gene and shown to promote transcription efficiently. A series of promoter deletion constructs was made, and results from transfection and subsequent chloramphenicol acetyltransferase assays suggested the presence of multiple cis-acting elements that contributed either positively or negatively to the transcription activity. Cotransfection competition experiments using subcloned cis-acting elements confirmed the existence of trans-acting factors interacting with these DNA fragments. In addition, a gel retardation assay was performed to demonstrate the physical binding of nuclear factors to certain fragments. The results complemented those of the deletion studies and led us to conclude that transcriptional regulation of the neu proto-oncogene involves at least one negative and three positive trans-acting factors interacting with different cis-acting elements along the neu gene promoter.

Transcriptional repression of the neu protooncogene by the adenovirus 5 E1A gene products.

Amplification/overexpression of the human neu protooncogene has been frequently found in human primary breast and ovarian cancers and is correlated with the number of axillary lymph nodes positive for metastasis in breast cancer patients. Identification of the factors controlling transcription of the neu gene is essential for understanding the mechanisms of neu gene regulation and its role in tumorigenicity. The adenovirus early region 1A (E1A) gene products are pleiotropic transcription regulators of viral and cellular genes and have been identified as a viral suppressor gene for metastasis. Here we demonstrate that transcription of neu can be strongly repressed by the E1A gene products. The 13S and 12S products of E1A gene are effective at repressing neu transcription and the transcriptional repression requires the conserved region 2 of the E1A proteins. The target for E1A repression was localized within a 139-base-pair DNA fragment in the upstream region of the neu promoter. In addition, competition experiments suggest that the sequence TGGAATG, within the 139-base-pair fragment, is an important element for the E1A-induced repression. These results indicate that E1A negatively regulates neu gene expression at the transcriptional level by means of a specific DNA element.

V-abl confers resistance and growth advantage to TNF-alpha in NIH3T3 cells.

Tumor necrosis factor (TNF-alpha) is a cytokine produced by macrophages and monocytes, and has been shown to have cytolytic, cytostatic or growth-stimulatory activity on transformed cells. However, the mechanism of these growth modulating activities of TNF-alpha is unknown. By studying the response of different oncogene-transformed NIH3T3 cells to TNF-alpha, we showed that the oncogene v-abl confers resistance to the cytostatic and cytolytic activities on TNF-alpha compared to the parental NIH3T3 cells. Most interestingly, v-abl expression also resulted in a growth-enhancing response to TNF-alpha at up to the highest dose of 6,400 units/ml. These altered properties were not due to the transformation event itself, since EJ-ras oncogene transformed NIH3T3 cells were more susceptible to TNF-alpha than the parental cells. Moreover, EMT-6, a mouse adenocarcinoma cell line, which responded similarly to NIH3T3 cells, did not show growth-enhancement at high TNF-alpha dosages. Though resistant to the direct cytotoxic activity of TNF-alpha, the v-abl transformed cell line was effectively killed by macrophages, as were the other cell lines. This suggests tumor cell killing by macrophages must involve mechanisms in addition to the secretion of TNF-alpha.

Effect of glucocorticoids on oncogene transformed NIH3T3 cells.

We studied the differential response of oncogene transformed NIH3T3 cells to glucocorticoids. As demonstrated for transformed human fibroblasts, the morphology of neu-, ras-, src- and sis-transformed mouse fibroblasts became more normal after glucocorticoid treatment. This change was not due to inhibition of the expression of oncogene mRNA or protein. However, the abl-transformed NIH3T3 cells were resistant to glucocorticoid-induced morphology change. These results indicate that the glucocorticoid-induced morphology change is specific to certain oncogene-transformed NIH3T3 cells. Transformed human fibroblasts generally have reduced amounts of cell surface fibronectin. When treated with glucocorticoids, they incorporate higher levels of fibronectin in their extracellular matrix, which correlates with their change in morphology. However, we found that, except for abl-transformed cells, the fibronectin level of the other oncogene transformed mouse cells was similar to non-transformed cells. Moreover, treatment of the neu-, ras-, src- and sis-transformed cells with glucocorticoids resulted in a change in morphology but no increase in cell surface fibronectin. These studies demonstrate that the glucocorticoid-induced morphological change of oncogene-transformed NIH3T3 cells is not due to enhanced expression of fibronectin. Therefore, other mechanisms are responsible for this glucocorticoid-induced phenotypic change of oncogene-transformed cells.