In cancer cells, elevated transcription factor-related Brn-3a regulator isolated from brain cDNA (Brn-3b) transcription factor enhances proliferation in vitro and increases tumour growth in vivo whilst conferring drug resistance and migratory potential, whereas reducing Brn-3b slows growth both in vitro and in vivo. Brn-3b regulates distinct groups of key target genes that control cell growth and behaviour. Brn-3b is elevated in >65% of breast cancer biopsies, but mechanisms controlling its expression in these cells are not known.
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As a transcription factor, Brn-3b regulates the expression of critical genes that control different cellular processes. For example, increased proliferation by Brn-3b may be associated with its ability to transactivate the promoters of genes required for cell cycle progression such as cyclin-dependent kinase 4 (CDK4) [4] and its regulatory partner cyclin D1 [5], which are required, whilst repressing breast cancer susceptibility gene 1 (BRCA1) [6], which is associated with cell cycle arrest in breast cancer cells. Invasiveness and drug resistance associated with Brn-3b in cancer cells are linked with its ability to transactivate genes such as the small heat shock protein 27 (HSP27) [7] whilst repressing promoters of genes encoding adhesion molecules, for example, γ-catenin/plakoglobin [8].
However, whilst the effects of increased Brn-3b in cancer cells have been characterised and many of its target genes have been studied, we do not know which factors contribute to the elevated Brn-3b mRNA and protein levels observed in breast cancer. In this study, we have cloned and analysed the regulatory region that controls Brn-3b gene expression in MCF-7 breast cancer cells. The results presented herein identify a proximal promoter present in the 5' sequences upstream of the Brn-3b gene which drives expression in MCF-7 cells. This promoter is transactivated by the growth factors nerve growth factor (NGF) and epidermal growth factor (EGF) and the hormone estradiol, all of which are known to promote the proliferation and/or survival of breast cancer cells. NGF and EGF increase promoter activity by signalling through the p42/p44-(ERK) mitogen-activated protein kinase (MAPK) pathway, whereas the effects of oestrogen are mediated via oestrogen receptor α (ERα) but not oestrogen receptor β (ERβ). We also show autoregulation by Brn-3b to increase its own expression. These findings suggest that increased transcription of Brn-3b in breast cancer cells is stimulated by growth factors and hormones that enhance proliferation and propagate through autoregulation.
Identification and cloning of transcription start site in transcription factor-related Brn-3a regulator isolated from brain cDNA (Brn-3b) promoter. (a) Homology plots (VISTA Genome Browser) showing regions of similarity in Brn-3b gene and 5' upstream sequences between human (top) and dog, horse and mouse (bottom) genome sequences. Regions of homology are indicated by peaks and grey shading, and positions of Brn-3b exons and intronic sequences are shown. (b) Schematic showing cloned BstX1 (B)/Stu1 (S)/Xho1 (X) (BSX) construct containing putative Brn-3b promoter and regulatory sequences or the BSX exon-intron-exon (BSXEIE) expression construct containing the promoter, regulatory and coding sequences, which were used for subsequent studies. Promoter and regulatory sequences are shown in grey-striped area. 5' noncoding sequences at the beginning of exon 1 are indicated by the black bar. The white stripe at the start of exon 2 represents unique sequences that are present in Brn-3b(s) transcripts, but not in Brn-3b(l) transcripts. The positions of restriction enzyme sites BstX1 (B), Stu1 (S) and Xho1 (X) used for cloning are also shown. (c) Luciferase activity of Brn-3b reporter constructs following transfection into MCF-7 cells is compared with baseline luciferase activity of the empty reporter vector. Values, shown as relative luciferase units (RLU),, were equalised with Renilla internal control (d) Schematic showing positions of putative start sites identified by in silico analysis. Initiator element and proximal TATA sequences are shown relative to ATG in exon 1, and putative intronic TATA sequences are indicated. Half-arrows show relative positions of primers used for polymerase chain reaction (PCR) assay following chromatin immunoprecipitation (ChIP) assay with α-TATA box binding protein (α-TBP) antibody (Ab) to analyse TBP binding to the different sites. (e) PCR products obtained when ChIP DNA (obtained with α-TBP Ab or secondary control Ab) was used for amplification with primers that flanked the upstream initiator elements (-1,048 bp) (A), -278TATA (B) or the intronic TA sequences (C). Primers for sequences within exon 2 (> 1 kb from ATG) were used for amplification in the negative control (D). Positive controls represent PCR products derived by using primers that amplified the known start site of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene using α-TBP or control ChIP DNA (E). Input represents amplification using one-tenth of DNA isolated before performing the ChIP assay. Right column (labeled 2nd Ab) shows the products obtained following amplification of control ChIP Ab (α-rabbit secondary Ab) using the indicated primers.
To identify factors that stimulate Brn-3b promoter activity and therefore gene expression in breast cancer cells, the BSX reporter construct, containing the putative Brn-3b promoter and regulatory sequences cloned into pGL2 basic reporter vector (see Materials and methods, and Figure 1b) was used in transfection studies. Figure 1c shows high basal activity from the Brn-3b promoter construct compared with empty pGL empty vector control, thereby confirming that these sequences were sufficient to promote reporter gene expression. The BSXEIE construct containing additional sequences, including the intron region, give rise to similar results (not shown).
To identify sites from which transcription may be initiated on this promoter, an in vivo ChIP assay was undertaken using an antibody to the TBP component of the basal transcriptional complex [13]. Primers were designed to amplify regions that flanked putative transcription start sites, as shown in Figure 1d (position in reference to first ATG), and referred to as upstream initiator sequence (at position -1048 to -1042) or proximal TATA-like sequence (located at -278 to -272). The primers used to amplify an intronic region with TA-like elements were also tested because this region was found to have an alternative promoter in the related Brn-3a gene, which has a genomic arrangement similar to that of Brn-3b. The primers for sequences in exon 2 were used as negative controls.
Figure 1e shows the PCR products obtained following amplification of α-TBP ChIP DNA using primers for different putative start sites in the promoter. Figure 1e (lane B) shows that primers flanking the putative proximal TATA site at -278 (-278TATA) produced a strong band that was not seen when these primers were used to amplify control ChIP DNA (secondary Ab). This product was comparable to the positive control PCR product obtained using primers that amplified the known start site in the GAPDH gene (Figure 1e, lane E), suggesting significant TBP binding to this proximal TATA-containing region of the promoter. In contrast, amplification of sequences spanning the putative upstream initiator element (Figure 1e, lane A) or intronic regions (Figure 1e, lane C) gave rise to faint bands. This may result either from weak binding of TBP to these regions or from variability in shear size of ChIP DNA. No bands were seen with primers amplifying exon 2 (negative control) (Figure 1e, lane D), indicating the specificity of the assay. The data therefore suggest significant binding of TBP to proximal TATA and possibly weak binding to initiator elements and sequences within the intron.
Analysing the transcription start sites of Brn-3b promoter. (a) Analysis of Brn-3b promoter (BSX) activity following the mutation of key sites to identify the transcriptional start site. Wild-type (WT) promoter in row 1 (right) is represented as 100%, and all mutations are expressed as percentages of the WT promoter. Schematic shows positions of mutations at key sites (indicated by x, left), for example, upstream initiator alone (row 2, right) or in combination with other sites (rows 7 to 9, right). The proximal -278TATA mutation is shown alone (row 3, right) or in combination (rows 7 and 9, right). The effects of mutation of different intronic TA alone (rows 4 to 6, right) or in combination (rows 8 to 10, right) are also shown. Values are equalised to internal control (Renilla luciferase), and the results represent data from three independent experiments expressed as means + SD. * is used to show statistical significance between different mutant promoter constructs and wild-type constructs. (b) Western blot analysis of Brn-3b protein expression in cellular extracts prepared from MCF7 cells transfected with BSXE1E expression constructs in which Brn-3b promoter drives expression of the Brn-3b gene. WT promoter activity is shown in left column. Middle column (Δ-278TATA shows the reduction in Brn-3b protein from expression constructs containing a mutation within the proximal -278TATA promoter of an otherwise, intact construct. Right column shows untransfected control cells with no reporter and therefore represents levels of endogenous Brn-3b protein in these cells.
Since -278TATA is necessary for transcriptional activity, we next tested whether altering this element was sufficient to reduce Brn-3b protein expression in these cells. For the studies, we used the BSXEIE constructs, in which the WT or mutant Brn-3b promoter (in which residues in -278TATA were altered in the otherwise intact promoter) was cloned upstream of its own coding sequence (comprising EIE) and therefore drives its own expression. Following transfection, protein extracts from cells transfected with WT or mutated -278TATA were used for immunoblotting to measure exogenous Brn-3b protein produced from the transfected BSXEIE construct compared with baseline expression (in empty vector transfected cells). Figure 2b (WT column) shows increased Brn-3b protein levels in cells expressing the WT construct (with intact -278TATA driving Brn-3b gene expression) compared with basal levels in untransfected control cells ("No Reporter" column). This was more evident for the longer Brn-3b(l) isoform because basal levels expressed in control cells are much lower compared with the shorter Brn-3b(s) isoform. However, mutation of -278TATA (in the otherwise intact construct) resulted in loss of this induction of Brn-3b protein (Δ-278TATA column) since levels were similar to endogenous expression in control cells. On the basis of the results of these different studies, we concluded that the proximal TATA located at position -278 from ATG (-278TATA) marks the transcription start site for Brn-3b transcription breast cancer cells. 2ff7e9595c
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