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.