The Structure And The Function Of The 5' Flanking Sequences Of Human BMPR2 Gene

The BMPR2 gene encodes the bone morphogenetic protein receptor typeⅡ(BMPR-Ⅱ), which is a member of the transforming growth factor-βreceptor (TGF-βR) superfamily. The proper function of the receptor is essential for BMP signaling, which is involved in a number of different cellular processes such as cell proliferation, apoptosis, differentiation, chemotaxis, angiogenesis and matrix production during embryogenic development as well as in adult life. Therefore, the roles that the BMP signaling pathway plays in carcinogenesis have been studied intensively in recent years. Moreover, the regulation of the expression of this gene is of particular interests since mutations in BMPR2 gene have been found in primary pulmonary arterial hypertension (PAH) and misexpression of this gene has been found in PAH/cancers. However, regulation of the expression of BMPRII is poorly understood so far. This study aimed to understand how this gene is regulated at the transcriptional level by identifying and analyzing the potential regulatory elements in the 5'-flanking regions of human BMPR2 gene. The results presented in this study suggest:1. The transcriptional start site (TSS) of human BMPR2 gene was found located at 1013 nucleotides upstream from the ATG translation initiation codon, using a combined strategy involving cDNA walking and 5'-RACE.2. The unusually long (1013 nucleotides ) 5' leader sequence of the BMPRII transcript has a high G+C content (62%). No putative IRES element was present in the 5' leader sequence, excluding a possible cap-independent translation initiation. According to the canonical "scanning model" of the eukaryotic translation initiation, the upstream open reading frame (uORF) containing adequate translation initiation sequence may be involved in the negative control of translation initiation from authentic translation initiation codon AUG.3. A 5.5K fragment of the 5' -flanking regions of human BMPR2 gene was cloned into vector pGL3-Basic to generate the recombinant pGL3-Basic-5.5K and was used in the following experiments.4. Six different deletion mutations of this 5.5K 5' flanking sequences were constructed by PCR using pGL3-Basic-5.5K as template and were subsequently cloned into the same vector, pGL3-Basic. The transcriptional activities of these promoter fragment were tested in HeLa cells, Lung adenocarcinoma cell line A549 and Hepatoma Cell Line SMMC-7721 by dual luciferase assay. The results suggested that the -1569bp～-1229bp (The first A nucleotide of the translation initiation codon ATG is numbered +1), the core promoter region, contain many putative transcription factor binding sites and thus may contain important elements for activating transcription. The region from-2333bp～-1570bp and the region from-5106bp～-2875bp may contain strong negative regulatory elements while the region from-2874bp～-2334bp may contain strong positive regulatory elements.5. The fragment encompassing -5106bp～-2875bp and the core promoter of BMPR2 gene were assembled into an integrated fusion fragment, which was cloned into pGL3-Basic for transcription activity analysis. The result suggested that the -5106bp～-2875bp region negatively regulated the transcription of the BMPR2 and this repression might associate with a cluster of Alu sequences.6. The proximal promoter region preceding the TSS lacked TATA box and CAAT box, but is rich in GC nucleotides and several possible GC box (SP1 binding sites) were observed. It suggested that the transcription of BMPR2 gene may be controlled by spl. Therefore, the eukaryotic expression vector pcDNA3.1(+)-SP 1 was constructed and contransfected with BMPR2 promoter-firefly luciferase gene reporter plasmid into HeLa cells. The result showed that the expression of spl could increase the promoter activity in a dose-dependent manner.7. Reporter luciferase constructs with specific mutations in two putative sp 1 binding sites were generated and the mutation of two SP1 sites caused a markedly decrease of promoter activity. In consistence, the electrophoresis mobility shift assay (EMSA) confirmed the binding specificity of the two putative spl sites.8. A GGC trinucleotides repeat that was 50 nucleotides downstream of the transcriptional start site of human BMPR2 gene was also noticed. The allele and genotype frequencies of this trinucleotides repeat were determined in 150 health volunteers of Chinese population. The results showed that the frequencies of GGC 12/GGC 12, GGC 12/GGC 11 and GGC 12/GGC8 were 98.67%(148/150), 0.67%(1/150)and 0.67%(1/150),respectively. The allele frequencies of GGC 12, GGC11 and GGC8 were 99.33%, 0.33% and 0.33%, respectively.9. The 12 GGC repeats in the fragment containing core promoter and 5' leader sequence was mutated and the luciferase expression assay showed that the mutant had a decreased transcriptional activity, suggesting that the importance of this 12 GGC repeats in normal expression of BMPR2 gene. 10. The GGC trinucleotides repeat and the flanking sequence was involved in a CpG island. The methylation of this CpG island was studied in human embryonic lung fibroblast (HELF) and human pulmonary giant cell carcinoma PGCL3. The results showed that this CpG island was hypermethylated in HELF and hypomethylated in PGCL3. In consistence, the expression of BMPR2 in HELF cell was lower than that in PGCL3 by using real-time fluorescence quantitative polymerase chain reaction, suggesting that the methylation of this CpG island may be involved in the transcriptional regulation of BMPR2 gene.