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Transcriptional Regulation of a Nasopharyngeal Carcinoma Tumor Suppressor Gene BRD7

Author: LiuHuaYing
Tutor: LiGuiYuan
School: Central South University
Course: Pathology and Pathophysiology
Keywords: BRD7 gene transcriptional regulation promoter cis-acting element trans-acting factor MicroRNA DNA methylation
CLC: R739.63
Type: PhD thesis
Year: 2007
Downloads: 414
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Abstract


Bromodomain is an evolutionally conserved domain that wasrecently identified in many living organisms. Bromodomain containingprotein has specific affinity to combine with acetylated lysines onN-terminal tails of histones. It has been demonstrated that bromodomainwas characteristics of proteins that regulated signal-dependenttranscriptional regulation through the mechanism that bromodomainproteins may modulate chromatin remodelling and facilitate the accessionof transcription factors to chromatin. BRD7 is a bromodomain gene thathas been recently cloned by cDNA RDA (cDNA RepresentationalDifference Analysis). It is down-expressed in Nasopharygeal Carcinoma(NPC) biopsies and their derived cell lines. Previous studies showed thatBRD7 is a cell cycle related transcription factor. It participates in cellcycle regulation by regulating some of the cell cycle associated genesthrough Ras/MEK/ERK and Rb/E2F pathways. Moreover, it can transmithistoric acetylation signal and modulate chromatin remodeling throughbinding to acetylated lysines of histone H3. Over-expression of BRD7inhibits cell growth and cell cycle progression of NPC cells, and partlyreverses malignant phenotype of NPC cells. To uncover the molecularmechanisms underlying down-expression of BRD7 in NPC cells, in thisstudy, we investigated the transcriptional regulation of BRD7.【Cloning, characterization and fine mapping of BRD7 promoter】Bioinformatics approaches provide a breakthrough point foranalyzing of transcriptional regulation. A 792 bp region spanning frompositions -375 to +416 was identified as potential promoter region ofBRD7 gene by using PromoterInspector, whereas a 252 bp region located positions from -393 to -141 was identified as BRD7 promoter by usingPromoterScan program. With human genomic DNA prepared fromhuman blood cells as templates, a fragment spanning from positions -711to +496 of BRD7 gene was amplified by PCR. This fragment expressesas strong promoter activity as SV40 promoter. Further analysis withdeletion constructs demonstrated that the region spanning from positions-404 to +46 is the proximal promoter of BRD7 gene, and that thefragment from positions -293 to -168 was indispensable for the basalpromoter activity of BRD7.To define minimal promoter of BRD7, a series of 5’, 3’ or internaldeletion constructs were generated from pGL3-404/+46, andcotransfected with the SV40β-galactosidase vectors into cultured cells.Luciferase assay revealed that the shortest 55 bp region from positions-266 to -212 is the minimal promoter of BRD7 gene, and that thisminimal promoter selectively functions in c-Myc-null cell lines【Identification and characterization of the cis-acting elements andtrans-acting factors in BRD7 promoter】Eukaryotic gene transcription is a remarkably intricate biochemicalprocess. An astounding number of protein factors were found to beresponsible for transcriptional control. Searches for binding sites oftranscription factors in BRD7 promoter were performed by usingMatInspector Professional program. No canonical TATA or CAAT boxeswere found, while several GC boxes and putative transcription bindingsites for KLF, Sp1, IFRF-2, AP2, MYC-MAX, E2F and E2F6 were foundin BRD7 promoter. The results of EMSA confirmed the specificoverlapping site of Sp1/MYC-MAX at -223/-198, consensus E2F-6 site at-243/-229 and E2F binding site at -183/-169. Supershift and ChIP assay revealed the specific binding of transcription factor c-Myc, E2F6 and Sp1with their corresponding binding sites in BRD7 minimal promoter.To explore the regulation role of transcription factor Sp1, c-Myc andE2F on BRD7 promoter activity and mRNA expression, we generated theexpression constructs of c-Myc, E2F6 and Sp1, and carded out a series ofrelated experiments. It was found that over-expression of c-Mycsignificantly inhibits BRD7 promoter activity and obviously reducesendogenous mRNA expression of BRD7 gene, while knockdown ofendogenous c-Myc increases more than ten fold of BRD7 promoteractivity and mRNA expression of BRD7 in NPC 5-8F cells. Additionally,MicroRNA chip results revealed that knockdown of endogenous c-Mycup-regulated 14 miRNA expression including hsa-miR-224, whichexpression was increased more than 66.49 fold, and down-regulated 7miRNA expression including hsa-miR-200c and hsa-miR-141. Onlineprediction results revealed that most of these miRNA target genes areinvolved in cell cycle and cellular apoptosis.Over-expression of transcription factor Sp1 increases BRD7promoter activity in a dose-dependant manner in NPC HNE1 and 5-8Fcells, but not enough to the promoter activity of the full-length promoter-404/+46, while mithramycin A, the specific competitor of Sp1, inhibitsthe promoter activity and endogenous expression of BRD7. Butover-expression of E2F6 has no effects on BRD7 minimal promoter.To further confirm the importance of the overlapping site ofMYC-MAX/Sp1 at -223/-198 and E2F site at -243/-229, reporterconstructs containing site mutant or deletion mutant of these two bindingsites were generated. It was found that site mutating of three keynucleotides (C/A, G/T, G/A) of the overlapping binding site of MYC-MAX/Sp1 increases 10000 fold of BRD7 promoter activity in NPC5-8F cells, whereas deletion of this overlapping region didn’t change thepromoter activity of pGL3-266,-212 either in COS7 or 5-8F cells.Similarly, mutation of four key nucleotides in E2F6 binding site didn’taffect the promoter activity of BRD7 gene, but deletion of E2F6 bindingsite significantly increased BRD7 promoter activity in NPC 5-8F cells.Moreover, immunoprecipitation along with immuno-localizationassay revealed the direct interaction between c-Myc and Sp1 in 5-8F cells,suggesting that c-Myc binds to Sp1 to cover or compete the domain ofSp1 for binding to overlapping site of Sp1/MYC-MAX at -223/-198 maybe another mechanism underlying the negative regulation effect of c-Mycon BRD7 promoter.【Methylation of BRD7 promoter suppresses its expression, whiledemethylation of BRD7 promoter increases its expression】Knock-down of c-Myc expression or over-expression of Sp1 is notenough to increase BRD7 promoter activity to that of the full-lengthpromoter -404/+46 or completely reverse BRD7 expression in NPC 5-8Fcells, indicating that other mechanisms such as methylation status,especially methylation of GC boxes (Sp1 sites), might be related totranscriptional regulation of BRD7 gene. A CpG island spanning frompositions -418 to -56 bp or from -374 to -4 bp was revealed by usingCpGplot and the CpGFinder program, respectively. The CpG islandspredicted by these two programs overlap with each other, and overlapwith BRD7 promoter. Methylation specific primers and unmethylationspecific primers were designed to detect the methylation status of BRD7promoter. It was found that BRD7 promoter was partly methylated in allanalyzed NPC cell lines, and that the methylation status of BRD7 promoter is reversely correlated with BRD7 mRNA expression.MSP-PCR sequencing results revealed ten methylated CpG sites in BRD7promoter region, among which one was included in Sp1 binding site at-353/-337, one in MYC-MAX binding site at -330/-317, one in Sp1binding site at -229/-198, one in E2F binding site at -243/-229, and theother two near the translation start site. As CpG methylase SssI cantransmit the -CH3 of SAM to Cytosine of CpG site, we incubated probesof oligonucleotide wt E2F6 (-243/-229) and wt Sp1/MYC-MAX(-223/-198) with SssI. EMSA results showed that DNA methylationinhibited the formation of DNA-protein complex. Luciferase and directGFP fluorescence assays showed that methylation of pGL3-404/+46 andpGL3-404/+46/GFP with SssI inhibits BRD7 promoter activity inanalyzed cell lines except for COS7 and BHK-21. 3.75μM of5’-aza-2’-deoxycytine (ADC) was sufficient to reverse the methylatedstatus of BRD7 promoter, and increased the mRNA and proteinexpression of BRD7 up to 66.7% and 63.3%, respectively. Results offlow cytometry analysis showed that 3.75μM ADC can inhibit cell cycleprogression of G2/M and S phase, and induce cellular apoptosis.Moreover, MicroRNA chip results revealed that 3.75μM ADCup-regulated 10 miRNA expression including hsa-miR-122a, whichexpression was increased more than 63.2 fold, and down-regulated 7miRNA expression including hsa-miR-200c and hsa-miR-203. Onlineprediction results showed that most of these miRNA target genes areinvolved in cell cycle and cellular apoptosis. More importantly, wedetected methylation status of BRD7 promoter in 36 cases of NPCpatients and 16 normal individuals, and found that the methylation rate ofBRD7 promoter in NPC patients is 100%, while 50% of weak promoter methylation was revealed in normal individuals.In summary, we have identified BRD7 promoter and finely mappedits positions, demonstrated the characteristics of BRD7 promoter,confirmed and investigated the functions of cis-acting elements andtrans-acting factors of BRD7 promoter, explored the effects of DNAmethylation on BRD7 promoter activity and mRNA expression, andfound that the methylation status of BRD7 promoter may be served asone of the biomarkers to distinguish NPC patients from normalindividuals. These results will help to better understand the biologicalfunctions of BRD7, elucidate the molecular mechanisms of BRD7involved in the carcinogenesis of NPC, provide evidences for thepromising clinical application of BRD7.

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CLC: > Medicine, health > Oncology > Department of Otolaryngology tumor > Pharyngeal tumors
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