In-depth Analysis Of BACE1Gene Minimal Promoter Structure

Objective: To determine the core region of BACE1gene promoter,identify the cis-acting regions and minimal promoter region that critical forthe transcription of BACE1, reveal the structure of the core promoter of theBACE1gene and study its function.Methods: On the basis of previous research, continue to constructluciferase reporter gene vector that contains differernt BACE1genepromoter region. Use the conventional methods to culture Human embryokidney cells (HEK293), transfect the luciferase reporter gene thatcontaining different BACE1gene promoter region with the transfectionreagent Lipofectamine2000(Invitrogen) in order to express the luciferasein the HEK293cells. Luciferase assay was performed with the dualluciferase assay reagent (Promega) after lysis the transfected cells with thepassive lysis buffer. The final activity ratios reflect the activity of BACE1gene promoter. To determinate the5’ and3’ boundary of minimal BACE1promoter, deletion or mutation reporter plasmids were constructed withplasmid p3BU-583/-400or p3BU-1149/-400as template and transfectedinto HEK293cells. To investigate whether the different regions have asynergistic effect on BACE1promoter activity, reporter plasmids withcombined single nucleotide mutations were constructed with plasmidp3BU-583/-400as template and transfected into HEK293cells.Results: Successfully constructed a series of plasmids that containeddifferent BACE1gene promoter region. The longest insert fragment is874 bp, contains the translation initiation site BACE1upstream sequence of1273to400, named as B1U-1273/-400, The results indicate that region-583to-574and-560to-540may have important and functional elementsrequired for promoter activity. It seems that-480is the3’end of regionrequired for BACE1minimal promoter activity and the region of-510to-480is important for BACE1promoter activity. The70base pairs regionbetween-550to-480bp may be the core promoter of BACE1gene. Themutations of578G/A and510A/C shown significant impact on theluciferase activity and suggested that near by each nucleotide of-578G or-510A there is a functional cis-acting region respectively. The twocis-acting regions TCE1and TCE2have a synergistic effect on BACE1promoter activity. TCE1is high conserved and served as a proximalactivator, the base number rather than the type between the TCE1and corepromoter is critical for the transcription. TCE2is critical for the basictranscription of BACE1and is like to be a core promoter element.Conclusion: We have found core promoter region of BACE1, andidentified two cis-acting elements near nucleotides-578G and-510A. Bothelements play a pivotal role and can function corporately in determiningBACE1gene transcription. The two cis-acting elements have a synergisticeffect on BACE1promoter activity. TCE1is high conserved and served as aproximal activator. TCE2is critical for the basic transcription of BACE1.BACE1core promoter does not contain typical core promoter elements.