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Two novel DNA motifs are essential for BACE1 gene transcription.

Xiang Y, Meng S, Wang J, Li S, Liu J, Li H, Li T, Song W, Zhou W - Sci Rep (2014)

Bottom Line: Two novel DNA motifs, designated TCE1 and TCE2, were found to be involved in activating the transcription of human BACE1 gene in a synergistic way.In conclusion, our studies have demonstrated that novel DNA motif TCE1 and TCE2 in human BACE1 gene promoter are two essential cis-acting elements for BACE1 gene transcription.Studies on how these two motifs being regulated by different stimuli could provide insights into the molecular mechanisms underlying AD pathogenesis and pharmaceutical potentials of targeting these motifs for AD treatment.

View Article: PubMed Central - PubMed

Affiliation: Ministry of Education Key Laboratory of Child Development and Disorders; Chongqing Key Laboratory of Translational Medical Research in Cognitive Development and Learning and Memory Disorders, Children's Hospital of Chongqing Medical University, 136 ZhongshanEr Lu, Yuzhong District, Chongqing 400014, China.

ABSTRACT
BACE1 gene encodes for β-Site amyloid β precursor protein (APP)-cleaving enzyme1, which is required for generating amyloid β protein(Aβ). Deposition of Aβ in brain plays an essential role in Alzheimer's Disease (AD) pathogenesis. BACE1 gene has a tissue-specific expression pattern and its expression is tightly regulated at transcriptional level. Core promoter is a minimal DNA sequence to direct transcription initiation and serves as a converging platform for the vast network of regulatory events. Here we identified the core promoter of human BACE1 gene, which is a 71 nucleotides region absent of typical known core promoter elements and is sufficient to initiate a basal transcription. Two novel DNA motifs, designated TCE1 and TCE2, were found to be involved in activating the transcription of human BACE1 gene in a synergistic way. Two single nucleotide mutations in these motifs completely abolished the promoter activity. In conclusion, our studies have demonstrated that novel DNA motif TCE1 and TCE2 in human BACE1 gene promoter are two essential cis-acting elements for BACE1 gene transcription. Studies on how these two motifs being regulated by different stimuli could provide insights into the molecular mechanisms underlying AD pathogenesis and pharmaceutical potentials of targeting these motifs for AD treatment.

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The cis-acting regions have a synergistic effect on activating BACE1 transcription.(a)(c) Schematic diagram of plasmid constructs containing the BACE1 gene promoter with double nucleotide mutations with the plasmid p3BU-583/-400 or p3BU-1149/-400 as template. The horizontal line indicates the BACE1 promoter region; horizontal arrow indicates transcriptional direction and the downward arrow indicates the BACE1 transcriptional start site. Open circle with letter in it indicates mutation. The box LUC represents the coding sequence of the luciferase reporter gene. The numbers indicate the endpoints of each construct, with +1 as the adenine of the physiological translational start codon of the BACE1 gene. (b) (d) HEK293 cells were transiently transfected reporter plasmids. Firefly luciferase activity was measured 24 h after transfection, and Renilla luciferase activity was used to normalize for transfection efficiency. Data are shown as percentage of control samples transfected with wild-type plasmid. The values represent means standard error of the mean (n = 3–6). # or * P< 0.05 by analysis of variance (ANOVA) with the posthocNewmann-Keuls test when comparing with control. (e) HEK293 cells were transiently transfected EGFP reporter plasmids. Fluorescence was observed 24 h after transfection.
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f3: The cis-acting regions have a synergistic effect on activating BACE1 transcription.(a)(c) Schematic diagram of plasmid constructs containing the BACE1 gene promoter with double nucleotide mutations with the plasmid p3BU-583/-400 or p3BU-1149/-400 as template. The horizontal line indicates the BACE1 promoter region; horizontal arrow indicates transcriptional direction and the downward arrow indicates the BACE1 transcriptional start site. Open circle with letter in it indicates mutation. The box LUC represents the coding sequence of the luciferase reporter gene. The numbers indicate the endpoints of each construct, with +1 as the adenine of the physiological translational start codon of the BACE1 gene. (b) (d) HEK293 cells were transiently transfected reporter plasmids. Firefly luciferase activity was measured 24 h after transfection, and Renilla luciferase activity was used to normalize for transfection efficiency. Data are shown as percentage of control samples transfected with wild-type plasmid. The values represent means standard error of the mean (n = 3–6). # or * P< 0.05 by analysis of variance (ANOVA) with the posthocNewmann-Keuls test when comparing with control. (e) HEK293 cells were transiently transfected EGFP reporter plasmids. Fluorescence was observed 24 h after transfection.

Mentions: To investigate whether the afore-mentioned different regions have a synergistic effect on BACE1 promoter activity, reporter plasmids containing single nucleotide mutations in both regions were constructed with plasmid p3BU-583/-400 as template and transfected into HEK293 cell. Compared with single nucleotide mutation of 578G/A (p3BU-583/-400M578A) or 580C/T (p3BU-583/-400M580T), double nucleotides mutation of 578G/A and 580C/T (p3BU-583/-400M580T578A) showed similar luciferase activity as single nucleotide mutation of 578G/A, suggesting that mutation of 580C/T has no additional effects on luciferase activity of plasmid p3BU-583/-400M578A (Fig. 3a,b). However, luciferase assay showed that double mutations 578G/A and 510A/C (p3BU-583/-400M578A510C) resulted in the strongest negative effect on luciferase activity and the luciferase activity of the double mutant plasmid was much lower than that of each single nucleotide mutation of 578G/A or 510A/C (Fig. 3a,b). Compared with single nucleotide mutation of 578G/A (p3BU-583/-400M578A) or 482T/A (p3BU-583/-400M482A), double nucleotides mutation of 578G/A and 482T/A (p3BU-583/-400M578A482A) showed similar luciferase activity as single nucleotide mutation of 578G/A, suggesting that mutation of 482T/A had no effects on promoter activity. The luciferase assay of the double mutant 510A/C and 482T/A (p3BU-583/-400M510C482A) also suggest that the mutation 482T/A had no significant effects on BACE1 gene transcription (Fig. 3a, b).


Two novel DNA motifs are essential for BACE1 gene transcription.

Xiang Y, Meng S, Wang J, Li S, Liu J, Li H, Li T, Song W, Zhou W - Sci Rep (2014)

The cis-acting regions have a synergistic effect on activating BACE1 transcription.(a)(c) Schematic diagram of plasmid constructs containing the BACE1 gene promoter with double nucleotide mutations with the plasmid p3BU-583/-400 or p3BU-1149/-400 as template. The horizontal line indicates the BACE1 promoter region; horizontal arrow indicates transcriptional direction and the downward arrow indicates the BACE1 transcriptional start site. Open circle with letter in it indicates mutation. The box LUC represents the coding sequence of the luciferase reporter gene. The numbers indicate the endpoints of each construct, with +1 as the adenine of the physiological translational start codon of the BACE1 gene. (b) (d) HEK293 cells were transiently transfected reporter plasmids. Firefly luciferase activity was measured 24 h after transfection, and Renilla luciferase activity was used to normalize for transfection efficiency. Data are shown as percentage of control samples transfected with wild-type plasmid. The values represent means standard error of the mean (n = 3–6). # or * P< 0.05 by analysis of variance (ANOVA) with the posthocNewmann-Keuls test when comparing with control. (e) HEK293 cells were transiently transfected EGFP reporter plasmids. Fluorescence was observed 24 h after transfection.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215305&req=5

f3: The cis-acting regions have a synergistic effect on activating BACE1 transcription.(a)(c) Schematic diagram of plasmid constructs containing the BACE1 gene promoter with double nucleotide mutations with the plasmid p3BU-583/-400 or p3BU-1149/-400 as template. The horizontal line indicates the BACE1 promoter region; horizontal arrow indicates transcriptional direction and the downward arrow indicates the BACE1 transcriptional start site. Open circle with letter in it indicates mutation. The box LUC represents the coding sequence of the luciferase reporter gene. The numbers indicate the endpoints of each construct, with +1 as the adenine of the physiological translational start codon of the BACE1 gene. (b) (d) HEK293 cells were transiently transfected reporter plasmids. Firefly luciferase activity was measured 24 h after transfection, and Renilla luciferase activity was used to normalize for transfection efficiency. Data are shown as percentage of control samples transfected with wild-type plasmid. The values represent means standard error of the mean (n = 3–6). # or * P< 0.05 by analysis of variance (ANOVA) with the posthocNewmann-Keuls test when comparing with control. (e) HEK293 cells were transiently transfected EGFP reporter plasmids. Fluorescence was observed 24 h after transfection.
Mentions: To investigate whether the afore-mentioned different regions have a synergistic effect on BACE1 promoter activity, reporter plasmids containing single nucleotide mutations in both regions were constructed with plasmid p3BU-583/-400 as template and transfected into HEK293 cell. Compared with single nucleotide mutation of 578G/A (p3BU-583/-400M578A) or 580C/T (p3BU-583/-400M580T), double nucleotides mutation of 578G/A and 580C/T (p3BU-583/-400M580T578A) showed similar luciferase activity as single nucleotide mutation of 578G/A, suggesting that mutation of 580C/T has no additional effects on luciferase activity of plasmid p3BU-583/-400M578A (Fig. 3a,b). However, luciferase assay showed that double mutations 578G/A and 510A/C (p3BU-583/-400M578A510C) resulted in the strongest negative effect on luciferase activity and the luciferase activity of the double mutant plasmid was much lower than that of each single nucleotide mutation of 578G/A or 510A/C (Fig. 3a,b). Compared with single nucleotide mutation of 578G/A (p3BU-583/-400M578A) or 482T/A (p3BU-583/-400M482A), double nucleotides mutation of 578G/A and 482T/A (p3BU-583/-400M578A482A) showed similar luciferase activity as single nucleotide mutation of 578G/A, suggesting that mutation of 482T/A had no effects on promoter activity. The luciferase assay of the double mutant 510A/C and 482T/A (p3BU-583/-400M510C482A) also suggest that the mutation 482T/A had no significant effects on BACE1 gene transcription (Fig. 3a, b).

Bottom Line: Two novel DNA motifs, designated TCE1 and TCE2, were found to be involved in activating the transcription of human BACE1 gene in a synergistic way.In conclusion, our studies have demonstrated that novel DNA motif TCE1 and TCE2 in human BACE1 gene promoter are two essential cis-acting elements for BACE1 gene transcription.Studies on how these two motifs being regulated by different stimuli could provide insights into the molecular mechanisms underlying AD pathogenesis and pharmaceutical potentials of targeting these motifs for AD treatment.

View Article: PubMed Central - PubMed

Affiliation: Ministry of Education Key Laboratory of Child Development and Disorders; Chongqing Key Laboratory of Translational Medical Research in Cognitive Development and Learning and Memory Disorders, Children's Hospital of Chongqing Medical University, 136 ZhongshanEr Lu, Yuzhong District, Chongqing 400014, China.

ABSTRACT
BACE1 gene encodes for β-Site amyloid β precursor protein (APP)-cleaving enzyme1, which is required for generating amyloid β protein(Aβ). Deposition of Aβ in brain plays an essential role in Alzheimer's Disease (AD) pathogenesis. BACE1 gene has a tissue-specific expression pattern and its expression is tightly regulated at transcriptional level. Core promoter is a minimal DNA sequence to direct transcription initiation and serves as a converging platform for the vast network of regulatory events. Here we identified the core promoter of human BACE1 gene, which is a 71 nucleotides region absent of typical known core promoter elements and is sufficient to initiate a basal transcription. Two novel DNA motifs, designated TCE1 and TCE2, were found to be involved in activating the transcription of human BACE1 gene in a synergistic way. Two single nucleotide mutations in these motifs completely abolished the promoter activity. In conclusion, our studies have demonstrated that novel DNA motif TCE1 and TCE2 in human BACE1 gene promoter are two essential cis-acting elements for BACE1 gene transcription. Studies on how these two motifs being regulated by different stimuli could provide insights into the molecular mechanisms underlying AD pathogenesis and pharmaceutical potentials of targeting these motifs for AD treatment.

Show MeSH
Related in: MedlinePlus