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A New G-Quadruplex with Hairpin Loop Immediately Upstream of the Human BCL2 P1 Promoter Modulates Transcription.

ABSTRACT: The abnormal overexpression of the BCL2 gene is associated with many human tumors. We found a new 28-mer G-quadruplex-forming sequence, P1G4, immediately upstream of the human BCL2 gene P1 promoter. The P1G4 is shown to be a transcription repressor using a promoter-driven luciferase assay; its inhibitory effect can be markedly enhanced by the G-quadruplex-interactive compound TMPyP4. G-quadruplex can readily form in the P1G4 sequence under physiological salt condition as shown by DMS footprinting. P1G4 and previously identified Pu39 G-quadruplexes appear to form independently in adjacent regions in the BCL2 P1 promoter. In the extended BCL2 P1 promoter region containing both Pu39 and P1G4, P1G4 appears to play a more dominant role in repressing the transcriptional activity. Using NMR spectroscopy, the P1G4 G-quadruplex appears to be a novel dynamic equilibrium of two parallel structures, one regular with two 1-nt loops and a 12-nt middle loop and another broken-strand with three 1-nt loops and a 11-nt middle loop; both structures adopt a novel hairpin (stem-loop duplex) conformation in the long loop. The dynamic equilibrium of two closely related structures and the unique hairpin loop conformation are specific to the P1G4 sequence and distinguish the P1G4 quadruplex from other parallel structures. The presence of P1G4 and Pu39 in adjacent regions of the BCL2 P1 promoter suggests a mechanism for precise regulation of BCL2 gene transcription. The unique P1G4 G-quadruplex may provide a specific target for small molecules to modulate BCL2 gene transcription.

SUBMITTER: Onel B

PROVIDER: S-EPMC5019542 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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A New G-Quadruplex with Hairpin Loop Immediately Upstream of the Human BCL2 P1 Promoter Modulates Transcription.

Onel Buket B Carver Megan M Wu Guanhui G Timonina Daria D Kalarn Salil S Larriva Marti M Yang Danzhou D

Journal of the American Chemical Society 20160222 8

The abnormal overexpression of the BCL2 gene is associated with many human tumors. We found a new 28-mer G-quadruplex-forming sequence, P1G4, immediately upstream of the human BCL2 gene P1 promoter. The P1G4 is shown to be a transcription repressor using a promoter-driven luciferase assay; its inhibitory effect can be markedly enhanced by the G-quadruplex-interactive compound TMPyP4. G-quadruplex can readily form in the P1G4 sequence under physiological salt condition as shown by DMS footprintin ...[more]

PMID: 26841249

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Project description:BACKGROUND: Alternative splicing of the locus A beta H-J-J generates functionally distinct proteins: the enzyme aspartyl (asparaginyl) beta-hydroxylase (AAH), truncated homologs of AAH with a role in calcium homeostasis humbug and junctate and a structural protein of the sarcoplasmic reticulum membranes junctin. AAH and humbug are over expressed in a broad range of malignant neoplasms. We have previously reported that this locus contains two promoters, P1 and P2. While AAH and humbug are expressed in most tissues under the regulation of the P1 promoter, AAH, junctin and junctate are predominantly expressed in excitable tissues under the control of the P2 promoter. We previously demonstrated that Sp transcription factors positively regulate the P1 promoter. RESULTS: In the present study, we extended the functional characterization of the P1 promoter of the A beta H-J-J locus. We demonstrated by quantitative Real-time RT-PCR that mRNAs from the P1 promoter are actively transcribed in all the human cell lines analysed. To investigate the transcription mechanism we transiently transfected HeLa cells with sequentially deleted reporter constructs containing different regions of the -661/+81 P1 nucleotide sequence. Our results showed that (i) this promoter fragment is a powerful activator of the reporter gene in HeLa cell line, (ii) the region spanning 512 bp upstream of the transcription start site exhibits maximal level of transcriptional activity, (iii) progressive deletions from -512 gradually reduce reporter expression. The region responsible for maximal transcription contains an E-box site; we characterized the molecular interactions between USF1/2 with this E-box element by electrophoretic mobility shift assay and supershift analysis. In addition, our USF1 and USF2 chromatin immunoprecipitation results demonstrate that these transcription factors bind the P1 promoter in vivo. A functional role of USF1/USF2 in upregulating P1-directed transcription was demonstrated by analysis of the effects of (i) in vitro mutagenesis of the P1/E-box binding site, (ii) RNA interference targeting USF1 transcripts. CONCLUSION: Our results suggest that USF factors positively regulate the core of P1 promoter, and, together with our previously data, we can conclude that both Sp and USF DNA interaction and transcription activity are involved in the P1 promoter dependent expression of AAH and humbug.

Dataset Information

A New G-Quadruplex with Hairpin Loop Immediately Upstream of the Human BCL2 P1 Promoter Modulates Transcription.

Publications

A New G-Quadruplex with Hairpin Loop Immediately Upstream of the Human BCL2 P1 Promoter Modulates Transcription.

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