Direct experimental evidence for quadruplex–quadruplex interaction within the human ILPR

Schonhoft, Joseph; Bajracharya, Rabindra; Dhakal, Soma; Yu, Zhongbo; Mao, Hanbin; Basu, Soumitra

doi:10.1093/nar/gkp181

Cited by 51 publications

(71 citation statements)

References 51 publications

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“…Located in the promoter region, G4 structures act as cis-regulatory elements 77 , modulating gene regulation bidirectionally. That is, G-quadruplex formed upstream of the gene can either enhance its transcription 78 or silence it 79 . We reasoned that the high density of CG nucleotides in the MAOA promoter might affect physical properties of the local DNA and lead to formation of quadruplex motifs.…”

Section: Resultsmentioning

confidence: 99%

Identification and characterization of putative methylation targets in theMAOAlocus using bioinformatic approaches

Shumay¹,

Fowler²

2010

Epigenetics

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Monoamine oxidase A (MAO A) is an enzyme that catalyzes the oxidation of neurotransmitter amines. A functional polymorphism in the human MAOA gene (high- and low- MAOA) has been associated with distinct behavioral phenotypes. To investigate directly the biological mechanism whereby this polymorphism influences brain function, we recently measured the activity of the MAO A enzyme in healthy volunteers. When found no relationship between the individual's brain MAO A level and the MAOA genotype, we postulated that there are additional regulatory mechanisms that control the MAOA expression. Given that DNA methylation is linked to the regulation of gene expression, we hypothesized that epigenetic mechanisms factor into the MAOA expression. Our underplaying assumption was that the differences in an individual's genotype play a key role in the epigenetic potential of the MAOA locus and, consequently, determine the individual's level of MAO A activity in the brain. As a first step towards experimental validation of the hypothesis, we performed a comprehensive bioinformatic analysis aiming to interrogate genomic features and attributes of the MAOA locus that might modulate its epigenetic sensitivity. Major findings of our analysis are the following: (1) the extended MAOA regulatory region contains two CpG islands (CGIs), one of which overlaps with the canonical MAOA promoter and the other is located further upstream; both CGIs exhibit sensitivity to differential methylation. (2) The uVNTR's effect on the MAOA's transcriptional activity might have epigenetic nature: this polymorphic region resides within the MAOA's CGI and itself contains CpGs, thus, the number of repeating increments effectively changes the number of methylatable cytosines in the MAOA promoter. An array of in silico analyses (the nucleosome positioning, the physical properties of the local DNA, the clustering of transcription-factor binding sites) together with experimental data on histone modifications and Pol 2 sites and data from the RefSeq mRNA library together suggest that the MAOA gene might have an alternative promoter. Based on our findings, we propose a regulatory mechanism for the human MAOA according to which the MAOA expression in vivo is executed by the generation of tissue-specific transcripts initiated from the alternative promoters (both CGI-associated) where transcriptional activation of a particular promoter is under epigenetic control.

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Section: Resultsmentioning

confidence: 99%

Identification and characterization of putative methylation targets in theMAOAlocus using bioinformatic approaches

Shumay¹,

Fowler²

2010

Epigenetics

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“…13 Although still controversial, 14 in vitro evidence points to the existence of high-order interactions between neighboring G-quadruplexes 15, 16 in single-stranded DNA (ss) sequences, such as those of telomeres, that can host multiple G-quadruplexes. This suggests that, like RNA or protein, tertiary DNA structures may also form.…”

Section: Introductionmentioning

confidence: 99%

“…This helps to explain the current controversial observations on the existence of DNA tertiary interactions. 14-16 …”

Section: Introductionmentioning

confidence: 99%

Tertiary DNA Structure in the Single-Stranded hTERT Promoter Fragment Unfolds and Refolds by Parallel Pathways via Cooperative or Sequential Events

et al. 2012

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The discovery of G-quadruplexes and other DNA secondary elements has increased the structural diversity of DNA well beyond the ubiquitous double helix. However, it remains to be determined whether tertiary interactions can take place in a DNA complex that contains more than one secondary structure. Using a new data analysis strategy that exploits the hysteresis region between the mechanical unfolding and refolding traces obtained by a laser-tweezers instrument, we now provide the first convincing kinetic and thermodynamic evidence that a higher order interaction takes place between a hairpin and a G-quadruplex in a single-stranded DNA fragment that is found in the promoter region of human telomerase. During the hierarchical unfolding or refolding of the DNA complex, a 15-nucleotide hairpin serves as a common species among three intermediates. Moreover, either a mutant that prevents this hairpin formation or the addition of a DNA fragment complementary to the hairpin destroys the cooperative kinetic events by removing the tertiary interaction mediated by the hairpin. The coexistence of the sequential and the cooperative refolding events provides direct evidence for a unifying kinetic partition mechanism previously observed only in large proteins and complex RNA structures. Not only does this result rationalize the current controversial observations for the long-range interaction in complex single-stranded DNA structures, but this unexpected complexity in a promoter element provides additional justification for the biological function of these structures in cells.

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“…32 Compared to other single molecule techniques such as fluorescence, 33 the force based approaches do not require bulky fluorophores that may alter native structures. In addition, they can provide mechanical information of structures, 24, 34 which is important not only for transcription, but also for other processes catalyzed by motor proteins such as DNA polymerases.…”

Section: Introductionmentioning

confidence: 99%

Coexistence of an ILPR i-Motif and a Partially Folded Structure with Comparable Mechanical Stability Revealed at the Single-Molecule Level

et al. 2010

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Investigation of i-motif is of high importance to fully understand the biological functions of G quadruplexes in the context of double stranded DNA. Whereas single molecule approaches have profiled G quadruplexes from a perspective unavailable by bulk techniques, there is a lack of similar literature on the i-motif in the cytosine (C) rich region complementary to G quadruplex forming sequences. Here, we have used laser tweezers to investigate the structures formed in 5′-(TGTCCCCACACCCC)2, a predominate variant in the insulin linked polymorphic region (ILPR). We have observed two species with the change in contour length (ΔL) of 10.4 (±0.1) and 5.1 (±0.5) nm, respectively. Since ΔL of 10.4 nm is located within the expected range for an i-motif structure, we assign this species to the i-motif. The formation of the i-motif in the same sequence has been corroborated by bulk experiments such as Br2 footprinting, circular dichroism, and thermal denaturation. The assignment of the i-motif is further confirmed by decreased formation of this structure (23 % to 1.3 %) with pH 5.5 7.0, which is a well established behavior for i-motifs. In contrast to the i-motif, the formation of the second species with ΔL of 5.1 nm remains unchanged (6.1±1.6 %) in the same pH range, implying that pH sensitive C:CH+ pairs may not contribute to the structure as significantly as those to the i-motif. Compared to the ΔGunfold of i-motif (16.0 ±0.8 kcal/mol), the decreased free energy in the partially folded structure (ΔGunfold 10.4 ± 0.7 kcal/mol) may reflect a weakened structure with reduced C:CH+ pairs. Both ΔL and ΔGunfold argue for the intermediate nature of the partially folded structure in comparison to the i-motif. In line with this argument, we have directly observed the unfolding of i-motif through the partially folded structure. The i-motif and the partially folded structure share similar rupture forces of 22-26 pN, which are higher than those that can stall transcription catalyzed by RNA polymerases. This suggests, from a mechanical perspective alone, that either of the structures can stop RNA transcription.

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Direct experimental evidence for quadruplex–quadruplex interaction within the human ILPR

Cited by 51 publications

References 51 publications

Identification and characterization of putative methylation targets in theMAOAlocus using bioinformatic approaches

Identification and characterization of putative methylation targets in theMAOAlocus using bioinformatic approaches

Tertiary DNA Structure in the Single-Stranded hTERT Promoter Fragment Unfolds and Refolds by Parallel Pathways via Cooperative or Sequential Events

Coexistence of an ILPR i-Motif and a Partially Folded Structure with Comparable Mechanical Stability Revealed at the Single-Molecule Level

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