Molecular population dynamics of DNA structures in a bcl-2 promoter sequence is regulated by small molecules and the transcription factor hnRNP LL

Cui, Yunxi; Koirala, Deepak; Kang, Hong Je; Dhakal, Soma; Yangyuoru, Philip M.; Hurley, Laurence H.; Mao, Hanbin

doi:10.1093/nar/gku185

Cited by 34 publications

(40 citation statements)

References 47 publications

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“…At higher pH, the signals become sharper. The presence of very broad signals at acidic pH reveals that Py39wt is folded into several i‐motif topologies . This feature is also clearly visible in the 1D 31 P NMR spectra of the oligonucleotide shown in Figure B, whereas a different conformation with different conformational dynamics is visible at pH 6.5 and becomes predominant at pH 7.5, as the 31 P signals sharpen.…”

Section: Resultsmentioning

confidence: 73%

Interaction of the N‐Terminal Tandem Domains of hnRNP LL with the BCL2 Promoter i‐Motif DNA Sequence

et al. 2017

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The human genome contains GC-rich sequences able to form tetraplex secondary structures known as the G-quadruplex and i-motif. Such sequences are notably present in the promoter region of oncogenes and are proposed to function as regulatory elements of gene expression. The P1 promoter of BCL2 contains a 39-mer C-rich sequence (Py39wt) that can fold into a hairpin or an i-motif in a pH-dependent manner in vitro. The protein hnRNP LL was identified to recognise the i-motif over the hairpin conformation and act as an activating transcription factor. Thus, the Py39wt sequence would act as an ON/OFF switch, according to the secondary structure adopted. Herein, a structural study of the interaction between hnRNP LL and Py39wt is reported. Both N-terminal RNA recognition motifs (RRM12) cooperatively recognise one Py39wt DNA sequence and engage their β-sheet to form a large binding platform. In contrast, the C-terminal RRMs show no binding capacity. It is observed that RRM12 binds to Py39wt regardless of the DNA conformation. We propose that RRM12 recognises a single-stranded CTCCC element present in loop 1 of the i-motif and in the apical loop of the hairpin conformation.

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Section: Resultsmentioning

confidence: 73%

Interaction of the N‐Terminal Tandem Domains of hnRNP LL with the BCL2 Promoter i‐Motif DNA Sequence

et al. 2017

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“…Together with partially folded structures, 42 this constitutes a rather complex array of observable structures in single-molecule mechanical unfolding experiments. Using an approach we have recently established to follow the population dynamics of individual DNA secondary structures with the statistical method PoDNano (Population Deconvolution at Nanometer resolution), 58,70 we first identified the size of different populations (measured in change in contour length [ΔL]; see We designed a medium-throughput assay based on a well-established FRET assay 71 to screen for compounds that might act as chaperones at the early stage of the folding process to shift the population species of the mutants in the 5-12 G-quadruplex back to the larger species. The WT strand containing the 5-12 G-quadruplex was labeled with FAM and TAMRA at each end for the FRET assay.…”

Section: Resultsmentioning

confidence: 99%

A Pharmacological Chaperone Molecule Induces Cancer Cell Death by Restoring Tertiary DNA Structures in Mutant hTERT Promoters

et al. 2016

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Activation of human telomerase reverse transcriptase (hTERT) is necessary for limitless replication in tumorigenesis. Whereas hTERT is transcriptionally silenced in normal cells, most tumor cells reactivate hTERT expression by alleviating transcriptional repression through diverse genetic and epigenetic mechanisms. Transcription-activating hTERT promoter mutations have been found to occur at high frequencies in multiple cancer types. These mutations have been shown to form new transcription factor binding sites that drive hTERT expression, but this model cannot fully account for differences in wildtype (WT) and mutant promoter activation and has not yet enabled a selective therapeutic strategy. Here we demonstrate a novel mechanism by which promoter mutations activate hTERT transcription, which also sheds light on a unique therapeutic opportunity. Promoter mutations occur in a core promoter region that forms tertiary structures consisting of a pair of G-quadruplexes involved in transcriptional silencing.We show that promoter mutations exert a detrimental effect on the folding of one of these Gquadruplexes, resulting in a nonfunctional silencer element that alleviates transcriptional repression. We have also identified a small drug-like pharmacological chaperone (pharmacoperone) molecule, GTC365, that acts at an early step in the G-quadruplex folding pathway to redirect mutant promoter G-quadruplex misfolding, partially reinstate the correct folding pathway, and reduce hTERT activity through transcriptional repression. This transcription-mediated repression effects cancer cell death through multiple routes including both induction of apoptosis through inhibition of hTERT's role in regulating apoptosis-related proteins and inducing senescence by decreasing telomerase activity and telomere length.We demonstrate the selective therapeutic potential of this strategy in melanoma cells that overexpress hTERT.3

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“…We propose further studies involving single-molecule laser tweezer experiments as previously described. 26,27,70 In this way we would first examine the effect of Nitidine on each strand of the Mid-region and its associated DNA secondary structure. This will be followed by an investigation of the duplex Mid-region, from which both structures can form, in the presence of hnRNP K. This set up will allow us to simultaneously examine the effect of G-quadruplex stabilization by Nitidine and the disruptive effect of Nitidine on the binding of hnRNP K.…”

Section: Discussionmentioning

confidence: 99%

Insight into the Complexity of the i-Motif and G-Quadruplex DNA Structures Formed in the KRAS Promoter and Subsequent Drug-Induced Gene Repression

et al. 2017

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Activating KRAS mutations frequently occur in pancreatic, colorectal, and lung adenocarcinomas. While many attempts have been made to target oncogenic KRAS, no clinically useful therapies currently exist. Most efforts to target KRAS have focused on inhibiting the mutant protein; a less explored approach involves targeting KRAS at the transcriptional level. The promoter element of the KRAS gene contains a GC-rich nuclease hypersensitive site with three potential DNA secondary structure-forming regions. These are referred to as the Near-, Mid-, and Far-regions, on the basis of their proximity to the transcription start site. As a result of transcription-induced negative superhelicity, these regions can open up to form unique DNA secondary structures: G-quadruplexes on the G-rich strand and i-motifs on the C-rich strand. While the G-quadruplexes have been well characterized, the i-motifs have not been investigated as thoroughly. Here we show that the i-motif that forms in the C-rich Mid-region is the most stable and exists in a dynamic equilibrium with a hybrid i-motif/hairpin species and an unfolded hairpin species. The transcription factor heterogeneous nuclear ribonucleoprotein K (hnRNP K) was found to bind selectively to the i-motif species and to positively modulate KRAS transcription. Additionally, we identified a benzophenanthridine alkaloid that dissipates the hairpin species and destabilizes the interaction of hnRNP K with the Mid-region i-motif. This same compound stabilizes the three existing KRAS G-quadruplexes. The combined effect of the compound on the Mid-region i-motif and the G-quadruplexes leads to downregulation of KRAS gene expression. This dual i-motif/G-quadruplex-interactive compound presents a new mechanism to modulate gene expression.

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Molecular population dynamics of DNA structures in a bcl-2 promoter sequence is regulated by small molecules and the transcription factor hnRNP LL

Cited by 34 publications

References 47 publications

Interaction of the N‐Terminal Tandem Domains of hnRNP LL with the BCL2 Promoter i‐Motif DNA Sequence

Interaction of the N‐Terminal Tandem Domains of hnRNP LL with the BCL2 Promoter i‐Motif DNA Sequence

A Pharmacological Chaperone Molecule Induces Cancer Cell Death by Restoring Tertiary DNA Structures in Mutant hTERT Promoters

Insight into the Complexity of the i-Motif and G-Quadruplex DNA Structures Formed in the KRAS Promoter and Subsequent Drug-Induced Gene Repression

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