16 research outputs found
A Mechanosensor Mechanism Controls the G‑Quadruplex/i-Motif Molecular Switch in the <i>MYC</i> Promoter NHE III<sub>1</sub>
<i>MYC</i> is overexpressed in many different cancer
types and is an intensively studied oncogene because of its contributions
to tumorigenesis. The regulation of <i>MYC</i> is complex,
and the NHE III<sub>1</sub> and FUSE elements rely upon noncanonical
DNA structures and transcriptionally induced negative superhelicity.
In the NHE III<sub>1</sub> only the G-quadruplex has been extensively
studied, whereas the role of the i-motif, formed on the opposite C-rich
strand, is much less understood. We demonstrate here that the i-motif
is formed within the 4CT element and is recognized by hnRNP K, which
leads to a low level of transcription activation. For maximal hnRNP
K transcription activation, two additional cytosine runs, located
seven bases downstream of the i-motif-forming region, are also required.
To access these additional runs of cytosine, increased negative superhelicity
is necessary, which leads to a thermodynamically stable complex between
hnRNP K and the unfolded i-motif. We also demonstrate mutual exclusivity
between the <i>MYC</i> G-quadruplex and i-motif, providing
a rationale for a molecular switch mechanism driven by SP1-induced
negative superhelicity, where relative hnRNP K and nucleolin expression
shifts the equilibrium to the on or off state
Variable temperature 1D <sup>1</sup>H NMR of c-MYC I-motif sequences.
<p>Imino proton regions of variable temperature 1D <sup>1</sup>H NMR spectra of C11T, C20T, C11/20/23T, and C11/14/20/23T at pH 5.5.</p
Imino proton assignments of C11T and C20T c-MYC I-motif.
<p>Imino proton assignments of C11T (A) and C20T (B) using 1D <sup>15</sup>N-filtered experiments on site-specific 6% <sup>15</sup>N-labeled oligonucleotides. Each site-specifically labeled cytosine is shown above its corresponding spectrum. The assignment of all cytosine imino protons is shown above the 1D spectra of the corresponding sequence. All samples are prepared at pH 5.5. NMR experiments were performed at 7°C except for the C7-labeled C20T which was performed at 1°C.</p
Variable pH 1D <sup>1</sup>H NMR of C11/14/20/23T c-MYC I-motif.
<p>Cytosine- and thymine- imino regions of 1D <sup>1</sup>H NMR spectra of C11/14/20/23T at various pHs at 7°C.</p
Folding structures of the c-MYC I-motifs.
<p>Schematic drawing of the folding structures of the c-MYC I-motifs formed in the C11T sequence (A), the C20T sequence (B), and the two equilibrating conformations in the C11/20/23T sequence (C). The C<sup>+</sup>-C base pairs are shown in white boxes. The dashed boxes indicate the possible C<sup>+</sup>-C base pairs that are in dynamic equilibrium and thus show weaker and broader C<sup>+</sup>-C imino peaks. (cytosine  =  yellow sphere, adenine  =  green sphere, thymine  =  blue sphere.)</p
Polypurine/polypyrimidine sequence located upstream (−89 to −43) of the promoter region of the VEGF gene
<p><b>Copyright information:</b></p><p>Taken from "Facilitation of a structural transition in the polypurine/polypyrimidine tract within the proximal promoter region of the human VEGF gene by the presence of potassium and G-quadruplex-interactive agents"</p><p>Nucleic Acids Research 2005;33(18):6070-6080.</p><p>Published online 20 Oct 2005</p><p>PMCID:PMC1266068.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Runs of guanines (GR-I through GR-V) are boxed. Binding sites of the transcriptional factors Egr-1 and Sp1 are underlined
CD spectra of the VEGF-Pu20T, d(TGCGCGCGT), in Tris–HCl buffer (20 mM, pH 7
<p><b>Copyright information:</b></p><p>Taken from "Facilitation of a structural transition in the polypurine/polypyrimidine tract within the proximal promoter region of the human VEGF gene by the presence of potassium and G-quadruplex-interactive agents"</p><p>Nucleic Acids Research 2005;33(18):6070-6080.</p><p>Published online 20 Oct 2005</p><p>PMCID:PMC1266068.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p>6) in the presence of increasing concentrations of KCl (0, 10, 50 and 100 mM). Each spectrum corresponds to four averaged scans taken at 25°C and is baseline corrected for signal contributions due to the buffer
The Transcriptional Complex Between the <i>BCL2</i> i‑Motif and hnRNP LL Is a Molecular Switch for Control of Gene Expression That Can Be Modulated by Small Molecules
In a companion paper (DOI: 10.021/ja410934b) we demonstrate that the C-rich
strand of the <i>cis-</i>regulatory element in the <i>BCL2</i> promoter element
is highly dynamic in nature and can form either an i-motif or a flexible
hairpin. Under physiological conditions these two secondary DNA structures
are found in an equilibrium mixture, which can be shifted by the addition
of small molecules that trap out either the i-motif (IMC-48) or the
flexible hairpin (IMC-76). In cellular experiments we demonstrate
that the addition of these molecules has opposite effects on <i>BCL2</i> gene expression and furthermore that these effects
are antagonistic. In this contribution we have identified a transcriptional
factor that recognizes and binds to the <i>BCL2</i> i-motif
to activate transcription. The molecular basis for the recognition
of the i-motif by hnRNP LL is determined, and we demonstrate that
the protein unfolds the i-motif structure to form a stable single-stranded
complex. In subsequent experiments we show that IMC-48 and IMC-76
have opposite, antagonistic effects on the formation of the hnRNP
LL–i-motif complex as well as on the transcription factor occupancy
at the <i>BCL2</i> promoter. For the first time we propose
that the i-motif acts as a molecular switch that controls gene expression
and that small molecules that target the dynamic equilibrium of the
i-motif and the flexible hairpin can differentially modulate gene
expression
Simultaneous Drug Targeting of the Promoter <i>MYC</i> G‑Quadruplex and <i>BCL2</i> i‑Motif in Diffuse Large B‑Cell Lymphoma Delays Tumor Growth
Secondary DNA structures
are uniquely poised as therapeutic targets
due to their molecular switch function in turning gene expression
on or off and scaffold-like properties for protein and small molecule
interaction. Strategies to alter gene transcription through these
structures thus far involve targeting single DNA conformations. Here
we investigate the feasibility of simultaneously targeting different
secondary DNA structures to modulate two key oncogenes, cellular-myelocytomatosis
(<i>MYC</i>) and B-cell lymphoma gene-2 (<i>BCL2</i>), in diffuse large B-cell lymphoma (DLBCL). Cotreatment with previously
identified ellipticine and pregnanol derivatives that recognize the <i>MYC</i> G-quadruplex and <i>BCL2</i> i-motif promoter
DNA structures lowered mRNA levels and subsequently enhanced sensitivity
to a standard chemotherapy drug, cyclophosphamide, in DLBCL cell lines.
In vivo repression of <i>MYC</i> and <i>BCL2</i> in combination with cyclophosphamide also significantly slowed tumor
growth in DLBCL xenograft mice. Our findings demonstrate concurrent
targeting of different DNA secondary structures offers an effective,
precise, medicine-based approach to directly impede transcription
and overcome aberrant pathways in aggressive malignancies
Insight into the Complexity of the i‑Motif and G‑Quadruplex DNA Structures Formed in the <i>KRAS</i> Promoter and Subsequent Drug-Induced Gene Repression
Activating <i>KRAS</i> 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 <i>KRAS</i> 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 <i>KRAS</i> 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 <i>KRAS</i> G-quadruplexes. The combined effect of the compound
on the Mid-region i-motif and the G-quadruplexes leads to downregulation
of <i>KRAS</i> gene expression. This dual i-motif/G-quadruplex-interactive
compound presents a new mechanism to modulate gene expression