The importance of negative superhelicity in inducing the formation of G-quadruplex and i-motif structures in the c-Myc promoter: implications for drug targeting and control of gene expression

Abstract

The importance of DNA supercoiling in transcriptional regulation has been known for many years, and more recently, transcription itself has been shown to be a source of this superhelicity. To mimic the effect of transcriptionally induced negative superhelicity, the G-quadruplex/i-motif-forming region in the c-Myc promoter was incorporated into a supercoiled plasmid. We show, using enzymatic and chemical footprinting, that negative superhelicity facilitates the formation of secondary DNA structures under physiological conditions. Significantly, these structures are not the same as those formed in single-stranded DNA templates. Together with the recently demonstrated role of transcriptionally induced superhelicity in maintaining a mechanosensor mechanism for controlling the firing rate of the c-Myc promoter, we provide a more complete picture of how c-Myc transcription is likely controlled. Last, these physiologically relevant G-quadruplex and i-motif structures, along with the mechanosensor mechanism for control of gene expression, are proposed as novel mechanisms for small molecule targeting of transcriptional control of c-Myc.

Figure 1

(A) Promoter structure of the c-Myc gene and location of the NHE III₁ region. (B) Sequences of the wild-type (Del4) and mutant (Del4-DM) plasmids and proposed equilibrium between supercoiled and partially relaxed forms of the plasmid.

Figure 2

In vitro footprinting of the wild-type and mutant c-Myc promoter regions with S1 nuclease. (A) Autoradiograms showing S1 nuclease cleavage sites on the G- and C-rich strands of the supercoiled Del4 plasmid. Plasmid DNA was incubated in the absence of salt (lanes 2) and in the presence of 100 mM KCl (lanes 3) at 37 °C overnight before digesting with S1 nuclease. (B) Densitometric scans of lanes 3 in (A), left and right panels. (C) Autoradiograms showing S1 nuclease cleavage sites on the G- and C-rich strands of the supercoiled Del4-DM plasmid. Plasmid DNA was incubated in the absence of salt (lanes 2) and in the presence of 100 mM KCl (lanes 3) at 37 °C overnight before digesting with S1 nuclease. (D) Densitometric scans of lanes 3 in (C), left and right panels.

Figure 3

DNase I footprinting of the wild-type c-Myc promoter region using the supercoiled Del4. The autoradiogram represents DNase I cleavage sites on the C-rich strand of the supercoiled Del4 plasmid in the absence of salt (lane 2) and in the presence of 100 mM KCl (lane 3). The vertical bar next to the gel indicates the polypyrimidine tracts. To the right of the gel is the sequence of the NHE III₁ with cytosine tracts 1−6 indicated by brackets.

Figure 4

In vitro footprinting of the wild-type and mutant c-Myc promoter regions with DMS. (A) Autoradiogram showing DMS modification sites on the G-rich strand of a supercoiled (SC) or linearized (L) form of the wild-type plasmid. Plasmid DNA was incubated in the absence of salt (lanes 1 and 3) and in the presence of 100 mM KCl (lanes 2 and 4) at 37 °C overnight before treatment with DMS. DMS modification sites were mapped using linear amplification by PCR with ³²P-labeled gene-specific primers. (B) Densitometric scans of the autoradiogram in (A). Asterisks indicate the adenine residues that are hypermethylated with DMS. The bars indicate the guanine repeats, which are numbered 1−6 from the 3′-end. (C) Autoradiogram showing DMS modification sites on the G-rich strand of a supercoiled mutant plasmid in the absence of salt (lane 1) and in the presence of 100 mM KCl (lane 2) at 37 °C overnight before treatment with DMS. (D) Densitometric scans of the autoradiogram in (C). The bars indicate the guanine repeats, which are numbered 1−6 from the 3′-end.

Figure 5

In vitro footprinting of the c-Myc promoter region with KMnO₄. Autoradiogram showing KMnO₄-reactive sites on the C-rich strand of the supercoiled Del4 plasmid. Plasmid DNA was incubated in the absence of salt (lane 1) and in the presence of 100 mM KCl (lane 2) at 37 °C overnight before incubation with KMnO₄. To the right of the gel is the sequence of the NHE III₁. Dotted lines indicate the KMnO₄ cleaved thymines, an asterisk marks the protected thymine site, and the numbered brackets denote the cytosine tracts.

Figure 6

(A) In vitro footprinting of the c-Myc promoter region with Br₂. The plasmid DNA was incubated in the absence of salt (lanes 1 and 2) and in the presence of 100 mM KCl (lane 3) at 37 °C overnight before incubation with Br₂. Arrows indicate enhanced reactivity to Br₂. (B) Expansion of the NHE III₁ region from (A) together with the densitometer scans of lane 3 and the G-sequencing lane. The horizontal bars indicate the six cytosine tracts numbered from the 5′ end. (C) Cartoon representation of the relative intensity of Br₂ cleavage of lane 3 from (A). Filled black circles = fully cleaved; half-filled black circles = moderately cleaved; quarter-filled black circles = weakly cleaved; filled yellow circles = fully protected. The brackets indicate the six cytosine tracts. (D) The major i-motif folding pattern deduced from the data in (B) and (C).

Figure 7

(A) Proposed equilibrating forms of the NHE III₁ produced under negative superhelicity. The resistance/sensitivity to S1 nuclease, DMS, KMnO₄, and Br₂ of the various forms is shown in the left panel. Requirements for transition to the single-stranded form or G-quadruplex/i-motif species are shown in the right panel. (B) Asymmetric positioning of the DMS-protected G-quadruplex (top bracket) and i-motif (bottom bracket) together with 14- and 5base overhangs. An asterisk marks the position of the G-to-A mutant base-pairs in the G-quadruplex loop isomer.

Figure 8

Cartoon showing a model for the mechanosensor mechanism and activation/silencing of the c-Myc promoter involving the FUSE and NHE III₁ in association with transcriptionally generated negative superhelicity (adapted from Figure 7 in ref. 44). (A) In the absence of serum, c-Myc transcription is turned off and the FUSE is masked by the nucleosome, and the binding sites for Sp1, CNBP, and hnRNP K are sequestered in the G-quadruplex/i-motif forms (G4/iM). The Pre-Promoter Escape Complex (PPC) remains paused within the promoter proximal regions (P1 and P2). (B) Upon addition of serum, topoisomerase I removes negative superhelicity to provide conditions amenable to conversion of secondary DNA structures in the NHE III₁ back to duplex or single-stranded forms. (C) Sp1 binds to the duplex NHE III₁, the FUSE-masking nucleosome is remodeled by SWI/SNF to expose FUSE, and the earliest factors bind to the promoter (YY1, E2F) to advance the PPC to a full Elongation Complex (EC). Negative superhelicity (– σ) generated by EC then melts the FUSE after nucleosomal remodeling. As negative superhelicity increases, duplex DNA is replaced by single-stranded DNA, and CNBP and hnRNP K bind to the NHE III₁. (D) Melted FUSE recruits FBP, which drives transcription up to peak levels by looping with TFIIH. (E) FBP activity leads to negative supercoil accumulation within the topologically closed FBP–TFIIH loop. High negative superhelicity fully melts FUSE and conscripts FIR through protein–protein and protein–DNA contacts. (F) As FIR represses transcription, the dynamic stress at FUSE falls, ejecting FBP. The FIR–TFIIH connection allows only basal transcription, so the polymerase at a low rate dissociates from TFIIH to become an EC. Without sufficient activation, the superhelical stress eventually dissipates, and the c-Myc promoter reverts back to the silent state in (A). For illustrative purposes, only the FUSE-masking nucleosome is shown.