The transcriptional complex between the BCL2 i-motif and hnRNP LL is a molecular switch for control of gene expression that can be modulated by small molecules

Abstract

In a companion paper (DOI: 10.021/ja410934b) we demonstrate that the C-rich strand of the cis-regulatory element in the BCL2 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 BCL2 gene expression and furthermore that these effects are antagonistic. In this contribution we have identified a transcriptional factor that recognizes and binds to the BCL2 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 BCL2 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.

Figure 1

Diagram of the BCL2 gene promoter region with the GC-rich element located directly upstream of the P1 promoter and targeting with IMC-48 and IMC-76. The C-rich i-motif-forming sequence is shown. Three and one-half sets of two intercalated hemiprotonated cytosine⁺–cytosine base pairs form the i-motif structure. The bases in bold correspond to the bases involved in base pairing within each of the structures. Here and in subsequent figures, the yellow, green, red, and blue circles represent the deoxynucleotides cytosine, adenine, guanine, and thymine, respectively. In the lower portion of the figure we also show the proposed partial hairpin that is in equilibrium with the i-motif and the proposed binding of IMC-48 and IMC-76 to the i-motif and partial hairpin, respectively, along with the proposed transcriptional consequences of targeting with IMC-48 and IMC-76.

Figure 2

Confirmation of hnRNP LL as a BCL2 i-motif–binding protein. (A) The effects of siRNA knockdown of hnRNP LL on the BCL2, kRAS, and PDGFR-β mRNA level in MCF-7 cells. 50 nM of hnRNP LL siRNA was added to MCF-7 cells for 72 h. GAPDH was used as an internal control (**P < 0.01). (B) Effect on concentration-dependent binding of hnRNP LL on the BCL2 i-motif–forming oligomer (Py39WT) by EMSA at pH 6.8. (C) Competition EMSA showing BCL2 i-motif–specific binding of hnRNP LL at pH 6.8. Nonlabeled (cold) oligomers were incubated with hnRNP LL on ice for 20 min and end-labeled Py39WT was added for 5 min. (D) Comparative K_d values for hnRNP LL binding to the biotin-Py39WT and biotin-Py39MutT at two different pH levels determined by SPR analysis.

Figure 3

EMSA and siRNA knockdown experiments demonstrate that hnRNP LL recognizes the i-motif through the lateral loops. (A) Folding pattern of the BCL2 i-motif showing the 5′ and 3′ lateral loops (II and V, respectively) and central loop (CL). (B) Competition EMSA showing selective binding of hnRNP LL to the two lateral loops of Py39WT with 8:5:7 loop folding pattern. Four different mutant sequences were used. Mut5′,3′L has mutations in two lateral loops, and Mut5′L and Mut3′L have mutations in the 5′ and 3′ loops, respectively (binding sequences are color coded to match i-motif folding pattern in A). MutCL has mutations in the central loop. The percents above the sets of histograms for Mut5′,3′L, Mut5′L, Mut3′L, and MutCL indicates the addition of free DNA for each concentration of cold oligomers divided by three. (C) A luciferase assay shows that knockdown with hnRNP LL siDNA was dependent on the wild-type sequence in the lateral loops of the i-motif. Three pGL3 constructs of wild-type, Mut5′,3′L, and MutCL were co-transfected with pRL-TK for normalization and siRNA to hnRNP LL for 72 h. Final relative luciferase activities were obtained by normalization of the ratio of firefly to renilla to siRNA-untreated control of each construct P values (****P < 0.0001, ***P < 0.001, ns: not significant) were determined by t-test analysis.

Figure 4

EMSA, Br₂ footprinting, and FRET show that hnRNP LL unfolds the BCL2 i-motif after binding. (A) CD analysis shows that binding of hnRNP LL produces a conformational change in the i-motif. hnRNP LL was preincubated with Py39WT or Py39MutT at pH 6.5 for 5 min at room temperature before measuring the CD. (B) Competition EMSA showing that 13 nt is the optimal length between two hnRNP LL binding sites for the binding of hnRNP LL. All oligomers are 39-mers. Competition EMSA experiments were conducted in a binding buffer (pH 6.8) for 20 min of preincubation of 250 nM of cold oligomers with hnRNP LL and subsequent 5 min incubation of end-labeled Py39WT. This represents about a 150 molar excess of cold DNA to labeled i-motif. The histogram below the gel shows the relative binding intensity from the EMSA gel. (C) Bromine footprinting of the BCL2 i-motif and hnRNP LL complex showing the conformational change of Py39WT induced by hnRNP LL. Py39WT and hnRNP LL were incubated for 5 min at room temperature, and bromine generated in situ was added for 30 min. Black and red plots are 0 and 10 μg of hnRNP LL, respectively. The peaks with the black dots correspond to those where maximum inhibition occurs and include C runs II and IV and the central loop. The right panel shows the folding pattern of the BCL2 i-motif with that region protected from Br₂ cleavage shown in the blue shading. Experimental conditions are described in the Methods section. (D) FRET experiments showing i-motif-specific unfolding activity by hnRNP LL. FAM/TAMRA dual-labeled probes were incubated at pH 6.5 or 7.9 with hnRNP LL at room temperature for 5 min, and then fluorescence intensity was measured at 495 nm (Ex.)/528 (Em.). Right panel shows the unfolding of the i-motif consistent with the fluorescence enhancement seen in the left panel (WT at pH 6.5). P values (**P < 0.01, ***P < 0.001) were determined by t-test analysis.

Figure 5

The consequences of sequestration of the flexible hairpin or the BCL2 i-motif by IMC-76 (A) and IMC-48 (B), respectively, on the binding of hnRNP LL to the i-motif. (A) EMSA analysis of the competition between IMC-76 and hnRNP LL for the i-motif (left) and densitometric analysis (right). (B) EMSA analysis of the cooperativity between IMC-48 and hnRNP LL for the i-motif (left) and densitometric analysis (right). IMC-76 or IMC-48 was incubated with Py39WT for 3 h, and hnRNP LL was added for 10 min at pH 6.5 before running the 6% native PAGE. Relative band intensities are plotted against IMC-76 or IMC-48 concentrations (right). Species 1 and 2 are proposed to be the i-motif and flexible hairpin, respectively.

Figure 6

Effect of IMC-76 and IMC-48 combined with knockdown of hnRNP LL on BCL2 mRNA levels. (A) Enhanced effect of IMC-76 treatment on the BCL2 mRNA levels after knockdown of hnRNP LL. (B) Restoration of BCL2 mRNA levels after treatment with IMC-48 following knockdown of hnRNP LL. After transfection of 50 nM of hnRNP LL siRNA into MCF-7 cells for 48 h, IMC-76 or IMC-48 was incubated for a further 24 h (***P < 0.001, **P < 0.01). The P value (**P < 0.01) was determined by one-way ANOVA analysis.

Figure 7

ChIP analysis of the effect of IMC-76 and IMC-48 given either singly (A and B) or in sequential order (IMC-76 followed by IMC-48) (C) on promoter occupancy of Sp1 and hnRNP LL. For single drug treatments (A and B), two concentrations (0.5 and 2 μM) of IMC-76 or IMC-48 with DMSO as a control were incubated with MCF-7 and BJAB cells, respectively, for 24 h. For sequential treatments (C), antagonism between IMC-76 and IMC-48 was shown through restoration of Sp1 and hnRNP LL promoter occupancy levels following administration of IMC-48 after prior knockdown with IMC-76 in MCF-7 cells. MCF-7 cells were treated with DMSO or 2 μM of IMC-76 for 24 h. In a similar way, other MCF-7 cells were treated first with 2 μM of IMC-76 and then with 2 or 4 μM of IMC-48 for a further 24 h. IP was performed with antibodies to Sp1 and hnRNP LL and IgG as a negative control and acetyl-histone H3 (AcH3) as a positive control. The P values (***P < 0.001, **P < 0.01) were determined by one-way ANOVA analysis.

Figure 8

Conformational transitions and biological consequences that occur following mutually exclusive binding of IMC-76, IMC-48, and hnRNP LL to the different equilibrating forms of the C-rich strand in the BCL2 promoter. Box A shows the two different major conformational states of the C-rich strand in the BCL2 promoter under different pH conditions. Acidic conditions drive formation of the i-motif, and at pH 6.6 there is a conformational mixture of the flexible hairpin and i-motif. Upon addition of IMC-76, the flexible hairpin form is sequestered (A to B), resulting in depletion of the populations of the i-motif species. Conversely, IMC-48 binds to the central loop of the BCL2 i-motif to sequester this species, and then the RRMs 1 and 2 of the hnRNP LL, which are closely spaced apart, are initially proposed to recognize and bind to both of the lateral loops (II and V) containing the CCCG and CGCC sequences, which are constrained in a single-stranded form (A to C). Following this recognition event, there are hnRNP LL–driven changes in the interhelical conformations such that the two lateral loops are forced apart so that the further spaced apart RRMs 2 and 3 or 4 are able to bind to the 5′ and 3′ CCCG and CGCC recognition sequences to form a stable complex (C to D). Last, hnRNP LL bound to the alternative conformation of the C-rich strand causes transcriptional activation of BCL2 (D to E). The consequence of competition between IMC-76 and hnRNP LL for the different conformational states of the C-rich strand depletes the population undergoing the transition A to C to D to E and repression of BCL2 gene expression. Alternatively, binding of IMC-48 to the BCL2 i-motif leads to an increased amount of i-motif that is bound by hnRNP LL and transcriptional activation (A to C to D).