Quadruplex structures of muscle gene promoter sequences enhance in vivo MyoD-dependent gene expression

Abstract

Gene promoters are enriched in guanine clusters that potentially fold into quadruplex structures. Such quadruplexes were implicated in the regulation of gene expression, plausibly by interacting with transcription factors. We showed previously that homodimers of the myogenic transcription factor MyoD bound in vitro most tightly bimolecular quadruplexes of promoter sequences of muscle-specific genes. By contrast, MyoD-E47 heterodimers formed tighter complexes with d(CANNTG) E-box motifs that govern muscle gene expression. Here, we show that DNA quadruplexes enhance in vivo MyoD and E-box-driven expression of a firefly luciferase (FL) reporter gene. HEK293 cells were transfected with FL expressing p4RTK-FL vector alone or together with MyoD expressing pEMSV-MyoD plasmid, with quadruplexes of alpha7 integrin or sarcomeric mitochondrial creatine kinase (sMtCK) muscle gene promoters or with a combination thereof. Whereas MyoD elevated by approximately 10-fold the levels of FL mRNA and protein, the DNA quadruplexes by themselves did not affect FL expression. However, together with MyoD, quadruplex DNA increased by approximately 35-fold the amounts of FL mRNA and protein. Without affecting its expression, DNA quadruplexes bound MyoD in the cells. Based on these results, we propose models for the regulation of muscle gene transcription by direct interaction of MyoD with promoter quadruplex structures.

Figure 1.

G′2 quadruplex α7 integrin DNA cooperates with MyoD to enhance the in vivo expression of an E-box driven FL reporter gene. HEK293 cells were transfected with FL expressing p4RTK-FL vector and normalizing RL expressing pCMV-RL plasmid alone or together with either 0.3 μg pEMSV-MyoD expression plasmid, with muscle-gene promoter quadruplexes or single-strands of α7 integrin DNA or with a combination thereof (see ‘Materials and Methods’ section). The cells were harvested and lysed 24 h post-transfection and RL-normalized FL activities were determined. The shown data represent average results of four independent experiments.

Figure 2.

G′2 quadruplex structures of α7 integrin or sMtCK DNA enhance in vivo MyoD-dependent expression of FL reporter gene. HEK293 cells were transfected with FL expressing p4RTK-FL vector and a normalizing RL expressing pCMV-RL plasmid alone or together with the indicated increasing amounts of the MyoD expressing pEMSV-MyoD plasmid without or with 1.2 μg of co-transfected G′2 quadruplex integrin or sMtCK DNA. Following growth for 24 h, the cells were lysed and RL-normalized FL activity and levels of RL-normalized FL mRNA were determined as described under ‘Materials and Methods’ section. The presented results are averages of three or four independent experiments.

Figure 3.

G′2 quadruplex sMtCK DNA does not affect the level of MyoD expression in cells. HEK293 cells were transfected with FL expressing p4RTK-FL vector, normalizing RL expressing pCMV-RL plasmid and the indicated amounts of the MyoD expressing pEMSV-MyoD plasmid without or with 1.2 μg G′2 quadruplex sMtCK DNA. The cells were harvested and lysed 24 h post-transfection and the amounts of expressed MyoD were assessed by western analysis as described under ‘Materials and Methods’ section. Shown is an autoradiogram of the immune blotted denaturing gel with molecular sizes of marker proteins indicated at the right hand.

Figure 4.

Proposed models for the enhancement of transcription by quadruplex DNA. (A) Model for a possible mode of action of quadruplex structures positioned in trans to MyoD and E-box-driven reporter gene. (B) Model for a possible mechanism of action of promoter quadruplex structures positioned in cis to E-box-driven muscle-specific gene. Yellow circles, MyoD; Blue circles, E-protein. See the text for detailed discussion.