Abstract 2078: A functional role for the MYC i-motif in transcription
Authors: Sutherland Caleb , Hurley Laurence 
Published: August 2015

Abstract
MYC is overexpressed in most types of tumors, but a means to selectively decrease its expression has yet to be found. Our recent findings on modulation of bcl-2 gene expression through protein interactions with the bcl-2 i-motif have provided a basis for further investigation of MYC gene control. It is proposed that the MYC i-motif could function in a similar molecular switch mechanism as in bcl-2. Binding sites for heterogeneous nuclear ribonucleoprotein K (hnRNP K) within the MYC promoter also exist in the i-motif-forming sequence. Circular dichroism and bromine footprinting confirmed that this DNA sequence is able to form an i-motif, and systematic mutation of the cytosine residues in this sequence has revealed a 5:5:5 loop configuration. Indeed, all loops of the i-motif, when folded into a 5:5:5 loop configuration, contain the hnRNP K consensus sequence (CCCT). Previous studies show that hnRNP K binds to this i-motif-forming sequence, but it was assumed to be single-stranded. Binding studies revealed that hnRNP K has more binding affinity to its consensus sequence in the i-motif compared to a mutant sequence where the i-motif cannot form. Further investigation of the MYC promoter revealed an additional two runs of cytosine seven bases downstream of the MYC i-motif. Biophysical studies showed that the additional two runs were not involved in i-motif formation; however, recent studies describe their importance for transcriptional activation. We found that hnRNP K preferred the longer 5CT sequence compared to the i-motif-forming 4CT sequence when using a competitive binding assay. Utilizing luciferase reporters containing either the 4CT or 5CT sequence validated that hnRNP K required both the i-motif and the fifth CT element for maximum transcriptional activation. Competition binding studies and bromine footprinting showed that hnRNP K bound to the downstream fifth CT element and the central and lateral loops of the i-motif. Additionally, we found that co-overexpression of Sp1 and hnRNP K induced a 10-fold increase in luciferase activity in the 5CT reporter only. We hypothesize that Sp1 continuously primes the promoter to initiate transcription, inducing more negative superhelicity and increasing the melting of duplex DNA. This increased melting grants hnRNP K's three KH domains access to the i-motif loops and the fifth CT element. Confirmation by ChIP analysis validated that Sp1 overexpression causes an increase in hnRNP K occupancy at the MYC promoter. These findings provide new insight into the mechanisms of MYC transcriptional control by the i-motif and G-quadruplex. We are employing drug discovery efforts that can target this molecular switch and inhibit MYC from being transcribed. The use of such interactive compounds is the first step in the development of new innovative approaches to treat cancers that have MYC overexpression.
Citation Format: Caleb Sutherland, Laurence Hurley. A functional role for the MYC i-motif in transcription. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2078. doi:10.1158/1538-7445.AM2015-2078