Caleb Sutherland Abstract
Caleb Sutherland
Ph.D. Candidate (graduated 08/24/2015)
Cancer Biology, GIDP
American Association for Cancer Research Annual Meeting 2015
Philadelphia, Pennsylvania
April 18-22, 2015
A functional role for the MYC i-motif in transcription
ABSTRACT
Abstract
MYC is overexpressed in most types of tumors, but a means to selectively decrease its expression is 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 5th CT element for maximum transcriptional activation. Competition binding studies and bromine footprinting showed that hnRNP K bound to the downstream 5th 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 5Th 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 into the development of new innovative approaches to treat cancers that have MYC overexpression.
Abstract (for Lay Audience)
Cancer cells harbor mutations that cause the cell to over produce proteins involved in cell growth, proliferation and avoidance of cell death. One of these proteins, known as MYC, is overexpressed in over 70 percent of cancer types. The research community has been able to show that inhibition of this protein can be a powerful strategy in treating cancer. Unfortunately efforts to directly drug and inhibit this protein have been slow and unsuccessful. Our lab proposes a new strategy where we target the DNA that is able to give rise to the protein. Their is however very little understanding to the complexity in which a protein is regulated at the DNA level. What we do know is that within the DNA that controls MYC expression there are two types of unique DNA sequences. These sequences have the ability to unwind from double stranded DNA and fold into unique structures known as the G-quadruplex and i-motif. We hypothesis that MYC regulatory proteins can bind to these structures and facilitate or repress protein expression, by instead using drugs that can bind these structures we can inhibit MYC from being made. The research I have conducted identified regulatory proteins and how they interact with these unique DNA sequences and alter MYC expression. Having a better grasp on how MYC is regulated at the DNA level can now provide the initial rationale to identify and develop drugs that can target these structures and silence MYC. We currently are employing drug discovery efforts to target these DNA structures. The use of such interactive compounds is the first step into the development of new innovative approaches to treat cancers that have MYC overexpression.