Defining the Role of Nucleolin on the Transcriptional Regulation of c-MYC through Modulation of the c-MYC NHE III1 Element.

Abstract

The activated product of the c-MYC proto-oncogene is one of the strongest known activators of carcinogenesis. It has been estimated that as many as one-seventh of all cancer deaths are associated with alterations in the c-MYC gene or its expression [1]. Therefore, understanding the regulation of c-MYC expression is a key factor in understanding carcinogenesis in many histologic classes of malignancy. The nuclease hypersensitive element (NHE) III₁ region of the c-MYC promoter has been shown to be particularly important in regulating c-MYC expression. Specifically, the formation of a G-quadruplex structure appears to promote repression of c-MYC transcription. In this dissertation, we investigate the role that nucleolin, a critical player in ribosome biogenesis and cell stress sensing, plays on the transcriptional regulation of the c-MYC promoter through its interaction with the c-MYC G-quadruplex structure. Our studies initiated with the design of a c-MYC G-quadruplex affinity column intended to trap potential c-MYC G-quadruplex-binding proteins that were then identified by LC-MS/MS. After careful examination of the literature of the list of potential c-MYC G-quadruplexbinding proteins, we realized that several of the proteins identified had been previously reported to interact directly with nucleolin. Consequently, we chose to focus our studies on nucleolin, as it could be a central regulator of the (NHE) III region. By performing chromatin immunoprecipitation in HeLa cells, we found that nucleolin indeed interacts with the c-MYC promoter region containing the NHE III₁ element. This binding activity was confirmed by both electromobility shift assay and polymerase stop assay. We provide evidence that nucleolin can induce the formation of the c-MYC G-quadruplex structure from single-stranded DNA, both in linear and circular DNA forms. We show that upon binding, nucleolin increases the stability of the c-MYC G-quadruplex structure leading to repression of c-MYC promoter activity. We also show that nucleolin binds with much higher affinity to G-quadruplex structures with topology similar to that of the parallel c-MYC G-quadruplex, such as those found in the VEGF and PDGF-A promoters; in comparison to G-quadruplexes found in telomeres or the c-MYB promoter, whose have significantly different topology. Interestingly, we also demonstrate that nucleolin binds with higher affinity to the c-MYC G-quadruplex than to its consensus RNA substrate, the nucleolin recognition element (NRE). Furthermore, we show that the C-terminal domain of nucleolin is critical for its interaction and stabilization of the c-MYC G-quadruplex structure. Lastly, we show that the binding of nucleolin to the (NHE) III region causes repression of c-MYC transcription. On the basis of these results, we propose that nucleolin may play an important role in the transcriptional regulation of c-MYC in vivo by inducing the formation of the c-MYC G-quadruplex structure.