The present studies were undertaken for the purpose of identifying new mechanisms by which signaling via the mTOR pathway regulates gene expression at the transcriptional level. Our starting point was a series of studies on two hepatic cell lines, one that is sensitive to the anti-proliferative effects of rapamycin and one that is not. We reasoned that the set of genes whose expression was modulated in response to rapamycin in both cell lines would represent genes whose control was independent of growth arrest. We further expected that examination of the promoter regions of these genes would allow for the identification of transcription factor binding domains for mTORC1-responsive transcription factors.
The results we obtained were unexpected. The only transcription factor binding sites that were identified contained E-boxes, the consensus sequence of which is CANNTG, with a palindromic canonical sequence of CACGTG 
. We interpreted this result as indicating that rapamycin-induced inhibition of signaling by mTORC1 might affect transcriptional control by the c-Myc network 
. Such a conclusion was supported by the commonality in the biological functions of mTOR and c-Myc. Both regulate cell growth, metabolism, gene expression, ribosome biogenesis and protein translation 
In order to test the hypothesis that there was a functional intersection between mTORC1 signaling and c-Myc regulation of gene expression, we interrogated the list of rapamycin-sensitive genes for their presence in a population of genes whose expression was regulated in response to the induction of c-myc
in rat fibroblasts. This approach to identifying c-Myc targets involved not just the detection of changes in expression but also a kinetic expression profiling analysis 
. By using this approach, we were able to assess not just the intersection between the populations of genes regulated by c-Myc and mTORC1, but also the upregulation versus downregulation of these genes. The results of this analysis demonstrated that c-Myc targets were indeed over-represented among the population of rapamycin-sensitive genes. With regard to direction of regulation, there was a modest concordance that was consistent with co-regulation by c-Myc and mTORC1, two positive regulators of cell growth and proliferation. Furthermore, these observations pertained to both the rapamycin-sensitive and rapamycin-resistant cell lines. The significance of these observations was supported by a similar analysis using a population of genes identified as targets for NFκB. In this case, no relationship with mTORC1 effects was identified.
Based on the intersection between the populations of genes regulated by mTORC1 and c-Myc, we hypothesized that c-Myc might be a direct or indirect participant in the pathway by which mTORC1 regulates gene expression. To test this hypothesis, we characterized rapamycin-induced changes in gene expression in the HO15.19 cell line. These cells were originally derived from TGR-1 cells, which were in turn derived from the Rat-1 fibroblast cell line 
. HO15.19, made null for c-myc
by homologous recombination 
, are an appropriate model system for studying the role of c-myc
in a variety of cell processes. In addition, the TGR-1 and HO15.19 cells represented a second model system in which we were able to confirm the intersection in the regulation of gene expression by mTORC1 and c-Myc.
To further assess the relationship between c-Myc and mTORC1 in the regulation of transcription, we examined the distribution of E-boxes among rapamycin responsive genes in the WB-F344 and WB311 hepatic cells. The significance of E-boxes lies in their role in c-Myc function in that c-Myc regulation of target genes appears to involve direct binding to E-boxes. Mutations in the E-boxes of two c-Myc target genes, nucleolin and BN51, result in the abrogation of transcriptional activation by c-Myc 
. Mutations in E-boxes are also associated with the removal of the repression of gene activation induced by the c-Myc binding partner Mad 
. Further, recent studies have shown that upregulated c-Myc targets in the HOMycER12 fibroblasts induced by OHT were enriched for E-boxes (manuscript in preparation, Sedivy et al.). We found that the E-box distribution along the promoter region of rapamycin-regulated genes in the WB-F344 and WB311 cells was similar and that they resembled the distribution observed in drosophila 
and humans 
. The E-box distribution in all of these cases tends to show location near the transcription start site. We found that the majority of promoters in rapamycin-regulated genes in the WB-F344 and WB311 cells contained one E-box. We did not find a difference in the number or distribution of E-boxes among genes that were upregulated versus downregulated in response to rapamycin. In summary, our data indicate that c-Myc and mTORC1 may overlap in their regulation of transcription via E-boxes, but that this overlap is independent of c-Myc itself.
Although c-Myc and mTOR are known to be central regulators of comparable biological processes, this is the first study, to our knowledge, that has identified a set of genes that are targets of both c-Myc and mTORC1. mTOR 
and c-Myc 
are known to regulate cellular metabolism and, more specifically, glucose metabolism 
. We found that rapamycin affected various metabolic and biosynthetic pathways in the TGR-1 and HO15.19 cells, similar to our observations in the WB-F344 and WB311 cells. The glycolysis/gluconeogenesis pathway was affected by rapamycin in all four cell lines with the gene for phosphoglycerate kinase-1 (Pgk1
) being downregulated in every case. Further study would be warranted to determine the significance of this observation.
With regard to the intersection between c-Myc and mTOR signaling, our pathway network reconstruction analyses indicated a role for c-myc
in mTORC1 action in the WB-F344, WB311 and TGR-1 cell lines. Prior studies have shown that rapamycin can downregulate both the transcription 
and translation 
in several cell types. It may be that the effect of rapamycin on the expression of c-myc
and cyclin D is not uniform and is cell type specific. Other studies examining the intersection between c-Myc and mTOR have demonstrated an ability of c-Myc to modulate signaling through the mTOR pathway via transcriptional downregulation of the tuberous sclerosis complex genes 
, which are negative regulators of mTOR-mediated translation activation. Finally, c-Myc has been shown to abrogate the regulation of translation by mTOR by controlling the expression and activity of the key translation regulator, 4EBP-1 
In summary, we have demonstrated an intersection between mTORC1 and c-Myc regulation of gene expression. This was observed in both hepatic cells and fibroblasts. However, control of gene expression by mTORC1 was independent of c-Myc, indicating that the intersection between these two systems is likely a reflection of their biological function, not their mechanism of gene expression control. Furthermore, our inability to detect the involvement of specific transcription factor binding domains in the regulation of gene expression by mTORC1 may indicate the involvement of epigenetic mechanisms, microRNAs or both in mTORC1 signaling.