Lubrano Lavadera, Miriam (2013) Myc-mediated transcription control of SAE1 and PUMA loci in cancer and reprogramming. [Tesi di dottorato]

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The Myc oncogene is the most studied transcription factor in biology. It plays an important role in development and cancer biology regulating cell cycle and apoptosis. The Myc oncogene is also implicated in a variety of other cellular processes such as self-renewal, differentiation and reprogramming in which its involvement is less understood. Despite growing evidence linking increase of Myc protein to stem-like phenotypes in cancer, the mechanism underlying Myc’s contribution to the acquisition of “stemness” at a molecular level it is not yet clear. To regulate its target genes, Myc binds to a specific sequence within DNA and mostly acts as a transcriptional activator. However, Myc down-regulates a small number of genes including PUMA and GADD45a the activity of which relies also upon FOXO3a transcription factor. In this study we have attempted i) to delineate the crosstalk between the Myc and FOXO3a pathways and ii) to suggest a molecular mechanism linking the increase of Myc protein and the consequent activation of its target genes to cell reprogramming. The RAT and hT-RPE cell lines expressing a 4-hydroxytamoxifen-inducible MycER chimera have been used to address the first task, demonstrating that following induction, Myc is rapidly recruited to PUMA and GADD45a. Furthermore, a concomitant switch in promoter occupancy from FOXO3a to PUMA was observed. Myc recruitment stimulates deacetylation of H3 and H4 histones and methylation of the lysine 9 in H3 on both PUMA and GADD45a promoters leading to their downregulation. PUMA is a close neighbor of SUMO-activating enzyme 1 (SAE1) and whilst studying the regulation of PUMA, we observed a peak of RNA polymerase II in the second intron of the PUMA gene, responsible for activation of SAE1. We found that Myc activates SAE1 transcription via direct binding to canonical E-boxes in the vicinity of its transcription start site. The downregulation of PUMA and GADD45a and the up-regulation of SAE1 have also been seen in the heterogeneous primary murine mammary progenitor cells following their reprogramming into stem cells induced by Myc. On the contrary, when expression of Myc leads to the acquisition of ‘stemness’ within a homogeneous population of murine progenitor cells we observed up-regulation of PUMA while SAE1 was not affected, suggesting that in these cells the regulation of PUMA remains under the transcriptional control of p53. Taken together, our data highlight a role for Myc in cell growth via the specific inhibition of FOXO3a-dependent transcription of PUMA and GADD45a and add a new insight in the context of Myc-driven oncogenesis. In fact, we show that, at least during Myc-mediated reprogramming of heterogeneous primary murine mammary progenitor cells, Myc activates directly SAE1 transcription. The activation of SAE1 has been previously shown by others as necessary for cancerous transformation.

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