Functional adaptation of c-Myc and its role in lymphoma-associated gene regulation

Abstract

The Myc proto-oncogene, is a highly disordered (lack of structure formation) transcription factor (TF), which can bind to its partner proteins and regulate different biological functions of the cells including proliferation, cell cycle, differentiation and apoptosis. In general, Myc deregulation is a major prime force in human tumors, which contributes to their uncontrolled cell proliferation, metastasis and tumor cell immortalizations. In particular, Myc expression influences the transcriptional profile of cells by promoting RNA Polymerase II gene transcription to produce mRNA, as well as the transcription of the rRNA and tRNA genes transcribed by RNA Polymerase I and III, respectively. Thus, controlling expression of ribosome components, required for protein synthesis, appears to be an important role of Myc in normal and cancer cells.

In this thesis, I have studied phylogenetic and molecular evolution of the Myc family proteins, for the first time exploiting their protein order/disorder properties and the extent of their conservation through the Metazoan and beyond. We systematically analyzed the predicted protein disorder profile of Myc family proteins using a range of different algorithms. Therefore, we showed that all Myc proteins are structurally disordered TFs and most of the interaction domains of c-Myc are within disordered regions. Moreover, Using Intrinsically Disorder Protein (IDP) profiles we established a new way to evaluate the evolution of TFs based on their disorder profile. Use of IDR predictions instead of protein sequences produced a better-supported phylogenetic tree of Myc proteins, including large clades containing c-Myc, MycN, MycL and dMyc proteins. In addition, we analyzed the effect of Burkitt’s lymphoma (BL) mutations on the disorder profile and suggested that these adaptive BL-Associated Mutations (BL-AM) could change the local conformation of c-Myc and thereby its functions.

Next, we studied Myc in the nucleolus, an adaptive context that has scarcely been studied, and its involvement in spatial chromatin domain organization of the rRNA genes. Accordingly, we found that Myc activation caused altered spatial organization of the mammalian rDNA by tethering the rDNA to the nucleolar scaffold/matrix via non-transcribed intergenic spacer sequences of the rDNA. In addition, in rat fibroblast cell lines we found that matrix associated rRNA genes are hypo-methylated on DNA sites in their upstream core promoter regions (CpG site at position -145).

Finally, I characterized lymphoma-associated gene expression induced by wild type Myc and how it is adapted in response to BL-AM (T58A and T58I). For this purpose, I established a cell system, consisting of low passage primary B-cells transduced with lentivirus expression constructs, in which wild type or BL-AM Myc could be induced to varying degrees by doxycycline in order to progressively promote a lymphoma-like phenotype. The transduced cells also constitutively over-expressed the BMI1 and BCLXL proteins to inhibit apoptosis. Progressive increase in Myc expression was associated with a progressive increase in cell proliferation, size and proportion of cells in S-phase for both wild type Myc and BL-AM, albeit with some differences between the different Myc proteins. RNA-Seq was used to measure cellular transcripts at seven different doxycycline concentrations for cells expressing wild type and BL-AM Myc. Generalized linear models (as implemented in the MaSigPro package (v3.3)) were then used to identify differentially regulated genes (DEG) with regard to Myc level and/ or mutant status. Thus, we found 4443 DEG common to all three-cell system as well as 543 DEG deregulated only in T58A and T58I cells. On the other hand, the results show DEG common between wild type and T58A (n=553) as well as between wild type and T58I (n=1062). Further analysis, identified 15 gene clusters with different patterns of differential gene expression and these genes were enriched in generally distinct sets of gene ontology terms, indicating little functional overlap between clusters. The data identify gene sets induced by Myc as the cells convert to lymphoma-like cells as well as gene sets where one or both BL-AM augment changes induced by wild type Myc.