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

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

Generating Random Samples Using Response Surface Methodology without need to Distribution of Parameters

Many of engineering problems have nonlinear or highly nonlinear limit state functions. Different approaches have been developed in calculating of failure probability in these problems. These methods calculate failure probability by generating random samples with a specific distribution. The Monte Carlo is one the most efficient and applicable method among these approaches. However, this method has some problems including need to calculating of variable distribution function parameters and inverse cumulative density function of variables. In order to solve these deficiencies, in the present research, an efficient method for generating samples is presented. Additionally, enhancing performance of Monte Carlo method and more accurate results by minimum computational cost for functions with very low failure probability can be regarded as other advantages of the proposed method. For evaluating performance of the proposed method, four engineering problems have been investigated and the obtained results for calculating of failure probability have been compared with available methods. By applying the proposed method, such main steps can be neglected and stable results with high accuracy can be gained in comparison with traditional methods in lower sample numbers too

Removal of Methylene Blue Dye from Aqueous Solutions Using Activated Fly Ash from Zarand Power Plant in Kerman

Adsorption is one of the major Processes for the removal of dyes from wastewater. This study investigates the removal of methylene blue dye, as an index color, using activated fly ash. For this purpose, fly ash from Zarand Power Plant was activated with formaldehyde in an acidic medium and used as the adsorbent. The parameters involved in the fly ash activation process (including temperature, time, formaldehyde content, as well as acid concentration and content) and those affecting the adsorption process were adjusted for optimized conditions. Experiments were performed with samples of real effluent from Baft Yazd Textile Plant.   Preparation of activated fly ash was accomplished in a formaldehyde to fly ash ratio of 0.25, an acid concentration of 8%, and an acid to fly ash ratio of 6 over 5 hours. Maximum removal efficiency of methylene blue from synthetic samples (with a concentration of 50mg/L) under optimal conditions (pH: 9, contact time: 30 minutes, temperature: 35 0c, and adsorbent dose: 3g/L) was equal to 99.07 ± 0.112%. Under these same conditions, an adsorption capacity of 16.5± 0.55 mg/g was achieved. The methylene blue removal efficiency from real samples under optimal conditions was 91.8±0.36%. Based on the findings of this study, modified fly ash from Zarand Power Plant may be recommended as an inexpensive adsorbent that can be used with a high removal efficiency for removing dyes from industrial effluents

Profile of functional amblyopia cases seen by optometrists in the Ministry of Health (MOH) Malaysia hospitals

Amblyopia is one of the most common causes of visual defi cit in children. Presently, in the Ministry of Health Malaysia hospitals, there is no documented data on the characteristic and profi le of amblyopia cases. This study was conducted to describe the profi le of new amblyopia cases seen by optometrists at the Ministry of Health (MOH) Hospitals. This study was a retrospective and multicenter study including all MOH hospitals with optometry clinics. Clinical record data of amblyopic patients aged 3 to 17 years old who were newly diagnosed between 1st August 2010 to 31st January 2011 and who fulfi lled the inclusion criteria were obtained. Data collected included demography, systemic history, ocular history and optometric fi ndings and diagnosis. Thirty eight MOH hospitals participated and a total of 301 patients were diagnosed with functional amblyopia within the study period. Mean age for these amblyopic patients was 7.70 + 0.16 years old. Boys were the predominant gender (57.1%) and Malay preceded the other races with a 65.4% occurrence. Mild amblyopia was found in 51.5% of the patients, 31.6% were with moderate amblyopia and only 16.9% of patients were severe amblyopia. The underlying amblyogenic causes assessed were ametropia (61.5%), anisometropia (25.2%), strabismus (9.3%) and stimulus deprivation (4.0%). Refractive error was discovered as the most common cause of amblyopia in this study. It is crucial for optometrists to detect this type of visual impairment and undertake an early optometric intervention

Origins of Myc Proteins – Using Intrinsic Protein Disorder to Trace Distant Relatives

<div><p>Mammalian Myc proteins are important determinants of cell proliferation as well as the undifferentiated state of stem cells and their activity is frequently deregulated in cancer. Based mainly on conservation in the C-terminal DNA-binding and dimerization domain, Myc-like proteins have been reported in many simpler organisms within and outside the Metazoa but they have not been found in fungi or plants. Several important signature motifs defining mammalian Myc proteins are found in the N-terminal domain but the extent to which these are found in the Myc-like proteins from simpler organisms is not well established. The extent of N-terminal signature sequence conservation would give important insights about the evolution of Myc proteins and their current function in mammalian physiology and disease. In a systematic study of Myc-like proteins we show that N-terminal signature motifs are not readily detectable in individual Myc-like proteins from invertebrates but that weak similarities to Myc boxes 1 and 2 can be found in the N-termini of the simplest Metazoa as well as the unicellular choanoflagellate, <i>Monosiga brevicollis</i>, using multiple protein alignments. Phylogenetic support for the connections of these proteins to established Myc proteins is however poor. We show that the pattern of predicted protein disorder along the length of Myc proteins can be used as a complementary approach to making dendrograms of Myc proteins that aids the classification of Myc proteins. This suggests that the pattern of disorder within Myc proteins is more conserved through evolution than their amino acid sequence. In the disorder-based dendrograms the Myc-like proteins from simpler organisms, including <i>M. brevicollis</i>, are connected to established Myc proteins with a higher degree of certainty. Our results suggest that protein disorder based dendrograms may be of general significance for studying distant relationships between proteins, such as transcription factors, that have high levels of intrinsic disorder.</p></div

Differential Transcriptional Reprogramming by Wild Type and Lymphoma-Associated Mutant MYC Proteins as B-Cells Convert to a Lymphoma Phenotype

The MYC transcription factor regulates a vast number of genes and is implicated in many human malignancies. In some hematological malignancies, MYC is frequently subject to missense mutations that enhance its transformation activity. Here, we use a novel murine cell system to (i) characterize the transcriptional effects of progressively increasing MYC levels as normal primary B-cells transform to lymphoma cells and (ii) determine how this gene regulation program is modified by lymphoma-associated MYC mutations (T58A and T58I) that enhance its transformation activity. Unlike many previous studies, the cell system exploits primary B-cells that are transduced to allow regulated MYC expression under circumstances where apoptosis and senescence pathways are abrogated by the over-expression of the Bcl-xL and BMI1 proteins. In such cells, transition from a normal to a lymphoma phenotype is directly dependent on the MYC expression level, without a requirement for secondary events that are normally required during MYC-driven oncogenic transformation. A generalized linear model approach allowed an integrated analysis of RNA sequencing data to identify regulated genes in relation to both progressively increasing MYC level and wild type or mutant status. Using this design, a total of 7569 regulated genes were identified, of which the majority (n = 7263) were regulated in response to progressively increased levels of wild type MYC, while a smaller number of genes (n = 917) were differentially regulated, compared to wild type MYC, in T58A MYC- and/or T58I MYC-expressing cells. Unlike most genes that are similarly regulated by both wild type and mutant MYC genes, the set of 917 genes did not significantly overlap with known lipopolysaccharide regulated genes, which represent genes regulated by MYC in normal B cells. The genes that were differently regulated in cells expressing mutant MYC proteins were significantly enriched in DNA replication and G2 phase to mitosis transition genes. Thus, mutants affecting MYC proteins may augment quantitative oncogenic effects on the expression of normal MYC-target genes with qualitative oncogenic effects, by which sets of cell cycle genes are abnormally targeted by MYC as B cells transition into lymphoma cells. The T58A and T58I mutations augment MYC-driven transformation by distinct mechanisms

Functionally significant amino acid substitutions in human c-Myc that occur in cancer.

1<p>Described in Chang et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075057#pone.0075057-Chang1" target="_blank">[42]</a>.</p

Multiple alignment and phylogenetic analysis of Uniprot Ref50 groups of Myc proteins.

<p><b>A.</b> Overview of the ClustalW 2.0 alignment of the representative Myc proteins. The gaps in the N-terminal part of the alignment are primarily due to the longer length of the fruit fly proteins in relation to Myc proteins from other species. <b>B.</b> Phylogentic tree showing relationships between the different Myc proteins. Bootstrap values are shown to indicate the level of support for the different parts of the tree topology shown. Well supported clades containing MycN and SMyc proteins (A), MycL proteis (B), c-Myc proteins (C) and dMyc proteins (D), as well as a less well supported clade containing Myc proteins from animals just outside the Craniata (E), are indicated.</p

Altered relative frequency of ELM category hits associated with ANCHOR regions in the transcript-copied MycL protein (P12525).

1<p>CLV = cleavage sites, LIG = ligand-binding sites, MOD = post-translational modification sites, TRG = targeting sites.</p>2<p>CHI squared test, 2 tailed, df = 3.</p

Predicted protein disorder profiles as an approach to identifying relationships between Myc proteins.

<p>(A) Dendrogram based on intrinsic disorder values calculated by VSL2P. Approximately unbiased bootstrap values (au) and conventional bootstrap values (bp) are shown as percent values in red and green, respectively. Node numbers (edge #) are shown in black and can be used to identify tabulated bootstrap values (au and bp) and associated standard error values in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075057#pone.0075057.s005" target="_blank">Table S4</a>. (B) Comparison of dendrograms made using different intrinsic disorder prediction methods. Heat map showing the level of correlation between dendrograms made with different disorder prediction algorithms.</p