The inhibitor of apoptosis (IAP) proteins traditionally regulate programmed cell death by binding to and inhibiting caspases. Recent studies have uncovered a variety of alternate cellular roles for several IAP family members. The cellular inhibitor of apoptosis 1 (cIAP1) protein, for instance, regulates different axes of the NF-κB signalling pathway. Given the extensive functions of NF-κB signalling in muscle differentiation and regeneration, I asked if cIAP1 also plays critical roles in skeletal muscle myogenesis. In a primary myoblast cell-culture system, genetic and pharmacological approaches revealed that loss of cIAP1 dramatically increases the fusion of myoblasts into myotubes. NF-κB signalling occurs along a classical and an alternative pathway, both of which are highly active in cIAP1-/- myoblasts. Suppression of the alternative pathway attenuates myotube fusion in wildtype and cIAP1-/- myoblasts. Conversely, constitutive activation of the alternative pathway increases myoblast fusion in wildtype myoblasts. cIAP1-/- mice have greater muscle weight and size than wildtypes, as well as an increased number of muscle stem cells. These results identify cIAP1 as a regulator of myogenesis through its modulation of classical and alternative NF-κB signalling pathways. Loss of the structural protein dystrophin in the mdx mouse model of Duchenne muscular dystrophy leads to chronic degeneration of skeletal muscle. The muscle pathology is strongly influenced by NF-κB signaling. Given the roles demonstrated for cIAP1 in cell culture and in vivo, I asked whether loss of cIAP1 would influence muscle pathology in the mdx mouse. To address this question, double-mutant mice were bred lacking both cIAP1 and dystrophin (cIAP1-/-;mdx). Histological analyses revealed that double-mutant mice exhibited reduced indications of damage on several measures, as compared to single-mutant (cIAP1+/+;mdx) controls. Unexpectedly, these reductions were seen in the “slow-twitch” soleus muscle but not in the “fast-twitch” extensor digitorum longus (EDL) muscle. The improvements in pathology of double-mutant solei were associated with reductions in muscle infiltration by CD68-expressing macrophages. Finally, the double-mutant mice exhibited improved endurance and resistance to damage during treadmill-running exercise. Taken together, these results suggest that loss of cIAP1, through its multiple regulatory functions, acts to improve myogenesis and increase muscle resistance to damage.

Platinum-based chemotherapies are effective agents in the treatment of a wide variety of human cancers. However, patients with recurrent disease can become resistant to platinum-based chemotherapy, leading to low overall survival rates. Activating transcription factor 3 (ATF3) is a stress-inducible gene that is a regulator of cisplatin-induced cytotoxicity. ATF3 protein expression was upregulated after cytotoxic doses of cisplatin treatment in a panel of cell lines. A chromatin immunoprecipitation assay showed that upon treatment with cisplatin, ATF3 directly bound to the CHOP gene promoter and this correlated with an increase in CHOP protein expression. In a 1200 compound library screen performed on cancer cell lines, disulfiram, a dithiocarbamate drug, was identified as an enhancer of the cytotoxic effects of cisplatin. This increased cytotoxic action was likely due to disulfiram and cisplatin’s ability to induce ATF3 independently through two separate mechanisms, namely the MAPK and integrated stress pathways. Furthermore, ATF3 protein and mRNA levels were variable amongst human ovarian and lung cancer tissues, suggesting the potential for basal expression of ATF3 to be predictive of cisplatin treatment response. Thus, understanding ATF3’s role in cisplatin-induced cytotoxicity will lead to novel therapeutic approaches that could improve this drug’s efficacy.

The recent discovery of adult neural precursor cells (NPCs) in the dentate gyrus and the subventricular zone of the lateral ventricles of most mammals holds much hope for the potential regeneration of damaged brain tissue. However, their use has been limited by their low numbers and relatively quiescent state, particularly in the aging brain. Previous studies from our laboratory have demonstrated a crucial role for the Rb/E2F pathway in the regulation and proliferation of NPCs, and the direct mechanistic involvement of E2F3 in regulating the pluripotency factor, Sox2. More recently, our investigations into the roles of E2F1 and E2F3 in during adult neurogenesis have revealed that loss of both these genes results in a dramatic loss of adult NPCs. Here, we have employed the Emx1-Cre and Nestin-CreERT2 transgenic models, to specifically delete E2F1 and E2F3 in the cerebral cortex and in NPCs in order to investigate the role of both these genes in embryonic neurogenesis. Our results suggest a switch in the requirement for both E2Fs 1 and 3 between embryonic and adult NPCs, demonstrated by a decrease in NPC proliferation and numbers starting only during late embryonic development and persisting through postnatal neurogenesis. These findings suggest that E2Fs 1 and 3 are essential for the maintenance of stem cells and neurogenesis in the adult brain. Moreover, their deletion results in defects in learning and memory. These studies reveal a crucial role for activating E2Fs in the long-term maintenance and proliferation of neural stem cells.

One of the greatest challenges in modern biology is to understand quantitatively the mechanisms underlying messenger Ribonucleic acid (mRNA) transcription within the cell. To this end, integrated functional genomics attempts to use the vast wealth of data produced by modern large scale genomic projects to understand how the genome is deployed to create a diversity of tissues and species. The expression levels of tens or hundreds of thousands genes are profiled at multiple time points or different experimental conditions in the genomic projects. The profiling results are deposited in large scale quantitative data files that are not possible to analyse without systematic computational methods. In particular, it is much more difficult to experimentally measure the concentration level of transcription factor proteins and their affinity for the promoter region of genes, while it is relatively easy to measure the result of transcription using experimental techniques such as microarrays. In the absence of such biological experiments, it becomes necessary to use in silico techniques to determine the transcription factor regulatory activities given existing gene expression profile data. It therefore presents significant challenges and opportunities to the computer science community. This PhD Project made use of one such in silico technique to determine the differences (if any) in transcription factor regulatory activities of different experimental conditions and time points.The research aim of the Project was to understand the transcriptional regulatory mechanism that controls the sophisticated process of gene expression in cells. In particular, differences in the downstream signalling from which transcription factors can play a role in predisposition to diseases such as Parasitic disease, Cancer, and Neuroendocrine disease. To address this question I have had access to large integrated genomics datasets generated in studies on parasitic disease, lung cancer, and endocrine (hormone) disease. The current state-of-the-art takes existing knowledge and asks "How do these data relate to what we already know?" By applying machine learning approaches the project explored the role that such data can play in uncovering new biological knowledge.

abstract: Vitellogenin (Vg) is an ancient and highly conserved multifunctional protein. It is primarily known for its role in egg-yolk formation but also serves functions pertaining to immunity, longevity, nutrient storage, and oxidative stress relief. In the honey bee (Apis mellifera), Vg has evolved still further to include important social functions that are critical to the maintenance and proliferation of colonies. Here, Vg is used to synthesize royal jelly, a glandular secretion produced by a subset of the worker caste that is fed to the queen and young larvae and which is essential for caste development and social immunity. Moreover, Vg in the worker caste sets the pace of their behavioral development as they transition between different tasks throughout their life. In this dissertation, I make several new discoveries about Vg functionality. First, I uncover a colony-level immune pathway in bees that uses royal jelly as a vehicle to transfer pathogen fragments between nestmates. Second, I show that Vg is localized and expressed in the honey bee digestive tract and suggest possible immunological functions it may be performing there. Finally, I show that Vg enters to nucleus and binds to deoxyribonucleic acid (DNA), acting as a potential transcription factor to regulate expression of many genes pertaining to behavior, metabolism, and signal transduction pathways. These findings represent a significant advance in the understanding of Vg functionality and honey bee biology, and set the stage for many future avenues of research. / Dissertation/Thesis / Doctoral Dissertation Evolutionary Biology 2019

CCAAT/Enhancer Binding Protein Alpha (C/EBPa) is transcription factor protein involved in the differentiation of many cell types, including granulocytes and pulmonary cells. Studies have found that downregulation of C/EBPa leads to tumor formation in the hematopoietic system, bones, lungs, liver, and other organs. Mutations and post translational modifications can also reduce the function of C/EBPa in humans, leading to cancers. Recent studies have made progress in treating acute promyelocytic leukemia (APL), an M3 subtype of acute myeloid leukemia (AML), by incorporating all trans retinoic acid (ATRA) in targeted treatments. ATRA increases C/EBPa expression levels, thus promoting cell differentiation and subsequent apoptosis of leukemia cells. Still, survival rates of AML patients are low. In patients diagnosed with AML subtypes M4 or higher, ATRA does not work. In addition, patients can become resistant to ATRA, making it essential to find an alternative therapy. Therefore, novel drug treatments are necessary. Through a high throughput screening method, we have determined a potent chemical compound, ICCB280, that can enhance C/EBPa expression levels. However, ICCB280’s effective concentration for cell differentiation is relatively high, so we performed a structural-activity relationship (SAR) analysis and discovered a more potent chemical, styryl quinazolinone CCAAT/Enhancer Binding Protein Compound 73 (CEBP- 73). We tested CEBP-73 with two cell lines, HL-60 and A549, which represent leukemia and lung cancer models, respectively. We found that CEBP-73 increased C/EBPa expression levels in a time-dependent, dose-responsive manner in both leukemia cells and lung cancer cells. In western blot analyses, while both ICCB280 and CEBP-73 upregulated C/EBPa protein expression, more protein was expressed in leukemia and lung cancer cells treated with CEBP-73 in a dose-dependent manner than in cells treated with ICCB280. Next, we investigated CEBP-73’s effectiveness in upregulating C/EBPa’s downstream genes. We observed enhanced expression of CEBPe (HL-60 specific downstream gene) in HL-60 cells, and enhanced SPC, NKX2-1 (codes for TTF-1), and HIF-1a (A549 specific downstream genes) expression levels in A549 cells. To investigate the mechanisms of increased C/EBPa expression, we asked whether expression of an extra coding CEBPA (ecCEBPA), a noncoding RNA for C/EBPa that prevents methylation at the CEBPA gene promoter site, will increase. We found that CEBP-73 increased not only C/EBPa expression, but also ecCEBPA in HL-60 and A549 cells. This is the first study to our knowledge that confirms styryl quinazolinone CEBP- 73 can increase ecCEBPA expression. To examine the effectiveness of CEBP-73 in vivo, EGFR-L858R-T790M (EGFRTL/CCSP-rtTA) mice were administered a vehicle solution (control), 1 mg/kg of CEBP-73, or 10 mg/kg CEBP-73. The results showed a trend in CEBP-73 concentrations; higher doses of CEBP-73 induce higher levels of C/EBPa expression in lung tissue. Fewer and smaller tumors were present in lungs treated with CEBP-73 than lungs treated with a control. These findings support the role of CEBP-73 in enhancing C/EBPa expression, including upregulation at the promoter region of the CEBPA gene and at downstream gene loci. In addition, the study’s results affirm the role of C/EBPa as an inducer of cell differentiation in leukemia and lung cancer by showing neutrophils with segmented lobes and granules, indications of cell maturity, in cells treated with compounds that enhanced C/EBPa expression. These data suggest that CEBP-73 could provide novel therapeutic approaches in treating leukemia and lung cancer and could potentially be modified to treat other cancers in targeted drug therapies.

Yin Yang 1 (YY1) is a sequence-specific DNA binding transcription factor that plays an important role in development and differentiation. It activates or represses many genes during cell growth and differentiation and is also required for the normal development of the mammalian embryo. Moreover, it has been shown that YY1 may function as a transcriptional initiator. In this dissertation, regulation of human YY1 is analyzed systematically at three levels: At the genomic level, one major transcriptional initiation site of the YY1 gene was mapped to 478 bp upstream of the ATG translational start site. The YY1 promoter was localized to within 277 bp upstream of the major transcriptional initiation site and was shown to contain multiple binding sites for transcriptional factor Sp1 but lack a consensus TATA box. Overexpression of the adenovirus E1A protein represses expression of the YY1 promoter. At the polypeptide level, the activity of YY1 is regulated through acetylation by p300 and PCAF and deacetylation by HDACs. YY1 was acetylated in two regions: both p300 and PCAF acetylated the central glycine/lysine-rich domain of residues 170- 200, and PCAF also acetylated YY1 at the C-terminal DNA-binding domain. Acetylation of the central region was required for the full transcriptional repression activity of YY1 and targeted YY1 for active deacetylation by HDACs. However, the C-terminal region of YY1 could not be deacetylated. Rather, the acetylated Cterminal region interacted with HDACs, which resulted in stable histone deacetylase activity associated with the YY1 protein. Furthermore, acetylation of the C-terminal domain decreased the DNA binding activity of YY1. At the protein complex level, YY1 was shown to form a complex with up to four different proteins consistently throughout different purification methods. These proteins are likely to have important regulatory roles in the transcriptional activity of YY1. Taken together, these findings will provide valuable information to our understanding of the regulatory mechanisms of transcription in general.

The goal of the ENCODE project has been to characterize regulatory elements in the human genome, such as regions bound by transcription factors (TFs), regions of open chromatin and regions with altered histone modifications. The ENCODE consortium has performed a large number of whole-genome experiments to measure TF binding, chromatin accessibility, gene expression and histone modifications, on a multitude of cell types and conditions in both human and mouse. In this dissertation I describe the analysis of numerous datasets comprising 66 epigenomes, chromatin accessibility and expression data across twelve tissues and seven time points, during mouse embryonic development. We defined chromatin states using histone modification data and performed integrative analysis on the states. We observed coordinated changes of histone mark signals at enhancers and promoters with gene expression. We detected evolutionary conserved bivalent promoters, selectively silencing ~3,400 genes, including hundreds of TFs regulating embryonic development. Second, I present a supervised method to predict TF binding across cell types, with features based on DNA sequence and patterns in DNase I cleavage data. We found that sequence and DNase read counts can outperform other features as well as state-of-the-art methods. I also describe our contribution to the ENCODE TF Binding DREAM challenge where we developed a method, using multiscale features and Extreme Boosting. Third, I describe methods, tools, and computational infrastructure that we have developed to handle large amounts of experimental data and metadata. These tools are fundamental to the selection and integration of large experimental datasets and are at the core of our pipelines, which are described in this dissertation. Finally, I present the protein docking server I developed, as well as algorithms and routines for post-processing predictions and protein structures. Collectively, this body of work encompasses computational approaches to the analyses of chromatin states, gene regulation, and the integration of large experimental datasets. / 2021-08-31T00:00:00Z

Background: Erythropoietin (EPO) can reduce myocardial ischemia/reperfusion (I/R) injury. However, the cellular mechanisms have not been elucidated entirely. The present study was to investigate whether transcription factor GATA-4 could be involved in EPO-induced cardioprotection when it was administered after ischemia, immediately before reperfusion. Methods and results: Male Balb/c mice treated with or without EPO were subjected to ischemia (45 min) followed by reperfusion (4 h). TTC staining showed that the infarct size in EPO-treated mice was significantly reduced compared with untreated I/R mice (P < 0.05). Echocardiography examination suggested that EPO administration significantly improved cardiac function following I/R. TUNEL assay indicated that EPO treatment decreased apoptosis. EPO administration also significantly increased the level of nuclear GATA-4 phosphorylation in the myocardium which was positively correlated with the reduction of myocardial infarction. In vitro hypoxia/re-oxygenation study showed that EPO treatment increased the levels of phospho-GATA-4 and decreased cardiomyocyte apoptosis. More significantly, blocking GATA-4 by transfection of a dominant-negative form of GATA-4 (dnGATA-4) abolished EPO-induced cardioprotective effects. Conclusion: EPO administration after ischemia, just before reperfusion induced cardioprotection and stimulated GATA-4 phosphorylation. Activation of GATA-4 may be one of the mechanisms by which EPO induced protection against myocardial I/R injury.

The Dlx homeobox genes encode homeodomain transcription factors that are involved in multiple developmental aspects. In the brain, these genes take part in neuronal migration and differentiation, more precisely in the migration and differentiation of GABAergic neurons. Dysfunctions in the GABAergic system can lead to various pathological conditions, where impaired inhibitory function is one of the main causes of several neuropathies characterized by neuronal hyperexcitability. The Dlx genes are organized as bi-gene clusters and highly conserved cis-regulatory elements have been previously characterized to be fundamental for the regulation of Dlx expression in developing embryos of different vertebrates. The activity of these regulatory elements and the Dlx genes has been well studied in developmental stages of mice and zebrafish, but little is known about their activity in the adult brain. The extensive neurogenesis that takes place in the adult zebrafish brain provides an ideal platform for the visualization of mechanisms involving dlx genes during adulthood and their possible involvement in adult neurogenesis. Here we show novel information concerning the expression of dlx1a, dlx2a, dlx5a and dlx6a in the adult zebrafish brain and provide insight into the identity of cells that express dlx. We also demonstrate the involvement of dlx genes in brain regeneration and through lineage tracing, their fate determination in the adult zebrafish brain. Analyses in the adult zebrafish has revealed that all four dlx paralogs are expressed in the forebrain and midbrain throughout adulthood and expression is found in almost all areas presenting continuous proliferation. Most dlx-expressing cells present GABAergic neuronal identity in the adult forebrain where, in some areas they were identified as the Calbindin subtype. In some areas of the midbrain, especially within the hypothalamus, many dlxexpressing cell co-localized with a marker for neural stem cells. However, cells expressing dlx iii genes did not co-localize with markers for proliferating cells or for glia. Investigations during brain regeneration in response to injury in the adult zebrafish brain has revealed that dlx5a expression decreases shortly after lesion and that the dlx5a/6a bi-gene cluster, more specifically, dlx5a, is up regulated during the peak of regeneration response proposing a possible role for dlx during regeneration in adults. Studies of lineage tracing have shown the progeny of dlx1a/2a-expressing cells in adults are located within small clusters in different areas of the adult brain where they seem to become mature neurons. Our observations provide a better understanding about the role of dlx genes during adulthood, further contributing to the general knowledge of the molecular pathways involved in adult neurogenesis and regeneration in the zebrafish adult brain.