The Epidermal Differentiation Complex (EDC) is a co-ordinately regulated locus that is evolutionarily conserved within mammals. It consists of a large number of genes, organised into clusters of gene families, which mainly encode structural constituents of the cornified envelope which replaces the plasma membrane of fully differentiated keratinocytes. It is thought that the developmental program of gene expression at the locus is regulated by specific changes in chromatin structure (Williams et al., 2002). To investigate this, I have characterised the chromatin structure of the EDC in cultured cell lines. These include a keratinocyte cell line, HaCaT cells, in which the locus is active and control cell lines where the locus is inactive. Chromatin is structured on a number of different levels, by the covalent modification of nucleosomes, the arrangement of nucleosomes into chromatin fibres and the arrangement of chromatin fibres into higher order structures within the interphase nucleus. I have assayed chromatin structure on all these levels using Chromatin Immunoprecipitation and Sucrose Gradient Sedimentation Analysis of Chromatin Fibre structure, partnered with oligonucleotide microarrays and Fluorescent In-Situ Hybridisation. By doing so I have examined the role each level of chromatin structure plays in regulating the human EDC and, characterised the relationships between the different levels across a large co-ordinately regulated locus in the human genome.

This study examined the mechanisms underlying the interactions between the natural polyphenol Resveratrol and HDAC inhibitors in both U937 myelomonocytic leukemia cell line and blood samples from AML patients and normal cord blood. Simultaneous exposure to Resveratrol and HDAC inhibitors (Vorinostat-SAHA or Panobinostat-LBH589) resulted in potentiating the lethality caused by any single agent of the combination, this interaction found to be synergistic at multiple concentrations. Exposing U937 cells to minimal toxic doses of Resveratrol and HDACIs results in release of mitochondrial pro-apoptotic proteins AIF and cytochrome c, pro-apoptotic caspase activation especially caspase-8, and induction of DNA damage. These events were associated with increase deacetylation of NF-κB and reactive oxygen species generation, as well as G0-G1 cell cycle arrest. Genetic knockdown of SIRT1 (a deacetylator of NF-κB that is upregulated by Resveratrol) resulted in significant increase in NF-κB acetylation and activity. However, SIRT1 knock down failed to protect U937 cells against combination-induced cell death, implying the possibility of the involvement of other mechanisms in inducing cell death rather than NF-κB deactivation only. Co-incubation of the antioxidant vi MnTBAP significantly reduced Resveratrol/HDACIs induced cell death, and resulted in a marked decrease in caspase-8, caspase-3, and PARP activation. Finally, the combined treatment of Resveratrol/HDACIs induce cell cycle changes possibly through Resveratrol action of blocking cell cycle in S phase exposing more cells to HDACIs lethality. Collectively, these finding indicate that the combined regimen of Resveratrol and HDAC inhibitors promote lethality in U937 cells and primary AML cells by a variety of mechanisms. The approved use of both agents in clinical setting make future clinical studies for development of this drug regimen a potential option in the battle with leukemia.

Genetic epidemiology has shown a large role for genetic influences on schizophrenia. However, the nature of the variants involved is debated. The common disease-common variant (CDCV) hypothesis suggests that schizophrenia is caused by common alleles with small effect sizes. According to the common disease-rare variant (CDRV) hypothesis, schizophrenia is caused by rare variants with large effect sizes. In recent years, evidence has been found for both common and rare variants in schizophrenia. Several SNPs have been associated with schizophrenia through genome-wide association studies (GWAS), supporting the CDCV hypothesis. In support of the CDRV hypothesis, individuals with schizophrenia have been found to have a higher burden of rare copy-number variants (CNVs). Also, several specific rare CNVs have been associated with schizophrenia. The exact mechanisms of these variants are unknown, but common and rare variants appear to affect many of the same pathways in the etiology of schizophrenia.

Genomic imprinting results in monoallelic parent-of-origin gene expression. Therian mammals show conclusive evidence for imprinting, while the evidence in Aves is conflicting. It’s unclear if Aves have the proteins necessary for establishment and maintenance of imprinting loci. Every examined avian orthologue to mammalian imprinted genes shows biallelic expression providing evidence for a lack of imprinting in Aves. While the known parent-of-origin quantitative trait loci in chicken do not overlap with differentiated methylated regions, further analysis with a larger sample size is required. No transcript in the chicken transcriptome at incubation day 4.5 shows parent-of-origin expression, providing strong evidence for a lack of imprinting at this stage of development. Investigating expression of the chicken transcriptome at additional developmental time points, and the transcriptome of other Aves would provide decisive evidence on the presence or lack of imprinting in Aves. Based on current knowledge, Aves lack imprinting as observed in mammals.

Smith-Magenis syndrome (SMS) is a genetic disorder caused by a deletion or mutation of the retinoic acid induced 1 (RAI1) gene on chromosome 17p11.2 that results in haploinsufficiency. SMS patients with a deletion account for 90% of the cases, while the other 10% have a mutation in RAI1. The syndrome is characterized by cognitive impairment, craniofacial abnormalities, sleep disturbances, developmental delay, obesity, and behavioral phenotypes. SMS is thought to affect 1:25,000 live births, although due to similar infantile phenotypes with Down syndrome and Prader-Willi syndrome, SMS may be mis- or under-diagnosed. In a study of 54 children, it was shown that by age 12, females with SMS are in the 90th weight percentile, while males reach the 90th percentile by age 14. It was also shown that viii teens and adults with Smith-Magenis syndrome commonly present with truncal obesity. In order to keep SMS patients healthy, to reduce the risk for future health problems associated with obesity, and to more fully understand the role of Rai1 in Smith-Magenis syndrome, it is first necessary to understand the mechanism by which SMS patients become obese. Mouse models of SMS provide a powerful tool for looking at potential mechanisms of obesity related to the haploinsufficiency of Rai1. Obesity in Smith-Magenis syndrome may result from a combination of I) behavioral, II) metabolic, and III) signaling mechanisms in which the haploinsufficiency of Rai1 causes deviations in critical pathways responsible for energy intake and expenditure. Data suggest that the Rai1+/- mice are obese and hyperphagic. Data also demonstrate that Rai1+/- mice do not have symptoms of metabolic syndrome associated with their obesity. Signaling mechanisms are deviated from normal in Rai1+/- mice, including leptin levels and the expression of Pomc, Mc4r, Bdnf, and Agrp. Treatment with ampakine drug may increase expression of Bdnf and help to control obesity in Rai1+/- mice.

Erythroid Krüppel-like factor (EKLF or KLF1) is a transcription factor with roles in embryonic and adult erythropoiesis. KLF1 knockout mouse embryos die due to severe anemia. Dominant human mutations in KLF1 can cause hereditary persistence of fetal hemoglobin. We show that KLF1 positively regulates β-globin and Bcl11A gene expression using KLF1 knockdown in in vitro-differentiated CD34+ human umbilical cord blood cells. -globin expression appears dependent on KLF1; it is increased with modest KLF1 knockdown but not in cells with low KLF1. KLF2 mRNA amounts are usually increased in KLF1 knockdown. KLF1 knockdown in CD34+ cells results in reduced colony forming ability. Interestingly, the expression of certain proliferation and cell cycle genes is reduced due to KLF1 knockout in mouse or knockdown in human erythroid cells. In conclusion, KLF1 is an important regulator of the β-globin locus and has roles in proliferation and cell cycle.

RNA degradation plays a fundamental role in maintaining cellular homeostasis, along with being a part of normal regulatory mechanisms, whether it occurs as a surveillance mechanism eliminating aberrant mRNAs or during RNA processing to generate mature transcripts. 3’-5’ exoribonucleases are essential mediators of RNA decay pathways, and one such evolutionarily conserved enzyme is polynucleotide phosphorylase (PNPase). The human homologue of this fascinating enzymatic protein (hPNPaseold-35) was cloned a decade ago in the context of terminal differentiation and senescence through a novel ‘overlapping pathway screening’ approach. Since then, significant insights have been garnered about this exoribonuclease and its repertoire of expanding functions. hPNPaseold-35 has progressed a long way from being just a 3’-5’ exoribonuclease to a functionally relevant molecule implicated in a multitude of diverse and important biological effects. hPNPaseold-35 plays central roles in diverse physiological processes including growth inhibition, senescence, mtRNA import, mitochondrial homeostasis, and RNA degradation, all while primarily being localized in the mitochondrial IMS (inter membrane space). hPNPaseold-35 also holds immense promise as a therapeutic agent due to its ability to degrade specific miRNA (miR-221) and mRNA (c-myc) species, and this property can be exploited in treating malignancies that are characterized by upregulation of harmful miRNA or mRNA molecules. But apart from these two targets, little is known about any other targets hPNPaseold-35 may degrade. Thus, the primary objective of this dissertation research was to identify targets other than c-myc or miR-221 that hPNPaseold-35 could directly degrade to discover newer and biologically relevant therapeutic targets for the treatment of hPNPaseold-35 –associated disease states. In order to do this we performed extensive microarray analyses following hPNPaseold-35 overexpression and depletion in mammalian cell lines, and were able to identify transcripts that could be potentially regulated by hPNPaseold-35 directly or indirectly. Apart from this we also analyzed the 3’UTR of c-myc in order to identify any specific sequence or secondary structural elements necessary for hPNPaseold-35 mediated degradation. Lastly, we identified certain residues in hPNPaseold-35 that have been under positive natural selection through evolution.

The Hsp90 chaperone complex associates with the telomerase enzyme, facilitating the assembly of the ribonucleoprotein complex. While previous data from our laboratory indicate that Hsp90 and p23 remain stably associated with (functionally active) telomerase, more recent experiments suggest that these chaperones associate with telomeres independent of telomerase, presumably through a specific interaction with telomere binding proteins. The current study examines the novel interactions between TRF2, TRF1, TIN2 and TPP1 and molecular chaperones (Hsp90, Hsp70, p23). In vitro and in cell experiments have shown an interaction between TRF1 and TRF2 and the molecular chaperones Hsp90 and Hsp70. Inhibition of Hsp90 using drugs that specifically block ATPase activity results in an increased association of TRF1 and TRF2 with Hsp90 to presumably stabilize the telomere associated proteins to the telomere. A definitive explanation as to the mechanisms underlying the chaperone/telomere associated protein interaction has yet to be determined and further studies examining chaperones’ contribution to telomere structure and function are underway. A better understanding of the telomeric proteins and Hsp90 and their roles in nuclear events is important, as both have extremely important functions in the cell. Our current working hypothesis is that chaperone proteins associate with TRF2, TRF1, TIN2 and TPP1 to facilitate telomeric protein-protein interactions and protein-telomere binding in both cancer and normal cells. The interaction between chaperones and telomere binding proteins may eventually provide a better understanding of telomeric structure and function. Defining the mechanisms of telomeric protein regulation is important in the development of new therapeutic approaches for targeting telomeres to induce dysfunction. Clinical trials are underway employing drugs targeting Hsp90 in cancer cells and given the results here, these Hsp90 compounds likely cause telomere alterations.

The DREAM complex assembles during G0/G1 when RB-like protein p130 recruits E2F4, DP1, and a core complex of five MuvB proteins to repress genes involved in cell cycle progression. In S-phase, the MuvB core dissociates from p130 and binds to BMYB transcription factor. Binding of the MuvB core to p130 requires phosphorylation of its subunit LIN52 at S28 residue by DYRK1A protein kinase. However, little is known about how the MuvB core interacts with p130 to form the DREAM complex, and how these interactions are manipulated throughout the cell cycle. In collaboration with Dr. Seth Rubin, we characterized the structural basis for DREAM assembly, and found that the LxSxExL sequence in LIN52 directly interacts with the LxCxE binding cleft within the pocket domain of p130. Furthermore, immunoprecipitation and proliferation assays revealed that mutating the LIN52 LxSxExL sequence to mimic the canonical LxCxE motif found in viral oncoproteins reduces cellular proliferation and stabilizes the DREAM complex in the presence of viral proteins. We addressed how the DREAM complex is disassembled upon cell cycle entry and found that CDK phosphorylation of p130 inactivates the DREAM complex by displacing p130 from the MuvB core. Under certain conditions, we found that BMYB and p130 simultaneously bind the MuvB core, while overexpression of BMYB disrupts DREAM assembly. Together, our study provides insight into the structural mechanisms of DREAM assembly and function, which can help identify novel approaches to halt tumor cell proliferation or dormancy.

DYRK1A is a protein kinase encoded by a gene implicated in Down syndrome pathogenesis. Loss of DYRK1A could promote oncogenic transformation. However, the regulation and substrates of DYRK1A are not fully understood. MudPIT proteomic analysis revealed novel DYRK1A interacting proteins with poorly characterized or even unknown functions. Therefore, the aim of this thesis was to understand the function of DYRK1A through the characterization of its interacting proteins. To achieve this aim, we established stable cell lines expressing these proteins and confirmed the interactions between DYRK1A and seven candidate binding partners. Furthermore, we found that all novel DYRK1A-interacting proteins also bind DCAF7, a previously reported DYRK1A-binding scaffold protein that binds to the N-terminus of DYRK1A. Using cyto-nuclear fractionation and immunostaining we found that DYRK1A-interacting proteins were present in different cellular compartments, suggesting that DYRK1A could play distinct roles in the cell depending on its localization. DYRK1A has been shown to regulate cell proliferation and actin cytoskeleton therefore we used cell proliferation assays and actin staining to determine the role of DYRK1A-interacting proteins in these processes. Here we report functional characterization of the interacting partners of DYRK1A and present cell-based models that will help to understand the function and regulation of this important protein kinase.