<p> Control of cell cycle progression is an important aspect of normal cell biology and is frequently disrupted in cancers. HCFC1 has been established as a critical component of cell cycle regulation, but the mechanism by which it mediates this function is not clearly understood. Recently HCFC1 has been shown to directly interact with the E2F family of proteins, which are key regulators of cell cycle progression, and bind promoters of cell cycle control genes. Interestingly, some of these promoters are also bound by THAP11, another HCFC1-interacting protein, which may be responsible for HCFC1 recruitment to these loci. </p><p> In this study, we analyzed existing genome-wide data and performed bench experiments at a large number of HCFC1 target genes to determine the mode of HCFC1 recruitment to cell cycle control genes. These analyses revealed that, on a genome-wide scale, HCFC1 is highly associated with THAP11 and ZNF143 transcription factors on chromatin, while the association with E2F1 &ndash; a member of the E2F protein family &ndash; is significantly weaker. Furthermore, the results of our study indicate that HCFC1 recruitment to chromatin is conditional on THAP11 and ZNF143, but not E2F1. Contrary to current literature, which suggests that THAP11 and ZNF143 may compete for binding to chromatin, our observations indicate that THAP11, ZNF143, and HCFC1 chromatin occupancy is mutually dependent. </p><p> Genome-wide and in vitro studies demonstrate that THAP11 and ZNF143 recognize overlapping DNA sequences. Given our results above, the mechanism of cooperative binding by these two proteins is unclear and warrants further investigation. To avoid possible artifacts associated with in vitro binding experiments, we used chromosomally-integrated synthetic constructs and CRISPR-Cas9-mediated approaches in intact cells to elucidate the role of DNA sequence in recruitment of the THAP11/ZNF143/HCFC1 complex and to establish its biological relevance. We show that the ACTACA submotif, shared by both THAP11 and ZNF143, directs recruitment of THAP11 and HCFC1 to ZNF143-occupied loci. Importantly, its position, spacing, and orientation relative to the ZNF143 core motif are critical for this action. Biologically, CRISPR-Cas9-mediated alteration of the ACTACA submotif in the endogenous OPHN1 promoter abolished its transcriptional activity and lead to lower RNA Polymerase II and histone H3 lysine 4 tri-methyl levels. Our in vivo approaches provide strong evidence for the molecular role of the ACTACA submotif in THAP11, ZNF143, and HCFC1 cooperative recruitment to chromatin and its biological necessity for target gene expression.</p>

Pyruvate carboxylase (Pyruvate: carbon dioxide ligase (ADP-forming), EC 6.4.1.1), is a biotin-dependent enzyme catalyzing a two step reaction ligating the carboxyl group to pyruvate. This enzyme was shown to be present in several tissues of ten different species of insects. Pyruvate carboxylase was purified from the thoraces of honeybee, Apis mellifera, and was found to be a tetramer consisting of 128 kDa subunits. The activity of this enzyme required acetyl-CoA, ATP, and Mg²⁺. The Kms of the enzyme for bicarbonate and pyruvate were similar to pyruvate carboxylases from other organisms. Pyruvate carboxylase was also purified from the yellow fever mosquito, Aedes aegypti. Two polypeptides of similar molecular weight (133 kDa and 128 kDa) were present and the N-terminal sequences of both were determined. The relative amounts of the 133 kDa and 128 kDa polypeptides were shown to differ in various tissues. The enzyme was found in all tissues examined and was concentrated in the thorax where the amount of the enzyme increased shortly after pupation. Pyruvate carboxylase in thoracic muscles of insects is likely to have an anaplerotic role. Pyruvate carboxylase was also found to be at high levels in the fat body preparations. Clones covering the complete cDNA of pyruvate carboxylase of Aedes aegypti were obtained. The 3942 bp nucleotide sequence including a 3585 bp coding region, was determined from these cDNA clones. The deduced 1195 amino acid sequence has a calculated Mr of 132, 200. Following a putative mitochondrial targeting sequence, three functional domains were identified including biotin carboxylase (BC), carboxyltransferase (CT), and biotin carboxyl carrier protein (BCCP). The mosquito pyruvate carboxylase amino acid sequence showed 55-75% identity to enzymes from other sources. The evolutionary relationship of pyruvate carboxylases among different organisms was analyzed. The evolution of domain structures of the biotin-dependent carboxylases including pyruvate carboxylase was also investigated. The above analysis provided evidence for the coevolution of BC and BCCP domains and early gene duplication events that shaped the family of biotin-dependent carboxylases.

The major developmental cue and the coordinating factor of metamorphic changes in insects is a group of steroid hormones, the ecdysteroids. Using subtractive hybridization I have isolated five cDNA clones that represent genes whose transcripts are up-regulated prior to pupal ecdysis in the nervous system of the tobacco hornworm, Manduca sexta, after a major ecdysteroid peak. Three of the genes, Mng10, Mng14 and tps9 are expressed primarily in the nervous system, the two other genes, esr16 and esr20 are specific to tracheal epithelial cells. In situ hybridizations showed that the transcripts of the two nervous system specific genes were localized to uniquely identifiable neurosecretory cells in the brain and ventral nervous system. The total number of cells expressing these genes in the CNS is 16-20. The third nervous system specific transcript is localized to small patches along the boundary between the central neuropil and neuronal cell bodies, in abdominal ganglia. The patches are smaller than nuclei, possibly mitochondria, and seem to be localized to large tracheal branches. Sequence analysis of the cDNA clones revealed that esr20, esr16 and Mng10 encoded a protein, with slight similarity to already identified sequences. These similarities, however, were insufficient to propose a function for the Manduca genes. Two of the sequences do not seem to encode a protein and are postulated to function as regulatory or structural RNAs. Statistical analysis of the coding and non-coding sequences showed a clear distinction between their behavior using the Zipf'test. Four of the five genes are regulated by ecdysteroids as predicted by the initial screen, transcript levels are reduced at high 20-HE concentrations. More detailed studies of the regulation of a tracheal-specific gene (esr20) showed that the accumulation of the mRNA requires first the presence then the subsequent absence of the ecdysteroids. The role of the ecdysteroids appears to be permissive and an additional factor is required to initiate the accumulation of the mRNA. The transcript of esr20 appears 16 hours before ecdysis and is removed at ecdysis by a mechanism that requires protein synthesis and possibly influences the stability of the transcript.

No description available.

No description available.

<p>The degree of intratumoral hypoxia is clinically correlated to poor response to therapy and increased incidence of distal spread in various cancer subtypes. Specifically, the transcription factor Hypoxia Inducible Factor-1alpha (HIF-1alpha), which is accumulated in cells in response to a hypoxic microenvironment, is implicated in poor disease outcome associated with intratumoral hypoxia. Using novel genetically engineered mouse models of primary soft tissue sarcoma, I show that in vivo genetic deletion of HIF-1alpha specifically in tumor cells 1) decreases the incidence of lung metastases by limiting sarcoma collagen deposition, and 2) improves sarcoma response to radiation therapy by limiting the inflammatory response and metabolic adaptations. These results define HIF-1alpha as a potential target for cancer therapy.</p> / Dissertation

<p>Glioblastoma multiforme (GBM, WHO grade IV astrocytoma) is the most common and lethal primary brain tumor in adults, with an average survival of slightly more than one year after initial diagnosis. GBMs display significant heterogeneity within the tumor mass, among which a subpopulation of cells called glioma-initiating cells (GICs) are responsible for tumorigenesis and resistance to conventional therapies. However, the molecular mechanisms underlying the distinct properties of GICs and non-GICs remain largely unknown. Besides the intrinsic molecular features of these GICs, specific tumor microenvironment, such as hypoxia, has been recognized to enhance the abilities of self-renewal and tumor initiation in GICs, while the mechanisms regulating responses of GICs to hypoxia remain poorly understood. </p><p>MicroRNAs are small non-coding RNAs that play important roles in the progression of various cancer types including GBM. To assess function of miRNAs in regulating GIC properties, we performed a candidate based miRNA qPCR array and found that miR-33a was up-regulated in GICs to promote GIC growth and self-renewal, consistent with its status as an onco-mir with a higher expression pattern associated with poor prognosis of GBM patients. Antagonizing miR-33a function in GICs led to reduced self-renewal and tumor progression in immune-compromised mice, whereas overexpression of miR-33a in non-GICs rendered them to display features associated with GICs. Mechanistically, miR-33a promoted the self-renewal of GICs via enhancing the activities of cAMP/PKA pathway and Notch signaling by targeting negative regulators of these two pathways, PDE8A and UVRAG. Together these findings reveal a miR-33a-centered signaling network that is required for GIC maintenance.</p><p>In the meantime, deregulated miRNAs under hypoxia in solid tumors have been uncovered to affect cancer cell adaptation to the stress through repressing oncogenes or tumor suppressors. We next dissected roles of miRNAs in affecting the responses of GICs to hypoxia. Through combination of a miRNA qPCR array and in vivo functional screen, we identified that miR-215 was critical for the GICs maintenance and GBM progression in the hypoxic stress via modulating the activities of multiple pathways. </p><p>In sum, this study has elucidated essential mechanisms in regulating the biological properties of GICs and their responses to the hypoxic stress through enhancing two distinct miRNA-mediated signal networks. Finding of this work will shed light on the development of specific therapies targeting GICs and the tumor microenvironment for GBM treatment.</p> / Dissertation

Myosins and tropomyosins represent two types of actin filament-associated proteins that often work together in contractile and motile processes in the cell. While the role of thin filament troponin-tropomyosin complexes in regulating striated muscle myosin II is well characterized, the role of tropomyosins in non-muscle myosin regulation is not well understood. Fission yeast has recently proved to be a useful model with which to study regulation of myosin motors by tropomyosin owing to its tractable genetics, well-defined actin cytoskeleton, and established actin biochemistry. A hallmark of type V myosins is their processivity -- the ability to take multiple steps along actin filaments without dissociating. However, the fission yeast type V myosin (Myo52) is a nonprocessive motor whose activity is enhanced by the sole fission yeast tropomyosin (Cdc8). The molecular mechanism and physiological relevance of tropomyosin-mediated regulation of Myo52 transport was investigated using a combination of in vitro and in vivo approaches. Single molecules of Myo52, visualized by total internal reflection fluorescence microscopy, moved processively only when Cdc8 was present on actin filaments. Small ensembles of Myo52 bound to a quantum dot, mimicking the number of motors bound to physiological cargo, also required Cdc8 for continuous motion. Although a truncated form of Myo52 that lacked a cargo-binding domain failed to support function in vivo, it still underwent actin-dependent movement to polarized growth sites. This result suggests that truncated Myo52 lacking cargo, or single molecules of wild-type Myo52 with small cargoes, can undergo processive movement along actin-Cdc8 cables in vivo. These findings outline a mechanism by which tropomyosin facilitates sorting of transport to specific actin tracks within the cell by switching on myosin processivity. To understand the broader implications of actomyosin regulation by tropomyosin we examined the role of two mammalian tropomyosins (Tpm3.1 and Tpm4.2) recently implicated in cancer cell proliferation and metastasis. As previously observed with Cdc8, Tpm3.1 and Tpm4.2 isoforms significantly enhance non-muscle myosin II (Myo2). Additionally, the mammalian tropomyosins enable Myo52 processive movement along actin tracks. In contrast to the positive regulation of Myo2 and Myo52, Cdc8 and the mammalian tropomyosins potently inhibit skeletal muscle myosin II, while having negligible effects on the highly processive mammalian myosin-Va. Thus, different motor outputs favoring functional specification within the same myosin class are possible in the presence of certain tropomyosins. In support of a conserved role for certain tropomyosins in regulating non-muscle actomyosin structures, Tpm3.1 rescued normal contractile ring dynamics, cytokinesis, and fission yeast cell growth in the absence of functional Cdc8. This work has broad implications with regard to regulation of non-muscle and muscle actomyosin function in complex cellular environments such as developing muscle tissue and metastatic cancer cells.

The role of cytosine modifications on nuclear transcription has been well characterized, but the function of DNA methylation in the mitochondrial genome has not been determined. Previous studies conducted by the Taylor laboratory have shown overexpression of the mitochondrial isoform of DNMT1 leads to strand-specific changes in gene expression. Here, we show that increased mtDNMT1 expression leads to an increase in the polycistronic transcript encoding the ND1 and Cox1 sequences. In order to understand the mechanistic basis of these changes, we investigated the effects of CpG methylation in the heavy strand promoter on transcription initiation and TFAM binding. Methylation was found to increase transcription initiation from HSP1 and result in larger TFAM:DNA complexes forming at lower protein concentrations. Our data suggest a functional role for cytosine methylation in the mitochondria, which we propose may have an effect on oxidative phosphorylation and cellular function.

CBP (CREB Binding Protein) is global transcriptional co-activator and histone acetyltransferase. CBP is involved in the modulation of the transcription of many genes via histone acetylation at the promoter regions of the target genes. Also, non-histone proteins and transcription factors can be acetylated by CBP to promote their transcriptional activation. In addition to its transcription co-activator role, CBP is involved in many other pathological and physiological cellular processes such as cell growth and differentiation, cell transformation and development, response to stress, cell cycle regulation and apoptosis. CBP and its paralogue p300, play double-edged roles in the regulation of p53, a well characterized tumor suppressor protein, via ubiquitination and acetylation activities. Prior work has shown that CBP and p300 contribute to the maintenance of physiologic p53 levels in unstressed cells via a cytoplasmic but not nuclear, p53-directed E4 polyubiquitin ligase activity, subsequently leading to p53 proteasomal degradation. Our previous work also revealed that CBP and p300 possess intrinsic cytoplasmic but not nuclear E3 autoubiquitination activity in the absence of cellular stress. The mechanism of the compartmentalized CBP/p300 ubiquitin ligase activities was not studied. In this thesis, I present insights gained from efforts to determine the regulation of CBP ubiquitin ligase activities in the cytoplasm versus nucleus, in the absence and presence of DNA damage stress. Chapter two discusses the effect of DNA damage on CBP E3 autoubiquitination activity and also addresses the differential post translational modifications between cytoplasmic and nuclear CBP, in the absence and presence of DNA damage. Aspects of the regulation of the compartmentalized CBP ubiquitin ligase activities in the absence of cellular stress were covered in chapter three. We employed Multidimensional Protein Identification Technology (MudPIT) and mass spectrometry analysis of purified cytoplasmic and nuclear CBP to identify nuclear and cytoplasmic CBP interacting proteins. MudPIT analysis revealed that Cell Cycle and Apoptosis Regulator protein (CCAR2), also known as Deleted in Breast Cancer 1 protein (DBC1), is a novel CBP-interacting protein, in the cytoplasmic and nuclear compartments. Functional analysis suggested that DBC1 directly regulates cellular compartmentalization of CBP E3 and p53-directed E4 ubiquitination activities. This work identifies the different regulatory mechanisms of differential CBP ubiquitin ligase activities in the absence and presence of DNA damage. Remarkably, DBC1 was identified as a novel binding partner of CBP and a critical regulator of CBP ubiquitination activities towards p53.This work may provide novel strategies for the development of cancer therapeutics against tumors maintaining wild type p53, which have deleted DBC1.