Functional Analysis of MrpL55, Vig and Mat1 Acting at the Crossroad of Cell Cycle, Transcription and Metabolism Regulation

Cell proliferation, transcription and metabolism are regulated by complex partly overlapping signaling networks involving proteins in various subcellular compartments. The objective of this study was to increase our knowledge on such regulatory networks and their interrelationships through analysis of MrpL55, Vig, and Mat1 representing three gene products implicated in regulation of cell cycle, transcription, and metabolism. Genome-wide and biochemical in vitro studies have previously revealed MrpL55 as a component of the large subunit of the mitochondrial ribosome and demonstrated a possible role for the protein in cell cycle regulation. Vig has been implicated in heterochromatin formation and identified as a constituent of the RNAi-induced silencing complex (RISC) involved in cell cycle regulation and RNAi-directed transcriptional gene silencing (TGS) coupled to RNA polymerase II (RNAPII) transcription. Mat1 has been characterized as a regulatory subunit of cyclin-dependent kinase 7 (Cdk7) complex phosphorylating and regulating critical targets involved in cell cycle progression, energy metabolism and transcription by RNAPII. The first part of the study explored whether mRpL55 is required for cell viability or involved in a regulation of energy metabolism and cell proliferation. The results revealed a dynamic requirement of the essential Drosophila mRpL55 gene during development and suggested a function of MrpL55 in cell cycle control either at the G1/S or G2/M transition prior to cell differentiation. This first in vivo characterization of a metazoan-specific constituent of the large subunit of mitochondrial ribosome also demonstrated forth compelling evidence of the interconnection of nuclear and mitochondrial genomes as well as complex functions of the evolutionarily young metazoan-specific mitochondrial ribosomal proteins. In studies on the Drosophila RISC complex regulation, it was noted that Vig, a protein involved in heterochromatin formation, unlike other analyzed RISC associated proteins Argonaute2 and R2D2, is dynamically phosphorylated in a dsRNA-independent manner. Vig displays similarity with a known in vivo substrate for protein kinase C (PKC), human chromatin remodeling factor Ki-1/57, and is efficiently phosphorylated by PKC on multiple sites in vitro. These results suggest that function of the RISC complex protein Vig in RNAi-directed TGS and chromatin modification may be regulated through dsRNA-independent phosphorylation by PKC. In the third part of this study the role of Mat1 in regulating RNAPII transcription was investigated using cultured murine immortal fibroblasts with a conditional allele of Mat1. The results demonstrated that phosphorylation of the carboxy-terminal domain (CTD) of the large subunit of RNAPII in the heptapeptide YSPTSPS repeat in Mat-/- cells was over 10-fold reduced on Serine-5 and subsequently on Serine-2. Occupancy of the hypophosphorylated RNAPII in gene bodies was detectably decreased, whereas capping, splicing, histone methylation and mRNA levels were generally not affected. However, a subset of transcripts in absence of Mat1 was repressed and associated with decreased occupancy of RNAPII at promoters as well as defective capping. The results identify the Cdk7-CycH-Mat1 kinase submodule of TFIIH as a stimulatory non-essential regulator of transcriptional elongation and a genespecific essential factor for stable binding of RNAPII at the promoter region and capping. The results of these studies suggest important roles for both MrpL55 and Mat1 in cell cycle progression and their possible interplay at the G2/M stage in undifferentiated cells. The identified function of Mat1 and of TFIIH kinase complex in gene-specific transcriptional repression is challenging for further studies in regard to a possible link to Vig and RISC-mediated transcriptional gene silencing.

Stress and developmental responses in Arabidopsis thaliana: regulation through the transcription factor-interacting protein RCD1

Plants constantly face adverse environmental conditions, such as drought or extreme temperatures that threaten their survival. They demonstrate astonishing metabolic flexibility in overcoming these challenges and one of the key responses to stresses is changes in gene expression leading to alterations in cellular functions. This is brought about by an intricate network of transcription factors and associated regulatory proteins. Protein-protein interactions and post-translational modifications are important steps in this control system along with carefully regulated degradation of signaling proteins. This work concentrates on the RADICAL-INDUCED CELL DEATH1 (RCD1) protein which is an important regulator of abiotic stress-related and developmental responses in Arabidopsis thaliana. Plants lacking this protein function display pleiotropic phenotypes including sensitivity to apoplastic reactive oxygen species (ROS) and salt, ultraviolet B (UV-B) and paraquat tolerance, early flowering and senescence. Additionally, the mutant plants overproduce nitric oxide, have alterations in their responses to several plant hormones and perturbations in gene expression profiles. The RCD1 gene is transcriptionally unresponsive to environmental signals and the regulation of the protein function is likely to happen post-translationally. RCD1 belongs to a small protein family and, together with its closest homolog SRO1, contains three distinguishable domains: In the N-terminus, there is a WWE domain followed by a poly(ADP-ribose) polymerase-like domain which, despite sequence conservation, does not seem to be functional. The C-terminus of RCD1 contains a novel domain called RST. It is present in RCD1-like proteins throughout the plant kingdom and is able to mediate physical interactions with multiple transcription factors. In conclusion, RCD1 is a key point of signal integration that links ROS-mediated cues to transcriptional regulation by yet unidentified means, which are likely to include post-translational mechanisms. The identification of RCD1-interacting transcription factors, most of whose functions are still unknown, opens new avenues for studies on plant stress as well as developmental responses.

Transcription factor GATA4 and apoptotic TRAIL pathway in granulosa cell function and tumors

Normal growth and development require the precise control of gene expression. Transcription factors are proteins that regulate gene expression by binding specific sequences of DNA. Abnormalities in transcription are implicated in a variety of human diseases, including cancer, endocrine disorders and birth defects. Transcription factor GATA4 has emerged as an important regulator of normal development and function in a variety of endoderm- and mesoderm- derived tissues, including gut, heart and several endocrine organs, such as gonads. Mice harboring a null mutation of Gata4 gene die during embryogenesis due to failure in heart formation, complicating the study of functional role of GATA4 in other organs. However, the expression pattern of GATA4 suggests it may play a role in the regulation of ovarian granulosa cell development, function and apoptosis. This premise is supported by in vitro studies showing that GATA4 regulates several steroidogenic enzymes as well as auto-, para- and endocrine signaling molecules important for granulosa cell function. This study assessed the in vivo role of GATA4 for granulosa cell function by utilizing two genetically modified mouse strains. The findings in the GATA4 deficient mice included delayed puberty, impaired fertility and signs of diminished estrogen production. At the molecular level, the GATA4 deficiency leads to attenuated expression of central steroidogenic genes, Steroidogenic acute regulatory protein (StAR), Side-chain cleavage (SCC), and aromatase as a response to stimulations with exogenous gonadotropins. Taken together, these suggest GATA4 is necessary for the normal ovarian function and female fertility. Programmed cell death, apoptosis, is a crucial part of normal ovarian development and function. In addition, disturbances in apoptosis have been implicated to pathogenesis of human granulosa cell tumors (GCTs). Apoptosis is controlled by extrinsic and intrinsic pathways. The intrinsic pathway is regulated by members of Bcl-2 family, and its founding member, the anti-apoptotic Bcl-2, is known to be important for granulosa cell survival. This study showed that the expression levels of GATA4 and Bcl-2 correlate in the human GCTs and that GATA4 regulates Bcl-2 expression, presumably by directly binding to its promoter. In addition, disturbing GATA4 function was sufficient to induce apoptosis in cultured GCT- derived cell line. Taken together, these results suggest GATA4 functions as an anti-apoptotic factor in GCTs. The extrinsic apoptotic pathway is controlled by the members of tumor necrosis factor (TNF) superfamily. An interesting ligand of this family is TNF-related apoptosis-inducing ligand (TRAIL), possessing a unique ability to selectively induce apoptosis in malignant cells. This study characterized the previously unknown expression of TRAIL and its receptors in both developing and adult human ovary, as well as in malignant granulosa cell tumors. TRAIL pathway was shown to be active in GCTs suggesting it may be a useful tool in treating these malignancies. However, more studies are required to assess the function of TRAIL pathway in normal ovaries. In addition to its ability to induce apoptosis in GCTs, this study revealed that GATA4 protects these malignancies from TRAIL-induced apoptosis. GATA4 presumably exerts this effect by regulating the expression of anti-apoptotic Bcl-2. This is of particular interest as high expression of GATA4 is known to correlate to aggressive GCT behavior. Thus, GATA4 seems to protect GCTs from endogenous TRAIL by upregulating anti-apoptotic factors such as Bcl-2.

Development stage-specific expression of fibroin in the silk worm Bombyx mori is regulated translationally

The contents of fibroin H RNA as a function of development have been quantitated in the posterior silk glands of Bombyx mori larvae on different days of 4th and 5th instars. The fibroin RNA levels increased during the feeding stages of larvae and the RNA got completely degraded during the interim moult. The patterns of accumulation of fibroin RNA were similar in both the instars. Although there was considerable increase in the fibroin RNA content during the 5th larval instar, the relative abundance of fibroin RNA in the total RNA was fairly constant during the 4th and 5th instars. The increased content of fibroin RNA in 5th instar was the consequence of an overall increase in transcription accompanying the development progress, rather than specific increase only in fibroin transcription. The contents of fibroin protein in the 4th and 5th instars of development have also been quantitated making use of a sensitive radioimmune assay with a purified, antifibroin antibody. There were substantial differences between 4th and 5th instars in the absolute fibroin contents as well as the relative proportion of fibroin in the total proteins. These results implied that although the fibroin gene was transcribed at the same efficiency during the 4th and 5th instars, the translational efficiency was much lower during the 4th instar. The extent of polyadenylation of fibroin RNA was similar in both instars. However, there was a two-fold increase in the polysome association of fibroin RNA in the 5th instar. Over and above this, there was substantial increase during the 5th instar in the contents of those tRNAs. (e.g. Gly, Ala and Ser) which are abundantly represented in fibroin and therefore directly related to the expression of fibroin. The increased polysome association of fibroin mRNA and the adequate supply of cognate tRNAs in the 5th instar, together contributes to the translational regulation of fibroin in a developmental stage-specific manner. Based on these observations, we propose that translational regulation plays a major role in the development stage-specific synthesis of fibroin in Bombyx mori.

Specificity in the regulation of eukaryotic gene transcription

The regulation of eukaryotic gene transcription poses major challenges in terms of the innumerable protein factors required to ensure tissue or cell-type specificity. While this specificity is sought to be explained by the interaction of cis-acting DNA elements and thetrans-acting protein factor(s), considerable amount of degeneracy has been observed in this interaction. Immunoglobulin heavy chain gene expression in B cells and liver-specific gene expression are discussed as examples of this complexity in this article. Heterodimerization and post-translational modification of transcription factors and the organization of composite promoter elements are strategies by which diverse sets of genes can be regulated in a specific manner using a finite number of protein factors

Co-evolution of specific amino acid in sigma 1.2 region and nucleotide base in the discriminator to act as sensors of small molecule effectors of transcription initiation in mycobacteria

The transcription from rrn and a number of other promoters is regulated by initiating ribonucleotides (iNTPs) and guanosine tetra/penta phosphate (p)ppGpp], either by strengthening or by weakening of the RNA polymerase (RNAP)-promoter interactions during initiation. Studies in Escherichia coli revealed the importance of a sequence termed discriminator, located between -10 and the transcription start site of the responsive promoters in this mode of regulation. Instability of the open complex at these promoters is attributed to the lack of stabilizing interactions between the suboptimal discriminator and the 1.2 region of sigma 70 (Sig70) in RNAP holoenzyme. We demonstrate a different pattern of interaction between the promoters and sigma A (SigA) of Mycobacterium tuberculosis to execute similar regulation. Instead of cytosine and methionine, thymine at three nucleotides downstream to -10 element and leucine 232 in SigA are found to be essential for iNTPs and pppGpp mediated response at the rrn and gyr promoters of the organism. The specificity of the interaction is substantiated by mutational replacements, either in the discriminator or in SigA, which abolish the nucleotide mediated regulation in vitro or in vivo. Specific yet distinct bases and the amino acids appear to have co-evolved' to retain the discriminator-sigma 1.2 region regulatory switch operated by iNTPs/pppGpp during the transcription initiation in different bacteria.

STAT-1 expression is regulated by IGFBP-3 in malignant glioma cells and is a strong predictor of poor survival in patients with glioblastoma Laboratory investigation

Object. Insulin-like growth factor binding proteins (IGEBPs) have been implicated in the pathogenesis of glioma. In a previous study the authors demonstrated that IGFBP-3 is a novel glioblastoma biomarker associated with poor survival. Since signal transducer and activator of transcription 1 (STAT-1) has been shown to be regulated by IGFBP-3 during chondrogenesis and is a prosurvival and radioresistant molecule in different tumors, the aim in the present study was to explore the functional significance of IGFBP-3 in malignant glioma cells, to determine if STAT-1 is indeed regulated by IGFBP-3, and to study the potential of STAT-1 as a biomarker in glioblastoma. Methods. The functional significance of IGFBP-3 was investigated using the short hairpin (sh)RNA gene knockdown approach on U251MG cells. STAT-1 regulation by IGFBP-3 was tested on U251MG and U87MG cells by shRNA gene knockdown and exogenous treatment with recombinant IGFBP-3 protein. Subsequently, the expression of STAT-1 was analyzed with real-time reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) in glioblastoma and control brain tissues. Survival analyses were done on a uniformly treated prospective cohort of adults with newly diagnosed glioblastoma (136 patients) using Kaplan-Meier and Cox regression models. Results. IGFBP-3 knockdown significantly impaired proliferation, motility, migration, and invasive capacity of U251MG cells in vitro (p < 0.005). Exogenous overexpression of IGFBP-3 in U251MG and U87MG cells demonstrated STAT-1 regulation. The mean transcript levels (by real-time RT-PCR) and the mean labeling index of STAT-1 (by IHC) were significantly higher in glioblastoma than in control brain tissues (p = 0.0239 and p < 0.001, respectively). Multivariate survival analysis revealed that STAT-1 protein expression (HR 1.015, p = 0.033, 95% CI 1.001-1.029) along with patient age (HR 1.025, p = 0.005, 95% CI 1.008-1.042) were significant predictors of shorter survival in patients with glioblastoma. Conclusions. IGFBP-3 influences tumor cell proliferation, migration, and invasion and regulates STAT-1 expression in malignant glioma cells. STAT-1 is overexpressed in human glioblastoma tissues and emerges as a novel prognostic biomarker.

Molecular mechanisms of interleukin-2 gene inducibility: developmental control and combinatorial action of transcription factors

Interleukin-2 is one of the lymphokines secreted by T helper type 1 cells upon activation mediated by T-cell receptor (TCR) and accessory molecules. The ability to express IL-2 is correlated with T-lineage commitment and is regulated during T cell development and differentiation. Understanding the molecular mechanism of how IL-2 gene inducibility is controlled at each transition and each differentiation process of T-cell development is to understand one aspect of T-cell development. In the present study, we first attempted to elucidate the molecular basis for the developmental changes of IL-2 gene inducibility. We showed that IL-2 gene inducibility is acquired early in immature CD4- CD8-TCR- thymocytes prior to TCR gene rearrangement. Similar to mature T cells, a complete set of transcription factors can be induced at this early stage to activate IL-2 gene expression. The progression of these cells to cortical CD4^+CD8^+TCR^(1o) cells is accompanied by the loss of IL-2 gene inducibility. We demonstrated that DNA binding activities of two transcription factors AP-1 and NF-AT are reduced in cells at this stage. Further, the loss of factor binding, especially AP-1, is attributable to the reduced ability to activate expression of three potential components of AP-1 and NF-AT, including c-Fos, FosB, and Fra-2. We next examined the interaction of transcription factors and the IL-2 promoter in vivo by using the EL4 T cell line and two non-T cell lines. We showed an all-or-none phenomenon regarding the factor-DNA interaction, i.e., in activated T cells, the IL-2 promoter is occupied by sequence-specific transcription factors when all the transcription factors are available; in resting T cells or non-T cells, no specific protein-DNA interaction is observed when only a subset of factors are present in the nuclei. Purposefully reducing a particular set of factor binding activities in stimulated T cells using pharmacological agents cyclosporin A or forskolin also abolished all interactions. The results suggest that a combinatorial and coordinated protein-DNA interaction is required for IL-2 gene activation. The thymocyte experiments clearly illustrated that multiple transcription factors are regulated during intrathymic T-cell development, and this regulation in tum controls the inducibility of the lineage-specific IL-2 gene. The in vivo study of protein-DNA interaction stressed the combinatorial action of transcription factors to stably occupy the IL-2 promoter and to initiate its transcription, and provided a molecular mechanism for changes in IL-2 gene inducibility in T cells undergoing integration of multiple environmental signals.

Structure-, stereochemistry-, and metal-regulated DNA binding/cleaving molecules

In the cell, the binding of proteins to specific sequences of double helical DNA is essential for controlling the processes of protein synthesis (at the level of DNA transcription) and cell proliferation (at the level of DNA replication). In the laboratory, the sequence-specific DNA binding/cleaving properties of restriction endonuclease enzymes (secreted by microorganisms to protect them from foreign DNA molecules) have helped to fuel a revolution in molecular biology. The strength and specificity of a protein:DNA interaction depend upon structural features inherent to the protein and DNA sequences, but it is now appreciated that these features (and therefore protein:DNA complexation) may be altered (regulated) by other protein:DNA complexes, or by environmental factors such as temperature or the presence of specific organic molecules or inorganic ions. It is also now appreciated that molecules much smaller than proteins (including antibiotics of molecular weight less than 2000 and oligonucleotides) can bind to double-helical DNA in sequence-specific fashion. Elucidation of structural motifs and microscopic interactions responsible for the specific molecular recognition of DNA leads to greater understanding of natural processes and provides a basis for the design of novel sequence-specific DNA binding molecules. This thesis describes the synthesis and DNA binding/cleaving characteristics of molecules designed to probe structural, stereochemical, and environmental factors that regulate sequence-specific DNA recognition.

Chapter One introduces the DNA minor groove binding antibiotics Netropsin and Distamycin A, which are di- and tri(N-methylpyrrolecarboxamide) peptides, respectively. The method of DNA affinity cleaving, which has been employed to determine DNA binding properties of designed synthetic molecules is described. The design and synthesis of a series of Netropsin dimers linked in tail-to-tail fashion (by oxalic, malonic, succinic, or fumaric acid), or in head-to-tail fashion (by glycine, β-alanine, and γ-aminobutanoic acid (Gaba)) are presented. These Bis(Netropsin)s were appended with the iron-chelating functionality EDTA in order to make use of the technique of DNA affinity cleaving. Bis(Netropsin)-EDTA compounds are analogs of penta(N-methylpyrrolecarboxamide)-EDTA (P5E), which may be considered a head-to-tail Netropsin dimer linked by Nmethylpyrrolecarboxamide. Low- and high-resolution analysis of pBR322 DNA affinity cleaving by the iron complexes of these molecules indicated that small changes in the length and nature of the linker had significant effects on DNA binding/cleaving efficiency (a measure of DNA binding affinity). DNA binding/cleaving efficiency was found to decrease with changes in the linker in the order β-alanine > succinamide > fumaramide > N-methylpyrrolecarboxamide > malonamide >glycine, γ-aminobutanamide > oxalamide. In general, the Bis(Netropsin)-EDTA:Fe compounds retained the specificity for seven contiguous A:T base pairs characteristic of P5E:Fe binding. However, Bis(Netropsin)Oxalamide- EDTA:Fe exhibited decreased specificity for A:T base pairs, and Bis(Netropsin)-Gaba-EDT A:Fe exhibited some DNA binding sites of less than seven base pairs. Bis(Netropsin)s linked with diacids have C₂-symmmetrical DNA binding subunits and exhibited little DNA binding orientation preference. Bis(Netropsin)s linked with amino acids lack C₂-symmetrical DNA binding subunits and exhibited higher orientation preferences. A model for the high DNA binding orientation preferences observed with head-to-tail DNA minor groove binding molecules is presented.

Chapter Two describes the design, synthesis, and DNA binding properties of a series of chiral molecules: Bis(Netropsin)-EDTA compounds with linkers derived from (R,R)-, (S,S)-, and (RS,SR)-tartaric acids, (R,R)-, (S,S)-, and (RS,SR)-tartaric acid acetonides, (R)- and (S)-malic acids, N ,N-dimethylaminoaspartic acid, and (R)- and (S)-alanine, as well as three constitutional isomers in which an N-methylpyrrolecarboxamide (P1) subunit and a tri(N-methylpyrrolecarboxamide)-EDTA (P3-EDTA) subunit were linked by succinic acid, (R ,R)-, and (S ,S)-tartaric acids. DNA binding/cleaving efficiencies among this series of molecules and the Bis(Netropsin)s described in Chapter One were found to decrease with changes in the linker in the order β-alanine > succinamide > P1-succinamide-P3 > fumaramide > (S)-malicamide > N-methylpyrrolecarboxamide > (R)-malicamide > malonamide > N ,N-dimethylaminoaspanamide > glycine = Gaba = (S,S)-tartaramide = P1-(S,S)-tanaramide-P3 > oxalamide > (RS,SR)-tartaramide = P1- (R,R)-tanaramide-P3 > (R,R)-tartaramide (no sequence-specific DNA binding was detected for Bis(Netropsin)s linked by (R)- or (S)-alanine or by tartaric acid acetonides). The chiral molecules retained DNA binding specificity for seven contiguous A:T base pairs. From the DNA affinity cleaving data it could be determined that: 1) Addition of one or two substituents to the linker of Bis(Netropsin)-Succinamide resulted in stepwise decreases in DNA binding affinity; 2) molecules with single hydroxyl substituents bound DNA more strongly than molecules with single dimethylamino substituents; 3) hydroxyl-substituted molecules of (S) configuration bound more strongly to DNA than molecules of (R) configuration. This stereochemical regulation of DNA binding is proposed to arise from the inherent right-handed twist of (S)-enantiomeric Bis(Netropsin)s versus the inherent lefthanded twist of (R)-enantiomeric Bis(Netropsin)s, which makes the (S)-enantiomers more complementary to the right-handed twist of B form DNA.

Chapter Three describes the design and synthesis of molecules for the study of metalloregulated DNA binding phenomena. Among a series of Bis(Netropsin)-EDTA compounds linked by homologous tethers bearing four, five, or six oxygen atoms, the Bis(Netropsin) linked by a pentaether tether exhibited strongly enhanced DNA binding/cleaving in the presence of strontium or barium cations. The observed metallospecificity was consistent with the known affinities of metal cations for the cyclic hexaether 18-crown-6 in water. High-resolution DNA affinity cleaving analysis indicated that DNA binding by this molecule in the presence of strontium or barium was not only stronger but of different sequence-specificity than the (weak) binding observed in the absence of metal cations. The metalloregulated binding sites were consistent with A:T binding by the Netropsin subunits and G:C binding by a strontium or barium:pentaether complex. A model for the observed positive metalloregulation and novel sequence-specificity is presented. The effects of 44 different cations on DNA affinity cleaving by P5E:Fe were examined. A series of Bis(Netropsin)-EDTA compounds linked by tethers bearing two, three, four, or five amino groups was also synthesized. These molecules exhibited strong and specific binding to A:T rich regions of DNA. It was found that the iron complexes of these molecules bound and cleaved DNA most efficiently at pH 6.0-6.5, while P5E:Fe bound and cleaved most efficiently at pH 7.5-8.0. Incubating the Bis(Netropsin) Polyamine-EDTA:Fe molecules with K₂PdCl₄ abolished their DNA binding/cleaving activity. It is proposed that the observed negative metalloregulation arises from kinetically inert Bis(Netropsin) Polyamine:Pd(II) complexes or aggregates, which are sterically unsuitable for DNA complexation. Finally, attempts to produce a synthetic metalloregulated DNA binding protein are described. For this study, five derivatives of a synthetic 52 amino acid residue DNA binding/cleaving protein were produced. The synthetic mutant proteins carried a novel pentaether ionophoric amino acid residue at different positions within the primary sequence. The proteins did not exhibit significant DNA binding/cleaving activity, but they served to illustrate the potential for introducing novel amino acid residues within DNA binding protein sequences, and for the development of the tricyclohexyl ester of EDTA as a superior reagent for the introduction of EDT A into synthetic proteins.

Chapter Four describes the discovery and characterization of a new DNA binding/cleaving agent, [SalenMn(III)]OAc. This metal complex produces single- and double-strand cleavage of DNA, with specificity for A:T rich regions, in the presence of oxygen atom donors such as iodosyl benzene, hydrogen peroxide, or peracids. Maximal cleavage by [SalenMn(III)]OAc was produced at pH 6-7. A comparison of DNA singleand double-strand cleavage by [SalenMn(III)]⁺ and other small molecules (Methidiumpropyl-EDTA:Fe, Distamycin-EDTA:Fe, Neocarzinostatin, Bleomycin:Fe) is presented. It was found that DNA cleavage by [SalenMn(III)]⁺ did not require the presence of dioxygen, and that base treatment of DNA subsequent to cleavage by [SalenMn(III)]⁺ afforded greater cleavage and alterations in the cleavage patterns. Analysis of DNA products formed upon DNA cleavage by [SalenMn(III)] indicated that cleavage was due to oxidation of the sugar-phosphate backbone of DNA. Several mechanisms consistent with the observed products and reaction requirements are discussed.

Chapter Five describes progress on some additional studies. In one study, the DNA binding/cleaving specificities of Distamycin-EDTA derivatives bearing pyrrole N-isopropyl substituents were found to be the same as those of derivatives bearing pyrrole N-methyl substituents. In a second study, the design of and synthetic progress towards a series of nucleopeptide activators of transcription are presented. Five synthetic plasmids designed to test for activation of in vitro run-off transcription by DNA triple helix-forming oligonucleotides or nucleopeptides are described.

Chapter Six contains the experimental documentation of the thesis work.

Global analysis of mRNA half-lives and de novo transcription in a dinoflagellate, Karenia brevis

Dinoflagellates possess many physiological processes that appear to be under post-transcriptional control. However, the extent to which their genes are regulated post-transcriptionally remains unresolved. To gain insight into the roles of differential mRNA stability and de novo transcription in dinoflagellates, we biosynthetically labeled RNA with 4-thiouracil to isolate newly transcribed and pre-existing RNA pools in Karenia brevis. These isolated fractions were then used for analysis of global mRNA stability and de novo transcription by hybridization to a K. brevis microarray. Global K. brevis mRNA half-lives were calculated from the ratio of newly transcribed to pre-existing RNA for 7086 array features using the online software HALO (Half-life Organizer). Overall, mRNA half-lives were substantially longer than reported in other organisms studied at the global level, ranging from 42 minutes to greater than 144 h, with a median of 33 hours. Consistent with well-documented trends observed in other organisms, housekeeping processes, including energy metabolism and transport, were significantly enriched in the most highly stable messages. Shorter-lived transcripts included a higher proportion of transcriptional regulation, stress response, and other response/regulatory processes. One such family of proteins involved in post-transcriptional regulation in chloroplasts and mitochondria, the pentatricopeptide repeat (PPR) proteins, had dramatically shorter half-lives when compared to the arrayed transcriptome. As transcript abundances for PPR proteins were previously observed to rapidly increase in response to nutrient addition, we queried the newly synthesized RNA pools at 1 and 4 h following nitrate addition to N-depleted cultures. Transcriptome-wide there was little evidence of increases in the rate of de novo transcription during the first 4 h, relative to that in N-depleted cells, and no evidence for increased PPR protein transcription. These results lend support to the growing consensus of post-transcriptional control of gene expression in dinoflagellates.

Waterborne exposure to fluorotelomer alcohol 6:2 FTOH alters plasma sex hormone and gene transcription in the hypothalamic-pituitary-gonadal (HPG) axis of zebrafish

Fluorotelomer alcohols (FTOHs) have shown estrogenic activity in vitro and in vivo, but the mechanism of this activity is not known. In this study, 18-week-old zebrafish (Danio rerio) were exposed to 0, 0.03, 0.3 and 3.0 mg/l 1H, 1H, 2H, 2H-perfluorooctan-1-ol (6:2 ETCH) for 7 days, and the effects on plasma sex hormone levels were measured followed by use of real-time PCR to examine selected gene expression in hypothalamic-pituitary-gonadal (HPG) axis and liver. Exposure to 6:2 FTOH significantly increased plasma estradiol (E2) and testosterone (T) levels in both males and females. Furthermore, the ratio of T/E2 was reduced in females while increased in males. In females, the increase of E2 was accompanied by up-regulated hepatic estrogenic receptor alpha (ER alpha) and vitellogenin (VTG1 and VTG3) expression. In males, the elevation of the T level is consistent with the up-regulation of cytochrome P450 c17 alpha-hydroxylase, 17, 20-lase (CYP17) and the down-regulation of cytochrome P450 aromatase A (CYP19A). The present study demonstrated that waterborne exposure to 6:2 FTOH alter plasma sex hormone levels and the ratio of T/E2, as well as the transcriptional profiles of some genes in the HPG axis and liver. The results suggested that FTOHs may disturb fish reproduction through endocrine disrupted activity. (C) 2009 Elsevier B.V. All rights reserved.

Effects of pure microcystin-LR on the transcription of immune related genes and heat shock proteins in larval stage of zebrafish (Danio rerio)

Microcystins (MCs) are cyanobacterial toxins in water blooms that have received increasing attention as a public biohazard for human and animal health. Previous studies were mainly focused on the toxic effects on adult fish, rather than juvenile or larvae, and the response of fish immune system were usually neglected. This paper presents the first data of the effects of microcystin-LR (MC-LR) on transcription of several genes essential for early lymphoid development (Rag1, Rag2, Ikaros, GATA1, Lck and TCR alpha) and heat shock proteins (HSP90, HSP70, HSP60, HSP27) in zebrafish larvae. Relative changes of mRNA transcription were analyzed by real time PCR. The transcription of Rag1, Rag2, Ikaros, GATA1, Lck and TCR alpha were up-regulated when following exposure to 800 mu g/L MC-LR, which may indicate that specific lymphocytes differentiation and TCR/lg arrangement are induced to counteract the toxic effects of MC-LR. It was also interesting to note the dramatically increased transcription of HSP90. HSP70, HSP60 and HSP27, which may indicate their important roles as molecular chaperones under oxidative stress. (C) 2009 Elsevier B.V. All rights reserved.

Context and conformation dictate function of a transcription antitermination switch

In bacteriophage, transcription elongation is regulated by the N protein, which binds a nascent mRNA hairpin ( termed boxB) and enables RNA polymerase to read through distal terminators. We have examined the structure, energetics and in vivo function of a number of N boxB complexes derived from in vitro protein selection. Trp18 fully stacks on the RNA loop in the wild-type structure, and can become partially or completely unstacked when the sequence context is changed three or four residues away, resulting in a recognition interface in which the best binding residues depend on the sequence context. Notably, in vivo antitermination activity correlates with the presence of a stacked aromatic residue at position 18, but not with N boxB binding affinity. Our work demonstrates that RNA polymerase responds to subtle conformational changes in cis-acting regulatory complexes and that approximation of components is not sufficient to generate a fully functional transcription switch.

cAMP stimulates transcription of the beta 2-adrenergic receptor gene in response to short-term agonist exposure.

In addition to conveying cellular responses to an effector molecule, receptors are often themselves regulated by their effectors. We have demonstrated that epinephrine modulates both the rate of transcription of the beta 2-adrenergic receptor (beta 2AR) gene and the steady-state level of beta 2AR mRNA in DDT1MF-2 cells. Short-term (30 min) exposure to epinephrine (100 nM) stimulates the rate of beta 2AR gene transcription, resulting in a 3- to 4-fold increase in steady-state beta 2AR mRNA levels. These effects are mimicked by 1 mM N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate (Bt2cAMP) or foskolin but not by phorbol esters. The half-life of the beta 2AR mRNA after addition of actinomycin D (46.7 +/- 10.2 min; mean +/- SEM; n = 5) remained unchanged after 30 min of epinephrine treatment (46.8 +/- 10.6 min; mean +/- SEM; n = 4), indicating that a change in transcription rate is the predominant factor responsible for the increase of beta 2AR mRNA. Whereas brief exposure to epinephrine or Bt2cAMP does not significantly affect the total number of cellular beta 2ARs (assessed by ligand binding), continued exposure results in a gradual decline in beta 2AR number to approximately 20% (epinephrine) or approximately 45% (Bt2cAMP) of the levels in control cells by 24 hr. Similar decreases in agonist-stimulated adenylyl cyclase activity are observed. This loss of receptors with prolonged agonist exposure is accompanied by a 50% reduction in beta 2AR mRNA. Transfection of the beta 2AR promoter region cloned onto a reporter gene (bacterial chloramphenicol acetyltransferase) allowed demonstration of a 2- to 4-fold induction of transcription by agents that elevate cAMP levels, such as forskolin or phosphodiesterase inhibitors. These results establish the presence of elements within the proximal promoter region of the beta 2AR gene responsible for the transcriptional enhancing activity of cAMP and demonstrate that beta 2AR gene expression is regulated by a type of feedback mechanism involving the second messenger cAMP.

Recovery from an acute infection in C. elegans requires the GATA transcription factor ELT-2.

The mechanisms involved in the recognition of microbial pathogens and activation of the immune system have been extensively studied. However, the mechanisms involved in the recovery phase of an infection are incompletely characterized at both the cellular and physiological levels. Here, we establish a Caenorhabditis elegans-Salmonella enterica model of acute infection and antibiotic treatment for studying biological changes during the resolution phase of an infection. Using whole genome expression profiles of acutely infected animals, we found that genes that are markers of innate immunity are down-regulated upon recovery, while genes involved in xenobiotic detoxification, redox regulation, and cellular homeostasis are up-regulated. In silico analyses demonstrated that genes altered during recovery from infection were transcriptionally regulated by conserved transcription factors, including GATA/ELT-2, FOXO/DAF-16, and Nrf/SKN-1. Finally, we found that recovery from an acute bacterial infection is dependent on ELT-2 activity.