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Item Beyond books : interactive lessons for the college biology classroom(2011-12) Londeore, Cynthia Fay; Jansen, Robert K., 1954-; Fischer, JaniceCollege level science is frequently taught as a recitation of facts in a lecture hall, and the students are expected to gain understanding and insight with their own study. Interactive learning is more effective than lecture based learning and more memorable for the students. Teaching with hands on models has been shown to specifically be beneficial in a college level molecular biology context. Included here is a guide for the instructor leading her through topic selection, activity development, and presentation to the class, as well as five complete and tested lesson plans with notes on alteration made and the reasons for them.Item Characterization of the XTH gene family in cotton(Texas Tech University, 2006-08) Lee, Joohyun; Allen, Randy D.; Auld, Dick L.; Zhang, Hong; Shi, Huazhong; Wright, Robert J.Cotton is the single most important crop in Texas agriculture, reaching a value of 4 billion dollars per year. Cotton fiber is the major product in cotton cultivation which is converted into economic revenue. Therefore, most research groups have focused on fiber development and improvement of fiber quality. The xyloglucan endotransglucosylase/hydrolase gene family has a unique role in plant cell wall elongation and development during plant growth. It can cut and paste xyloglucan fragments into xyloglucan hemicellulose matrix in Type I plant cell wall, allowing cell wall extension and loosening while bearing tension for normal cell metabolism. XTH genes encode enzymes that may play a role in determining fiber length and other fiber qualities during cotton fiber development. Both the structures and functions of the XTH family have been well studied in Arabidopsis. In this research the cotton XTH genes were cloned by using the sequence information of Arabidopsis XTHs. A cotton boll cDNA library and a cotton genomic BAC library were screened and used for cotton XTH gene cloning. The clones of cotton XTHs were classified to three distinctive groups based on their 3ŒUTR sequences. The three groups were denoted as GhXTH-G1, G2, and G3. The sequences of abnormal transcripts of GhXTH-G1 showed transcription termination in the middle of introns, producing shortened forms of mRNAs. In addition, two GhXTH-G1 genes were analyzed and assigned to A and D sub genomes. Real time quantitative RT-PCR was used to study tissue and development specific patterns of XTH gene expression. GhXTH-G3 appears to be the most important cotton XTHs in cotton fiber development. Also, hormone treatment results indicated that brassinolide (BL), gibberellin (GA3), naphthalene acetic acid (NAA) induced GhXTH expression in developing cotton fiber.Item Characterization of the XTH gene family in cotton(2006-08) Lee, Joohyun; Allen, Randy D.; Auld, Dick L.; Wright, Robert J.; Shi, Huazhong; Zhang, HongCotton is the single most important crop in Texas agriculture, reaching a value of 4 billion dollars per year. Cotton fiber is the major product in cotton cultivation which is converted into economic revenue. Therefore, most research groups have focused on fiber development and improvement of fiber quality. The xyloglucan endotransglucosylase/hydrolase gene family has a unique role in plant cell wall elongation and development during plant growth. It can cut and paste xyloglucan fragments into xyloglucan hemicellulose matrix in Type I plant cell wall, allowing cell wall extension and loosening while bearing tension for normal cell metabolism. XTH genes encode enzymes that may play a role in determining fiber length and other fiber qualities during cotton fiber development. Both the structures and functions of the XTH family have been well studied in Arabidopsis. In this research the cotton XTH genes were cloned by using the sequence information of Arabidopsis XTHs. A cotton boll cDNA library and a cotton genomic BAC library were screened and used for cotton XTH gene cloning. The clones of cotton XTHs were classified to three distinctive groups based on their 3ŒUTR sequences. The three groups were denoted as GhXTH-G1, G2, and G3. The sequences of abnormal transcripts of GhXTH-G1 showed transcription termination in the middle of introns, producing shortened forms of mRNAs. In addition, two GhXTH-G1 genes were analyzed and assigned to A and D sub genomes. Real time quantitative RT-PCR was used to study tissue and development specific patterns of XTH gene expression. GhXTH-G3 appears to be the most important cotton XTHs in cotton fiber development. Also, hormone treatment results indicated that brassinolide (BL), gibberellin (GA3), naphthalene acetic acid (NAA) induced GhXTH expression in developing cotton fiber.Item Defining the role of Mtf1 and N-terminal domain of Rpo41 in transcription initiation and replication(2012-05) Chang, Hae Ryung; Yin, Yuhui Whitney; Hackert, Marvin L.; Jayaram, Makkuni; Johnson, Kenneth; Molineux, IanMitochondrion is an organelle found in the eukaryotic cell. It is responsible for essential metabolic processes as well as ATP production via oxidative phosphorylation (OXPHOS). The mitochondrion contains DNA that encodes for several subunits in the OXPHOS system as well as rRNA and tRNA for translation. It also has its own replication, transcription and translation machinery. Proper maintenance of the mitochondrial DNA is critical for the cell’s health. Saccharomyces cerevisiae mitochondrial transcription system has been a great model system for its ease of genetic manipulation as well as having conserved RNA polymerases across species. The polymerases are homologues to T7 RNA polymerase, but have longer N-terminal domain and require transcription factor(s). The reason for the extra domain as well as the need for an accessory factor is still unclear. This study reveals the role of Rpo41 N-terminal domain (NTD) as well as clarifies the role of Mtf1, the transcription factor, in transcription initiation. Rpo41 is the 153 kDa catalytic subunit, and Mtf1 is 40 kDa, the transcription factor of the yeast mitochondria. We have shown that Mtf1 is required for correct promoter sequence recognition as well as inhibition of incorrect initiation. Although it was thought that Rpo41 has intrinsic promoter recognition capability, we have shown that Rpo41 can initiate transcription on a pre-melted DNA, even if it is not the consensus promoter sequence. N-terminal truncation mutant studies showed that the NTD of Rpo41 is also required for correct transcription initiation. On linear duplex DNA, N-terminal truncation of 321 amino acids has little effect when Mtf1 is present. On pre-melted DNA, it shows opposite trend from the wild-type. 160 N-terminal amino acid residue truncation shows little activity, whereas Mtf1 increases activity, even on non-promoter initiation sites. We further investigated properties of Rpo41 in replication. A link between mitochondrial transcription and replication has been suggested before, where Rpo41 functions as the leading strand primase. Our studies show that Rpo41 can indeed function as the leading and lagging strand primase, and explains why Rpo41 is able to initiate transcription on non-promoter sites. N-terminal truncation resulted in loss of primase activity, which shows that NTD is required for replication.Item Genome-wide approaches to explore transcriptional regulation in eukaryotes(2014-05) Park, Daechan; Iyer, Vishwanath R.; Marcotte, Edward M; Paull, Tanya T; Miller, Kyle M; Stevens, Scott WTranscriptional regulation is a complicated process controlled by numerous factors such as transcription factors (TFs), chromatin remodeling enzymes, nucleosomes, post-transcriptional machineries, and cis-acting DNA sequence. I explored the complex transcriptional regulation in eukaryotes through three distinct studies to comprehensively understand the functional genomics at various steps. Although a variety of high throughput approaches have been developed to understand this complex system on a genome wide scale with high resolution, a lack of accurate and comprehensive annotation transcription start sites (TSS) and polyadenylation sites (PAS) has hindered precise analyses even in Saccharomyces cerevisiae, one of the simplest eukaryotes. We developed Simultaneous Mapping Of RNA Ends by sequencing (SMORE-seq) and identified the strongest TSS and PAS of over 90% of yeast genes with single nucleotide resolution. Owing to the high accuracy of TSS identified by SMORE-seq, we detected possibly mis-annotated 150 genes that have a TSS downstream of the annotated start codon. Furthermore, SMORE-seq showed that 5’-capped non-coding RNAs were highly transcribed divergently from TATA-less promoters in wild-type cells under normal conditions. Mapping of DNA-protein interactions is essential to understanding the role of TFs in transcriptional regulation. ChIP-seq is the most widely used method for this purpose. However, careful attention has not been given to technical bias reflected in final target calling due to many experimental steps of ChIP-seq including fixation and shearing of chromatin, immunoprecipitation, sequencing library construction, and computational analysis. While analyzing large-scale ChIP-seq data, we observed that unrelated proteins appeared to bind to the gene bodies of highly transcribed genes across datasets. Control experiments including input, IgG ChIP in untagged cells, and the Golgi factor Mnn10 ChIP also showed the strong binding at the same loci, indicating that the signals were obviously derived from bias that is devoid of biological meaning. In addition, the appearance of nucleosomal periodicity in ChIP-seq data for proteins localizing to gene bodies is another bias that can be mistaken for false interactions with nucleosomes. We alleviated these biases by correcting data with proper negative controls, but the biases could not be completely removed. Therefore, caution is warranted in interpreting the results from ChIP-seq. Nucleosome positioning is another critical mechanism of transcriptional regulation. Global mapping of nucleosome occupancy in S. cerevisiae strains deleted for chromatin remodeling complexes has elucidated the role of these complexes on a genome wide scale. In this study, loss of chromodomain helicase DNA binding protein 1 (Chd1) resulted in severe disorganization of nucleosome positioning. Despite the difficulties of performing ChIP-seq for chromatin remodeling complexes due to their transient and dynamic localization on chromatin, we successfully mapped the genome-wide occupancy of Chd1 and quantitatively showed that Chd1 co-localizes with early transcription elongation factors, but not late transcription elongation factors. Interestingly, Chd1 occupancy was independent of the methylation levels at H3K36, indicating the necessity of a new working model describing Chd1 localization.Item Identification and characterization of a positive regulatory region for activation induced cytidine deaminase mediated gene conversion in chicken B cells(2009-12) Kim, Yonghwan, 1975-; Tian, Ming, Ph. D.; Tucker, Philip W.; Paull, Tanya T.; Iyer, Vishwanath; Yin, WhitneyB cells have unique machinery to make up a large pool of antibody repertoire. After V(D)J recombination in early B cell development, the rearranged immunoglobulin genes are further diversified by somatic hypermutation (SHM), gene conversion (GC) and class switch recombination (CSR). Acitvation induced cytidine deaminase (AID) is a key initiating factor for SHM, GC and CSR. A majority of research data supports the model that AID modifies Ig genes at the DNA level by deaminating cytosines to uracils. The mutagenic activity of AID is largely restricted to Ig genes to avoid genomic instability in general. The specificity cannot be attributed to the primary sequence of the Ig genes since unrelated DNA is mutated by AID in the context of Ig genes. A clue to this problem is that AID function is dependent on transcription. Since not all transcribed genes are mutated by AID, there must be something special about the transcription of Ig genes, and the reasoning has prompted extensive analysis of Ig promoters and enhancers. We addressed this question in chicken B cell line DT40. We identified a 2.4-kilobase regulatory region which is important for AID function both within and outside of Ig locus. This regulatory region contains binding sites for multiple transcription factors. Mutation of these binding sites impairs AID mediated gene conversion. In addition, ablation of NF-κB family member, c-Rel and p50, reduces the AID targeting function of this regulatory region. Since the implicated transcription factors have been reported to associate with histone acetylases, the regulatory region may function by facilitating the access of AID to target DNA. To test this hypothesis, we used the I-SceI endonuclease and dam methylase as probes for chromatin structure. We found that the regulatory region does not increase chromatin accessibility to these probes. In fact, the regulatory region appears to interfere with the cleavage of target DNA by I-SceI. Another possible role of the regulatory region could be direct recruitment of AID to Ig genes. To test this hypothesis, we utilized Dam identification method. Surprisingly, we found that the regulatory region facilitates AID targeting to the Igλ locus.Item Identification of the Influenza A nucleoprotein sequence that interacts with the viral polymerase(2011-08) Marklund, Jesper Karl; Krug, Robert M.; Marcotte, Edward M.; Sawyer, Sara L.; Stevens, Scott W.; Sullivan, Christopher S.; Wilke, Claus O.Influenza A is a negative stranded RNA virus with a segmented genome. Once the virus infects a cell it must replicate its full length viral genomic RNA (vRNA) through a positive sense complementary intermediate RNA (cRNA) as well as transcribe viral messenger RNA (mRNA) using the vRNA as a template. The regulation of whether the viral polymerase replicates the genome by synthesizing cRNA, or produces mRNA in order to make viral protein involves, the viral nucleoprotein (NP). We tried to find the sequence residues of NP that directly interact with the viral polymerase. We mutated to alanine several residues on NP that are surface exposed on recently solved crystal structures as well as those thought to be oriented toward the viral polymerase complex in cryo-EM studies. As a first screen, we tested these mutants in a mini-genome assay where the NP stimulation of the viral polymerase can be studied in transfected cells. Through this screen we found that the NP mutants that hindered its ability to stimulate polymerase activity the most were located in a loop between two alpha helixes in the head domain of NP located at residues 203 to 209. Specifically, the NP single mutants of R204, W207, and R208 were inactive in the mini-genome assay. Using RT-PCR we found that the cRNA to vRNA step of replication is severely inhibited by these mutations. Immunoprecipitation using transfected cells showed that the NP mutants lost the ability to bind all three polymerase subunits. This indicates that this loss of polymerase binding may be the reason the NP mutant fails to stimulate polymerase activity. To make sure that this loss of polymerase stimulation was not due to altering other functions of NP we made sure that the protein had proper cellular localization, oligomerization, and RNA binding abilities. Using immuniflourescence we found that mutant NP localized to the nucleus just like wild type. In order to test RNA binding and oligomerization we tested NP purified from a baculovirus expressing system. Using fluorescence polarization we found that NP binds single stranded RNA with similar affinity to wild type. Using gel filtration we found that mutant NP forms oligomers just like wild type. Using covariation analysis of how different positions in an amino acid alignment change relative to each other we predicted possible binding sites between NP and the three polymerase subunits PA, PB1 and PB2. Due to more complete crystal structure data we focused on the PA-NP interaction and found that covariation aided in finding binding sequence residues on PA but not NP. Another outcome of developing the covariation method was developing a program to view broad primary structure changes in large sequence alignments. This method has been informative in evaluating how amino acid positions in influenza have changed over time, as well as what defines specific residues as belonging to human or avian viruses.Item Improved Algorithms for Discovery of Transcription Factor Binding Sites in DNA Sequences(2012-02-14) Zhao, XiaoyanUnderstanding the mechanisms that regulate gene expression is a major challenge in biology. One of the most important tasks in this challenge is to identify the transcription factors binding sites (TFBS) in DNA sequences. The common representation of these binding sites is called ?motif? and the discovery of TFBS problem is also referred as motif finding problem in computer science. Despite extensive efforts in the past decade, none of the existing algorithms perform very well. This dissertation focuses on this difficult problem and proposes three new methods (MotifEnumerator, PosMotif, and Enrich) with excellent improvements. An improved pattern-driven algorithm, MotifEnumerator, is first proposed to detect the optimal motif with reduced time complexity compared to the traditional exact pattern-driven approaches. This strategy is further extended to allow arbitrary don?t care positions within a motif without much decrease in solvable values of motif length. The performance of this algorithm is comparable to the best existing motif finding algorithms on a large benchmark set of samples. Another algorithm with further post processing, PosMotif, is proposed to use a string representation that allows arbitrary ignored positions within the non-conserved portion of single motifs, and use Markov chains to model the background distributions of motifs of certain length while skipping these positions within each Markov chain. Two post processing steps considering redundancy information are applied in this algorithm. PosMotif demonstrates an improved performance compared to the best five existing motif finding algorithms on several large benchmark sets of samples. The third method, Enrich, is proposed to improve the performance of general motif finding algorithms by adding more sequences to the samples in the existing benchmark datasets. Five famous motif finding algorithms have been chosen to run on the original datasets and the enriched datasets, and the performance comparisons show a general great improvement on the enriched datasets.Item Mechanisms of transcriptional activation of estrogen responsive genes in breast cancer cells(2009-06-02) Chen, Chien-ChengEstrogen receptor (ER) acts as a ligand-activated transcription factor that regulates the expression of genes. The genomic mechanisms of ER action include ligand-induced dimerization of ER which binds estrogen responsive elements (EREs) in the promoters of target genes. There are also nongenomic mechanisms of ER action which are associated with membrane bound or cytosol ER-dependent activation of various protein-kinase cascades which also influence expression of target genes. Egr-1 is an immediate-early gene induced by 17?-estradiol (E2) in the rodent uterus and breast cancer cells. Deletion analysis of the Egr-1 promoter identified a minimal E2-responsive region that contained serum response element (SRE3) which bound Elk-1 and serum response factor (SRF) in gel mobility shift assays. Hormone-responsiveness of Egr-1 in MCF-7 cells was specifically inhibited by PD98059, a MAPKK inhibitor, but not by LY294002, an inhibitor of PI3-K. These results contrasted with the hormone-dependent activation of the SRE in the c-fos promoter, which was inhibited by both PD98059 and LY294002, suggesting that Egr-1, like c-fos, is activated through non-genomic pathways of estrogen action but through activation of different kinases. COUP-TFs are orphan nuclear receptors expressed in a variety of tissues where they regulate biological functions and organogenesis. In this study, we investigated coactivation of ER? by COUP-TF1 in cell lines transiently cotransfected with the pERE3 construct. COUP-TFI coactivated ER?-mediated transactivation, but unlike many other coactivators, COUP-TFI also enhanced transactivation of ER? when cells were cotransfected with the TAF1-ER? mutant or the 19c-ER? mutant. These data indicate that helix 12 of ER? is not required for coactivation by COUP-TFI when AF-1 of ER? is intact. However, when the AF-1 of ER? is deleted, the intact AF-2 function is required for coactivation by COUP-TFI. Analysis of multiple COUP-TFI deletion mutants showed that the DNA-binding domain and C-terminal region of COUP-TFI were important for coactivation of ER?. Point mutations of the DNA-binding domain of COUP-TFI resulted in loss of interactions with ER?, suggesting that the DNA-binding domain of COUP-TFI is important for its coactivation activity facilitating interactions with ER?. These results demonstrate that COUP-TFI coactivated ER? through a non-classical LXXLL-independent pathway.Item Multiple roles for the zebrafish transcriptional activator SBF/Staf(2009-05-15) Halbig, Kari MicheleEukaryotic transcriptional activators stimulate transcription of genes otherwise expressed at low levels. The typical activator operates by binding to specific sites on DNA with its activating region contacting the multiprotein machinery that directs transcription. SBF/Staf is a transcriptional activator that binds to the SPH element found in the promoters of genes for snRNAs and genes that code for mRNAs. SBF/Staf binds to SPH through a reiterated zinc finger DNA binding domain and also contains two distinct activation domains, one for snRNA genes and one for mRNA genes. To test the role of SBF/Staf in vivo, morpholino antisense oligos were used to knock down SBF/Staf expression in zebrafish. A high percentage of developing zebrafish embryos exhibited abnormalities. Co-injection of a synthetic mRNA construct rescued the morpholino-induced knockdown. Furthermore, both the mRNA and snRNA activation domains have significant roles in the function of SBF/Staf because when each domain was removed separately, partial rescue of the knockdown phenotype was obtained. When both domains were removed, no rescue of the phenotype was observed. Unexpectedly, knockdown of SBF/Staf expression in zebrafish embryos caused an increase in steady-state levels of all endogenous mRNAs tested, as well as transcripts produced from co-injected U6 maxigenes. However, quantitative RT-PCR analysis showed a relatively smaller increase in the steady-state levels of several mRNAs from genes that contain a SPH element in their promoters. In zebrafish U6 genes, the SPH element is in the unique location of being next to the TATA box, instead of ~220 bp upstream of the start site as in mammals. To determine the significance of the proximally-located SPH element for transcription of the zebrafish U6 snRNA gene, the SPH element was mutated. Transcription of a zebrafish U6 maxigene was reduced to 20.6% in transfected ZF4 cells and 26.8% in injected embryos, compared to that of the U6 maxigene with a normal promoter. This work indicates a more global role of SBF/Staf in mRNA gene transcription, instead of only activating the transcription of snRNA and a few mRNA genes, leading to an increased importance of the role of SBF/Staf in transcriptional control.Item Negative regulation of the myogenic pathway in the neuronal cell line RT4-B8(Texas Tech University, 1997-12) Auvenshine, Ronald ChristopherNeurons and skeletal muscle cells may share a common lineage, although once thought to arise from distinct precursor pools. While the majority of skeletal muscle is derived from mesoderm, a small percentage arises from the ectodermally derived neural crest, which also gives rise to the entire peripheral nervous system. In addition, cells which express the skeletal muscle-specific marker myf-5 have been identified in the mouse central nervous system early in development. Finally, certain types of neural tumors give rise to myoblasts when cultured. Studies in our laboratory indicate that based on the endogenous expression of MyoD, the peripheral neurotumor derived neuronal cell line, RT4-B8, may provide a unique model to study this lineage relationship. In vivo, the MyoD family of basic helix-loop-helix transcription factors has been shown to play a pivotal role in the regulation of skeletal muscle myogenesis. Significantly, the ectopic expression of MyoD in many non-muscle cell types can activate the entire myogenic program. The focus of this study was to gain an understanding of the regulation of MyoD function in a neuronal cell type. Our results demonstrate that despite the expression of both MyoD mRNA and protein, the myogenic pathway was not activated in RT4-B8. This finding is unique in that, to our knowledge, no other cell has been reported to express endogenous MyoD and lack the ability to activate the myogenic pathway. The inability of RT4-B8 cells to activate the myogenic pathway was demonstrated by immunoblot analyses, which revealed that the muscle-specific myogenin, desmin, or a-sarcomeric actin genes were not expressed in RT4-B8. In addition, myotubes did not form when RT4-B8 cells were grown in culture conditions that induced the differentiation of C2C12 mouse myoblasts. The MyoD expressed by RT4-B8 was wild type with respect to the size of the protein and length of the mRNA. Finally, MyoD protein was present in the nuclei of RT4- B8 cells, and capable of binding to DNA in vitro. The results of the experiments conducted in this study suggest that the endogenous expression of MyoD is not sufficient to activate the myogenic pathway in RT4-B8. We therefore conclude that the block to myogenesis in RT4-B8 may lie downstream of the expression of MyoD.Item The effects of environmental pollutants on steroidogenesis and steroidogenic acute regulatory protein expression(Texas Tech University, 2001-05) Walsh, Lance PatrickMany environmental pollutants disrupt male reproductive function, inhibit steroid hormone biosynthesis, reduce serum testosterone levels, and decrease sperm counts. Although a disruption in steroidogenesis may underlie toxicant-induced reproductive toxicity, the mechanism by which these compounds inhibit steroid production remains unclear. The steroidogenic acute regulatory (StAR) protein mediates the rate-limiting and acutely-regulated step in steroidogenesis, the transfer of cholesterol from the outer to the inner mitochondrial membrane where die cytochrome P450 side chain cleavage (P450scc) enzyme initiates the synthesis of all steroid hormones. We hypothesized that environmental toxicants block steroidogenesis by disrupting StAR protein expression for several reasons: first, in contrast to the steroidogenic enzymes which have long half-lives and are chronically regulated, StAR protein is not an enzyme, is acutely regulated, its active form is highly labile and it must be continuously synthesized for steroid production to persist. Second, because StAR protein mediates the rate-limiting step in steroidogenesis, steroid production is very sensitive to disruptions in its expression. Finally, recent studies suggest that some environmental pollutants inhibit steroidogenesis by reducing cholesterol availability, implicating StAR protein as a potential target for these compounds. Using mouse MA-10 Leydig tumor cells, we studied the effects of acute exposure (<4 h) to the organochlorine insecticide lindane (y-hexachlorocyclohexane; HCH), the organophosphate insecticide Dimethoate, the herbicide Roundup, and the imidazole antifungal drugs econazole and miconazole on: (1) steroid production, (2) total cellular protein synthesis, (3) StAR and steroidogenic enzyme activity and expression, and (4) protein kinase A (PKA) activity. Our studies demonstrate that these toxicants reversibly inhibited steroidogenesis without causing cell toxicity or affecting PKA activity. While lindane and Dimethoate reduced StAR protein expression by reducing StAR mRNA levels. Roundup, econazole and miconazole reduced StAR protein levels post-transcriptionally. Although Roundup and Dimethoate inhibited P450scc activity, a reduction in StAR protein expression alone could account for the effects of these compounds on steroidogenesis, suggesting that the inhibition of P450scc activity in these instances has little physiological significance, its activity being distal to StAR function. Our studies strongly suggest that several different classes of environmental pollutants inhibit steroidogenesis by reducing StAR protein expression, thus supporting our hypothesis.Item Transcriptional control of epithelial morphogenesis(2013-05) Chung, Mei-I; Wallingford, John B.How tissues and organs develop into their final shape during embryogenesis is a fascinating and long-standing question in developmental biology. Tissue morphogenesis is driven by a variety of events at the cellular level and individual cell shape change is one of the central morphogenetic engines. Thus, it is crucial to understand what signals specify the correct cell behavior in specific groups of cells during development. For my doctoral studies, I have focused on two cell shape change events, apical constriction and cilia assembly. First, we present data demonstrating that Shroom3 is essential for cell shape changes and morphogenesis in the developing vertebrate gut, where Shroom3 transcription requires the Pitx1 transcription factor. We identified a Pitx-responsive regulatory element in the genomic DNA upstream of Shroom3, and showed that Pitx proteins directly activated Shroom3 transcription in Xenopus. Moreover, we showed that ectopic expression of Pitx proteins was sufficient to induce Shroom3-dependent cytoskeletal reorganization and epithelial cell shape change. These data demonstrated new breadth to the requirements for Shroom3 in morphogenesis, and also provided a cell-biological mechanism for Pitx transcription factors action during morphogenesis. Next, we focused on understanding the transcriptional regulation of ciliogenesis. We first showed that Rfx2 transcription factor broadly controlled ciliogenesis, and by RNA- and ChIP-sequencing, we showed that Rfx2 directly regulated a wide range of genes encoding diverse ciliogenic machinery. Finally, in addition to ciliogenesis regulation, a large number of non-ciliary genes in our Rfx2 dataset led us to identify a novel role of Rfx2 in controlling insertion of multi-ciliated cells into the overlying mucociliary epithelium. Moreover, we showed here that Slit2, a target of Rfx2, was involved in multi-ciliated cell movements, possibly through mediating cortical E-cadherin level. This work allowed us to begin building a genetic network controlling multi-ciliated cells in mucociliary epithelium. Together, we showed a transcriptional regulation of apical constriction driving gut morphogenesis and a comprehensive transcriptional network that governs multi-ciliated cell development.Item Transcriptional mechanisms that produce BK channel-dependent drug tolerance and dependence(2011-12) Li, Xiaolei, Ph. D.; Atkinson, Nigel (Nigel S.)Tolerance to anesthetic drugs is mediated partially by homeostatic mechanisms that attempt to restore normal neural excitability. The BK-type Ca2+-activated K+ channel, encoded by the slo gene, plays an important role in this neural adaptation. In Drosophila, a single sedative dose of the organic solvent anesthetic benzyl alcohol induces dynamic spatiotemporal changes in histone H4 acetylation across the slo regulatory region and leads to slo induction and tolerance. Mutations ablating the expression of slo also block the acquisition of tolerance, whereas activating the expression of a slo transgene results in resistance to drug sedation. Moreover, artificially inducing histone acetylation with the histone deacetylase inhibitor causes similar acetylation changes, slo induction, and functional tolerance to the drug. Histone acetylation changes occur over two highly conserved non-coding DNA elements, 6b and 55b, of the slo control region. To investigate the function of these two elements, I generated individual knockout mutants by gene targeting. Both knockout alleles are backcrossed into the CS wild type background. The 6b element seems to repress slo induction after drug sedation, because the 6b knockout allele overreacts to the drug. Compared to the wild type, 6b knockout allele shows a much greater slo message induction after drug sedation, it also displays stronger enhancements in seizure susceptibility and following frequency. In addition, the 6b deletion causes a persistent tolerance for at least a month, while tolerance only lasts about 10 days in wild type flies. My investigation also indicates that the 55b element limits basal slo expression in muscle. Finally, to investigate if the particular histone acetylation spikes are required for drug-induced slo induction and tolerance, I tether the histone-modifying enzymes, HDAC or HAT, to the 6b and 55b DNA elements, respectively. I observe that the positioning of an HDAC on these two elements blocks drug-induced slo induction and the development of tolerance. Therefore, histone acetylation across slo control region is required for the activation of slo and the acquisition of tolerance.