Browsing by Subject "CTCF"
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Item An Investigation of Insulator Proteins in Mosquito Genomes(2013-08-02) Johanson, MichaelTransgenic mosquitoes are beneficial for the design and implementation of various pathogen control programs. However, low and variable expression of transgenes caused by position effects is a hindrance to the characterization and effective use of transgenes in mosquito species. The use of insulator sequences to flank transgenes may have the ability to overcome position effects caused by the genomic environment surrounding the insertion site. CTCF is a multifunctional protein, conserved from humans to Drosophila. Its role as an enhancer blocker in the Drosophila bithorax complex and its proximal binding to other insulator proteins on Drosophila chromosomes makes it a good candidate for identifying insulator sequences throughout the mosquito genome that may be used to improve mosquito transgenesis. Its multi-functionality as a transcription factor and genome organizer also makes CTCF worthy of investigation for an improved understanding of the regulation of the mosquito genome. This study uses chromatin immunoprecipitation with an An. gambiae CTCF antibody followed by Illumina deep sequencing (ChIP-Seq) to identify regions of CTCF binding throughout the An. gambiae genome. A subset of the CTCF binding site peaks was validated using ChIP-PCR. Another subset of this data set, including the ChIP-PCR validated peaks, was input into the motif finding tool, AlignACE, in order to identify a CTCF binding site consensus. Four motifs were identified, none of which were found in more than 11.9% of the ChIP-Seq data set. These results lead us to conclude that An. gambiae CTCF binds to a wider variety of sequences compared to Drosophila CTCF. This work also includes a comparison of the expression profiles of the dipteran insulator proteins, Su(Hw) and CP190, with that of CTCF across multiple life stages in Ae. aegypti. The results of this study suggest the possibility of genomic colocalization, as has been recently discovered in Drosophila. The identification of CTCF binding site peaks throughout the An. gambiae genome provides a large data set of potential insulator sequences that may be used to improve mosquito transgenesis, and provide a new model for the study of CTCF function in a species with medical significance.Item CTCF Contributes to the Regulation of the Ribosomal DNA in Drosophila melanogaster(2012-02-14) Guerrero, PaolaThe 35S rDNA gene clusters on the X and Y chromosomes of Drosophila melanogaster are repeats of approximately 150 to 225 copies. Each are transcribed as a single unit by RNA Polymerase I and modified into the 18S, 5.8S, 2S and 28S ribosomal rRNAs. Reduction in the array copy number results in a bobbed phenotype, characterized by truncated bristles and herniations of abdominal cuticle, due to a decrease in protein production. In some copies within the arrays, R1 and R2 retrotransposable elements are inserted in a conserved region of the 28S gene which represses the transcription of a functional rRNA. Inserted arrays are transcribed at very low levels, but it is not clear how they are identified for repression. Similarly, a subset of uninserted arrays are silenced, and the epigenetic mechanism controlling how this decision is made it is also unknown. The CCCTC binding factor (CTCF) is a boundary element binding protein and a transcriptional regulator found in the nucleolus of differentiated mammalian cells, whose localization requires poly (ADP-ribosyl)ation. We investigated whether CTCF might be involved in the regulation of rDNA expression in Drosophila. Our data show that CTCF is found at the nucleolus of both polytene and diploid nuclei, and we have identified binding sites in the 28S gene, R1 and R2 elements by a bioinformatic approach. ChIP data indicate that CTCF binds only to the site in the R1 retrotransposon. Reduction of CTCF or members of the poly(ADP-ribosyl)ation pathway by RNAi in S2 cells causes an increase in the amount of 35S rDNA gene, R1, and R2 transcripts. In flies, CTCF and PARG mutant alleles show disrupted nucleoli and increased rRNA transcripts. Mutant alleles of CTCF suppress variegation of a P-element inserted in a 35S rDNA array, but not of elements inserted elsewhere in the genome. Consistent with a role for CTCF in rRNA regulation, we found that during oogenesis CTCF is recruited to the nucleolus of nurse cells at early stages when the demand of ribosomes is low and it leaves this compartment in later stages when the cell increases rRNA production. We conclude from these studies that CTCF acts as a regulation of rDNA transcription by RNA polymerase I.Item Genome-wide target identification of sequence-specific transcription factors through ChIP sequencing(2011-05) Lee, Bum Kyu; Iyer, Vishwanath R.; DeLozanne, Arturo; Marcotte, Edward; Tucker, Philip; Stevens, ScottThe regulation of gene expression at the right time, place, and degree is crucial for many cellular processes such as proliferation and development. In addition, in order to maintain cellular life, cells must rapidly and appropriately respond to various environmental stimuli. Sequence-specific transcription factors (TFs) can recognize functional regulatory DNA elements in a sequence-specific manner so that they can regulate only a specific group of genes, a process which enables cells to cope with diverse internal and external stimuli. Human has approximately 1,400 sequence-specific TFs whose aberrant expression causes a wide range of detrimental consequences including developmental disorders, diseases, and cancers; therefore, it is pivotal to identify the binding sites of each sequence-specific TF in order to unravel its roles in and mechanisms of gene regulation. Even though some TFs have been intensively studied, the majority of TFs still remain to be studied, particularly the tasks of identifying their genome-wide target genes and deciphering their biological roles in specific cellular contexts. Many questions remain unanswered: how many sites on the human genome a sequence-specific TF can bind; whether all TF-bound sites are functional; how a TF achieves binding specificity onto its targets; how and to what extent a TF is involved in gene regulation. Comprehensive identification of the binding sites of sequence-specific TFs and follow-up molecular studies including gene expression microarrays will provide close answers to these questions. Chromatin Immunoprecipitation coupled with recently developed high-throughput sequencing (ChIP-seq) allows us to perform genome-scale unbiased identification of the binding sites of sequence-specific TFs. Here, to gain insight into gene regulatory functions of TFs as well as their influences on gene expression, we conducted, in diverse cell lines, genome-wide identification of the binding sites of several sequence-specific TFs (CTCF, E2F4, MYC, Pol II) that are involved in a wide range of biological functions, including cell proliferation, development, apoptosis, genome stability, and DNA repair. Analysis of ChIP-seq data provided not only comprehensive binding profiles of those TF across the genome in diverse cell lines, but also revealed tissue-specific binding of CTCF, MYC, and Pol II as well as combinatorial usage among these three factors. Analyses also showed that some CTCF binding sites were inherited from parents to children and regulated in an individual-specific as well as allele-specific manner. Finally, genome-wide target identification of several TFs will broaden our understanding of the gene regulatory roles of these sequence-specific TFs.Item Promoters, enhancers and insulators for improved mosquito transgenesis(Texas A&M University, 2006-10-30) Gray, Christine ElizabethLow level and variable transgene expression plague efforts to produce and characterize transgenic lines in many species. When transformation efficiency is high, productive transgenic lines can be generated with reasonable effort. However, most efforts to date in mosquitoes have resulted in suboptimal levels of transformation. This, coupled with the large space and intensive labor requirements of mosquito colony maintenance makes the optimization of transformation in mosquitoes a research priority. This study proposes two strategies for improving transgene expression and transformation efficiency. The first is to explore exogenous promoter/enhancer combinations to direct expression of either the transgene itself, or the transposase required for insertion of the transgene into the genome. An extension of this strategy is to investigate the use of a powerful viral transactivating protein and its cognate enhancer to further increase expression of these targets. The second strategy involves the identification of an endogenous boundary element for use in insulating transgenes and their associated regulatory elements. This would mitigate the inappropriate expression or silencing of many transgenes inserted into ??????unfavorable?????? genomic environments as a consequence of an inability to specifically target the integration of transposons currently used in mosquito transgenesis. The IE1 transactivating protein and its cognate enhancer from a baculovirus were shown to significantly increase expression of a reporter gene from three different promoters in cultured mosquito cells. Other heterologous enhancer/promoter combinations resulted in minimal increases or insignificant changes in expression. Orthologues of the vertebrate insulator-binding factor, CTCF, were cloned and characterized in two mosquito species, Aedes aegypti and Anopheles gambiae. The expression profile of mosquito CTCF is consistent with its role as a putative insulatorbinding protein. Preliminary binding site studies reveal a C/G-rich binding site consistent with that known in vertebrates and indicate that CTCF may bind widespread sites within mosquito genomes.