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search.filters.applied.f.advisor: Storici, Francesca × End date: 2017 × Start date: 2013 × Item Type: Publication ×

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Now showing 1 - 6 of 6
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Understanding pathways regulating liver versus pancreas fate decision and beta-cell regeneration

2017-08-02 , Xu, Jin

The liver and pancreas originate from common endodermal progenitors in early development. It has been shown that Bone morphogenetic protein 2b (Bmp2b) signaling is essential for determining the fate of these bipotential progenitors towards the liver or pancreas. Our goal was to identify Bmp2b-mediated gene regulatory networks and to delineate their underlying mechanisms governing the liver versus pancreas fate decision. From microarray assays, we found four and a half LIM domains 1b (fhl1b) as a novel target of Bmp2b signaling. We observed that fhl1b is primarily expressed in the prospective liver primordium. By loss- and gain-of-function analyses combined with a single cell lineage tracing technique, we showed that fhl1b favors the specification of liver and suppresses the induction of pancreatic cells. Diabetes is characterized by compromised glucose regulation. Both type 1 and type 2 diabetes patients suffer from losing functional β-cells along the progression of the disease. Our goal was to expand the understanding on pathways potentially promoting β-cell regeneration. First, given its function in suppressing pancreas induction with concomitant stimulation of liver, we conducted loss- and gain-of-function studies of fhl1b using a zebrafish model of type 1 diabetes. We conclude that fhl1b regulates the regeneration of β-cells mainly via modulating ductal progenitor-to-β-cell neogenesis. In addition, we identified TBK1/IKKε inhibitors (TBK1/IKKε-Is) as enhancers of β-cell regeneration through a chemical screening. We demonstrated that these inhibitors enhance β-cell-specific proliferation and further investigated the gene regulatory networks mediating mitogenic effects of TBK1/IKKε-Is. In collaboration with Dr. Oyelere and Dr. García at Georgia Institute of Technology, we further explored the specificity and potency of these inhibitors in mammalian systems (rat and human islet culture in vitro and a mouse model of type 1 diabetes in vivo) to complement our findings in zebrafish. We showed the conserved effects of increasing function and proliferation of β-cells in mammalian systems.

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Protein-assisted targeting of genes in yeast and human cells

2013-06-28 , Ruff, Patrick

This work was designed as a proof-of-principle concept or prototype to show the effect of protein-assisted targeting of DNA to specific genomic loci. Two strategies were employed to deliver the DNA with the aim that once inside the cell the DNA would be delivered to the target sequence by the assistance of a protein. In our case, the chosen protein was the site-specific meganuclease I-SceI. The first strategy described herein was to bind the targeting DNA to I-SceI by the use of a fusion protein between I-SceI and a known DNA-binding domain, the GAL4-DBD. The second strategy involved using a DNA aptamer to I-SceI to link the targeting DNA and I-SceI. Testing in vivo revealed that in our human cells (HEK-293) single-stranded DNA was more efficient at gene targeting than double-stranded DNA. In order for the first strategy to work, we needed to have some region of double-stranded DNA. We found that in human cells, it was better for gene targeting to have that double-stranded DNA on the 5’ side of our targeting DNA. We also used gel shift assays to confirm binding by our candidate DNA-binding domain, the GAL4-DBD. We were unable to detect expression of the fusion protein of I-SceI and the GAL4-DBD. For the second strategy we were able to construct an aptamer to I-SceI using a variant of the systematic evolution of ligands by exponential enrichment (SELEX). The I-SceI aptamer was synthesized as part of a longer DNA molecule containing homology to a target locus. Using this chimeric oligonucleotide (part aptamer, part DNA repair region) testing was done in both yeast and human cells. Aside from instances where the aptamer’s secondary structure may have been compromised, the aptamer containing oligonucleotide stimulated repair at a rate 2 to 15-fold higher than the non-selected control sequence. These experimental results show that by delivering targeting DNA within close proximity to the site of modification, gene targeting frequencies can be increased.

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Screening for the Functionality of RNA Templated Repair of Double-Strand Breaks in Saccharomyces Cerevisae

2017-05 , Gordon, Katherine

Double-strand breaks (DSBs) in DNA are detrimental, as they can cause mutations and genomic rearrangements, which in turn leads to cancer and other diseases. Recent research reveals that DSBs can be repaired by RNA-templated homologous recombination. However, RNA-templated repair of DSBs is not well understood. In order to better understand the mechanism of RNA-templated repair of DSBs, the current research aims to identify the proteins that facilitate the repair. The research utilizes a system wherein RNA-templated repair of DSBs is known to occur. A yeast overexpression plasmid library was produced in order to test the ability of fragments of the yeast genome to facilitate RNA-templated repair of a DSB when these are highly expressed in the yeast cells. In order to test the ability of added gene fragments to facilitate RNA-templated repair of DSBs, the experimental candidates were exposed to galactose in order to induce a DSB, to activate the transcription of the overexpressed gene fragment, and to initiate the transcription of anti-sense RNA used to repair the break. The candidates were then moved to medium without histidine in order to assess the frequency of repair. Once a large number of colonies (~50,000) are screened, we expect to identify several proteins that facilitate RNA-templated repair of DSBs. Identifying the specific genes that facilitate this repair mechanism will assist in characterizing the functionality of the RNA-templated DNA repair mechanism. Identifying these genes will also allow for better predictions for how this same phenomenon may occur in human cells.

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Gene targeting at and distant from DNA breaks in yeast and human cells

2013-04-02 , Stuckey, Samantha Anne

Here we developed multiple genetic systems through which genetic modifications driven by DNA breaks caused by the I-SceI nuclease can be assayed in the yeast Saccharomyces cerevisiae and in human cells. Using the delitto perfetto approach for site-directed mutagenesis in yeast, we generated isogenic strains in which we could directly compare the recombination potential of different I-SceI variants. By genetic engineering procedures, we generated constructs in human cells for testing the recombination activity of the same I-SceI variants. Both in yeast and human cells we performed gene correction experiments using oligonucleotides (oligos) following modification and/or optimization of existing gene targeting protocols and development of new ones. We demonstrated that an I-SceI nicking enzyme can stimulate recombination on the chromosome in S. cerevisiae at multiple genomic loci. We also demonstrated in yeast that an I-SceI-driven nick can activate recombination 10 kb distant from the initial site of the chromosomal lesion. Moreover we demonstrated that an I-SceI nick can stimulate recombination at the site of the nick at episomal and chromosomal loci in human cells. We showed that an I-SceI double-strand break (DSB) could trigger recombination up to 2 kb distant from the break at an episomal target locus in human cells, though the same was not observed for the nick. Overall, we demonstrated the capacity for I-SceI nick-induced recombination in yeast and human cells. Importantly, our findings reveal that the nick stimulates gene correction by oligos differently from a DSB lesion, as determined by genetic and molecular analyses in yeast and human cells. This research illustrates the promise of targeted gene correction following generation of a nick.

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Ribonucleotides in yeast genomic DNA are targets of RNase H2 and nucleotide excision repair

2013-12-13 , Shetty, Lahari

Ribonucleotides can be incorporated into the yeast genome through a variety of mechanisms, including through DNA polymerazation, DNA priming, and oxidative damage. Ribonucleotides contain a reactive 2’ hydroxyl group on the sugar, which can distort the DNA double helix and lead to defective replication and transcription and ultimately mutagenesis. Ribonucleotide excision repair (RER) has been found to remove ribonucleotides through the enzyme RNase H2, though the in vivo substrate specificity is not known. Nucleotide excision repair (NER) removes bulky lesions formed in DNA, however its role in the extraction of ribonucleotides has not yet been determined in eukaryotes. Previously developed oligonucleotide-driven gene correction assays in Saccharomyces cerevisiae, or baker’s yeast, have shown that paired and mispaired rNMPs embedded into genomic DNA, if not removed, serve as templates for DNA synthesis and can result in a genetic alteration. We implemented this assay to examine whether RNase H2 and NER can target specific rNMPs in DNA. Our results deliver new evidence that RNase H2 specifically recognizes isolated paired and mispaired rNMPs embedded in yeast genomic DNA and that the NER mechanism can recognize an isolated paired rNMP as damage during DNA double-strand break repair in yeast.

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Ribonucleotides embedded in yeast genomic DNA are targets of RNase H2 and the nucleotide excision repair system

2013-04 , Gombolay, Alli

Increasing evidence suggests that ribonucleotides may represent one of the most common non-standard nucleotides found in genomic DNA. Therefore, it is important to understand the extent to which ribonucleotides alter genomic integrity and the cellular mechanisms that are responsible for removing them. We developed oligonucleotide-driven gene correction assays in the yeast Saccharomyces cerevisiae to show that, if not removed, mispaired and paired ribonucleotides embedded in genomic DNA serve as templates for DNA synthesis and could cause genetic change. We found that RNase H type 2 targets single paired and mispaired ribonucleotides, as well as a stretch of two or three ribonucleotides embedded in DNA, and the nucleotide excision repair system can target single paired ribonucleotides as damage.

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