Browsing by Subject "Chromatin Immunoprecipitation"

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Transcriptional Regulation of Intestinal Stem Cell Lineage in Drosophila
(2017-04-17) Lan, Qing; Jiang, Jin; Kraus, W. Lee; Sadek, Hesham A.; Jiang, Huaqi
The question of how somatic stem cells respond to tissue needs is always intriguing, since aberrant somatic stem cell behaviors may lead to adult tissue degeneration or tumorigenesis. Here, this thesis focuses on the transcriptional regulation of a somatic stem cell lineage: the intestinal stem cell in Drosophila adult gut. The Drosophila adult gut is a dynamic organ. It is maintained by hundreds of somatic gut stem cell evenly distributed throughout the gut epithelium. These multi-potent somatic stem cells undergo self-renewal and differentiation to replenish two mature gut cell types: the absorptive enterocytes and secretory entero-endocrine cells. Through an RNAi screen targeting transcription factors required for stem cell-mediated acute gut regeneration, two novel transcription factors, the FoxA family Fork head (Fkh) and SoxE family sox100b (dSox9), were uncovered and functionally characterized in this thesis. During gut regeneration, transcription factor Fkh and dSox9 are required for stem cell proliferation. During gut homeostasis, Fkh maintains stemness and prevents progenitor from precocious differentiation; dSox9 controls lineage differentiation through Jak-Stat pathway. To further probe mechanisms underlying gut stem cell physiology, ChIP-Seq technique was applied to map chromatin binding sites of gut stem cell regulators (HA tagged) in stem/progenitor cells of dissected fly guts, including transcription factors (FoxA family/Fkh, SoxE family/dSox9, bHLH family/Da), niche pathway downstream factors (Jak-Stat pathway/Stat92E, BMP pathway/Mad, Notch pathway/Su(H), JNK pathway/Kay), and transcriptional regulators (Mediator/Med20, p300/Nej). A set of shared ChIP-Seq peak regions likely functions as enhancers to drive gene expression in gut stem/progenitor cells. This thesis leads to the speculation of a transcriptional network that maintains gut stem/progenitor cell normal physiology in adult Drosophila.
Ubiquitin, the Proteasome, and Dynamics at the Protein/DNA Interface
(2006-05-16) Nalley, Kip A.; Kodadek, Thomas J.
Recently it has been discovered that a mutant species of Gal4, that contains a three amino acid change in a surface loop of the DNA binding domain, does not occupy the GAL 1/10 promoter under Gal4 inducing conditions as measured by Chromatin Immunoprecipitation (ChIP) assays. However, this protein, Gap71, occupies the promoter similarly to Gal4 under non-inducing (poised) conditions. Additionally this protein was found to be poorly ubiquitylated in vitro under conditions where Gal4 is ubiquitylated. In order to determine the mechanisms involved in the protein destabilization I have examined the properties of the individual mutations that comprise Gap71. These experiments have revealed that serine 22 is a site of phosphorylation of the Gal4 DBD and that lysine 23 is essential for S22 phosphorylation, possibly acting as part of the kinase recognition site. Mutation of either residue blocks Gal4 DBD phosphorylation, its subsequent ubiquitylation and compromises the ability of the activator to bind promoter DNA in vivo. These data represent the first report of an essential phosphorylation event for this paradigmatic transcription factor. In addition, experiments were done to directly measure the dynamics of the Gal4 /DNA complex. To measure the dynamics I have exploited the system developed by Dr. D. Picard and others using the Gal4 DNA binding domain fused to the estrogen receptor ligand binding domain. Each of these constructs has been shown to be inactive until the addition of estradiol, when they are released and bind the Gal4 UAS. These constructs allow me to temporally control the appearance of a large quantity protein that is able to compete with the endogenous Gal4 for the UAS sites in the genome. Under non-inducing conditions, the results are consistent with a rapidly exchanging complex. However, upon induction, the Gal4-promoter complexes "lock in" and exhibit long half-lives of one hour or more. Furthermore, pharmacological inhibition of proteasome-mediated proteolysis had little or no effect of Gal4-mediated gene expression. These studies show that proteasome-mediated turnover is not a general requirement for transactivator function and, when considered in the context of previous studies, that different transactivator-promoter complexes can have widely different lifetimes.