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Title: The role of Egr1 and Egr2 in adipocyte biology
Author: Boyle, K.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2007
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Two related transcription factors, early growth and response gene 1 (Egr1) and Egr2, were investigated here for their role in relaying insulin’s transcriptional effects. Both proteins were transiently induced by insulin in serum-starved 3T3-L1 adipocytes. Bioinformatic analysis of the promoters of insulin-induced genes in adipocytes revealed a propensity for the presence of Egr1 consensus binding sites, especially among transcription factors. By using chromatin immunoprecipitation it was demonstrated that insulin induced Egr1 bound to the promoters of a number of these transcription factors and thereby likely contributes to a previously unidentified transcriptional cascade. Further analysis of the function of Egr1 and Egr2 in adipocytes would benefit from adipocytes-specific approaches to modulate their expression. The endogenous RNA interference pathway is commonly harnessed to mediate knockdown of target gene expression. However, 3T3-L1 adipocytes are refractory to conventional means of either siRNA transfection or short hairpin RNA (shRNA) expression. A strategy was thus developed allowing inducible shRNA expression in adipocytes. The Cre-LoxP system was chosen for tight regulation of inducible expression.  Incorporation of a loxP-flanked DNA cassette inside the shRNA loop permitted Cre-inducible shRNA expression and target gene knockdown. Concurrently, a strategy for Cre-inducible exogenous gene expression was developed. Therefore, tightly controlled systems for the time-specific investigation of gene function were established. The 3T3-L1 cell line is a valuable model for the study of adipocytes differentiation, a poorly understood process in vivo. The development of a strategy capable of investigating Egr1 and Egr2 function in a time-specific manner permitted investigation of these genes during 3T3-L1 differentiation. Treatment of post-confluent 3T3-L1 preadipocytes with an established hormonal cocktail, consisting of isobutyl-methylxanthine (IBMX), dexamethasone and insulin, induces the adipocytes differentiation program, which requires the sequential induction of the number of transcription factors. It was found that Egr1 and Egr2 displayed a degree of reciprocity in their expression patterns during differentiation, such that when Egr2 expression was maximal Egr1 was very low, and vice versa. Cre-inducible expression of Egr1 in confluent preadipoctyes inhibited, whereas inducible expression of Egr2 promoted, adipocytes differentiation. Conversely, RNAi-mediated knockdown of Egr1 expression potentiated, whereas knockdown of Egr2 inhibited, adipocytes differentiation. Egr2 acts, in part, to mediate the effects of insulin on the differentiation program.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available