Date of Graduation

1997

Document Type

Dissertation/Thesis

Abstract

The chicken very low density apolipoprotein II (apoVLDLII) gene encodes an egg yolk protein that is synthesized in the liver in response to estrogen. Regulation of this gene is mediated by the estrogen receptor via two estrogen response elements (EREs) located in the promoter. Following the administration of estrogen, there is a four hour lag period prior to the detection of apoVLDLII mRNA. To examine the events that occur during this lag period, in vivo footprinting and run-on transcription assays were performed. Time courses of the transcription of the apoVLDLII gene were done using the transcription run-on assay. The antisense RNA probes used in this assay represented the {dollar}5\\sp\\prime ,{dollar} middle and {dollar}3\\sp\\prime{dollar} regions of the apoVLDLII gene. Following a single injection of 17 {dollar}\\beta{dollar}-hydroxy estradiol, Estrogen dependent initiation occurred in one hour followed by non-processive elongation at three hours. Efficient elongation, however, was delayed for 24 hr. Treatment with estrogen plus cycloheximide doubled the rate of initiation, but abolished elongation. These findings establish two classes of estrogen-inducible RNA polymerase II elongation coactivators that appear at 3 and 24 hours following the administration of estrogen. To determine the events that precede each increase in the rate of transcription, chromatin structural changes were monitored by hypersensitive site (HS) analysis, DMS and DNase I in vivo footprinting on genomic DNA amplified by ligation-mediated PCR. HS analysis revealed that the appearance of estrogen-induced HS accompany initiation of transcription. These hormone-induced HS were maintained throughout transcription, and decayed after 10 weeks, long after transcription level had gone down to undetectable levels. The maintenance of these hormone induced HS was independent of transcription. Therefore gene silencing is not only a simple matter of open and closed conformations. DMS in vivo footprinting results demonstrated rapid chromatin remodeling within the EREs, in consistent with estrogen-induced occupancy of the ERE by the estrogen receptor. Dramatic chromatin alterations within the apoVLDLII promoter coincided with each increase in the rate of transcription. CHX altered the estrogen induced changes observed in the EREs. Estrogen plays a critical role in regulating chromatin structural alterations.

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