IN VIVO ANALYSES OF HYPERTROPHY MEDIATED GENE ACTIVATION

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DESCRIPTION (adapted from the applicant's abstract): It is hypothesized that the mechanism of hypertrophy mediated gene activation can be determined studying transcriptional regulation of BCK using the in vivo model (injection of DNA constructs into the adult heart). Analyses reveal that increased binding activity of a 60 kD nuclear factor (HRF) to a novel 33 base pair enhancer (HRE) mediate BCK induction in pressure overload hypertrophy. Hypotheses: 1) the HRE, HRF, and HRE/HRF complex mediate BCK gene induction in hypertrophy in vivo; and 2) hypertrophy induction of HRE is via a combination of AngII-PKC-MAPkinase and JAK/STAT mediated increased in HRF binding availability. Specific Aim 1 seeks to characterize the HRE and HRF using EMSA, DNA footprint, and transient transfection (via injection of the adult rat heart) to define the specific bases and combinatorial elements responsible. To establish the HRE as a bona fide hypertrophy response element, transgenic analyses are proposed. Specific Aim 2 is designed to characterize and clone the HRF. The AngII-PCK-MAP and JAK-STAT signaling pathways activated in vivo and their relationship with HRE/HRF mediated gene activation will be defined in Specific Aim 3 using immunoblot, EMSA, and transient transfection analyses.