IN VIVO ANALYSES OF HYPERTROPHY MEDIATED GENE ACTIVATION

描述(改编自申请人的摘要)：假设 可以确定肥大介导的基因激活的机制 利用体内模型研究BCK的转录调控 (将DNA结构注入成人心脏)。分析表明， 60kD核因子(HRF)与新型33的结合活性增强 碱基对增强子(HRE)介导压力超负荷时BCK的诱导 肥大。假设：1)HRE、HRF和HRE/HRF复合体介导BCK 体内肥大的基因诱导；2)HRE肥大的诱导 是通过AngiI-PKC-MAPKinase和JAK/STAT介导的增加 在HRF结合可用性中。具体目标1试图描述HRE的特征 使用EMSA、DNA足迹和瞬时转染法(通过注射)的HRF 以定义特定的碱基和组合 有责任的人员。将HRE确定为真正的肥厚 响应元件、转基因分析等。具体目标2是 设计用来表征和克隆HRF。Angii-PCK-MAP和JAK-STAT 体内激活的信号通路及其与HRE/HRF的关系 介导的基因激活将在使用免疫印迹的特定目标3中定义， EMSA和瞬时转染分析。

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.