Mechanisms of TGFB Signal Transduction

描述（由申请人提供）：骨形态发生蛋白（BMPs）代表TGFbeta生长因子家族的最大子集，其作用范围从早期发育期间的骨形态发生到中胚层的腹化。bmp发挥如此多样的作用和组织特异性的机制尚不清楚。本提案的长期目标是通过探索bmp的空间和时间效应来了解bmp能够产生特定基因表达事件的分子机制。我们最近克隆了一种新的bmp介导基因转录因子，叫做OAZ。初步数据支持OAZ通过与至少两个伙伴Smads1/4或Olf-1/EBF的不同蛋白质相互作用来调节不同的基因。需要验证的具体假设是，OAZ与这些不同的蛋白质相互作用导致独特转录复合物的组装和差异基因调控。新的研究结果表明，OAZ激活了Smad6基因，这是一种公认的BMP信号拮抗剂，支持通过OAZ进行BMP信号传导的潜在的新型负反馈回路。在Specific Aim 1中，我们将通过(a)确认OAZ结合的小鼠Smad6启动子中的序列元件，(b)通过BMPs和野生型或显性阴性OAZ表达激活Smad1的时间过程研究OAZ诱导Smad6的功能意义，以及(c)鉴定新的OAZ哺乳动物靶基因来验证这一点。我们以OAZ为诱饵，在酵母双杂交筛选中发现了两个新的OAZ辅因子Asc-1/TRIP4和Parp1。在Specific Aim 2中，我们将通过以下方式研究OAZ-Asc-1复合物的功能意义：(a)阐明Asc-1通过过表达反意义Asc-1构建体抑制Asc-1表达在xvent2活化中的生理作用，(b)确定Asc-1作为OAZ转录伙伴的功能作用，以及(c)表征OAZ-Asc-1复合物是否组装在已知的BMP靶基因启动子（Smad6, Msx-1， Msx-2和Id1）上。最后,在针对性3中,我们将研究聚(ADP-ribose)聚合酶的生理作用,Parp1, OAZ通过研究代数余子式的(a)的机制Parp1-mediated poly-ADP-ribosylation积极调节Xvent-2基因的转录,(b)是否已知蛋白质的OAZ转录复杂(OAZ Smads,和/或Asc-1)由Parp1 poly-ADP-ribosylated,和(c) Parp1是否参与已知BMP的规定目标基因启动子(Smad6、Msx-1 Msx-2,和Id1)。这些研究应该为tgf - β介导的基因表达调控提供重要的见解，这与正常生理和各种疾病状态直接相关。

DESCRIPTION (provided by applicant): Bone morphogenetic proteins (BMPs) represent the largest subset of the TGFbeta family of growth factors, with actions that range from osteomorphogenesis to ventralization of mesoderm during early development. The mechanisms by which BMPs exert such diverse effects and tissue-specificity are poorly understood. The long-term objective of this proposal is to understand molecular mechanisms by which BMPs are able to generate specific gene expression events by exploring spatial and temporal effects of BMPs. We recently cloned a novel transcription factor for BMP-mediated genes, called OAZ. Preliminary data support that OAZ regulates different genes by distinct protein interactions with at least two partners, Smads1/4 or Olf-1/EBF. The specific hypothesis to be tested is that OAZ interactions with such distinct proteins leads to assembly of unique transcriptional complexes and differential gene regulation. New results show that OAZ activates the Smad6 gene, a well-recognized antagonist of BMP signaling, supporting a potential novel negative feedback loop for BMP signaling via OAZ. In Specific Aim 1, we will test this by (a) confirming the sequence element in the mouse Smad6 promoter to which OAZ binds, (b) examining the functional significance of Smad6 induction by OAZ with time-course studies of Smad1 activation by BMPs and wild type or dominant negative OAZ expression, and (c) identifying novel mammalian target genes for OAZ. We have identified two novel cofactors of OAZ, Asc-1/TRIP4 and Parp1 in yeast two-hybrid screens using OAZ as a bait. In Specific Aim 2, we will study the functional significance of the OAZ-Asc-1 complex by (a) elucidating a physiological role of Asc-1 in activation of Xvent-2 by inhibiting Asc-1 expression by overexpression of antisense-Asc-1 construct, (b) determining the functional role of Asc-1 as a transcription partner of OAZ, and (c) characterizing whether the OAZ-Asc-1 complex assembles on known BMP target gene promoters (Smad6, Msx-1, Msx-2, and Id1). Finally, in Specific Aim 3, we will study a physiological role of poly (ADP-ribose) polymerase, Parp1, as a cofactor of OAZ by studying (a) the mechanism by which Parp1-mediated poly-ADP-ribosylation positively regulates transcription of Xvent-2 gene, (b) whether or not known proteins of the OAZ transcriptional complex (OAZ, Smads, and/or Asc-1) are poly-ADP-ribosylated by Parp1, and (c) whether Parp1 is involved in the regulation of known BMP target gene promoters (Smad6, Msx-1, Msx-2, and Id1). These studies should provide important insights into TGFbeta-mediated regulation of gene expression, which is of direct relevance to both normal physiology and a variety of disease states.