Mechanisms of TGFB Signal Transduction

描述（由申请方提供）：骨形态发生蛋白（BMP）代表TGF β家族生长因子的最大子集，其作用范围从早期发育期间的骨形态发生到中胚层的腹侧化。骨形成蛋白发挥如此多样的作用和组织特异性的机制知之甚少。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介导的基因的转录因子，称为OAZ。初步数据支持OAZ通过与至少两个伴侣Smads 1/4或Olf-1/EBF的不同蛋白质相互作用来调节不同的基因。有待检验的具体假设是，OAZ与这些不同蛋白质的相互作用导致独特的转录复合物和差异基因调控的组装。新的结果表明，OAZ激活Smad 6基因，一种公认的BMP信号拮抗剂，支持通过OAZ的BMP信号的潜在的新的负反馈回路。在具体目标1中，我们将通过以下方式进行测试：（a）确认OAZ结合的小鼠Smad 6启动子中的序列元件，（B）通过BMP激活Smad 1和野生型或显性阴性OAZ表达的时程研究，检查OAZ诱导Smad 6的功能意义，以及（c）鉴定OAZ的新型哺乳动物靶基因。我们已经确定了两个新的辅因子OAZ，Asc-1/TRIP 4和Parp 1在酵母双杂交筛选使用OAZ作为诱饵。在具体目标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）通过过表达反义Asc-1构建体来抑制Asc-1表达，从而阐明Asc-1在Xvent-2活化中的生理作用，（B）确定Asc-1作为OAZ的转录伴侣的功能作用，和（c）表征OAZ-Asc-1复合物是否在已知的BMP靶基因启动子（Smad 6、Msx-1、Msx-2和Id 1）上组装。最后，在具体目标3中，我们将研究聚乙烯的生理作用通过研究（a）Parp 1介导的多聚ADP核糖基化正向调节Xvent-2基因转录的机制，（B）OAZ转录复合物的已知蛋白质是否（OAZ、Smads和/或Asc-1）被Parp 1聚ADP核糖基化，和（c）Parp 1是否参与已知BMP靶基因启动子（Smad 6、Msx-1、Msx-2和Id 1）的调节。这些研究将为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.