Histone Methyltransferases and TGF-beta Signaling

描述（由申请人提供）：这是一份R21申请，响应PA 02-100“激素调控基因表达中的复合物形成”。tgf - β配体超家族通过受体丝氨酸-苏氨酸激酶传递信号，受体丝氨酸-苏氨酸激酶利用可易位转录因子Smads向下游传递信号。在细胞核中，Smads与dna结合蛋白和转录辅助因子结合，调控靶基因的表达。tgf - β和BMP信号的丧失对癌症具有重要意义，因为一些人类癌症已被证明在其发展过程中失去了tgf - β信号能力，并且至少在一种遗传性癌症综合征中显示了BMP受体的突变。最近发现靶基因转录调控的一个重要组成部分是抑制，而抑制的丧失对正常发育和体内平衡是有害的。我们已经确定了BMP调节的Smad蛋白与Suv家族的组蛋白甲基转移酶的关联。这些蛋白甲基化赖氨酸9上的组蛋白H3并导致转录抑制或沉默。在小鼠中，Suv蛋白的缺失与b细胞淋巴瘤的自发发展有关。因此，Smads与Suv的关联为tgf - β信号通路调控的基因转录沉默提供了一种机制，这可能在癌症中很重要。在本研究中，我们将进一步在生化和功能水平上表征Smad蛋白与Suv的相互作用。在Specific Aim 1中，我们将以SuvH1和Smadl为原型，确定Suv和Smad蛋白相互作用的分子细节。我们将绘制每个蛋白质上的相互作用域，并确定在抑制复合体中是否存在可能的转录伙伴。在特异性目标2中，我们将使用已知的BMP信号抑制启动子的报告基因检测来描述相互作用的功能意义。我们还将确定参与指定转录抑制的顺式元件，并使用染色质免疫沉淀和其他检测确定Smads和Suv蛋白是否与DNA相互作用。这些研究将为进一步分析Suv-Smad在转录沉默中的相互作用机制提供基础。

DESCRIPTION (provided by applicant): This is a R21 application in response to PA 02-100 "Complex Formation in Hormonal Regulation of Gene Expression." The TGF-beta superfamily of ligands signal through receptor serine-threonine kinases that transmit their signal downstream using translocatable transcription factors called Smads. In the nucleus, Smads associate with DNA-binding proteins and transcriptional co-factors to regulate target gene expression. Loss of TGF-beta and BMP signaling have important implications for cancer, as several human cancers have been shown to have lost TGF-beta signaling capability during their development, and mutation of a receptor for BMPs has been shown in at least one hereditary cancer syndrome. It has lately become apparent that a significant component of transcriptional regulation of target genes is repression and that loss of repression is harmful to normal development and homeostasis. We have identified association of BMP regulated Smad proteins with histone methyltransferases of the Suv family. These proteins methylate histone H3 on lysine 9 and cause transcriptional repression or silencing. In the mouse, loss of Suv proteins is associated with the spontaneous development of B-cell lymphoma. The association of Smads with Suv thus provides a mechanism for transcriptional silencing of genes regulated by TGF-beta signaling pathways that may be important in cancer. In this proposal, we will further characterize the interaction of Smad proteins with Suv at both the biochemical and functional level. In Specific Aim 1 we will determine the molecular details of the interaction of Suv and Smad proteins using SuvH1 and Smadl as prototypes. We will map the interaction domains on each protein and determine if likely transcriptional partners are present in the repression complex. In Specific Aim 2 we will characterize the functional significance of the interaction using reporter gene assays of a promoter known to be repressed by BMP signaling. We will also determine the cis elements involved in specifying transcriptional repression and determine if Smads and Suv proteins interact on DNA using chromatin immunoprecipitation and other assays. These studies will provide the foundation into further analysis of the mechanism of Suv-Smad interaction in transcriptional silencing.