Cell Cycle Control and Tumor Suppressors

当p33ING1(ING1)被证明与P53在物理和功能上相互作用时，我们假设p33ING1是编码P53共转录因子基因家族的先证者成员，从而启动了对ING1相关基因的搜索。通过使用LHC中已经建立的方法，我们已经确定了四个相关基因(p47ING3、p29ING4和p28ING5；ING2-5)。我们正在系统地研究ING基因家族中的每一个成员。例如，我们之前已经报道，与ING1b不同，ING1的另一种剪接形式ING2是由DNA损伤剂依托泊苷或新卡西诺抑素诱导的。ING1b或ING2通过诱导G1-S细胞周期检查点和细胞凋亡，以P53依赖的方式负性调节细胞的生长和存活。ING2可显著增强P53的转录反式激活活性。此外，ING2的表达增加了P53在赖氨酸-382位的乙酰化。这些结果最近得到了其他人的证实和推广，即ING2是一个核磷脂酰肌醇受体。综上所述，ING2是一种DNA损伤诱导基因，通过增强P53的乙酰化来负向调节细胞的增殖。我们最近对ING2的siRNA敲除的数据也表明，ING2可以促进场景表型的诱导。 在与Karl Riabowol的合作中，我们发现ING家族的一部分成员在同一细胞类型的平行实验中强烈抑制人甲胎蛋白(AFP)启动子的活性，但刺激p21(WAF1)启动子的活性，与P53的作用相似。P47(ING1a)亚型也抑制AFP启动子活性，但与其他ING亚型不同，它抑制p21(WAF1)启动子。P47(ING3)可上调p21(WAF1)启动子活性，但对AFP启动子活性无影响。ING1b和ING2还抑制了Hep3B P53缺失细胞系中的AFP启动子，而P53共表达增强了这种转录抑制。ING对AFP基因转录的抑制强烈依赖于与肝细胞核因子1(HNF1)转录因子结合的AT基序。事实上，电泳迁移率改变分析证实HNF1与AT基序结合，但令人惊讶的是，我们发现与这些AT基序结合的ING1复合体没有HNF1蛋白。ING1和P53均能抑制AFP转录并诱导p21的表达，而P53蛋白的负调控因子hSIR2对AFP启动子的作用与HDAC1相反，并且与HDAC1一样抑制p21启动子的活性。此外，我们还发现p33(ING1b)与hSIR2物理上相互作用，逆转其诱导AFP启动子的能力，并诱导Lys(373)和/或Lys(382)上P53残基的乙酰化。这些发现为p33(ING1b)至少通过两种机制抑制AFP转录提供了新的证据，其中一种机制包括p53。第一种是通过与AT基序结合并排除HNF1结合，同时可能针对启动子区域的HAT活性；第二种是通过结合和抑制hSIR2对P53蛋白去乙酰化的能力来增加活性的乙酰化P53的水平。 我们发现ING3-5也参与了细胞应激的P53反应通路。我们的初步数据表明，ING家族的不同成员在物理和功能上都与组蛋白去乙酰化酶(如Sir2)或组蛋白乙酰转移酶(如p300)的多蛋白复合体相互作用，并通过染色质重塑共同调节P53介导的转录。

When p33ING1 (ING1) was shown to physically and functionally interact with p53, we hypothesized that p33ING1 was the proband member of a family of genes encoding p53 co-transcription factors, and thus, initiated a search for ING1-related genes. By using methodologies already established in the LHC, we have identified four related genes (p47ING3, p29ING4 and p28ING5; ING2-5). We are systematically investigating each of these members of the ING gene family. For example, we have previously reported that unlike ING1b, an alternatively spliced form of ING1, ING2, is induced by the DNA-damaging agents, etoposide or neocarzinostatin. ING1b or ING2 negatively regulate cell growth and survival in a p53-dependent manner through the induction of G1-S cell cycle checkpoints and apoptosis. ING2 strongly enhances the transcriptional transactivation activity of p53. Furthermore, ING2 expression increases the acetylation of p53 at lysine-382. These results have been recently confirmed and extended by others, i.e., ING2 is a nuclear phosphoinositide receptor. Taken together, ING2 is a DNA damage-inducible gene that negatively regulates cell proliferation through the activation of p53 by enhancing its acetylation. Our recent data from SiRNA knockdown of ING2 also indicates that ING2 can contribute to the induction of the scenescent phenotype. In collaboration with Karl Riabowol, we found that a subset of ING family members strongly repressed human alpha-fetoprotein (AFP) promoter activity, but stimulated the p21(WAF1) promoter in parallel experiments in the same cell type, similar to the effects of p53. The p47(ING1a) isoform also repressed AFP promoter activity, but in contrast to other ING isoforms, it repressed the p21(WAF1) promoter. p47(ING3) upregulated p21(WAF1) promoter activity, but it did not have any effect on the AFP promoter. ING1b and ING2 also repressed the AFP promoter in Hep3B p53-null cell lines, and p53 coexpression enhanced this transcriptional repression. Suppression of AFP gene transcription by ING was strongly dependent on AT-motifs that bind to the hepatocyte nuclear factor 1 (HNF1) transcription factor. Indeed, electrophoretic mobility shift assays confirmed that HNF1 binds to AT-motifs, but we found, surprisingly, that the ING1 complexes binding to these AT-motifs were devoid of HNF1 protein. Both ING1 and p53 were able to suppress AFP transcription and cause p21 induction; hSIR2, a negative regulator of the p53 protein, showed the opposite effects on the AFP promoter and, like HDAC1, repressed p21 promoter activity. In addition, we found that p33(ING1b) physically interacts with hSIR2, reverses its ability to induce the AFP promoter, and induces acetylation of p53 residues at Lys(373) and/or Lys(382). These findings provide novel evidence that p33(ING1b) represses AFP transcription by at least two mechanisms, one of which includes p53. The first is by binding to the AT-motif and excluding HNF1 binding while possibly targeting HAT activity on promoter regions, and the second is by increasing the levels of active, acetylated p53 via binding and inhibiting the ability of hSIR2 to deacetylate p53 protein. We have discovered that ING3-5 also participate in the p53 response pathway to cellular stress. Our preliminary data indicate that different members of the ING family physically and functionally interact with multiprotein complexes of either histone deacetylases, e.g., SIR2, or histone acetyltransferase, e.g., p300, and coregulate p53-mediated transcription by chromatin remodeling.