The role of Inil in chromatin development and cancer

Inil 在染色质发育和癌症中的作用

• 批准号: 6464449
• 负责人: STEPHEN JONES
• 金额: $ 34.68万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6464449
• 关键词: DNA damage; DNA repair; biotransformation; cell cycle; cell growth regulation; cell proliferation; chromatin; embryonic stem cell; enzyme activity; enzyme mechanism; gene expression; gene mutation; gene targeting; laboratory mouse; p53 gene /protein; protein protein interaction; protein structure function; tumor suppressor genes; tumor suppressor proteins

DESCRIPTION: (provided by applicant) Inil is a subunit of the mammalian, ATP-dependent, SWI/SNF chromatin remodeling complexes. These enzymes play a key role in cell growth by altering chromatin structure to render normally condensed DNA accessible to cellular machinery, an important preliminary step to facilitate gene expression, cell division, and development. Subunits of the SWI/SNF enzymes interact with Rb and BRCA-l, known tumor suppressor proteins. The recent identification of Ini1 mutations in familial malignant rhabdoid tumors has further heightened interest in the link between SWI/SNF complexes and tumor suppression. Previous work from our labs has identified and characterized mammalian SWI/SNF complexes as possessing ATP dependent chromatin remodeling activities and has identified a role for SWI/SNF chromatin remodeling in differentiation of muscle as well as other differentiation events. Recently, we have generated mice deficient for Ini and have found that Inil is required for proper early embryogenesis, and tumor formation in Inil-haploinsufficient mice has identified Inil as a bonajide tumor suppressor in mice. The research described in this proposal will draw upon the strengths of the two investigators, utilizing a combination of genetic, biochemical, and molecular approaches to elucidate the mechanistic role of Inil in transformation and differentiation. Additionally, these studies will examine the role of Ini chromatin remodeling enzymes. Mice heterozygous for Inil will be treated with DNA damaging agents, and will be bred to mice bear mg inactivated alleles of the p53 gene or the Rb gene to investigate the link between the Inil-based SWI/SNF activity and DNA damage repair pathways, and to explore the role of the two major tumor suppressor pathways in Inil-mediated tumor suppression. Mice bearing a conditional allele of Ini1 will also be generated via gene targeting experiments in ES cells. The specificity of the site of tumorigenesis in the Inil-deficient mice is striking; 100 percent of the tumors arise on the face, head, and neck of the mouse, whereas in humans bearing an inactivated Ini1 gene, most of the tumors arise in the kidney and/or brain. Interestingly, these structures are all derived from neural crest progenitor cells during development. In order to determine if tumorigenesis in the Inil mice is due to a defect in neural crest cell growth or differentiation, the conditional Inil mice will be bred with mice expressing Cre specifically in neural crest cells. The conditional Inil mice also will be crossed with mice expressing the Cre recombinase in cardiac and skeletal muscle in order to provide in vivo evidence for a link between SWI/SNF activity and muscle cell differentiation. Lastly, we will purify SWI/SNF complexes from Inil-deficient cells. Isolated purified protein will be characterized for subunit composition and the ability to interact with tumor suppressor proteins. Furthermore, we will assess the ability of purified complexes/subunits lacking Ini1 to remodel chromatin structure and induce specific gene activation events in vitro. These proposed experiments should serve not only to define the role of Inil in differentiation and chromatin remodeling, but should also provide much insight into the mechanisms of Inil-mediated tumor suppression.

描述：（由申请人提供）INIL是哺乳动物的亚基， ATP依赖性，SWI/SNF染色质重塑复合物。这些酶可以发挥钥匙 通过改变染色质结构以正常渲染，在细胞生长中的作用 凝结的DNA可访问蜂窝机械，这是一个重要的初步步骤 促进基因表达，细胞分裂和发育。亚基 SWI/SNF酶与RB和BRCA-L相互作用，BRCA-L，已知的肿瘤抑制蛋白。 最近对家族性恶性肿瘤中INI1突变的鉴定 肿瘤进一步提高了对SWI/SNF复合物之间的联系的兴趣 和抑制肿瘤。我们实验室的先前工作已经确定， 将哺乳动物SWI/SNF复合物表征为具有ATP染色质 重塑活动，并确定了SWI/SNF染色质的作用 肌肉分化以及其他分化的重塑 事件。最近，我们产生了不足的INI的小鼠，发现 适当的早期胚胎发生和肿瘤形成需要INIL 无肌无蛋白的小鼠已将INIL识别为可疑的肿瘤抑制剂 在老鼠中。该提案中描述的研究将借鉴优势 在两个研究人员中，利用遗传，生化和 分子方法阐明了INIL的机理作用 转变和差异化。此外，这些研究将检查 INI染色质重塑酶的作用。小鼠杂合的inil会 用DNA破坏剂处理，并将繁殖到小鼠bear mg p53基因或RB基因的灭活等位基因研究链接 在基于INIL的SWI/SNF活动和DNA损伤修复途径之间 探索两个主要肿瘤抑制途径在INIL介导的 肿瘤抑制。带有条件等位基因的小鼠也将是 通过基因靶向实验在ES细胞中产生。特异性 在缺陷型小鼠中的肿瘤发生部位令人震惊。 100％ 肿瘤出现在鼠标的脸，头部和颈部，而在人类中 带有灭活的INI1基因，大多数肿瘤出现在肾脏和/或 脑。有趣的是，这些结构都来自神经rest 发育过程中的祖细胞。为了确定是否在 INIL小鼠是由于神经Crest细胞生长的缺陷或 分化，有条件的小鼠将用表达的小鼠繁殖 CRE专门在神经rest细胞中。有条件的小鼠也将是 与表达心脏和骨骼肌肉中CRE重组酶的小鼠交叉 为了提供体内证据，证明SWI/SNF活动与 肌肉细胞分化。最后，我们将从 缺陷型细胞。孤立的纯化蛋白将被表征为 亚基组成和与肿瘤抑制蛋白相互作用的能力。 此外，我们将评估缺乏纯化的复合物/亚基的能力 INI1重塑染色质结构并诱导特定的基因激活事件 体外。这些提出的实验不仅应用于定义角色 分化和染色质重塑的INIL，但也应提供 深入了解INIL介导的肿瘤抑制的机制。