Hypoxia Signaling, Chromatin Remodeling and Nickel Carcinogenesis

缺氧信号传导、染色质重塑和镍致癌作用

基本信息

批准号：
7138704
负责人：
Max Costa
金额：
$ 40.14万
依托单位：
NEW YORK UNIVERSITY SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-08-16 至 2010-06-30
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): The long-range goals of this grant proposal are to study the involvement of the hypoxia signaling pathway upon chromatin remodeling, gene silencing, and cell transformation. Hypoxia or agents that mimic hypoxia cause a global loss of histone acetylation and increase the methylation of H3 lysine 9 which is the chromatin mark of gene silencing for the individual nucleosomes. Methylation of H3 lysine 9 leads to DNA methylation and inherited gene silencing. We will study the ability of hypoxia and agents that activate hypoxia signaling (NiCI2, CoCI2, deferoxamine, and DMOG) to induce cell transformation in wild-type or in HIF-1alpha knockout mouse embryo fibroblast (MEF). We will transfect a normal HIF-1alpha construct into MEF cells with a knockout of this gene to study whether we can restore their ability to be transformed by hypoxia and agents that mimic hypoxia. We will also stably transfect mutated HIF-1alpha constructs that will constitutively express HIF in its stabile and or stable/active form into MEF HIF+/- cells and study the effect of stabilized and/or activated HIF- 1alpha on anchorage-independent growth and tumor formation in nude mice. We will investigate global changes in H3K9 acetylation, H3K9 methylation, G9a activity (enzyme that dimethylates H3K9) and DNA methylation in MEF with intact or knockout of HIF-1alpha following exposure to hypoxia or agents that mimic hypoxia. We will also utilize the expression levels of DHFR and DNA mismatch repair gene Mlh1 which are down-regulated by hypoxia signaling to study gene specific effects of chromatin remodeling induced by hypoxia signaling using CHIP assays targeted to their promoters with antibodies specific to H3K9 dimethylation and/or H3K9 acetylation. We have obtained mouse embryonic stem cell wild-type (WT), G9a knockout (G9a-/-) and G9a-/- stably transfected with wild-type G9a (G9a-/- + G9a WT), and we will utilize these cells to address the role of G9a in mediating histone methylation H3K9 and the effects of hypoxia signaling on DNA methylation and DHFR/MIh expression. We will study the reversibility and stability of MEF cells that have already been transformed via the hypoxia pathway to chromatin remodeling agents and R59949 (HIF inhibitor). These studies will allow us to understand the importance of hypoxia signaling and the HIF-1alpha transcription factor in promoting neoplastic cell transformation.

描述（由申请人提供）：该赠款建议的远程目标是研究缺氧信号通路在染色质重塑，基因沉默和细胞转化时的参与。低氧或模仿缺氧导致组蛋白乙酰化的全局丧失并增加H3赖氨酸9的甲基化的药物，这是单个核小体的基因沉默的染色质标记。 H3赖氨酸9的甲基化导致DNA甲基化和遗传基因沉默。我们将研究激活缺氧信号传导（NICI2，COCI2，脱氧胺和DMOG）的缺氧和药物的能力，诱导野生型或HIF-1Alpha敲除小鼠胚胎成纤维细胞（MEF）中的细胞转化。我们将使用该基因的敲除将正常的HIF-1Alpha构建体转染到MEF细胞中，以研究我们是否可以恢复其通过模仿缺氧的缺氧和药物转化的能力。我们还将稳定地转染突变的HIF-1Alpha构建体，这些构建体将以稳定和/或稳定/活性形式组成地表达HIF，并研究MEF HIF +/-细胞，并研究稳定和/或激活的HIF-1Alpha对锚固小鼠中独立的HIF-1Alpha对锚固不依赖的生长和肿瘤形成的影响。我们将研究H3K9乙酰化，H3K9甲基化，G9A活性（将H3K9二甲基二甲基化的酶）和DNA甲基化的全球变化，在暴露于低氧或模拟低氧的药物后，HIF-1Alpha完整或敲除HIF-1Alpha的全球变化。我们还将利用DHFR和DNA不匹配修复基因MLH1的表达水平，这些基因MLH1被低氧信号传导下调，以研究使用针对其启动子靶向H3K9二甲基化和/或H3K9乙酰化的抗体的芯片靶向促进剂的芯片靶向的缺氧信号传导诱导的染色质重塑的特异性作用。我们已经获得了小鼠胚胎干细胞野生型（WT），G9A敲除（G9A - / - ）和G9A - / - 稳定地用野生型G9A（G9A - / - - / - - + G9A WT）转染，我们将利用这些细胞来解决G9A在介导的Hytana Methation H3K9和H3K9的介导作用中的作用。 DHFR/MIH表达。我们将研究已经通过缺氧途径转化为染色质重塑剂和R59949（HIF抑制剂）的MEF细胞的可逆性和稳定性。这些研究将使我们能够了解缺氧信号传导和HIF-1Alpha转录因子在促进肿瘤细胞转化中的重要性。