The FIAT-ATF4 interaction and the control of bone mass

描述(申请人提供)：我们已经克隆了FIAT(抑制ATF4介导的转录)，一种亮氨酸拉链蛋白，它与ATF4相互作用，抑制依赖ATF4的骨钙素(OCN)基因的转录。在成骨细胞中表达FIAT的转基因小鼠骨量减少，OCN基因转录降低。因此，FIAT调节骨量的增加，并作为成骨功能的转录抑制因子。由于达到最大峰值骨量可以预防骨质疏松，因此更好地了解峰值骨量的分子决定因素是至关重要的。我们假设FIAT抑制碱性结构域-亮氨酸拉链(BZIP)转录因子的转录活性，如ATF4或AP-1家族成员，以调节体内的骨量。为了验证拟议的假设，我们将：(1)分析FIAT在成骨细胞形成过程中的表达模式；(2)对FIAT-ATF4相互作用的结构功能进行分析；(3)利用RNA干扰技术抑制FIAT基因在成骨细胞中的表达；(4)在小鼠体内使FIAT基因失活；(5)确定FIAT是否能与bZIP因子c-jun、Fra-1或Delta-FosB进行二聚体反应，以抑制其转录活性。在成骨细胞成熟过程中，将使用免疫共沉淀法(Co-I.P.)评估Fiat-ATF4异源二聚体的相对丰度。并将使用染色质免疫沉淀平行监测ATF4与OCN启动子的结合。CO-I.P.和定点突变的瞬时转染分析将鉴定FIAT和ATF4中的功能亮氨酸拉链，并研究RSK2的磷酸化在FIAT-ATF4相互作用中的作用。我们将使用RNAi来抑制培养的成骨细胞中FIAT基因的表达。将通过测量Fiat的表达、OCN基因转录的开始和水平、OCN启动子被ATF4占据、I型胶原合成和矿化来评估Fiat RNA敲除的影响。我们将使用Cre/lox技术在小鼠身上使Fiat基因失活。突变小鼠的骨表型将使用DEXA、微型计算机断层扫描、组织形态计量学、实时聚合酶链式反应和生物力学测试进行分析。最后，将使用蛋白质相互作用和瞬时转染试验来确定Fiat是否可以与其他bZIP分子二聚化以抑制其活性。这些实验将阐明菲亚特在体内调节骨量积累的分子机制。

DESCRIPTION (provided by applicant): We have cloned FIAT (Factor Inhibiting ATF4-mediated Transcription), a leucine zipper protein that interacts with ATF4 to repress ATF4-dependent transcription of the osteocalcin (OCN) gene. Transgenic mice expressing FIAT in osteoblasts have reduced bone mass and lower OCN gene transcription. Thus FIAT regulates bone mass accrual and acts as a transcriptional repressor of osteoblastic function. Since achieving a maximal peak bone mass is protective against osteoporosis, a better understanding of the molecular determinants of peak bone mass is critical. We hypothesize that FIAT represses the transcriptional activity of basic domain-leucine zipper (bZIP) transcription factors, such as ATF4 or AP-1 family members, to regulate bone mass in vivo. To test the proposed hypothesis, we will: (1) Analyze the expression pattern of FIAT during osteoblastogenesis; (2) Perform structure-function analysis of the FIAT-ATF4 interaction; (3) Inhibit FIAT gene expression in osteoblasts using RNA interference; (4) Inactivate the FIAT gene in mice; (5) Determine if FIAT can dimerize with the bZIP factors c-Jun, Fra-1, or delta-FosB to inhibit their transcriptional activity. The relative abundance of FIAT-ATF4 heterodimers will be assessed during osteoblast maturation using co-immunoprecipitation (Co-I.P.) and the binding of ATF4 to the OCN promoter will be monitored in parallel using chromatin immunoprecipitation. Co-I.P. and transient transfection assays with site-specific mutants will identify the functional leucine zippers within FIAT and ATF4 and examine the role of phosphorylation by RSK2 on the FIAT-ATF4 interaction. We will use RNAi to inhibit FIAT gene expression in cultured osteoblasts. The impact of FIAT RNA knockdown will be assessed by measuring FIAT expression, onset and level of OCN gene transcription, OCN promoter occupancy by ATF4, type I collagen synthesis, and mineralization. We will inactivate the FIAT gene in mice using the Cre/lox technique. The bone phenotype of the mutant mice will be analyzed using DEXA, micro-computed tomography, histomorphometry, Real-Time PCR, and biomechanical testing. Finally, protein interaction and transient transfection assays will be used to determine if FIAT can dimerize with other bZIP molecules to inhibit their activity. These experiments will elucidate the molecular mechanisms through which FIAT regulates bone mass accrual in vivo.