The FIAT-ATF4 interaction and the control of bone mass

描述（由申请人提供）：我们克隆了FIAT（抑制ATF 4介导的转录的因子），一种亮氨酸拉链蛋白，其与ATF 4相互作用以抑制骨钙素（OCN）基因的ATF 4依赖性转录。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调节骨量增加，并作为成骨细胞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症，因此更好地了解峰值骨量的分子决定因素至关重要。我们假设FIAT抑制碱性结构域亮氨酸拉链（bZIP）转录因子（如ATF4或AP-1家族成员）的转录活性，以调节体内骨量。为了验证所提出的假设，我们将：（1）分析FIAT在成骨细胞发生过程中的表达模式;（2）进行FIAT-ATF4相互作用的结构-功能分析;（3）使用RNA干扰抑制FIAT基因在成骨细胞中的表达;（4）灭活小鼠中的FIAT基因;（5）确定FIAT是否可以与bZIP因子c-Jun、Fra-1或delta-FosB二聚化以抑制它们的转录活性。将在成骨细胞成熟期间使用免疫共沉淀（Co-I.P.）并使用染色质免疫沉淀平行监测ATF 4与OCN启动子的结合。使用位点特异性突变体的共I.P.和瞬时转染测定将鉴定FIAT和ATF 4内的功能性亮氨酸拉链，并检查RSK 2磷酸化对FIAT-ATF 4相互作用的作用。我们将使用RNAi抑制FIAT基因在培养的成骨细胞中的表达。FIAT RNA敲除的影响将通过测量FIAT表达、OCN基因转录的开始和水平、ATF 4对OCN启动子的占用、I型胶原蛋白合成和矿化来评估。我们将使用Cre/lox技术在小鼠中表达FIAT基因。将使用DEXA、显微计算机断层扫描、组织形态计量学、实时PCR和生物力学测试分析突变小鼠的骨表型。最后，将使用蛋白质相互作用和瞬时转染测定来确定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FIAT调节体内骨量增加的分子机制。

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.