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. ? ? ?
