AbstractThe ATF4 transcription factor is a key regulator of osteoblast differentiation that controls osteocalcin gene transcription and type I collagen protein synthesis. We have characterized factor‐inhibiting ATF4‐mediated transcription (FIAT), a leucine zipper protein that dimerizes with ATF4 to form inactive dimers that cannot bind DNA. Overexpression of FIAT in osteoblasts of transgenic mice inhibited osteocalcin gene transcription and reduced osteoblastic activity, leading to osteopenia (Yu et al. [2005] J Cell Biol 169:591–601). We therefore hypothesized that inhibition of FIAT would enhance ATF4 activity, leading to increased osteocalcin transcription, type I collagen synthesis, and mineralization. We used small interfering RNAs (siRNA) to knockdown FIAT in pools of MC3T3‐E1 cells stably transfected with 1.3 kb of the mouse osteocalcin gene promoter driving expression of luciferase. Stable expression of the FIAT siRNA sequence inhibited FIAT expression without significantly affecting the level of total or Ribosomal S6 Kinase‐2‐phosphorylated ATF4 protein. Occupancy of the osteocalcin proximal promoter by ATF4 was increased and transcription of the osteocalcin‐promoter‐dependent luciferase reporter showed earlier onset and increased levels. Similarly, endogenous osteocalcin gene expression was enhanced in primary osteoblasts transfected with the FIAT siRNA. FIAT knockdown cells also displayed higher expression of bone sialoprotein, increased type I collagen protein synthesis, and enhanced mineralization. These data suggest that inhibition of FIAT expression increases ATF4 activity and confirm the important role of FIAT in osteoblast function. J. Cell. Biochem. 106: 186–192, 2009. © 2008 Wiley‐Liss, Inc.