SUMOylated αNAC Potentiates Transcriptional Repression by FIAT

Abstract

The transcriptional coregulator αNAC (Nascent polypeptide associated complex And Coregulator alpha) and the transcriptional repressor FIAT (Factor Inhibiting ATF4-mediated Transcription) interact but the biological relevance of this interaction remains unclear. The activity of αNAC is extensively modulated by post-translational modifications (PTMs). We identified a novel αNAC PTM through covalent attachment of the Small Ubiquitin-like MOdifier (SUMO1). Recombinant αNAC was a SUMO1 target in in vitro SUMOylation assays and we confirmed that αNAC is conjugated to SUMO1 in cultured osteoblasts and in calvarial tissue. The amino acid sequence of αNAC contains one copy of the composite "phospho-sumoyl switch" motif that couples sequential phosphorylation and SUMOylation. We found that αNAC is selectively SUMOylated at lysine residue 127 within the motif and that SUMOylation is enhanced when a phosphomimetic mutation is introduced at the nearby serine residue 132. SUMOylation did not alter the DNA-binding capacity of αNAC. The S132D, hyper-SUMOylated αNAC mutant specifically interacted with histone deacetylase-2 (HDAC2) and enhanced the inhibitory activity of FIAT on ATF4-mediated transcription from the Osteocalcin gene promoter. This effect required binding of SUMOylated αNAC to the target promoter. We propose that maximal transcriptional repression by FIAT requires its interaction with SUMOylated, HDAC2-interacting αNAC.

Dephosphorylation of the transcriptional cofactor NACA by the PP1A phosphatase enhances cJUN transcriptional activity and osteoblast differentiation

“…4A), several ribosome-associated proteins interacting with NACA, including ribosomal protein L38 (RPL38). More importantly, among the previously known transcriptional partners of NACA, we observed interaction with TXLNG (FIAT) and TXLNA (47)(48)(49)(50)(51)(52). Taken together these observations are supportive of the fidelity of our purification method.…”

Altered gene dosage confirms the genetic interaction between FIAT and αNAC

“…We previously confirmed the interaction of endogenous FIAT and αNAC proteins in osteoblasts and demonstrated a functional role for this interaction in osteoblast function, related to maximal repression of ATF4-mediated transcription of the Ocn gene (Hekmatnejad et al, 2014). In the present study, we attempted to assess the physiological relevance of the interaction between αNAC and FIAT by creating a mouse model of combined Fiat and Naca heterozygous deficiency.…”

“…This interaction was independently confirmed using over-expression of epitope-tagged proteins in heterologous cell systems (Yoshida et al, 2005). Our recent work has confirmed that endogenous, post-translationally modified αNAC functionally interacts with the FIAT protein in osteoblastic cells to maximally repress ATF4-mediated Ocn gene transcription (Hekmatnejad et al, 2014). We set out to provide evidence of the physiological relevance of these findings through manipulation of Fiat and Naca dosage in genetically modified mice, with particular focus on skeletal development.…”