Endocrine Abstracts

Marshall-Smith syndrome (MSS) is a congenital disorder affecting skeletal and neural development, due to mutations in the nuclear factor I/X (NFIX) gene. NFIX encodes a ubiquitously expressed transcription factor that regulates the expression of viral and cellular genes. To identify novel genes that are misregulated by NFIX mutations, RNA sequencing and proteomics analyses were performed on mouse embryonic fibroblast (MEF) cells derived from a representative Nfix mutant mouse model for MSS (Nfix^Del2/Del2) and wild-type mice. This revealed that cellular retinoic acid binding protein 2 (Crabp2) was upregulated at both the RNA and protein levels (2.59-fold and 2.83-fold, P < 0.012, respectively). Validation studies using qRT-PCR and Western blot analyses confirmed that Crabp2 was upregulated at the RNA and protein levels (2.4-fold and 5-fold, P < 0.0001, respectively) in Nfix^Del2/Del2 MEFs compared to wild-type MEFs and that 60% of the 5 MSS patients’ fibroblasts had altered CRABP2 transcript (P < 0.05) and protein (P < 0.001) levels, compared to 3 normal fibroblasts. We identified a putative nuclear factor I (NFI) binding site, to which NFIX binds, in the CRABP2 5’ untranslated region (UTR). To investigate its effect on promoter activity, luciferase reporter constructs under the transcriptional control of either the wild-type or mutant (with a mutated or deleted NFI binding site) CRABP2 promoter were transfected into monkey kidney fibroblast (COS-7) cells. Mutation and deletion of the NFI binding site resulted in 1.3-fold (P < 0.001) and 0.6-fold (P < 0.0001) change in luciferase expression compared to the wild-type promoter, respectively. Furthermore, co-transfection of COS-7 cells with wild-type CRABP2 promoter-driven luciferase reporter constructs and N-terminal-FLAG tagged wild-type and MSS-mutant NFIX cDNA constructs showed that the MSS-associated NFIX mutants significantly increased luciferase reporter activity at the CRABP2 promoter (1.5-fold, P < 0.05) compared to wild-type NFIX. Thus, our results suggest that NFIX directly regulates the activity of the CRABP2 promoter and alters CRABP2 expression in MSS patients.