Control of Fiat (factor inhibiting ATF4‐mediated transcription) expression by Sp family transcription factors in osteoblasts

Abstract

FIAT (factor inhibiting ATF4-mediated transcription) represses Osteocalcin gene transcription and inhibits osteoblast activity by heterodimerizing with ATF4 to prevent it from binding DNA. It thus appears important to identify and characterize the molecular mechanisms that control Fiat gene expression in osteoblasts. In silico sequence analysis identified a canonical GC-box within a 1,400 bp region of the proximal Fiat gene promoter. Electrophoretic mobility shift assays (EMSA) with MC3T3-E1 osteoblastic cells nuclear extracts indicated that the transcription factors Sp1 and Sp3, but not Sp7/OSTERIX, bound this proximal GC-box. Chromatin immunoprecipitation confirmed interaction of the two transcription factors with the Fiat promoter GC-element in living osteoblasts. Transient transfection studies showed that Sp1 dose-dependently activated the expression of a Fiat-luciferase reporter construct while both the long or short isoforms of Sp3 dose-dependently inhibited transcription from the Fiat reporter construct. Transfection of an Sp7/OSTERIX expression vector did not affect expression of the Fiat-luciferase reporter. Co-transfection of increasing amounts of the Sp3 expression vector in the context of maximal Sp1-dependent Fiat-luciferase activation led to dose-dependent repression of the expression of the reporter. Using RNA knockdown, we measured a reduction in steady-state Fiat expression when Sp1 was inhibited, and a reciprocal increase upon Sp3 knockdown. In parallel, treatment of osteoblasts with WP631, which prevents Sp1/DNA interactions, strongly inhibited the expression of Fiat and reduced the occupancy of the Fiat promoter proximal GC-box by Sp1. Taken together, our results suggest an interplay between Sp1 and Sp3 as a mechanism involved in the control of Fiat gene expression in osteoblasts.

Two functional loci in the promoter of EPAS1 gene involved in high-altitude adaptation of Tibetans

“…The decreased inhibition of IKZF1 on EPAS1-C/insertion/T promoter as compared with that on EPAS1-C/deletion/C promoter ( Figure 2A ) might be caused by the steric hindrance of other transcriptional factors binding to the 40-bp insertion fragment. Sp1 is a zinc finger transcription factor that binds to GC-rich motifs of many promoters acting as an activator 32 or a repressor 33 , which is involved in many cellular processes, including cell differentiation, cell growth, apoptosis 34 , immune responses 35 and so on. Sp1 contains three spliced transcript variants encoding different isoforms (isoform 1–3).…”

A neomorphic variant in SP7 alters sequence specificity and causes a high-turnover bone disorder

“…Based on similarities with other SP family members, SP7 was first thought to bind GC-rich sequences 1 , 9 – 11 which are present in the promoters of many genes important for osteoblast functions 12 , 13 , such as Col1a1 and Col1a2 14 . However, other studies have shown evidence for a lack of binding preference for a GC-rich sequence 15 . More recently, Hojo et al showed that SP7 differs from other SP proteins in that it has a lower binding affinity for GC-rich sequences, and instead interacts with DLX5 and with AT-rich promoter/enhancer sequences to transactivate genes in osteoblasts 16 .…”

The role of microRNAs in bone remodeling

“… 92 , 94 , 95 Factors inhibiting ATF4-mediated transcription (Fiat) have been found to suppress ATF4 activity and, co-expressed in osteoblasts 96 , 97 and modulated by the Sp family, to exclude Osx. 98 In the matrix mineralization stage, ATF4 is positively regulated, to a large extent, by c-Jun N-terminal kinase (JNK), affecting the expression of OCN and BSP. 99 Moreover, ATF4 can also bind to the promoter of RANKL and can ultimately inhibit bone resorption.…”