Characterization of a silencer element in the first exon of the human osteocalcin gene

Abstract

Osteocalcin, the major non-collagenous protein in bone, is transcribed in osteoblasts at the onset of extracellular matrix mineralization. In this study It was demonstrated that sequences located in the first exon of the human osteocalcin gene possess a differentiation-related osteocalcin silencer element (OSE). Osteocalcin was rendered transcribable in UMR-106 cells and proliferating normal osteoblasts after deletion of the -3 to +51 region. Site-specific mutagenesis of this region revealed that a 7 bp sequence (TGGCCCT) (+29 to +35) is critcal for silencing function. Mobility shift assays demonstrated that a nuclear factor bound to the OSE. The OSE binding protein was present in proliferating normal pre-osteoblasts and in UMR-106 and ROS 17/2.8 osteosarcoma cells, but was absent from post-proliferative normal osteoblasts. The binding protein was inhibited by fragments containing the +29/+35 sequence, but not by other promoter fragments or by the consensus oligomers of unrelated nuclear factors AP-1 and Spl. DNase I footprinting demonstrated that the OSE binding-protein protected the +17 to +36 portion of the first exon, consistent with the results of mapping studies and competitive mobility shift assays. It Is hypothesized that this silencer is activated by complexing of the OSE binding protein to the OSE during the osteoblast proliferation stage and that the OSE binding protein is down-regulated at the onset of extracellular matrix mineralization.

Structural analysis and characterization of tissue and hormonal responsive expression of the avian bone sialoprotein (BSP) gene

“…Of greatest interest to mention are the homeobox sequences Msx2 (Hox 8.1) and the COL II silencer sequences which have been shown to be functionally active in regulating the expression of the mammalian osteocalcin gene [Towler et al, 1994;Hoffmann et al, 1994;Frenkel et al, 1993;Li et al, 1995]. The demonstrated apparent function of these elements is also suggested in the studies reported here since their deletion causes a six fold increase in the activity of the bsp promoter.…”

“…Furthermore the level of inhibition by the silencers in chicken bsp is similar to that of the rat COL II silencer elements [Savagner et al, 1989]. In reference to the effects of this element in the regulation of a bone specific gene, it is also of interest to note that these silencer elements also play a role in the regulation of the osteocalcin gene activity [Frenkel et al, 1993;Li et al, 1995]. Since the highest expression of bsp/CAT in fibroblast is still significantly lower than that in osteoblast, these data suggest the existence of a bone-specific enhancer in this segment of the promoter sequence.…”

Transcriptional Regulation of the Rat Renin Gene by Regulatory Elements in Intron I

“…This represents the central and most conserved part of several transcriptional silencers found in a variety of genes including the exon 1 silencer of the rat osteocalcin gene. 32 In the human homolog of the same gene, exon 1 carries another silencer element (TGGCCCT), 33 which resembles the sequence TGGCCTC (ϩ163 to ϩ169 bp) located in the center of the palindromic protein-binding region of renin intron I. It has to be clarified whether the same transcription factors bind the exon 1 elements of the osteocalcin gene or another described silencer and the DNA-element found in the 5Ј-end of rat renin intron I.…”

Activity of the Osteocalcin Promoter in Skeletal Sites of Transgenic Mice and during Osteoblast Differentiation in Bone Marrow-Derived Stromal Cell Cultures: Effects of Age and Sex*

“…Despite some compelling evidence supporting this idea (51)(52)(53), several observations appear inconsistent with a role for the osteocalcin silencer in the developmental control of this gene: 1) potent silencing is mediated by osteocalcin intragenic sequences in both transiently (53,54) and stably (our unpublished data) transfected ROS 17/2.8 cells, which represent a mature osteoblastic phenotype; and 2) the 1.7-kb osteocalcin promoter construct, lacking the intragenic negative regulatory sequences, is silent during the early stages of osteoblast differentiation in marrow-derived stromal cell cultures. absence of transcriptional activity in osteoprogenitor cells and during the early proliferative stages, has been well documented in calvaria-derived osteoblast cultures (reviewed in Ref.…”