APBP-1, a DNA/RNA-binding protein, interacts with the chick aggrecan regulatory region.
Expression of the extracellular proteoglycan aggrecan is both cell-specific and developmentally regulated. Previous studies identified six functionally defined cis elements in the aggrecan promoter region which were shown to repress aggrecan gene expression (1). Using competition electrophoretic mobility shift assays (EMSAs) we have now identified in nuclear extracts a functional repressor cis element, (T/C)TCCCCT(A/C)RRC, which occurs at multiple locations within the chick aggrecan regulatory region. We purified the factor that binds to this cis element and established that it, APBP-1 (aggrecan promoter-binding protein-1), is a 19-kDa protein that has significant homology to CIRP (cold inducible RNA-binding protein). Recombinantly expressed APBP-1 mimics the native cis element-trans factor interaction in EMSAs. In situ hybridization demonstrates that aggrecan and APBP-1 RNA expression are restricted to complementary tissues in the developing limb, and Northern blot analysis of chick limb bud mRNA shows that APBP-1 mRNA expression is inversely correlated with aggrecan mRNA expression. Functional analyses by transient transfections and Northern blot analyses suggest APBP-1 has the capacity to repress aggrecan expression, indicating that this factor may be important regulator of aggrecan gene expression.
Pirok
Edward W
EW
Department of Pediatrics, University of Chicago, Chicago, Illinois 60637, USA.
Domowicz
Miriam S
MS
Henry
Judith
J
Wang
Youli
Y
Santore
Matthew
M
Mueller
Melissa M
MM
Schwartz
Nancy B
NB
D053674:Aggrecans; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins / Q000737:chemistry*; D005786:Gene Expression Regulation; D017403:In Situ Hybridization; D037181:Lectins, C-Type / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011509:Proteoglycans / Q000737:chemistry*; D012313:RNA / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000502:physiology*; D014158:Transcription, Genetic; D014162:Transfection
D053674:Aggrecans; D004268:DNA-Binding Proteins; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D009841:Oligonucleotides; D011509:Proteoglycans; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012313:RNA; D004247:DNA