Heterogeneous nuclear ribonucleoprotein K represses transcription from a cytosine/thymidine-rich element in the osteocalcin promoter.
HnRNP K (heterogeneous nuclear ribonucleoprotein K) was biochemically purified from a screen of proteins co-purifying with binding activity to the osteocalcin promoter. We identify hnRNP K as a novel repressor of osteocalcin gene transcription. Overexpression of hnRNP K lowers the expression of osteocalcin mRNA by 5-fold. Furthermore, luciferase reporter assays demonstrate that overexpression of hnRNP K represses osteocalcin transcription from a CT (cytosine/thymidine)-rich element in the proximal promoter. Electrophoretic mobility-shift analysis reveals that recombinant hnRNP K binds to the CT-rich element, but binds ss (single-stranded), rather than ds (double-stranded) oligonucleotide probes. Accordingly, hnRNP K antibody can supershift a binding activity present in nuclear extracts using ss sense, but not antisense or ds oligonucleotides corresponding to the CT-rich -95 to -47 osteocalcin promoter. Importantly, addition of recombinant hnRNP K to ROS 17/2.8 nuclear extract disrupts formation of a DNA-protein complex on ds CT element oligonucleotides. This action is mutually exclusive with hnRNP K's ability to bind ss DNA. These results demonstrate that hnRNPK, although co-purified with a dsDNA-binding activity, does not itself bind dsDNA. Rather, hnRNP K represses osteocalcin gene transcription by inhibiting the formation of a transcriptional complex on the CT element of the osteocalcin promoter.
Stains
Joseph P
JP
Division of Bone and Mineral Diseases, Washington University School of Medicine, and Barnes-Jewish Hospital, St. Louis, MO, USA.
Lecanda
Fernando
F
Towler
Dwight A
DA
Civitelli
Roberto
R
D000818:Animals; D001482:Base Composition / Q000235:genetics*; D001483:Base Sequence / Q000235:genetics; D045744:Cell Line, Tumor; D003596:Cytosine / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D034641:Heterogeneous-Nuclear Ribonucleoprotein K / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000473:pathology; D015675:Osteocalcin / Q000235:genetics*; D012516:Osteosarcoma / Q000473:pathology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D012097:Repressor Proteins / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics*
D004277:DNA, Single-Stranded; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D012097:Repressor Proteins; D014157:Transcription Factors; D015675:Osteocalcin; D003596:Cytosine; D004247:DNA; D013936:Thymidine