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 activiyv 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.
- Heterogeneous nuclear ribonucleoprotein K
- Transcription regulation
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology