Identification of heterogeneous nuclear ribonucleoprotein K as a transactivator for human low density lipoprotein receptor gene transcription

Abstract

hnRNP K, a member of the family of heterogeneous ribonucleoproteins, is known to exert various functional roles in the nucleus, cytoplasm, and mitochondria to affect different cellular processes including chromatin remodeling, transcription, splicing, and translation. Here we report, for the first time, that hnRNPK is specifically involved in human LDL receptor (LDLR) gene transcription in HepG2 cells. We show that depletion of hnRNPK by siRNA transfection reduces the expression of LDLR mRNA and protein by more than 50% as measured by quantitative real-time PCR and Western blot analysis. Importantly, we show that the decay rate of LDLR mRNA is not affected by hnRNP K siRNA transfection, whereas the LDLR promoter activity is significantly decreased. Furthermore, overexpression of hnRNP K increased the LDLR promoter activity by the luciferase reporter assay. By utilizing a series of mutational and deletional constructs of LDLR promoter luciferase reporters, we mapped the K-responsive element to the repeat 3 (R3) sequence of the LDLR promoter. Electrophoretic mobility shift assays show that the K protein binds to a single-stranded DNA probe containing the CT-rich element of R3, which is in contrast to the requirement of double-stranded DNA for Sp1 to bind to R3. Finally, chromatin immunoprecipitation assays reveal a direct interaction of hnRNP K with the LDLR promoter in intact HepG2 cells. These new findings provide strong evidence demonstrating that hnRNP K is an important transactivator for human LDLR gene transcription. This work sheds new light on our current understanding of how LDLR gene expression is controlled at the transcriptional level.