hDREF Regulates Cell Proliferation and Expression of Ribosomal Protein Genes

Abstract

Although ribosomal proteins (RPs) are essential cellular constituents in all living organisms, mechanisms underlying regulation of their gene expression in mammals remain unclear. We have established that 22 out of 79 human RP genes contain sequences similar to the human DREF (DNA replication-related element-binding factor; hDREF) binding sequence (hDRE) within 200-bp regions upstream of their transcriptional start sites. Electrophoretic gel mobility shift assays and chromatin immunoprecipitation analysis indicated that hDREF binds to hDRE-like sequences in the RP genes both in vitro and in vivo. In addition, transient luciferase assays revealed that hDRE-like sequences act as positive elements for RP gene transcription and cotransfection of an hDREF-expressing plasmid was found to stimulate RP gene promoter activity. Like that of hDREF, expression of RP genes is increased during the late G₁ to S phases, and depletion of hDREF using short hairpin RNA-mediated knockdown decreased RP gene expression and cell proliferation in normal human fibroblasts. Knockdown of the RPS6 gene also resulted in impairment of cell proliferation. These data suggest that hDREF is an important transcription factor for cell proliferation which plays roles in cell cycle-dependent regulation of a number of RP genes.

We are grateful to S. Taketani for the plasmid carrying the human metallothionein 2A gene promoter, T. Kiyono for HFF cells, M. Fujita for RNAs from WI38 cells, and H. Miyoshi and A. Miyawaki for lentivirus vectors. We thank M. Moore for critical reading of the manuscript.

This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) and the 21st Century COE Program. D.Y. was supported by a research fellowship from the JSPS for Young Scientists during the performance of the studies.