Transcriptional Pausing Caused by NELF Plays a Dual Role in Regulating Immediate-Early Expression of the junB Gene

Abstract

Human 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole sensitivity-inducing factor (DSIF) and negative elongation factor (NELF) negatively regulate transcription elongation by RNA polymerase II (RNAPII) in vitro. However, the physiological roles of this negative regulation are not well understood. Here, by using a number of approaches to identify protein-DNA interactions in vivo, we show that DSIF- and NELF-mediated transcriptional pausing has a dual function in regulating immediate-early expression of the human junB gene. Before induction by interleukin-6, RNAPII, DSIF, and NELF accumulate in the promoter-proximal region of junB, mainly at around position +50 from the transcription initiation site. After induction, the association of these proteins with the promoter-proximal region continues whereas RNAPII and DSIF are also found in the downstream regions. Depletion of a subunit of NELF by RNA interference enhances the junB mRNA level both before and after induction, indicating that DSIF- and NELF-mediated pausing contributes to the negative regulation of junB expression, not only by inducing RNAPII pausing before induction but also by attenuating transcription after induction. These regulatory mechanisms appear to be conserved in other immediate-early genes as well.

We thank Sachiko Okabe, Michiko Tatsuno, Junko Kato, Sachiko Okamoto, and Tetsu Yung for technical assistance and manuscript preparation and members of the Handa lab for helpful suggestions. We also thank George Orphanides for providing a ChIP protocol.

This study was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to T.W. and H.H.) and a grant from NEDO (to H.H.). This study was also supported in part by the Tokyo Tech. Award for Challenging Research to T.W. and a grant from the 21st Century COE Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan. M.A. is a research fellow of the Japan Society for the Promotion of Science.