HCTR9, a Component of Paf1 Complex, Participates in the Transcription of Interleukin 6-responsive Genes through Regulation of STAT3-DNA Interactions

Min-Young Youn,Hyun-Seok Yoo,Min-Jung Kim,Sun-Young Hwang,Yongwook Choi,Stephen V Desiderio,Joo-Yeon Yoo

doi:10.1074/jbc.m705411200

Abstract

PAF, which is composed of Paf1, Cdc73, Ctr9, Leo1, and Rtf1, is a novel complex with multiple functions in transcription-related activities. The PAF complex interacts with histone-modifying enzymes and RNA polymerase II to regulate transcription. With general transcription regulatory potential in yeast, Hyrax/Cdc73 has been reported to associate with beta-catenin to control Wnt/Wg signal-specific transcription in Drosophila. Here, we present the first evidence of IL-6 signal-specific transcriptional regulation by SH2BP1/CTR9 in mammals. Upon LPS injection of mice, we observed transient induction of the mammalian PAF complex in the liver. Inhibition of CTR9 specifically abrogated expression of IL-6-responsive genes, but had no effect on genes constitutively expressed or induced by interferon-beta, TNFalpha, or IL-1beta. The PAF complex was found in the promoter regions of IL-6-responsive HP and FGGgamma, but not in the promoter region of constitutively active GAPDH. Transcriptional activation by STAT3 was inhibited when CTR9 siRNA was introduced, whereas transcriptional activation was enhanced by mCtr9 overexpression. IL-6-activated Stat3 was found to co-localize and interact with CTR9. In CTR9-depleted cells, decreased STAT3 association with the promoter regions, as well as impaired K4-trimethylation of histone H3 in the coding regions, of target genes was observed. These data suggest that CTR9 participates in the transcription of IL-6-responsive genes through the regulation of DNA association of STAT3 and modification of histone methylation.

Highlights

Innate immune responses grant the host early protection against infectious agents, resulting in immediate activation of specialized immune cells and production of inflammatory cyto
SH2BP1/CTR9 Controls DNA Binding of STAT3 to the Promoter Region of Target Genes—We examined whether CTR9
Cells were stimulated with IL-6 and STAT3 binding to promoter regions of target genes was determined by Chromatin Immunoprecipitation (ChIP) in cells transfected with control or CTR9-specific siRNA

Summary

EXPERIMENTAL PROCEDURES

Plasmids—A full-length cDNA of mouse Ctr was obtained in the laboratory of S. Full-length cDNAs of mouse Cdc and Paf were obtained by RT-PCR from livers of LPS-treated mice and cloned into the pENTR/TOPO or pcDNA3.1/myc mammalian expression plasmids. RNA Isolation and Analysis—Total RNA was extracted from livers of LPS-treated mice or HepG2 cells using the TRIzol Reagent (Invitrogen). Cells were collected in lysis buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholic acid, 0.1% SDS, 0.1 mg/ml phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 5 ␮g/ml aprotinin, 2 ␮g/ml pepstatin, 5 ␮g/ml leupeptin, and 1 mM benzamidine), and the chromatin was sonicated three times for 40 s each at setting 5.0 using a Branson 250 Sonicator with a microtip. At 36-h post-transfection, cells were treated with IL-6 plus IL-6sR (10 ng/ml each), or OSM (10 ng/ml), for 12 h, and a dual luciferase assay was performed according to the manufacturer’s instructions (Promega).

Normalized Intensity

RESULTS

Relative Luc Activity Relative Luc Activity

ACTIN Input

DISCUSSION