Regulation of Nuclear Receptor Transcriptional Activity by a Novel DEAD Box RNA Helicase (DP97)

Abstract

We have identified a novel DEAD box RNA helicase (97 kDa, DP97) from a breast cancer cDNA library that interacts in a hormone-dependent manner with nuclear receptors and represses their transcriptional activity. DP97 has RNA-dependent ATPase activity, and mapping studies localize the interacting regions of DP97 and nuclear receptors to the C-terminal region of DP97 and the hormone binding/activation function-2 region of estrogen receptors (ER), as well as several other nuclear receptors. Repression by DP97 maps to a small region (amino acids 589 -631) that has homology to a repression domain in the corepressor protein NCoR2/SMRTe. This region of DP97 is necessary and sufficient for its intrinsic repression activity. The N-terminal helicase region of DP97 is, however, dispensable for its transcriptional repressor activity. The knockdown of endogenous cellular DP97 by antisense DP97 or RNA interference (siRNA for DP97) results in significant enhancement of the expression of estradiol-ER-stimulated genes and attenuation of the repression of genes inhibited by the estradiol-ER. This implies that endogenous DP97 normally dampens stimulation and intensifies repression of estradiol-ER-regulated genes. Our findings add to the growing evidence that RNA helicases can associate with nuclear receptors and function as coregulators to modulate receptor transcriptional activity.Nuclear receptors comprise a superfamily of transcription factors that activate or repress gene transcription in a manner that is dependent on the nature of the hormonal ligand and coregulator proteins (coactivators or corepressors) that are recruited to the ligand-receptor complex (1). Among the steroid hormone receptors, estrogen receptors (ERs) 1 mediate the diverse stimulatory and repressive biological actions of estrogens and antiestrogens. These ligands, which are naturally occurring as well as synthetic, display a spectrum of activities ranging from full agonist to full antagonist that are reflective of changes in receptor conformation engendered by the ligand and the distinct coactivator/corepressor proteins recruited. These observations have led to the designation of some of these ligands as selective ER modulators (SERMs) (2, 3). Many coactivator proteins have been identified, and these assemble into several dynamic multiprotein complexes (1, 4 -7). These coactivator complexes include the SRC/p160 family of proteins, CREB-binding protein (CBP) and/or p300, and other factors that are recruited in a temporally ordered fashion (4) and up-regulate nuclear receptor activity, at least in part, through enhanced histone acetyltransferase activity (5-7). ATPdependent chromatin remodeling complexes, such as BRG1/ hBrm, and the TRAP-DRIP-ARC complex, which act sequentially or combinatorially, also enhance gene transcription by facilitating RNA polymerase II recruitment to promoters (4).In contrast to the coactivators, far fewer corepressors are known. Most fully characterized are NCoR (nuclear receptor corepressor) and SMRT (silencing mediator of retinoi...

Estrogen receptor corepressors -- a role in human breast cancer?

“…To date, the RRM has only been shown to be important for ERα corepression in the case of RTA (Norris et al 2002). For SAFB1 (SM Townson, K Kang, AV Lee & S Oesterreich; unpublished observations) and DP97 (Rajendran et al 2003), for example, repressor activity and RRM-and DEAD box-motif-containing regions, respectively, are physically and functionally separable. This is clearly an evolving field which might also benefit from reconsideration of dogmas.…”

“…Realizing the limitations of these experimental conditions, investigators have begun to study the effects of cofactors on the expression of endogenous estrogen-regulated genes. For example, MTA1 (Mazumdar et al 2001) and MTA1s (Kumar et al 2002) overexpression leads to decreased expression of the estrogen-induced genes c-myc and pS2, and siRNA-mediated depletion of DP97 results in increased estrogen induction of pS2 and WISP2 (Rajendran et al 2003).…”

“…FKHR also contains an LXXLL sequence, but this motif does not seem to be required for interaction with ERα (Qin & Schiff, unpublished observations). Other corepressors such as REA (Delage-Mourroux et al 2000), and DP97 (Rajendran et al 2003) …”

“…Yeast two-hybrid screens have often been applied to identify ERα corepressors, such as the repressor of ERα activity (REA) (bait: AF-2 with point mutation L540Q; library source: MCF-7) (Montano et al 1999), the repressor of tamoxifen transcriptional activity (RTA) (bait: N-terminus amino acids 51-149; library source: HeLa) (Norris et al 2002), the ligand-dependent corepressor (LCoR) (bait: LBD in the presence of estradiol; library source: fetal kidney and prostate) (Fernandes et al 2003), the DEAD box RNA helicase (DP79) (bait: LBD complexed with the antiestrogen tamoxifen; library source: MCF-7) (Rajendran et al 2003), and the SMRT/HDAC1-associated repressor protein SHARP (bait: SMRT, library source: mouse embryo E17) (Shi et al 2001). The orphan nuclear receptor SHP (short heterodimer partner) was originally isolated in a yeast two-hybrid screen using several conventional and orphan members of the receptor superfamily, including RAR and TR (Seol et al 1996), and was subsequently shown to interact with and repress ERα (Seol et al 1998, Johansson et al 2000.…”

Krüppel-like factor 5 mediates the transforming activity of oncogenic H-Ras

“…Sequences specific for Egr1 is a mixture of two oligonucleotides, 5 0 -AACAUCGCUCUGAAUAAUGAG-3 0 and 5 0 -AAACG-CAAGAGGCAUACCAAA-3 0 , that correspond to aa 312-339 and 1224-1242, respectively, in the Egr1 coding region (GenBank accession number NM_007913). Double-stranded siRNA oligonucleotides and an NS, 'scramble' duplex (Kullmann et al, 2002;Rajendran et al, 2003), used as control, were synthesized at Dharmacon, Inc. (Lafayette, CO, USA). The cells were grown to 40% confluence in six-well plates before transfection with siRNA using DMRIE-C reagent (Invitrogen) for 4 h in Opti-MEM I Reduced Serum Medium (Invitrogen).…”