The transcriptional cofactor complex CRSP is required for activity of the enhancer-binding protein Sp1

Abstract

Activation of gene transcription in metazoans is a multistep process that is triggered by factors that recognize transcriptional enhancer sites in DNA. These factors work with co-activators to direct transcriptional initiation by the RNA polymerase II apparatus. One class of co-activator, the TAF(II) subunits of transcription factor TFIID, can serve as targets of activators and as proteins that recognize core promoter sequences necessary for transcription initiation. Transcriptional activation by enhancer-binding factors such as Sp1 requires TFIID, but the identity of other necessary cofactors has remained unknown. Here we describe a new human factor, CRSP, that is required together with the TAF(II)s for transcriptional activation by Sp1. Purification of CRSP identifies a complex of approximate relative molecular mass 700,000 (M(r) approximately 700K) that contains nine subunits with M(r) values ranging from 33K to 200K. Cloning of genes encoding CRSP subunits reveals that CRSP33 is a homologue of the yeast mediator subunit Med7, whereas CRSP150 contains a domain conserved in yeast mediator subunit Rgr1. CRSP p200 is identical to the nuclear hormone-receptor co-activator subunit TRIP2/PBP. CRSPs 34, 77 and 130 are new proteins, but the amino terminus of CRSP70 is homologous to elongation factor TFIIS. Immunodepletion studies confirm that these subunits have an essential cofactor function. The presence of common subunits in distinct cofactor complexes suggests a combinatorial mechanism of co-activator assembly during transcriptional activation.

Glucosamine activates the plasminogen activator inhibitor 1 gene promoter through Sp1 DNA binding sites in glomerular mesangial cells.

“…Thus, glucosamine may also have augmented transcriptional activation by Sp1 or affected interactions between Sp1 and other proteins. There are 4 transcriptional activation domains in Sp1 that interact with transcriptional cofactors, including subunits of the TF II D and CRSP complexes (70)(71)(72)(73). Recognized interactions between Sp1 and Smads (74), AP-1 (78), retinoblastoma protein (60,76), cyclin D (70), CBP/p300 (through progesterone receptors) (77), or other Sp1 molecules to form higher-order multimers (71) could all have been affected by glucosamine and merit further investigation.…”

p300 plays a role in p16INK4a expression and cell cycle arrest

“…Specific site mutations for the three Sp1 sites were introduced into the p16 promoter by a two-step PCR procedure as described previously (Han et al, 2006a). The expression constructs of Sp1 and Gal4-Sp1 were the gifts from Dr Robert Tjian (Department of Molecular and Cell Biology, University of California, Berkeley) (Ryu et al, 1999). The expression vectors containing the wild-type (wt) p300 (pCIp300) and its HAT-deletion derivative (pCI-p300, HATD1472-1522) were generously provided by Dr Joan Boyes (Institute of Cancer Research, UK).…”

Regulation of RNA polymerase II activity by CTD phosphorylation and cell cycle control

“…The CDK8-cyclin C complex was subsequently shown to be associated with the RNAP II holoenzyme in yeast (Liao et al, 1995) and in mammalian cells (Ossipow et al, 1995;Maldonado et al, 1996). More recently, CDK8 and cyclin C were also identi®ed as components of mammalian mediator-and SRB protein-containing co-activator complexes that can both positively and negatively affect (activated) RNAP II transcription activation in vitro (Sun et al, 1998;Boyer et al, 1999;Gu et al, 1999;Hampsey and Reinberg, 1999;Na È a È r et al, 1999;Rachez et al, 1999;Ryu et al, 1999;Malik and Roeder, 2000). Overall protein levels and catalytic activity of CDK8-cylin C do not¯uctuate signi®cantly during the cell cycle .…”