The human retinoblastoma susceptibility gene promoter is positively autoregulated by its own product.

K Park,J Choe,N.E Osifchin,D.J Templeton,P.D Robbins,S.J Kim

doi:10.1016/s0021-9258(17)37572-5

Abstract

The product of the retinoblastoma susceptibility gene is a 105-kDa protein that has properties of a cell cycle regulatory factor. Previous reports indicated that two distinct DNA-binding factors, RBF-1 and ATF, play an important part in the transcription of the human retinoblastoma gene (Rb). Recently, we demonstrated that pRb activates expression of the human transforming growth factor-beta 2 gene through ATF-2. Since the human Rb gene promoter also contains an ATF-2-like binding site, we examined whether pRb can regulate its own expression through ATF-2. Here we report that overexpression of Rb stimulates Rb promoter activity through the ATF binding site in a variety of different cell types. Mutation of the ATF binding site of the Rb promoter abolishes the Rb autoinduction. We have also determined that the carboxyl-terminal domain of pRb is responsible for the Rb autoinduction through ATF-2. Rb autoinduction may be important for maintaining the action of pRb during cell growth, and loss of autoinductibility may contribute to retinoblastoma.

Highlights

The product of the retinoblastoma susceptibility ge1n9e91; Helin et al, 1992; Kaelin et a l . , 1992: Shan et al, 1992; is a 106-kDa protein that has propertiesof a cell cycle Kim et al, 1992a, 1992b; Gu et al, 1993; Hagemeier et a l . , regulatory factor
Two naturally occurring pointmutations in thperoautoinductionmay be important for maintaining the amco-ter region of the human retinoblastoma gene have been tion of pRb during cell growth, and loss of autoinduc- identified which result in decreased expression of the Rb gene ibility may contribute to retinoblastoma
In cause cell cycle arrest inmid- to late GI phase(Goodrich et al, this report, we provide evidence that Rb promoter activity is

Summary

MATERIALS AND METHODS

Plasmids-ChimericpromoterXATplasmids, pRbl, pRb2, pRb3, pRb4, pRb5, pRbEiATFmtp,Rb6, and pRb7, were constructed by ligating human Rb promoter fragments (Sakaiet al., 1991),produced by polymerase chain reaction, into the promoterless CAT-containing plasmid, Recent studies have demonstrated that pRb binds cellular pCAT (Promega). One additional chimeric promoter/CAT plasmid was constructed by ligation of the ATF site of the human Rb promoter, produced by polymerase chain reaction amplification,thtoe region of the human TGF-p2 promoter correspondingto the 131-basepair sequence between nucleotides -68 and +63, and into the SmaI site of pGEM4-SVOCAT (O’Reillyet al., 1992) andverified as above. PLCPRF/5 (human hepatoma) cells were grown in minimal essential medium supplemented responsible for its stimulation by pRb, a series of human Rb with 10% fetal bovine serum. The transcription of all 5' deletion constructs expression plasmid,pSVGH, and determinationof secreted growthhormone in the medium, prior to harvesting for CAT activity(Nichols Institute, San Juan Capistrano,CA). The analysisof bindpositions -193 to -1852 (Sakai et al, 1991).To determine if this ATF site is involved in pRb-mediated transcriptional regulation, we mutated the siteby making a 3-base substitution (5'GTGCCCCTT-3') in the pRb5 promoter.

RESULTS

AAGTGACGTTTT -68

DISCUSSION