The Human Retinoblastoma Susceptibility Gene Promoter Is Positively ...

Val. 269. No. 8, Issue of February 25, pp. 6083-6088,1994 Printed in U.S.A.

THEJOURNALOF BIOWCICUCHEMISTRY

The Human Retinoblastoma Susceptibility Gene Promoter Is Positively Autoregulatedby Its Own Product* (Received forpublication, July 9, 1993, and in revised form, October 15, 1993)

Keunchil Park, Jennifer Choe, Nicole E. OsifchinS, Dennis J. Templeton§,Paul D. RobbinsS, and SeongJin Kimll From the Laboratory of Chemoprevention, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, the Wepartment of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, and the §Institute of Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106

1991; Helin et al., 1992; Kaelin et a l . , 1992: Shan et al., 1992; The product of the retinoblastoma susceptibility gene is a 106-kDa protein that has properties of a cell cycle Kim et al., 1992a, 1992b; Gu et al., 1993; Hagemeier et a l . , regulatory factor. Previous reports indicated that two 1993). Most of these interactions have been demonstrated in distinct DNA-binding factors, RBF-1 and ATF’, play an vitro, but three,E2F, Elf-1, and MyoD, have also been observed important part in the transcription of the human reti- in vivo(Helin et al., 1992; Kaelin et al.,1992; Wang et al., 1993; noblastoma gene (Rb). Recently,we demonstrated that Gu et al., 1993). These results suggest that pRb might act asa pRb activates expression ofthehumantransforming transcriptionalregulator. pRb inhibitsthetranscription of growth factor-b2 gene through ATF-2. Since thehuman genes involved in growthcontrol (Wagnerand Green,1991) and Rb gene promoter also contains an ATF-2-like binding can regulate expression of several genes, including transformsite, we examined whether pRb can regulate its o w n ing growth factor-01 (TGF-01) and -02, c-fos, and insulin-like expression throughATF-2. Here we report that overex- growth factor I1 genes (Robbins et a l . , 1990; Kim et al., 1991, pression of Rb stimulates Rb promoter activity through 1992a, 1992b). Because TGF-ps inhibit proliferation of many the ATF binding site in a varietyof different cell types. cell types and arrest growth in the late GI phase of the cell Mutation of the ATF binding site of the Rb promoter cycle, induction of TGF-Ps by pRb may play an important role abolishes the Rb autoinduction. We havealsodetermined that the carboxyl-terminal domainof pRb is re- in cell cycle control. Recently, two naturally occurring pointmutations in the prosponsible for the Rb autoinduction through ATF-2.Rb moter region of the human retinoblastoma gene have been autoinduction may be important for maintaining the action of pRb during cell growth, and loss of autoinduc- identified which result in decreased expression of the Rb gene (Sakai et al., 1991). These mutations are associated with heibility may contribute to retinoblastoma. reditaryretinoblastoma,suggestingthat a quantitative decrease in the expression of the Rb gene can contribute t o the Inactivation of the retinoblastoma gene product (pRb)’ has development of retinoblastoma. These mutations occurred in been associated with theetiology of a subset of human tumors the promoter regions that bind RBF-1,Spl, andATFs, suggest(Friend et al., 1986, 1987; Fung et al., 1987; Lee et al., 1987, ing that these transcription factors might play an important 1988; Harbour et al., 1988; Horowitz et al., 1989, 1990). The role in the expression of the human Rb gene. Since pRb is a transcriptional regulator that can regulate protein product of the retinoblastomasusceptibility gene (pRb) thus appears toplay an importantrole in the negative regula- activity of both S p l (Kim et a l . , 1992b) and ATF-2 (Kim et al. own promoter. In tion of cell proliferation. It has been suggested that pRb can 1992a1,it is possible that pRb can regulate its cause cell cycle arrest in mid- to late GI phase(Goodrich et al., this report, we provide evidence that Rb promoter activity is 1991).Whereas underphosphorylatedforms of pRb are the pri- directly stimulated by its own gene product through the ATF mary species seen in the Goand G1 phases of the cell cycle, the binding site. This positive autoregulatory loop is likely to amprotein undergoes phosphorylation at multiple sites as cells plify responses togrowth signals, traverse theG1/S boundary (Miharaet al., 1989; Ludlow et al., MATERIALS AND METHODS 1990; Laiho et al., 1990). This suggests that it is the underPlasmids-ChimericpromoterXATplasmids, pRbl, pRb2, pRb3, phosphorylated form of pRb that is involved in negativegrowth pRb4, pRb5, pRbEiATFmt,pRb6, and pRb7, were constructed by ligating control. Moreover, products of certain DNA virus oncogenes human Rb promoter fragments (Sakaiet al., 1991), produced by polybind to the underphosphorylatedform of pRb. merase chain reaction, into the promoterless CAT-containing plasmid, Recent studies have demonstrated that pRb binds cellular pCAT (Promega). The 3’ oligonucleotide used in all amplifications cortranscription factors (Rustgi et al., 1991; Defeo-Jones et al., responded to the 21-base pair sequence starting at -48 from the ATG codon of the human Rb gene to which an XbaI site and four random nucleotides were added. Using these oligonucleotides, fragments were * This work was supported in part by United States Public Health amplified according to the standard protocol of the GeneAmp kit (PerService Grant CA-55227 from the National Cancer Institute (to P.D. R.). The costs of publication of this article were defrayed in part by the kin-Elmer). Correct sequence of the polymerase chain reaction-amplipayment of page charges. This article must therefore be hereby marked fied fragments was verified by DNA sequence analysis. One additional chimeric promoter/CAT plasmid was constructed by “advertisement” inaccordance with 18 U.S.C.Section 1734 solelyto ligation of the ATF site of the human Rb promoter, produced by polyindicate this fact. to region of the human TGF-p2 ll To whom correspondence should be addressed: Laboratory of Che- merase chain reaction amplification,the moprevention, NCI, NIH, Bldg. 41, Rm. B1106, Bethesda, MD 20892. promoter correspondingto the 131-basepair sequence between nucleotides -68 and +63, and then into the SmaI site of pGEM4-SVOCAT Tel.: 301-496-5391; Fax: 301-496-8395. The abbreviations used are: pRb, retinoblastomagene product; TGF, (O’Reillyet al., 1992) andverified as above. transforming growth factor; CAT,chloramphenicol acetyltransferase; The human Rb expression vector phRB and control plasmid have ATF, activating transcriptionfactor. been described (Robbins et al., 1990).Rb deletion constructswere also

6083

Rb Its Regulates

6084 CCL-64 pJ3D phRB

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FIG.1. Inducibility of human RblCATchimeric gene by human Rb. The pRb5 Rb promoter construct (10 pg) was cotransfected with 20 pg of Rb plasmid (phRb),or the control plasmid (pJ30)into each of the cell lines by the calcium phosphate coprecipitation method. Forty-eight hours after transfection, the cells were harvested, andCAT activity was determined. Transfection efficiency was normalized as described under "Materials and Methods." Results of a representative experiment are shown.

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described previously (Qian et al., 1992). The expression plasmids pECEFIG.2. Mapping of the promoter element mediating Rb autoATF-1 and pECE-ATF-2 were kindly provided by Michael Green. Cell Culture-CCL-64 (mink lung epithelial), Saos-2 (human osteo- induction. At the top, an extended map of the promoter region of the genic sarcoma), and C-33A(human cervical carcinoma) cells were grown human Rb gene is shown. To analyze the inducibility by Rb, 10 pg of each plasmid DNA was cotransfected with 20 pg of the Rb expression in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containplasmid (phRb)or the control plasmid (pJ3fl)into CCL-64 cells; after 48 ing 10% fetal bovine serum. PC-3 (human prostate adenocarcoma)cells h, cells were harvested, and CAT enzymeactivitywasdetermined. were grown in Dulbecco's modified Eagle's m e d i u d - 1 2 medium Results of a representative experiment are shown. supplemented with 10% fetal bovine serum. 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. deletion constructs was cotransfected into CCG64 cells with a Transfections wereperformed by the calcium phosphate precipitation method. Following incubation with calcium phosphate precipitates and human Rb expression plasmid or control plasmid. Basal exglycerol shock, the cells were incubated for 48 h. After 48 h, cells were pression of pRbl was low to undetectable, suggesting that seharvested, and extracts were assayed for CAT activity according to quences between -686 and -586 might contain a negative eleGorman et al. (1982). CAT enzyme activity was normalized for trans- ment. The activity of pRb2 (-586 to -48) was induced 7-fold by fection efficiency by cotransfection of 1.0 pg of a human growth hormone pRb (Fig. 2). The transcription of all 5' deletion constructs expression plasmid,pSVGH, and determinationof secreted growthhordown to -197 was stimulated by pRb, whereas the pRb inducmone in the medium, prior to harvesting for CAT activity (Nichols ibility dropped almost to the basal level when the 5' deletion Institute, San Juan Capistrano,CA). Mobility Shift Assays-GST-ATFI and GST-ATF2 fusion proteins extended to -186, suggesting that thepRb-responsive element were kindly provided by Susan Wagner and Michael Green. Purified is located between -197 and -186. The human Rb promoter E2F protein was provided by Hans Huber. Binding reaction mixtures contains a consensus ATF binding site (5'-GTGACGT'M"3') at contained 50 mM Tris, pH 7.5, 50 m~ NaCl, 5 mM MgC12, 1 m~ dithio- positions -193 to -1852 (Sakai et al., 1991). To determine if this threitol, 1 mM EDTA, 5% glycerol, 100 p~ ZnCI2, and 10,000 cpm of ATF site is involved in pRb-mediated transcriptional regula"'P-labeled probe. The oligonucleotide probes used in the gel shift analysis were prepared by annealing the complementary strands and end tion, we mutated the siteby making a 3-base substitution (5'labeling using large fragmentDNA polymerase I. The analysisof bind- GTGCCCCTT-3') in the pRb5 promoter. This mutant promoter ing complexes was done by electrophoresis on a 5% 0.5 x TBE (50 mM (pRb5 ATF mt) was not activated by pRb (Fig. 3, lane 4 ) . This Tris, 50 mM boric acid, 1 m~ EDTA) polyacrylamide gel. Gels were dried result suggests that pRb stimulates transcriptionof the human directly and autoradiographed. Rb promoter through anATF binding site. RESULTS

To demonstrate further theinvolvement of the ATF binding site in the autoinduction of human Rb, the human Rb ATF sequence was ligated to the region of the human TGF-p2 promoter between-68 and +63, which contains the TATA sequence but not the TGF-P2 ATF sequence (pRbATF-pB2-68) (Fig. 4; OReilly et al., 1992). We demonstrated previously that pRb activates transcriptionof the humanTGF-p2 gene through the transcription factor ATF-2 (Kim et al., 1992a). The ATF binding site (5"GCACGTCA-3') in the TGF-p2 gene is located a t positions -74 to -67 (Kim et al., 1992). We transfected pRbATFpB2-68, the TGF-P2 promoter constructs, pB2-77, which contains an ATF binding site as a positive control and pB2-40, which does not contain an ATF sequence, into CCL-64 cells with or without the Rb expression plasmid. Both pB2-77 and pRbATF-pB2-68 showed an increase in CAT activity by pRb,

Induction of Rb Dunscription by the Rb Protein-To determine if Rb protein is able to regulate its own transcription, we examined pRb-mediated regulation of the humanRb promoter in five different cell lines. Regulation of human Rb-CAT gene constructs wasmonitored following transfection of a human Rb promoter construct containing sequences between -197 and -48 into cells together with either vector plasmid DNA (pJ3n) or this sameplasmid carrying the human Rb cDNA(phRb) (Fig. 1, lanes 1-10). In three independentexperiments, we observed an average3-6-fold increase in CAT activity withpRb, depending on the cell types. A previous report (Hamel et al., 1992) showed that indifferentiated p19 (embryonic carcinoma) cells, overexpression of Rb represses Rb promoter activity. However, we have not observed repression of the human Rb promoter in the cell lines. Osifchin, N., Ohtani-Fujita, N., Fujita, T., Kim, S.-J., Sakai, T., and To identify the specific sequences in the humanRb promoter Robbins, P. D. (1994) J. Biol. Chem. 269,6383-6389.

Rb Its Regulates

O w n Promoter

6085

whereaslittleincreasein CAT activitywasobservedwhen pB2-40 was cotransfected witha n Rb expression plasmid. This suggests that the Rb promoter sequence is functioning analogous to the TGF-P2ATF-2 binding site. The ATF Site in the Human Rh Promoter Is a Relatively High Affinity Binding Site for ATF-2-ATFs are a family of transcription factors that posses homologous DNA binding domains (Hai et al., 1989). We demonstrated previously that pRb can interact with the ATF-2 protein in nuclear extracts and that pRb can specifically stimulate ATF-2-mediated transcription. To investigate whetherATF-2 is also involved inthe autoinduction of the human Rb promoter, we examined whether ATF-2 is

able to bind with high affinity to the Rb-ATF site. Using equal amounts of bacterially expressed GST-ATF1 and GST-ATF2 fusion proteins, we demonstrated ina mobility shift assay that both fusion proteins bound to the ATF site of the human Rb promoter (Fig.5A, lanes 1,2,5, and 6 ) .However, the GST-ATF2 fusion protein bound to the ATF site of the humanRb promoter (Fig. 5A, lanes 5 and 6 ) with higher affinity thana GST-ATF1, indicating that the ATF site in the human Rb promoter is a relatively high affinity binding site for ATF-2. The 3-nucleotide substitution mutant (-199/-183 mt) failed to bind either GSTATFl or GST-ATF2 (Fig. 5A, lanes 4 and 8 ) . To investigate further the role of ATF-1 and ATF-2 in the transcription of the human Rb promoter, cotransfection experiments with expression plasmids containing ATF-1 or ATF-2 cDNAs wereperformed. CCL-64 cell linesweretransfected with pRb5 in the presence or absence of pECE-ATF-1 or pECEEGAAGTGACGTT~ ATF-2 (Fig. 5B ). Cotransfection with ATF-2 resulted in a modATF est 3-4-foldincreasein CAT expressionin CCL-64cells, whereas cotransfection with ATF-1 resulted in a decrease in G~GAAGTGCCCCTT? CAT expression. ATF ml. The ATF-2 binding site in the human Rb promoter is directly upstream from a putative E2F binding site. pRb has been dempRb5 ~Rb5 ATF mt. onstrated to bind to E2F in vivo to regulate its activity nega" I .. tively. Thus ATF-2 and E2F thatare positively and negatively f -b pJ3R regulated by pRb, respectively, bind to adjacent sequences.To + + phR0 investigate whether E2F protein binds to the E2F site in the human Rb promoter, we repeated the mobility shift assay with a purified E2F protein. Fig.6 shows that purified E2F protein bound with similar affinities to the E2F sites of the humanRb and the adenovirus E2 promoters (Fig. 6, lanes 1 and 6), showing that the Rb E2F site is a high affinity binding site for purified E2F protein. This binding was specifically competed for by the adenovirus-E2F and Rb-E2F competitors, but not by the Rb ATF/Spl (-205 to -186) and the Rbp53 (-63 to -88)2 competitors. This result suggests that E2F could be involved in YO Acetylation 38.3 4.7 4.3 2.5 mediatingnegativeregulation by Rb in certain cell types 1 2 3 4 FIG.3. Mutation in the ATF site abolishes the Rb autoinduc- (Hamel et al., 1992). ibility. The humanRb ATF sequence was mutatedat threenucleotides. The Carboxyl-terminalRegion of the Retinoblastoma Protein Construction of the pRb5 ATF mt was described under "Materials and Is Responsible for the Autoinduction-Recently, pRb has been Methods." These constructs were cotransfected with either phRb or shown to interact with E2F, and two regions of pRb are repJ3f1 intoCCL-64 cells. pSVGH (1 pg) was added as an internalcontrol To investigate if pRb for transfection efficiency. Each experiment was repeated at least three quired for E2F binding (Qian et al., 1992). times. induces its own transcription through the same regions of pRb "

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FIG.4. Autoinduction of human Rb is mediated throughthe ATF binding site. The promoter constructs generated for promoter/CAT reporter plasmids are shown a t t h top. e Construction of the plasmids pB2-77 and pB240 was described previously ( O h i l l y et al., 1992).The plasmid pRbATF-pB2-68 was generated by polymerase chain reaction amplification using the Rb ATF-containing oligonucleotides andpB2-68 a s a template.Activity of the constructs was assayed in CCL-64 cells. Each of theexperiments was repeated at least three times.

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Rb Regulates Its O w n Promoter

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FreeFIG. 6. Purified E2F binds to the E2F site of the human Rb promoter. Purified E2F protein was used in mobility shift assay of E2F site-containing oligonucleotides. The adenovirus E2F probe contains sequences from -71 to -32 of the adenovirus E2 promoter. The Rb E2F probe is derived from sequences -197 to -175 of the humanRb promoter (5'-AGCTCG"TCCCGCGG?TGG-3'). The Rb ATF/Spl and Rb p53 probes are 5'-AGCTGACGCCGCGGGCGGAAGTGACG"TCCCGS' and 5'-AGCTAGAGGACGGGGCGTGCCCCGACGTGC-3',respectively.

3

domain and contains several sites of phosphorylation of pRb (Lees et al., 1991). T w o other mutantsof the carboxyl terminus of pRb, deleting codons 614-662 (DLl1) (within the second E1A 0 bindingdomain), andtheother deleting codons 839-892 0 2 5 50 (DLE), were found to be consistently diminished but partially pg DNA FIG.5. The ATF site in the human Rb promoter is a relatively active in pRb5 induction. Thesethree mutantswere previously high affinity binding site for ATF-2. Panel A, bacterially expressed found to be unable to complex to the E2F transcription factor fusion proteins GST-ATF1 (lanes 1-4) and GST-ATF2 (lanes 5 4 )were (Qian et al., 1992). Three other mutants, affecting either the used in mobility shift assays of ATF site-containing oligonucleotides. The Rb probe is derived from sequences from -199 to -183 ofthe human first or part of the second E1A binding domains, or codons 817-839, were fully able to induce pRb5, despite the fact that Rb promoter. Specificity of GST-ATF1 or GST-ATF2 binding was assayed by competition (50-fold molar excess) withthe wild-type Rb ATF they were previously found to lack binding to E2F and to be site -199/-183 (lanes 7 and 7 ) or binding to a mutantoligonucleotides unable toinduce growth arrest in transfected cells (Qian et al., -199/-183 mt (lanes 4 and 8).The sequences of the oligonucleotides 1992). The contrast between protein sequences important for used are shown. Panel B , Rb promoter activity inthe presence ofATF-1 E2F transcription factor binding and transcriptional stimulaand ATF-2. The average CAT activity after normalization to growth hormone of pRb5 from CCL-64 cells cotransfected in the presence or tion of pRb5 demonstrates that autostimulation of the pRb5 absence of two concentrationsof pECE-ATFI (open bar)or pECE-ATF2 promoter by pRb does not require direct interaction between (filled bar).The values represent ratios of the activity obtained with pRb and E2F. Further, this result suggests that different tranpRb5 in the absenceof cotransfectant and are an average of two to three scription factors interact with pRb through distinctsequences. cotransfections with similar results.

2

1.0

required for E2F binding, the pRb5 promoterICAT construct was transfected into CCL-64 cells together with deletion mutants of pRb expression vectors. A schematic representation of deletion mutants of pRb expression vectors used is shown in the top panel of Fig. 7 (Qian et al., 1992). Cotransfection of cells with the Rb promoter/CAT reporter construct (pRb5) and the wild-type pRb resultedininduction ofCAT activity 6-fold higher than thevector control (Fig. 7). Most of the smalldeletion mutants tested retain the ability of wild-type pRb to induce the pRb5 promoter, including each of the amino-terminal mutants, affecting codons 37-614, and the"spacer" separating the two E1A binding domains. One mutant, deleting codons 775817, is essentially devoid of pRb5 inducing activity. This mutant is located immediately following the second E1A binding

DISCUSSION

The retinoblastoma susceptibility geneproduct Rb is a negative regulator of cell growth whose inactivation is associated with the etiology of a subset of human tumors. How Rb functions to constrain cell proliferation is unclear, but it has been demonstrated that Rb can function as a transcriptional regulator. The observed transcriptional regulation by Rb is mediated through theregulation of activity of specific transcription factors. Rb has been shown to interact specifically with certain transcription factors to modulate their activity ineither a positive or negative manner. In particular, Rb has been shown to bind and repress E2F-mediated transcription, whereas it can bind to and stimulate ATF-2-mediated transcription. Given that Rb is a transcriptional regulator, it was of interest to

6087

Rb Regulates Its O w n Promoter ‘337.89 A89-140 Ala-202

A202-249 A249-309 A309-343 A343-389 A389-580

A580-614 A614-662 A662-775 ~775-a17 A817-839 Aa39-892 A892-926

(DL11 IDUJ IDL3J (DL41 (DL51 (DL61 (DL71 (DL91 (DL101 IDLlll (DL121 IDL13J (DL141 (DL15) (DL16)

HA litope

CONSTRUCTS

FIG.7. Mapping of the region of the human Rb protein responsiblefor the autoinducibility.A schematic representation of deletion mutants of pRb expression vectors used inthis study is shown at the top. The constructionof these plasmids was described elsewhere (Qian et al., 1992). The average CAT activity after normalization to growth hormone obtained from CCL-64 cells is graphically represented. The values represent ratios of the activity obtained with the pRb5 and are an averageof three to four experiments with similar results.

(Kim et al., determine if Rb was able to regulate itsown expression. Thus certain cell types andnegatively regulated in others we have examined the ability of the product of the retinoblas- 1991). We have mapped the domains of pRb responsible for confertoma susceptibility gene to autoregulate its own promoter acring activation of the Rb promoter through ATF-2 using a panel tivity using a transient cotransfection assay. We have demonstrated that infive different cell types, coex- of Rb mutants (Qianet al., 1992). Our finding that thecarboxyl pression of Rb stimulated Rb promoter activity 3-6-fold. The terminus of pRb is sufficient to induce the ATF-2-mediated stimulation was observed both in cells containing a normal Rb transcription correlates with functions of pRb shown by two (CCL-64) or a defective Rb (Saos-2). Deletion analysis of the Rb other groups. Goodrich et al. (1991) reported that a bacterially promoter has identified an ATF binding site in the promoter as expressed fusion protein encoding the carboxyl terminus of pRb growth of microinjected cells. &in et al. (1992) important for the stimulation by Rb. Several different lines of is able to inhibit evidence suggest that it is ATF-2 that functionally interacts have recently reported that a carboxyl-terminal fragment of with Rb-ATF site. First, the Rb ATF binding site is similar t o pRb expressed by the strong cytomegalovirus promoter is sufthe ATF binding site in the TGF-p2 promoter that we have ficient to reduce colony formation in transfected Saos-2 cells. demonstrated previously t o bind t o ATF-2 (Kim et al., 1992a). Our results also suggest that the amino-terminal fragmentof Second, insertion of the Rb ATF-2 site in place of the TGF-P2 pRb is not required t o induce the Rb promoter activity. We demonstrated recently that pRb regulates S p l transcripATF binding site stillallows for activation of the TGF-P2 promoter by Rb. Third, ATF-2 binds to the Rb ATF binding site tion (Kim et al., 1992b). Our unpublished data also demoncarboxyl-terminal portionof the pRb is required with high affinity. Fourth, expression of ATF-2 in vivo stimu- strate that the lates Rb promoter activity. Taken together these results di- for the regulation of S p l transcription. Similarly, the carboxyl rectly implicate ATF-2 as an important factor for mediating terminus has been shown to bind the D-type cyclin, c-my, myoD, and Elf-1 in vitro (Dowdy et al., 1993; Ewen et al., 1993; positive autoregulation by Rb. It hasbeen reported previously that Rb is able to regulate itsRustgi et al., 1992; Gu et al. 1993; Wang et al., 1993). These expression in P19 cells negatively (Hamel et al., 1992). Al- results suggest that the carboxyl terminus of pRb is the general though we have not observed negative regulation of the Rb target for transcriptional regulatory factors. promoter by Rb, we have demonstrated thata putative binding Several growth factors including platelet-derived growth facsite for E2F directly adjacent to the ATF-2 binding site in the tor (Paulsson et al., 19871, TGF-a (Coffey et al., 1988), and Rb promoter indeed binds E2Fwith high affinity. This E2F site TGF-P1 (Van Obberghen-Schilling et al., 1988; Kim et al., 1990) may be important for conferring negative regulation by Rb in also autoregulate the expression of their mRNAs, resulting in certain cell types, whereas the ATF-2 might be involved in increased secretion of the respective peptides. Such autoinducpositive regulation by Rb in other cell types. Similar to the Rb tion can amplify responses to these growth factors during depromoter, we have identified ATF, SP1, and E2F binding sites velopment or in disease processes such as carcinogenesis. The in the TGF-P1 promoter that is positively regulated by Rb in transcription factor j u n proto-oncogene is also positively auto-

Rb Regulates Its Own Promoter

6088

regulated by its product, JudAP-1 (Angel et al., 1988). This positive regulatory loop is likely to be responsible for prolonging the transientsignal. Rb autoregulation may alsobe responsible for amplification of negative growth signals. Many observations indicate that pRb must act in some way as a transducer of signals that cause the cell to stop growing. Since pRb is a nuclear protein, it may regulate a bank of responder genes including c-fos, c-myc, and TGF-Ps whose expression somehow influences growth and differentiation. Therefore, an autoregulatory loop may be very important for pRb maintain sufficient levels to transduce thenegative growth signals efficiently. The finding that oncogenic germ line mutations in S p l and ATF sites in the humanRb gene also resulted in hereditary retinoblastoma because of a quantitative decrease in the expression of the Rb gene (Sakai et al., 1992) suggests that autoinduction of the Rb through an ATF site might be important for the biological function of Rb. Acknowledgments-We thank M. Sporn, A. Roberts, and D. Romeo for discussions and readingof the manuscript.We also thank R. Allison and L. Mullen for oligonucleotide synthesis. REFERENCES

Hai, T.,Liu, F., Coukos, W. J., and Green, M. (1989)Genes & Deu. 3,2083-2090 Hamel, P. A,, Gill, R. M., Phillips, R. A., and Gallie, B. L. (1992)Mol. Cell.Biol. 12,

3431-3438 Harbour, J . W., Lai, S . L., Whang, P. J., Gazdar, A. F., Minna, J. D., and Kaye, F. J. (1988)Science 241, 353-357 Helin, K., Lees, J. A., Vidal, M., Dyson, N., Harlow, E., and Fattaey,A. (1992)Cell

70,337-350 Horowitz, J. M.,Yandell, D. W., Park, S. H., Canning, S., Whyte, P., Buchkovich, K., Harlow, E., Weinberg, W. A,, and Dryja, T. P. (1989)Science 243, 937-940 Horowitz, J. M., Park, S. H., Bogenmann, E., Cheng, J.-C.,Yandell, D. W., Kaye, E J., Minna, J. D., Dryja, T. P., and Weinberg, R. A. (1990)Proc. Natl. Acad. Sci. U. S. A. 87,2775-2779 Kaelin, W. G., Jr., Krek, W., Sellers, W. R., DeCaprio, J. A., Ajchenbaum, F., Fuchs, C. S., Chittenden, T., Li, Y., Farnham, P. J., Blanar,M.A., Livingston, D. M., and Flemington, E. K. (1992)Cell 70,351364 Kim, S.J., Angel, P., Lafyatis, R., Hattori, K., Kim, K. Y., Sporn, M. B., Karin, M., and Roberts, A.B. (1990)Mol. Cell. Biol. 10,1492-1497 Kim, S.J., Lee, H.-D., Robhins, P. D., Busam, K , Sporn, M. B., and Robert, A. B. (1991)Proc. Natl. Acad. Sci. U. S. A . 88, 3052-3056 Kim, S.-J., Wagner, S., Liu, F., OReilly, M.A., Rohhins,P. D., and Green,M. (1992a) Nature 358, 331-334 Kim, S.J., Onwuta, U. S., Lee, Y. I., Li, R., Botchan, M. R., and Rohhins, P. D. (1992b)Mol. Cell. Biol. 12, 2455-2463 Laiho, M., DeCaprio, J. A,, Ludlow, J. W., Livingston, D.M., and Massague, J. (1990)Cell 62, 175-185 Lee, E. Y., To, H., Shew,J. Y., Bookstein, R., Scully, P., and Lee, W. H. (1988)Science

241,218-221 Lee, W. H., Bookstein, R., Hong, F., Young, L. H., Shew, J. Y., and Lee, E. Y.-H. P. (1987)Science 235, 1394-1399 Lees, J. A,, Buchkovich, K. J., Marshak, D. R., Anderson, C. W., and Harlow, E. EMBO J . 10. 42794290 (19911 ~, Ludlow, J. W., Shon, J., Pipas,J . M., Livingston, D. M., and DeCaprio, J. A. (1990) Cell 387396 Mihara, K., Cao, X. R., Yen, A., Chandler, S., Driscoll, B., Murphree, A. L., TAng, A., and Fung, Y. K. (1989)Science 246, 1300-1303 OReilly, M. A,, Geiser, A. G., Kim, S.J., Bruggeman, L. A,, Luu, A. X.,Roberts, A. B., and Sporn, M. B. (1992)J. Biol. Chem. 267, 1993S19943 Paulsson, Y., Hammacher, A., Heldin, C. H., and Westermark, B. (1987)Nature ~~

Angel, P., Hattori, K., Smeal, T., and Karin, M. (1988)Cell 55, 875-885 Coffey, R. J., Jr., Derynck, R., Wilcox, J. N., Bringman, T. S., Goustin, A. S., Moses, H. L., and Pittelkow, M. R. (1987)Nature 328, 817-820 Defeo-Jones, D., Huang, P. S., Jones, R. E., Haskell, K. M., Vuocolo, G. A., Hanobik, M. G., Huher, H. E., and Oliff, A. (1991)Nature 352,251-254 Dowdy, S . F., Hinds, P. W., Louie, K., Reed, S. I., Arnold, A,, and Weinberg, R. A. (1693)CeZl 73,499-511 Ewen, M. E., Sluss, H. K., Sherr, C. J., Matsushime, H., Kato, J.-Y., andLivingston, D. M. (1993)Cell 73,487497 Friend, S . H., Bernards, R., Rogelj, S., Weinberg, R. A., Rapaport, J . M., Albert, D. M., and Dryja, T.P. (1986)Nature 323,643-646 Friend, S. H., Horowitz, J. M., Gerber, M. R., Wang, X.-F,, Bogenmann, E., Lee, F. R., and Weinberg, R. A. (1987)Proc. Natl. Acad. Sei. U. S. A. 84,9059-9063 Funa, Y. K., Murphree, A. L., T'Ang, A,, Qian, J., Hinrichs, S . H., and Bebedict,W. FT(1987)Science 236, 1675-1661 Goodrich, D. W., Wang, N. P., Qian, Y.-W., Lee, E. Y.-H. P., and Lee, W.-H. (1991) Cell 67, 293-302 Gorman, C. M., Moffat, L. F., and Howard, B. H. (1982)Mol. Cell. B i d . 2,1044-

1051 Gu, W., Schneider, J. W., Condorelli, G., Kaushal, S., Mahdavi, V., and NadalGinard, B. (1993)Cell 72,309324 Hagemeier, C., Bannister, A. J., Cook, A., and Kouzarides, T.(1993)Proc. Natl. Acad. Sci. U. S. A. 90,1580-1584

~

~~~

~

~

60.

328.715-717 Qian, Y., Luckey, C., Horton, L., Esser, M., and Templeton, D. J. (1992)Mol. cell. Biol. 12,5363-5372 Qin, X:Q., Chittenden, T., Livingston, D. M., and Kaelin, W. G. (1992)Genes & Deu.

6,953-964 Rohhins, P. D., Horowitz, J. M., and Mulligan, R. C. (1990)Nature 346, 668-671 Rustgi, A. K., Dyson, N. J., and Bernards, R. (1991)Nature 362,541-544 Sakai, T.,Ohtani, N., McGee, T.L., Rohbins, P. D., and Dryja, T. P. (1991)Nature

353,

83-86

Shan, B., Zhu, X., Chen, P.-L., Derfee, T., Yong, Y., Sharp, D., and Lee, W.-H. (1992) Mol. Cell. Biol. 12, 562&5631 Van Obberghen-Schilling, E.,Roche, N. S., Flanders, K. C., Sporn, M. B., and Roberts, A. B. (1988)J . Eiol. Chem. 263, 7741-7746 Wagner, S., and Green, M. (1991)Nature 352, 189-190 Wang, C.-Y., Petryniak, B., Thompson, C.B. Kaelin, W. G., and Leiden, J. M. (1993) Science 260, 1330-1335