DNA hypermethylation/boundary control loss identified in retinoblastomas associated with genetic and epigenetic inactivation of the RB1 gene promoter.

DNA hypermethylation events occur frequently in human cancers, but less is known of the mechanisms leading to their initiation. Retinoblastoma, an intraocular cancer affecting young children, involves bi-allelic inactivation of the RB1 gene (RB-/-). RB1 encodes a tumour suppressing, cell cycle regulating transcription factor (pRB) that binds and regulates the RB1 core and other E2F responsive promoters with epigenetic functions that include recruitment of histone deacetylases (HDACs). Evidence suggests that bi-allelic epigenetic inactivation/hypermethylation of the RB1 core promoter (PrE-/E-), is specific to sporadic retinoblastomas (frequency~10%), whereas heritable RB1 promoter variants (Pr-/+, frequency~1-2%) are not associated with known epigenetic phenomena. We report heritable Pr-/- retinoblastomas with the expected loss of pRB expression, in which hypermethylation consistent with distal boundary displacement (BD) relative to normal peripheral blood DNAs was detected in 4/4 cases. In contrast, proximal BD was identified in 16/16 RB-/- retinoblastomas while multiple boundaries distal of the core promoter was further identified in PrE-/E-and PrE-/E+ retinoblastomas. However, weak or no DNA hypermethylation/BD in peripheral blood DNA was detected in 8/9 Pr-/+ patients, with the exception, a carrier of a microdeletion encompassing several RB1 promoter elements. These findings suggest that loss of boundary control may be a critical step leading to epigenetic inactivation of the RB1 gene and that novel DNA methylation boundaries/profiles identified in the RB1 promoter of Pr-/- retinoblastomas, may be the result of epigenetic phenomena associated with epimutation in conjunction with loss of pRB expression/binding and/or RB1 promoter interactions with boundary control elements.

Identification of germline and somatic mutations affecting the retinoblastoma gene.

Retinoblastoma (RB) is a malignant tumor of developing retina that arises when abnormalities resulting in loss of function affect both alleles of the gene at the retinoblastoma locus (RB1) on chromosome 13q. The majority of RB tumors do not show gross alterations in a 4.7-kb fragment (4.7R), which is a candidate RB1 gene. To search for more subtle mutations, the ribonuclease protection method was used to analyze 4.7R messenger RNA from RB tumors. Five of 11 RB tumors, which exhibit normal 4.7R DNA and normal-sized RNA transcripts, showed abnormal ribonuclease cleavage patterns. Three of the five mutations affected the same region of the messenger RNA, consistent with an effect on splicing involving an as yet unidentified 5' exon. The high frequency of mutations in 4.7R supports the identification of 4.7R as the RB1 gene. However, the unusual nature of some of the abnormalities of 4.7 R alleles indicates that the accepted sequence of genetic events involved in the genesis of RB may require reevaluation.

Genetic origin of mutations predisposing to retinoblastoma.

Retinoblastoma is one of several human tumors to which predisposition can be inherited. Molecular genetic analysis of several nonheritable cases has led to the hypothesis that this tumor develops after the occurrence of specific mitotic events involving human chromosome 13. These events reveal initial predisposing recessive mutations. Evidence is presented that similar chromosomal events occur in tumors from heritable cases. The chromosome 13 found in the tumors was the one carrying the predisposing germline mutation and not the homolog containing the wild-type allele at the Rb-1 locus. These results suggest a new approach for identifying recessive mutant genes that lead to cancer and a conceptual basis for accurate prenatal predictions of cancer predisposition.

The retinoblastoma protein and cell cycle regulation.

Although the precise function of the retinoblastoma gene product, p110RB1, remains unknown, recent data suggest that it plays a role in the control of cellular proliferation by regulating transcription of genes required for a cell to enter or stay in a quiescent or G0 state, or for progression through the G1 phase of the cell cycle. However, it is difficult to rationalize the expression of p110RB1 in a wide range of tissues with the fact that mutations in the RB1 gene initiate cancers in a limited number of tissues.

Cumulative effect of phosphorylation of pRB on regulation of E2F activity.

The product of the retinoblastoma susceptibility gene, pRB, is a nuclear phosphoprotein that controls cell growth by binding to and suppressing the activities of transcription factors such as the E2F family. Transactivation activity is inhibited when E2F is bound to hypophosphorylated pRB and released when pRB is phosphorylated by cyclin-dependent kinases (CDKs). To determine which of 16 potential CDK phosphorylation sites regulated the pRB-E2F interaction, mutant pRB proteins produced by site-directed mutagenesis were tested for the ability to suppress E2F-mediated transcription in a reporter chloramphenicol acetyltransferase assay. Surprisingly, no one CDK site regulated the interaction of pRB with E2F when E2F was bound to DNA. Instead, disruption of transcriptional repression resulted from accumulation of phosphate groups on the RB molecule.

A bipartite nuclear localization signal in the retinoblastoma gene product and its importance for biological activity.

The retinoblastoma gene product, p110RB1, appears to regulate cell growth by modulating the activities of nuclear transcription factors. The elements that specify the transport of p110RB1 into the nucleus have not yet been explored. We now report the identification of a basic region, KRSAEGGNPPKPLKKLR, in the C terminus of p110RB1, which has sequence similarity to known bipartite nuclear localization signals (NLSs). A two-amino-acid mutation introduced into this putative NLS [to give mutant NLS(NQ)] or deletion of the entire NLS (delta NLS) abrogated exclusive nuclear localization, yielding proteins which were distributed either equally throughout the cell or predominantly in the cytoplasm. A mutant protein [NLS(NQ)/delta 22] containing both the mutated NLS and a deletion of exon 22, previously shown to disrupt the interaction of p110RB1 with several cellular transcription factors and oncoproteins, accumulated only in the cytoplasm. When fused to the C terminus of Escherichia coli beta-galactosidase, the RB1 NLS directed this protein to the nucleus, indicating that the motif is not only necessary but also sufficient for nuclear transport. Neither NLS(NQ) nor delta NLS was hyperphosphorylated in vivo, but both retained their abilities to interact, in vitro, with simian virus 40 large T antigen, adenovirus E1a, and the cellular transcription factor E2F. When transfected at multiple copy number, the NLS mutant alleles displayed reduced biological activity, measured by inhibition of growth of the osteogenic sarcoma cell line Saos-2, which has no wild-type RB1. Naturally occurring mutations and deletions in exon 25 of RB1 which disrupt the NLS may lead to partial or complete inactivation of p110RB1 and may be responsible for some retinoblastoma and other tumors.

Transcriptional repression of the E2-containing promoters EIIaE, c-myc, and RB1 by the product of the RB1 gene.

The protein product of the retinoblastoma susceptibility gene, p110RB1, is a nuclear phosphoprotein [W.H. Lee, J.Y. Shew, F.D. Hong, T.W. Sery, L.A. Donoso, L.J. Young, R. Bookstein, and E.Y. Lee, Nature (London) 329:642-645, 1987] with properties of a cell cycle regulator (K. Buchkovich, L.A. Duffy, and E. Harlow, Cell 58:1097-1105, 1989; P.L. Chen, P. Scully, J.Y. Shew, J.Y. Wang, and W.H. Lee, Cell 58:1193-1198, 1989; J.A. DeCaprio, J.W. Ludlow, D. Lynch, Y. Furukawa, J. Griffin, H. Piwnica-Worms, C.M. Huang, and D.M. Livingston, Cell 58:1085-1095, 1989; and K. Mihara, X.R. Cao, A. Yen, S. Chandler, B. Driscoll, A.L. Murphree, A. TAng, and Y.K. Fung, Science 246:1300-1303, 1989). Although the mechanism of action of p110RB1 remains unknown, several lines of evidence suggest that it plays a role in the regulation of transcription. We now show that overexpression of p110RB1 causes repression of the adenovirus early promoter EIIaE and the promoters of two cellular genes, c-myc and RB1, both of which contain E2F-binding motifs. Mutation of the E2 element in the c-myc promoter abolishes p110RB1 repression. We also demonstrate that a p110RB1 mutant, which is refractory to cell cycle phosphorylation but intact in E1a/large T antigen-binding properties, represses EIIaE with 50- to 80-fold greater efficiency than wild-type p110RB1. These data provide evidence that hypophosphorylated p110RB1 actively represses expression of genes with promoters containing the E2F-binding motif (E2 element).

Mutations in the RB1 gene and their effects on transcription.

Inactivation of both alleles of the RB1 gene during normal retinal development initiates the formation of a retinoblastoma (RB) tumor. To identify the mutations which inactivate RB1, 21 RB tumors isolated from 19 patients were analyzed with the polymerase chain reaction or an RNase protection assay or both. Mutations were identified in 13 of 21 RB tumors; in 8 tumors, the precise errors in nucleotide sequence were characterized. Each of four germ line mutations involved a small deletion or duplication, while three somatic mutations were point mutations leading to splice alterations and loss of an exon from the mature RB1 mRNA. We were unable to detect expression of the mutant allele in lymphoblasts of three bilaterally affected patients, although the mutation was present in the genomic DNA and transcripts containing the mutations were obvious in the RB tumors in the absence of a normal RB1 allele. The variations in the level of expression of mutant transcripts suggest deregulation of RB1 transcription in the absence of a functional RB1 gene product.

Hyperphosphorylation of the retinoblastoma gene product is determined by domains outside the simian virus 40 large-T-antigen-binding regions.

With the murine retinoblastoma (RB) cDNA, a series of RB mutants were expressed in COS-1 cells and the pRB products were assessed for their ability (i) to bind to large T antigen (large T), (ii) to become modified by phosphorylation, and (iii) to localize in the nucleus. All point mutations and deletions introduced into regions previously defined as contributing to binding to large T abolished pRB-large T complex formation and prevented hyperphosphorylation of the RB protein. In contrast, a series of deletions 5' to these sites did not interfere with binding to large T. While some of the 5' deletion mutants were clearly phosphorylated in a cell cycle-dependent manner, one, delta Pvu, failed to be phosphorylated depsite binding to large T. pRB with mutations created at three putative p34cdc2 phosphorylation sites in the N-terminal region behaved similarly to wild-type pRB, whereas the construct delta P5-6-7-8, mutated at four serine residues C terminal to the large T-binding site, failed to become hyperphosphorylated despite retaining the ability to bind large T. All of the mutants described were also found to localize in the nucleus. These results demonstrate that the domains in pRB responsible for binding to large T are distinct from those recognized by the relevant pRB-specific kinase(s) and/or those which contain cell cycle-dependent phosphorylation sites. Furthermore, these data are consistent with a model in which cell cycle-dependent phosphorylation of pRB requires complex formation with other cellular proteins.

Direct transcriptional repression by pRB and its reversal by specific cyclins.

It was recently shown that the E2F-pRB complex is a negative transcriptional regulator. However, it was not determined whether the whole complex or pRB alone is required for repression. Here we show that pRB and the related protein p107 are capable of direct transcriptional repression independent of E2F. When fused to the DNA binding domain of GAL4, pRB or p107 represses transcription of promoters with GAL4 binding sites. Thus, E2F acts as a tether for pRB or p107 but is not actively involved in repression of other enhancers. This function of pRB maps to the pocket and is abrogated by mutation of this domain. This result suggests an intriguing model in which the pocket has a dual function, first to bind E2F and second to repress transcription directly, possibly through interaction with other proteins. We also show that direct transcriptional repression by pRB is regulated by phosphorylation. Mutations which render pRB constitutively hypophosphorylated potentiate repression, while phosphorylation induced by cyclin A or E reduces repression ninefold.

Infrequent genomic rearrangement and normal expression of the putative RB1 gene in retinoblastoma tumors.

Retinoblastoma (RB) tumors develop when both alleles of a gene (RB1) are mutated and unable to function normally. Recently, Friend et al. [S. H. Friend, R. Bernards, S. Rogelj, R. A. Weinberg, J. M. Rapaport, D. M. Albert, and T. P. Dryja, Nature (London) 32:643-646, 1986] reported the cloning of a gene, 4.7R, with some properties expected for the RB1 gene, namely, a high frequency (30%) of genomic rearrangements in tumors and absence of message in all RB tumors examined. To extend the characterization of this gene, we used 4.7R probes to search for genomic rearrangements of DNA and to study the expression of the 4.7R gene in RB tumors, osteosarcoma (OS) tumors arising in RB patients, and other normal and malignant tissues. In 34 previously unreported RB and OS tumors arising in RB patients, we observed only four (12%) with genomic abnormalities. Transcripts of 4.7R were present in 12 of 17 RB tumors, 2 of 2 OS tumors, and all non-RB tumors and normal tissues tested. We were unable to confirm the high frequency of truncated messages of 4.7R in RB tumors reported by Lee et al. (W. H. Lee, R. Bookstein, F. Hong, L. J. Young, J. Y. Shaw, and E. Y. Lee, Science 235:1394-1399, 1987) and Fung et al. (Y. K. Fung, A. L. Murphree, A. Tang, J. Qian, S. H. Hinrichs, and W. F. Benedict, Science 236:1657-1661, 1987) but did confirm the presence of a truncated transcript in the RB cell line Y79. Of the RB and RB-related OS tumors which appeared normal on Southern blots, 2 of 26 or 12% had abnormal transcripts, giving a combined frequency of 22% abnormalities in the 4.7R gene detectable by Southern and Northern (RNA) blot analyses.

Hereditary haemorrhagic telangiectasia: mutation detection, test sensitivity and novel mutations.

BACKGROUND: Hereditary haemorrhagic telangiectasia (HHT) is a genetic disorder present in 1 in 8000 people and associated with arteriovenous malformations. Genetic testing can identify individuals at risk of developing the disease and is a useful diagnostic tool. OBJECTIVE: To present a strategy for mutation detection in families clinically diagnosed with HHT. METHODS: An optimised strategy for detecting mutations that predispose to HHT is presented. The strategy includes quantitative multiplex polymerase chain reaction, sequence analysis, RNA analysis, validation of missense mutations by amino acid conservation analysis for the ENG (endoglin) and ACVRL1 (ALK1) genes, and analysis of an ACVRL1 protein structural model. If no causative ENG or ACVRL1 mutation is found, proband samples are referred for sequence analysis of MADH4 (associated with a combined syndrome of juvenile polyposis and HHT). RESULTS: Data obtained over the past eight years were summarised and 16 novel mutations described. Mutations were identified in 155 of 194 families with a confirmed clinical diagnosis (80% sensitivity). Of 155 mutations identified, 94 were in ENG (61%), 58 in ACVRL1 (37%), and three in MADH4 (2%). CONCLUSIONS: For most missense variants of ENG and ACVRL1 reported to date, study of amino acid conservation showed good concordance between prediction of altered protein function and disease occurrence. The 39 families (20%) yet to be resolved may carry ENG, ACVRL1, or MADH4 mutations too complex or difficult to detect, or mutations in genes yet to be identified.

Development of retinoblastoma in the absence of telomerase activity.

BACKGROUND: The length and stability of telomeres (essential functional structures at the end of eukaryotic chromosomes) have been implicated in the control of cell lifespan. Most somatic cells lack telomerase, the enzyme that synthesizes telomeric DNA, and their telomeres shorten with cell division. Cells immortalized in vitro, on the other hand, express telomerase and maintain their telomeres. Telomerase activity has also been detected in the large majority of tumors from a variety of cancers. These observations have suggested that telomere maintenance is required for unlimited cell proliferation and that telomerase is a marker for cell immortality in vitro and in vivo. PURPOSE: We investigated whether telomerase is activated during the development of retinoblastoma. This is a childhood eye cancer associated with a limited number of mutations in an embryonic tissue and thus likely to develop in cells that have long telomeres. The ease of detection of retinoblastoma makes it possible to screen relatively small tumors before extensive proliferation of the malignant cells. METHODS: We measured telomerase activity in 34 samples of retinoblastoma, four retinoblastoma-derived cell lines, and six cell lines derived from other cancers. Only three of the cell lines from other cancers expressed the retinoblastoma protein. Telomerase activity was assayed by a polymerase chain reaction protocol in extracts prepared from tumors or cell lines. The level of enzyme activity in cell extracts was quantified at several protein concentrations and expressed relative to that in a positive control, after normalization for the amount of protein. Telomere length was measured by Southern blot hybridization of genomic DNA with a telomere-specific probe. Average values of telomere length in telomerase-positive and telomerase-negative tumors and in cell lines were compared by two-sided, two-sample Student's t test. RESULTS: No telomerase activity was detected in 17 (50%) of 34 retinoblastomas. Assays of cell lines derived from other cancers revealed no association between the presence or the level of the enzyme activity and the expression of the retinoblastoma protein. Telomeres were significantly longer in telomerase-negative tumors than in telomerase-positive tumors (P = .0008). CONCLUSIONS: Our results indicate that retinoblastoma can develop when telomeres are still relatively long and in the absence of telomerase. Telomerase activity associated with short telomeres is, however, observed in 50% of the retinoblastomas and in retinoblastoma-derived cell lines. IMPLICATIONS: Telomerase may not be a marker for acquisition of the malignant phenotype in the case of tumors that are derived from cells with long telomeres and that are associated with a low number of mutations.

Reproducible growth in tissue culture of retinoblastoma tumor specimens.

Retinoblastoma is a unique embryonic tumor which frequently arises because of an autosomal dominantly inherited mutation. Study of the genetic changes associated with retinoblastomas requires techniques that allow proliferation of fresh tumor specimens in tissue culture. However, until the present study, there were no reported methods for routinely obtaining in vitro growth of fresh retinoblastoma tumor cells. When placed on selected fibroblast feeder layers, 65% of fresh retinoblastoma specimens grew; but in the absence of fibroblasts, the same specimens died. Individual fibroblast strains from normal donors, retinoblastoma patients, and mice were effective feeder layers. A fibroblast strain that provided good feeder function for one tumor generally supported growth of other tumors also. Direct contact was required between the tumor cells and the fibroblast; conditioned media alone did not support tumor growth and immobilization of tumor cells in semisolid media above fibroblasts failed to give colony growth. Cultured tumor cells injected into the eyes of athymic nude mice formed tumors histologically characteristic of retinoblastomas.

Failure of RB1 to reverse the malignant phenotype of human tumor cell lines.

In addition to retinoblastoma and osteosarcoma, mutation of both alleles of the RB1 gene occurs frequently in several other types of tumors. In order to evaluate the role of RB1 in cancer, the wild type RB1 gene was introduced into the RB1-deleted breast cancer cell line MDA-468-S4 and retinoblastoma cell lines WERI-Rb1 and Y-79. The RB1 complementary DNA was under control of the inducible murine metallothionein promoter in MDA-468-S4 and the thymidine kinase promoter in the retinoblastoma lines. The protein, p110RB1, produced from the exogenously introduced gene appeared normal by immunoprecipitation, Western blot analysis, and nuclear localization and also showed normal cell cycle-dependent phosphorylation and an ability to bind to E1a protein. No changes in growth rate or morphology were observed in either of the reconstituted cell types. Expression of p110RB1 in MDA-468-S4 did not affect anchorage-independent growth when measured by colony formation in soft agar. Although the ability of WERI-Rb1 cells expressing p110RB1 to form colonies in methylcellulose was reduced, the reconstituted retinoblastoma cell lines formed intraocular tumors in immunodeficient mice with the same efficiency as the RB1-negative parent cell lines and the tumors produced by the RB1-reconstituted cells continued to express p110RB1. These experimental results suggest that the malignant phenotype is little affected by the replacement of p110RB1 and that RB1 is a relatively weak tumor suppressor gene.

Developmental basis of retinal-specific induction of cancer by RB mutation.

Understanding why children with RB mutations specifically develop retinoblastoma will contribute to the understanding of the fundamental principles of cancer. Only a subset of developing retinal cells are at risk for developing cancer when RB is mutant because rod photoreceptor and bipolar cells never normally express RB. Retinoblastomas are observed to arise commonly in the inner nuclear layer, where they can show features attributed to outer nuclear layer cells (photoreceptors). The best-studied function of RB is control of the cell cycle, and the usual tissue consequence of loss of RB is apoptosis. Perhaps the specificity of RB mutation for retinal cancer resides in the dependency of this tissue on programmed cell death to achieve a precise architecture of individual types of interconnecting neurons. The additional mutations that are present in all retinoblastoma, such as the i(6p) marker chromosome, may interrupt signals that normally would induce apoptosis when RB is absent. A combination of loss of cell cycle control and loss of signals that delete extra cells would result in retinoblastoma.

Mutations of the p53 gene do not occur in testis cancer.

To characterize the role of p53 in the development of testis cancer, we looked for mutations in the coding sequences of the p53 gene. DNA was obtained both from familial and sporadic testis cancer specimens, as well as from peripheral blood from members of a testis cancer kindred. Mutations in the p53 gene were screened using a combination of constant denaturant gel electrophoresis and single-strand conformational polymorphism analysis, 2 screening methods that can detect single base changes. Abnormalities detected by these methods were confirmed by sequencing of the corresponding cloned polymerase chain reaction products. All conserved regions of the p53 coding sequences were examined, encompassing all previously reported sites of mutations. No mutations were found in any of 22 germ cell cancers of the testis or in the germline DNA of 17 members of the testis cancer family. This is in striking contrast to most other human cancers, in which mutations of p53 are the most commonly described molecular event associated with tumorigenesis. We conclude that dysfunction of tumor suppressor gene or genes other than p53 may prove to play an important role in the development of germ cell cancers of the testis.

Multidrug resistance protein (MRP) expression in retinoblastoma correlates with the rare failure of chemotherapy despite cyclosporine for reversal of P-glycoprotein.

Failure of chemotherapy associated with expression of the multidrug resistance protein p170 frequently occurs in retinoblastoma (RB). Despite using cyclosporine, which inhibits p170 and improves our chemotherapy results, rare failures occur. In nonmetastatic primarily enucleated RBs, we show expression of p170 in 3 of 18 samples and expression of multidrug resistance protein (MRP), the second protein associated with resistance to chemotherapy, in 1 of 18 samples. All three RBs that failed chemotherapy without cyclosporine expressed MRP with p170. All three RBs that were enucleated immediately when chemotherapy failed despite the addition of cyclosporine expressed only MRP. One RB enucleated 2 years after failing chemotherapy with cyclosporine, despite radiation and salvage chemotherapy, expressed both p170 and MRP. Two metastatic RBs that expressed both p170 and MRP at diagnosis and at recurrence failed chemotherapy without cyclosporine, whereas one metastatic RB that expressed neither protein was cured by chemotherapy without cyclosporine. MRP may result in failure of chemotherapy despite the elimination of p170-expressing clones by cyclosporine.

Molecular cloning and characterization of the mouse RB1 promoter.

We report the isolation and characterization of the mouse RB1 promoter and surrounding DNA sequences, and the identification of elements required for basal transcriptional activity. The mouse RB1 promoter, like the human homologue, has a high G + C content, constitutes a CpG island and is devoid of typical TATA and CAAT boxes. The first 235 base-pairs upstream of the translation initiation codon in the mouse promoter exhibit 80% sequence homology with the human sequence. This homology includes a region which contains putative binding sites for the transcription factors Sp1, ATF and E2F/DRTF1. Four major transcription initiation sites were identified downstream of this conserved region. Mutational analysis revealed that the Sp1 and ATF binding sites, but not the putative E2F/DRTF1 binding site, are critical for promoter activity. Complete disruption of the putative Sp1 and ATF sites abrogated transcription, whereas the introduction of point mutations, previously identified in the Sp1 and ATF sites in two low penetrance retinoblastoma families, reduced promoter activity in a cell type specific manner. Less reduction in activity occurred in retinoic acid induced differentiated P19 cells and NIH3T3 mouse fibroblasts than in undifferentiated embryonal carcinoma P19 cells. Activity of the RB1 promoter was found to be stimulated in retinoic-acid induced differentiated P19 cells compared to undifferentiated P19; this stimulation required intact Sp1 and ATF sites but not the putative E2F/DRTF1 binding site. Our results indicate that basal level of RB1 expression is governed by Sp1 and ATF.

Regions controlling hyperphosphorylation and conformation of the retinoblastoma gene product are independent of domains required for transcriptional repression.

Cell cycle-dependent phosphorylation appears to control the function of the retinoblastoma gene product, p110RB1. We have previously shown that both the N-terminal portion and the portion corresponding to exon 23 are important in the hyperphosphorylation of p110RB1. To gain further insight into the domains involved in these phosphorylations, we have created a series of mutations in the murine cDNA and investigated their effects on phosphorylation, protein conformation and transcription of genes controlled by an E2 element. Three series of p110RB1 constructs were made: (1) mutations and/or deletions in the cluster of potential p34cdc2 consensus sequences of exon 23, (2) deletions for the entire region C-terminal to the large T antigen (TAg)-binding domains or (3) mutations in eight potential p34cdc2 sites, including four in exon 23. Following transfections into COS cells, phosphorylation, TAg binding and protein conformation were evaluated. Constructs were also tested for their ability to repress transcription from the adenovirus early promoter. All mutant proteins bound to SV40 TAg. Mutation of Ser-781, Ser-787 and Ser-788 had no detectable effect on the phosphorylation pattern of p110RB1, producing both the fast-migrating, hypophosphorylated and slower migrating, hyperphosphorylated bands on SDS-PAGE gels. However, mutation or deletion of Ser-800 and Ser-804 resulted in proteins which failed to show the characteristic shift in molecular weight associated with the hyperphosphorylated form of p110RB1. A protein mutated only at Pro-805, the residue adjacent to Ser-804, behaved similarly to mutants with both Ser-800 and Ser-804 altered; mutation of Pro-801 had no effect on the mobility of the hyperphosphorylated species. While differences were seen in the ability of these proteins to be hyperphosphorylated, the wild-type function of repression of adenovirus early promoter activity was retained. These results are consistent with the large shift in apparent molecular weight of p110RB1 upon hyperphosphorylation being due to phosphorylation of Ser-804. Phosphorylation of this p34cdc2 consensus sequence and the subsequent conformational change in p110RB1 detected in denaturing gels is not necessary, however, for the phosphorylation-dependent modulation of p110RB1-TAg interaction.