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.

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.

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).

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.

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.

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.