Retinoblastoma-family protein

EVOLUTIONARY HOMOLOGS

Table of contents

Other Retinoblastoma protein interactions

In C. elegans, the formation of the hermaphrodite vulva is induced by an RTK/Ras signaling pathway. The vulva is generated from six multipotent ventral ectodermal blast cells, P3.p-P8.p. Each of these six P(3-8).p cells can potentially adopt either the 1° vulval cell fate, the 2° vulval cell fate, or the 3° nonvulval cell fate. During wild-type development, a signal from the gonadal anchor cell induces the nearest P(3-8).p cell, P6.p, to adopt the 1° fate and the adjacent P5.p and P7.p cells to adopt the 2° fate. The cells furthest from the anchor cell, P3.p, P4.p, and P8.p, adopt the uninduced 3° fate. Vulval induction acts through a signaling pathway, which includes an EGF-like ligand; a receptor tyrosine kinase, Ras and MAP kinase, to regulate the activities of the ETS transcription factor LIN-1 and the winged-helix transcription factor LIN-31 (Lu, 1998 and references).

Vulval induction is negatively regulated by the synthetic multivulva (synMuv) genes. Loss-of-function mutations in these genes result in a multivulva (Muv) phenotype as a consequence of the expression of vulval cell fates by the P3.p, P4.p, and P8.p cells. The Muv phenotype of these mutants requires mutations in two genes. Specifically, these synMuv mutations fall into two classes, referred to as A and B. Animals carrying a class A and a class B mutation have a Muv phenotype, while animals carrying one or more mutations of the same class have a wild-type vulval phenotype. These mutations appear to define two functionally redundant pathways that negatively regulate the expression of vulval cell fates (Lu, 1998).

Four class A genes (lin-8, lin-15A, lin-38, and lin-56) and ten class B genes (lin-9, lin-15B, lin-35, lin-36, lin-37, lin-51, lin-52, lin-53, lin-54, and lin-55) have been identified. lin-15 encodes both A and B activities in two nonoverlapping transcripts. lin-15A, lin-15B, lin-9, and lin-36 encode novel proteins. Two genes, an Rb related protein and its binding partner, have been characterized in one of these pathways. lin-35 encodes a protein similar to the tumor suppressor Rb and the closely related proteins p107 and p130. lin-53 encodes a protein similar to RbAp48, a mammalian protein that binds Rb. In mammals, Rb and related proteins act as regulators of E2F transcription factors, and RbAp48 may act with such proteins as a transcriptional corepressor. It is proposed that LIN-35 and LIN-53 antagonize the Ras signaling pathway in C. elegans by repressing transcription in the vulval precursor cells of genes required for the expression of vulval cell fates (Lu, 1998).

It is proposed that the class B synMuv genes inhibit vulval induction by a conserved mechanism: LIN-35 Rb forms a complex with a sequence-specific transcription factor, presumably an E2F-like protein, and recruits a corepressor complex containing HDA-1, LIN-53 p48, and other proteins to turn off the transcription of vulval specification genes via E2F-binding sites. In the wild type, in the P3.p, P4.p, and P8.p cells, synMuv gene activity antagonizes the basal activity of the RTK/Ras pathway by repressing transcription of vulval genes. As a result, those cells adopt the nonvulval 3° fate. However, in P5.p, P6.p, and P7.p the antagonistic effect of synMuv gene activity is inactivated or can be overcome by the activated RTK/Ras pathway, thereby releasing transcriptional repression and permitting the expression of vulval fates. In the P(3-8).p cells of a synMuv mutant, repression cannot occur and all six P(3-8).p cells express vulval fates, resulting in a Muv phenotype. The synMuv genes do not appear to exert their effects by regulating cell cycle progression of the P(3-8).p cells, since all six of these cells have very similar cell cycle profiles. The synMuv genes must act genetically upstream of or in parallel to the Ras pathway. Action in parallel would be consistent with recent findings from studies of mammalian cells: dominant-negative Ras and Ras neutralizing antibodies induce an Rb-dependent block in DNA synthesis and G1 arrest, suggesting that Rb functions to inhibit mitogenesis downstream of or in parallel to Ras (Lu, 1998 and references).

The tumor suppressor retinoblastoma protein (pRB) plays an important role in the production and maintenance of the terminally differentiated phenotype of muscle cells. pRB inactivation, through either phosphorylation, binding to T antigen, or genetic alteration, inhibits myogenesis. Moreover, inactivation of pRB in terminally differentiated cells allows them to reenter the cell cycle. In addition to its involvement in the myogenic activities of MyoD, pRB is also required for the cell growth-inhibitory activity of this myogenic factor. pRB and MyoD directly bind to each other, both in vivo and in vitro, through a region that involves the pocket and the basic-helix-loop-helix domains, respectively. All the results obtained are consistent with the proposal that the effects of MyoD on the cell cycle and of pRB on the myogenic pathway result from the direct binding of the two molecules (Gu, 1993).

The Saccharomyces cerevisiae SNF2/SWI2 protein is essential for the regulated expression of a variety of genes. A human SWI2/SNF2 homolog, hBrm, is a positive participant in glucocorticoid-receptor-mediated transcription, but its mechanism of action is not known. The retinoblastoma protein, RB, has also been shown to stimulate the transcription of several genes, although the target for RB has not been identified in any of these transcriptional events. RB is shown to upregulate glucocorticoid-receptor-mediated transcription. The effect of either RB or hBrm is dependent on the presence of the other. Furthermore, RB and hBrm interact with one another in vitro and in vivo. These results highlight a new role for RB, which is to interact with hBrm in order to potentiate glucocorticoid-receptor-activated transcription (Singh, 1995).

The tumor suppressor retinoblastoma protein (RB) plays a central role in cellular growth regulation, differentiation, and apoptosis. Phosphorylation of RB results in a consequent loss of its ability to inhibit cell cycle progression. However, what remains unclear is the way in which RB phosphorylation might be regulated in apoptotic or postmitotic cells, such as neurons. Neuronal Cdc2-like kinase (Nclk), composed of Cdk5 and a neuronal Cdk5 activator [p25(nck5a)], can bind and phosphorylate RB. Since RB has been shown recently to associate with D-type G1 cyclins and viral oncoproteins through a common peptide sequence motif of LXCXE, Nclk binding may be mediated by a related sequence motif (LXCXXE) found in p25(nck5a). In vitro binding of bacterially expressed p25(nck5a) to a GST-RB fusion protein has been demonstrated. GST-RB and reconstituted Cdk5.p25(nck5a) coprecipitate, and GST-RB is phosphorylated by bacterially expressed Cdk5.p25(nck5a) kinase and by Cdk5.p25(nck5a) kinase purified from bovine brain. Immunoprecipitation of RB from embryonic mouse brain homogenate results in the coprecipitation of Cdk5. Cdk5 kinase activity is maximal during late embryonic development, the period of the greatest programmed cell death in developing neurons. Taken together, these results suggest that Nclk can bind to and phosphorylate RB in vitro and in vivo. It is inferred that Nclk may play an important role in regulating the activity of RB in the brain, including perhaps in neurons undergoing apoptosis (Lee, 1997).

pRb restricts cellular proliferation by affecting the gene expression of all three classes of nuclear RNA polymerases. To elucidate the molecular mechanisms underlying pRb-mediated repression of ribosomal DNA (rDNA) transcription by RNA polymerase I, the effect of pRb was analyzed in a reconstituted transcription system. pRb, but not the related protein p107, acts as a transcriptional repressor by interfering with the assembly of transcription initiation complexes. The HMG box-containing transcription factor UBF is the main target for pRb-induced transcriptional repression. UBF and pRb form in vitro complexes involving the C-terminal part of pRb and HMG boxes 1 and 2 of UBF. The interactions between UBF and TIF-IB and between UBF and RNA polymerase I, respectively, are not perturbed by pRb. However, the DNA binding activity of UBF to both synthetic cruciform DNA and the rDNA promoter is severely impaired in the presence of pRb. These studies reveal another mechanism by which pRb suppresses cell proliferation, namely, by direct inhibition of cellular rRNA synthesis (Voit, 1997).

To decipher the mechanism of Rb function at the molecular level, a number of Rb-interacting proteins have been systematically characterized, among these, a clone termed C5 that encodes a protein of 1,978 amino acids with an estimated molecular mass of 230 kDa. The corresponding gene was assigned to chromosome 14q31, the same region where genetic alterations have been associated with several abnormalities of thyroid hormone response. The protein uses two distinct regions to bind Rb and thyroid hormone receptor (TR), respectively, and was named Trip230. Trip230 binds to Rb independently of thyroid hormone while it forms a complex with TR in a thyroid hormone-dependent manner. Ectopic expression of the protein Trip230 in cells, but not a mutant form that does not bind to TR, specifically enhances TR-dependent transcriptional activity. Coexpression of wild-type Rb, but not mutant Rb that fails to bind to Trip230, inhibits such activity. These results not only identify a coactivator molecule that modulates TR activity, but also uncover a role for Rb in a pathway that responds to thyroid hormone (Chang, 1997).

The specific loss of pRB or p107 together with p130 disrupts the normal development of only a very limited spectrum of tissues. These developmental defects have been attributed primarily to deregulation of E2F activity and consequent uncontrolled proliferation. It was hypothesized, however, that the tissue-specific nature of these defects may also reflect deregulation of pRB-family associated factors that are specifically involved in determining cell fate. The pRB-family members are here reported to interact with transcription factors that contain paired-like homeodomains such as MHox, Chx10 and Pax-3 (Drosophila homolog: Paired). The interaction between the pRB-family and the paired-like homeodomain proteins was initially identified in a yeast two-hybrid screen where the N-terminal portion of p130 was used to isolate interacting factors from an embryonic mouse library. This interaction has been confirmed by in vitro binding and co-immunoprecipitation assays. Co-expression of Pax-3 dependent pRB, p107 or p130 with Pax-3 causes repression of activated transcription from the c-met promoter. These data demonstrate that the pRB-family proteins can modulate the activity of factors which specifically control cell fate and/or differentiation as well as controlling cell cycle regulators (Wiggan, 1998).

The retinoblastoma protein (Rb) binds to transcription factor E2F and blocks transactivation by E2F. The Rb-E2F complex binds to promoters and actively represses transcription of cell cycle genes. The small pocket region of Rb constitutes the repressor motif of Rb. There are two sequences within the pocket, termed A and B, that are conserved across species as well as in the Rb-related proteins p107 and p130. The repressor motif in Rb is formed by interaction of these domains. As with Rb, domains A and B from the pocket region of p107 also interact to form a repressor motif. And the domains from Rb and p107 are at least somewhat interchangeable in the formation of the repressor motif and in growth suppression. The interaction between domains A and B, and thus repressor activity, is blocked by the hyperphosphorylation catalyzed by G1 cyclin-dependent kinases, resulting in derepression of cell cycle genes and transition from G1 to S phase of the cell cycle (Luo, 1998 and references).

The adenovirus E1A protein both activates and represses gene expression to promote cellular proliferation and inhibit differentiation. A cellular protein has been identifed and characterized that antagonizes transcriptional activation and cellular transformation by E1A. This protein (termed CREG, for cellular repressor of E1A-stimulated genes) shares limited sequence similarity with E1A and binds both the general transcription factor TBP and the tumor suppressor pRb in vitro. In transfection assays, CREG represses transcription and antagonizes 12SE1A-mediated activation of both the adenovirus E2 and cellular hsp70 promoters. CREG also antagonizes E1A-mediated transformation, since expression of CREG reduces the efficiency with which E1A and the oncogene ras cooperate to transform primary cells. Binding sites for E2F, a key transcriptional regulator of cell cycle progression, are required for repression of the adenovirus E2 promoter by CREG, and CREG inhibits activation by E2F. Since both the adenovirus E1A protein and transcriptional activation by E2F function to promote cellular proliferation, the results presented here suggest that CREG activity may contribute to the transcriptional control of cell growth and differentiation (Veal, 1998).

Rb also associates with histone deacetylase. This association serves to repress transcription by promoting formation of nucleosomes that inhibit transcription. However, mSin3A, a corepressor that binds to the transcription factor Mad and appears to tether MAD to histone deacetylase, in contrast to other repressors, is not detected in the complex between Rb and histone deacetylase. Interaction between domain A and B in the Rb pocket forms a site for association with histone deacetylase. Recruitment of histone deacetylase by either Rb or Mad results in a decrease in acetylated histone H3 associated with the promoter in vivo, consistent with the idea that this recruitment indeed results in deacetylation of histones bound to the promoter. This Rb-mediated recruitment of histone deacetylase can only repress a subset of promoters and transcription factors. Repression of the adenovirus major late promoter by Rb and Mad is dependent on histone deacetylase activity, while repression of the tyrosine kinase promoter and the SV40 enhancer by Rb is independent of histone deacetylase activity. The activity of other promoters and transcription factors appears resistant to recruitment of histone deacetylase, but these promoters and transcription factors are still blocked by Rb through direct inhibition of these transcription factors. Thus, Rb can block transcription through two separate mechanisms (direct action and recruitment of histone deacetylase), and both mechanisms are required to account for the pattern of promoters repressed by Rb. Surprisingly, even though Rb and p107 appear to share significant structural similarity within the pocket repressor motif, p107 does not interact with histone deacetylase and does not depend on histone deacetylase activity to repress transcription. The results demonstrate fundamental differences in the mechanism of transcriptional repression by Rb and p107 and suggest that p107 may only have a subset of the repressor activities of Rb (Luo, 1998).

The retinoblastoma protein (Rb) acts as a critical cell-cycle regulator; loss of Rb function is associated with a variety of human cancer types. Rb binds to members of the AP-1 family of transcription factors, including c-Jun, and stimulates c-Jun transcriptional activity from an AP-1 consensus sequence. The interaction involves the leucine zipper region of c-Jun and the B pocket of Rb as well as a C-terminal domain. The complexes are found in terminally differentiating keratinocytes and cells entering the G1 phase of the cell cycle after release from serum starvation. The human papillomavirus type 16 E7 protein, which binds to both c-Jun and Rb, inhibits the ability of Rb to activate c-Jun. The results provide evidence of a role for Rb as a transcriptional activator in early G1 and as a potential modulator of c-Jun expression during keratinocyte differentiation. Transient transfection assays were performed to determine what effect expression of Rb has on Jun-mediated transcription driven by a single AP-1 consensus binding site from the collagenase gene promoter. In both primary human keratinocytes and CV-1P cells, addition of exogenously expressed c-Jun causes, on average, a 10-fold increase in luciferase activity over endogenous levels with the vector alone. When c-Jun and Rb are co-transfected, transactivation increases another 5- to 6-fold when compared with the participation of c-Jun alone. Wild-type Rb as well as Rb small protein (RbSP) with and without a mutation at amino acid 706 are able to activate transcription, while the N-terminal domain (pRbNT amino acids 1-329), which does not bind c-Jun, is unable to activate c-Jun. c-Jun is auto-regulated through an AP-1 site; similar activation by Rb was observed using a region of the c-Jun promoter containing the AP-1 site (Nead, 1998).

Pax-5 codes for the transcription factor BSAP, which plays an important role in midbrain patterning, B cell development, and lymphoma formation. Pax-5 is known to control gene expression by recognizing its target genes via the NH2-terminal paired domain and by regulating transcription through a COOH-terminal regulatory module consisting of activating and inhibitory sequences. The central region of Pax-5 contains a sequence with significant homology to the first alpha-helix of the paired-type homeodomain. This partial homeodomain has been highly conserved throughout vertebrate evolution because it is found not only in Pax-5 but also in the related Pax-2 and Pax-8 members of the same Pax subfamily. The partial homeodomain binds the TATA-binding protein (TBP) and retinoblastoma (Rb) gene product. Both TBP and Rb were shown by coimmunoprecipitation experiments to directly associate with Pax-5 in vivo. The conserved core domain of TBP and the pocket region as well as COOH-terminal sequences of Rb are required for interaction with the partial homeodomain of Pax-5 in in vitro binding assays. Furthermore, Pax-5 is specifically bound only by the underphosphorylated form of Rb. These data indicate that Pax-5 is able to contact the basal transcription machinery through the TBP-containing initiation factor TFIID, and that its activity can be controlled by the cell cycle-regulated association with Rb (Eberhard, 1999).

Previous work has demonstrated the critical role for transcription repression in quiescent cells through the action of E2F-Rb or E2F-p130 complexes. Recent studies have shown that at least one mechanism for this repression involves the recruitment of histone deacetylase. Nevertheless, these studies also suggest that other events likely contribute to E2F/Rb-mediated repression. Using a yeast two-hybrid screen to identify proteins that specifically interact with the Rb-related p130 protein, it has been demonstrated that p130, as well as Rb, interacts with a protein known as CtIP. This interaction depends on the p130 pocket domain, which is important for repression activity, as well as an LXCXE sequence within CtIP, a motif previously shown to mediate interactions of viral proteins with Rb. CtIP interacts with CtBP, a protein named for its ability to interact with the C-terminal sequences of adenovirus E1A. Recent work has demonstrated that the Drosophila homolog of CtBP is a transcriptional corepressor for Hairy, Knirps, and Snail. Both CtIP and CtBP can efficiently repress transcription when recruited to a promoter by the Gal4 DNA binding domain, thereby identifying them as corepressor proteins. Moreover, the full repression activity of CtIP requires a PLDLS domain that is also necessary for the interaction with CtBP. It is proposed that E2F-mediated repression involves at least two events, either the recruitment of a histone deacetylase or the recruitment of the CtIP/CtBP corepressor complex (Meloni, 1999).

E-cadherin plays a pivotal role in the biogenesis of the first epithelium during development, and its down-regulation is associated with metastasis of carcinomas. Inactivation of RB family proteins by simian virus 40 large T antigen (LT) in MDCK epithelial cells results in a mesenchymal conversion associated with invasiveness and a down-regulation of c-Myc. Reexpression of RB or c-Myc in such cells allows the reexpression of epithelial markers, including E-cadherin. Both RB and c-Myc specifically activate transcription of the E-cadherin promoter in epithelial cells but not in NIH 3T3 mesenchymal cells. This transcriptional activity is mediated in both cases by the transcription factor AP-2. In vitro AP-2 and RB interaction involves the N-terminal domain of AP-2 and the oncoprotein binding domain and C-terminal domain of RB. In vivo physical interaction between RB and AP-2 has been demonstrated in MDCK and HaCat cells. In LT-transformed MDCK cells, LT, RB, and AP-2 were all coimmunoprecipitated by each of the corresponding antibodies, and a mutation of the RB binding domain of the oncoprotein inhibits its binding to both RB and AP-2. Taken together, these results suggest that there is a tripartite complex between LT, RB, and AP-2 and that the physical and functional interactions between LT and AP-2 are mediated by RB. Moreover, they define RB and c-Myc as coactivators of AP-2 in epithelial cells and shed new light on the significance of the LT-RB complex, linking it to the dedifferentiation processes occurring during tumor progression. These data confirm the important role for RB and c-Myc in the maintenance of the epithelial phenotype and reveal a novel mechanism of gene activation by c-Myc (Batsche, 1998).

The nuclear matrix is defined as the insoluble framework of the nucleus and has been implicated in the regulation of gene expression, the cell cycle, and nuclear structural integrity via linkage to intermediate filaments of the cytoskeleton. A novel nuclear matrix protein, NRP/B (nuclear restricted protein/brain), has been isolated that contains two major structural elements: a BTB domain-like structure in the predicted NH2 terminus, and a "kelch motif" in the predicted COOH-terminal domain. NRP/B mRNA (5.5 kb) is predominantly expressed in human fetal and adult brain with minor expression in kidney and pancreas. During mouse embryogenesis, NRP/B mRNA expression is upregulated in the nervous system. The NRP/B protein is expressed in rat primary hippocampal neurons, but not in primary astrocytes. NRP/B expression is upregulated during the differentiation of murine Neuro 2A and human SH-SY5Y neuroblastoma cells. Overexpression of NRP/B in these cells augments neuronal process formation. Treatment with antisense NRP/B oligodeoxynucleotides inhibits the neurite development of rat primary hippocampal neurons as well as the neuronal process formation during neuronal differentiation of PC-12 cells. Since the hypophosphorylated form of retinoblastoma protein (p110[RB]) is found to be associated with the nuclear matrix, and overexpression of p110(RB) induces neuronal differentiation, whether or not NRP/B is associated with p110(RB) was investigated. Both in vivo and in vitro experiments demonstrate that NRP/B can be phosphorylated and can bind to the functionally active hypophosphorylated form of p110(RB) during neuronal differentiation of SH-SY5Y neuroblastoma cells induced by retinoic acid. These studies indicate that NRP/B is a novel nuclear matrix protein, specifically expressed in primary neurons, that interacts with p110(RB) and participates in the regulation of neuronal process formation (Kim, 1998).

Recent evidence indicates that cell cycle regulatory molecules are involved in neuronal differentiation. During neuronal differentiation, cyclin-dependent kinase (CDK)1 activities decline, and phosphorylation of the p110RB is reduced, leading to the appearance of a p110RB-containing E2F DNA-binding complex. Neuronal differentiation can be induced by overexpression of CDK inhibitor p27KIP or p110RB, suggesting that loss of p110RB phosphorylation is an important determinant for neuronal differentiation. Cyclin dependent kinase-2 (CDK2) overexpression inhibits the NGF-induced differentiation of PC-12 cells. CDK5 expression and kinase activity are correlated with the extent of differentiation of neuronal cells in the developing brain. NRP/B is the first nuclear matrix protein to be identified that is expressed specifically in primary neurons, that interacts with p110RB, and participates in the regulation of neuronal process formation. The results of this study suggest that NRP/B might be involved in cell cycle withdrawal after commitment to differentiation, or alternatively, that NRP/B might be involved in the regulation of neuronal cell differentiation by interfering with the function of cell cycle regulatory proteins, such as p110RB. (Kim, 1998 and references).

The underphosphorylated, active form of the Rb tumor suppressor binds to E2F and represses its transcription activity, leading to cell cycle arrest. The E2F-Rb complex also binds directly to the promotor of some cell cycle genes and actively represses transcription in part through the action of histone deacetylase. E2F can be liberated from the Rb tumor suppressor by a number of viral oncoproteins, including the large T antigens of SV40. Inactivation of Rb by Simian virus 40 (SV40) large T antigen is one of the central features of tumorigenesis induced by SV40. Both the N-terminal J domain and the LxCxE motif of large T antigen are required for inactivation of Rb. The crystal structure of the N-terminal region (residues 7-117) of SV40 large T antigen bound to the pocket domain of Rb reveals that large T antigen contains a four-helix bundle; residues from helices alpha2 and alpha4 and from a loop containing the LxCxE motif participate in the interactions with Rb. The two central helices and a connecting loop in large T antigen have structural similarities with the J domains of the molecular chaperones DnaJ and HDJ-1, suggesting that large T antigen may use a chaperone mechanism for its biological function. However, there are significant differences between large T antigen and the molecular chaperones in other regions and these differences are likely to provide the specificity needed for large T antigen to inactivate Rb (Kim, 2001).

Developmental control of bone tissue-specific genes requires positive and negative regulatory factors to accommodate physiological requirements for the expression or suppression of the encoded proteins. Osteocalcin (OC) gene transcription is restricted to the late stages of osteoblast differentiation. OC gene expression is suppressed in nonosseous cells and osteoprogenitor cells and during the early proliferative stages of bone cell differentiation. The rat OC promoter contains a homeodomain recognition motif within a highly conserved multipartite promoter element (OC box I) that contributes to tissue-specific transcription. The CCAAT displacement protein (CDP), a transcription factor related to the Cut homeodomain protein in Drosophila, may regulate bone-specific gene transcription in immature proliferating osteoblasts. Using gel shift competition assays and DNase I footprinting, CDP/cut is shown to recognize two promoter elements (TATA and OC box I) of the bone-related rat OC gene. Overexpression of CDP/cut in ROS 17/2.8 osteosarcoma cells results in repression of OC promoter activity; this repression is abrogated by mutating OC box I. Gel shift immunoassays show that CDP/cut forms a proliferation-specific protein/DNA complex in conjunction with cyclin A and p107, a member of the retinoblastoma protein family of tumor suppressors. These findings suggest that CDP/cut may represent an important component of a cell signaling mechanism that provides cross-talk between developmental and cell cycle-related transcriptional regulators to suppress bone tissue-specific genes during proliferative stages of osteoblast differentiation (van Gurp, 1999).

In cultured mammalian cells the histone methylase SUV39H1 and the methyl-lysine binding protein HP1 functionally interact to repress transcription at heterochromatic sites. Lysine 9 of histone H3 is methylated by SUV39H1, creating a binding site for the chromo domain of HP1. SUV39H1 and HP1 are both involved in the repressive functions of the retinoblastoma (Rb) protein. Rb associates with SUV39H1 and HP1 in vivo by means of its pocket domain. SUV39H1 cooperates with Rb to repress the cyclin E promoter. In fibroblasts that are disrupted for SUV39, the activity of the cyclin E and cyclin A2 genes are specifically elevated. Chromatin immunoprecipitations show that Rb is necessary to direct methylation of histone H3, and is necessary for binding of HP1 to the cyclin E promoter. These results indicate that the SUV39H1-HP1 complex is not only involved in heterochromatic silencing but also has a role in repression of euchromatic genes by Rb and perhaps other co-repressor proteins (Nielsen, 2001).

The Rb protein functions as a repressor, at least partly, through the recruitment of histone deacetylase activity. Whether histone methylation might also be involved in Rb-mediated repression is considered in this study, since the SUV39H1 methylase has repressive potential. To establish whether Rb can associate with histone-methylase activity, a glutathione S-transferase (GST)-Rb fusion was incubated with nuclear extract, and any bound methylase activity was assayed on bulk histones as a substrate. GST-Rb can purify histone-methylase activity, whereas GST fusions to transcriptional activators such as P/CAF, E2F1, p53 and ATF2 do not. The Rb-associated methylase activity is specific for histone H3 and does not recognize the GAR substrate for arginine methylases (Nielsen, 2001).

An antibody directed against Rb can precipitate histone-methylase activity that is specific for histone H3. This methylase binds the pocket domain of Rb because tumor-derived mutations in the pocket (F706C), or truncations of the pocket (928 and 737), abolish binding to the methylase. The Rb-associated methylase has specificity for Lys 9 of histone H3 (Nielsen, 2001).

The SUV39H1 protein possesses lysine methylase activity, which resides within its conserved SET domain. As this enzyme has specificity for Lys 9 of histone H3 an investigation was carried out to see whether SUV39H1 could be the methylase associated with Rb. A GST-Rb fusion can bind to transfected, hemagglutinin (HA)-tagged SUV39H1. Endogenous Rb also associates with endogenous SUV39H1, as shown by a co-immunoprecipitation analysis (Nielsen, 2001).

Whether SUV39H1 can act as co-repressor with Rb was investigated. SUV39H1 represses the activity of a promoter bearing GAL4 sites in a concentration-dependent manner in vivo, but only when Gal4-Rb is present at the promoter. The co-repressor functions of SUV39H1 can also be seen on the cyclin E promoter, a natural target for Rb-mediated repression. This promoter can be stimulated by E2F and is not affected by SUV39H1 alone. Under limiting conditions, where Rb represses E2F activity slightly, the SUV39H1 enzyme can further repress E2F activity in cooperation with Rb. When the methylase domain of SUV39H1 is removed, the resulting SUV39H1SET is unable to mediate repression. These results suggest that SUV39H1 uses its methylase activity to repress the cyclin E promoter when it is targeted there by Rb (Nielsen, 2001).

SUV39H1 is known form a complex with the HP1 protein. Recently, HP1 function has been placed downstream of SUV39H1 histone methylation, since HP1 recognizes specifically, and binds to, histone H3 methylated at Lys 9. This mechanistic link prompted an investigation of the role of HP1 in Rb/SUV39H1-mediated repression. Rb and HP1 can interact in a two-hybrid screen in yeast, and it has been shown that there is an LXCXE motif (X is any amino acid) in HP1. It was therefore asked if HP1 binds to Rb in mammalian cells. A GST-HP1 fusion can bind Rb that is present in nuclear extracts; Rb and HP1 can associate in vivo, as determined by co-immunoprecipitation analysis. An LXCXE motif peptide can compete for the binding of histone H3 methylase activity to Rb, but does not affect the binding of H3 methylase activity to HP1, which is consistent with the finding that the methylase activity is associated with the Rb pocket (Nielsen, 2001).

Whether HP1 can recognize methylated lysines while associated with Rb was tested. To address this, a histone H3 peptide methylated at Lys 9 was used as an affinity resin. Recombinant Rb does not bind to this methylated peptide, but it can do so efficiently in the presence of recombinant HP1. This result confirms that HP1 can bind directly to Rb and that it can recognize Rb and methylated lysine simultaneously. A similar experiment was attempted using nuclear extracts as the source of protein. The H3 peptide methylated at Lys 9 binds to HP1, SUV39H1 and Rb, as detected by Western blotting (Nielsen, 2001).

These results suggest that an Rb-regulated promoter such as cyclin E should be associated with HP1. To test this chromatin immunoprecipitation analysis of the cyclin E promoter was performed. A nucleosome encompassing the cyclin E initiation site (cyclin Epr) that is known to be deacetylated is associated with HP1 in fibroblast cells of mouse embryos. Since the cyclin Epr nucleosome binds HP1, whether this nucleosome contains histone H3 that is methylated at Lys 9 was examined. To test this an antibody was produced that recognizes histone H3 when methylated at Lys 9. In Rb+/+ cells the cyclin Epr nucleosome contains methylated histone H3 and is associated with HP1. However, in Rb-/- cells histone H3 methylation and HP1 binding is significantly reduced. Thus, in the presence of Rb, methylase activity and HP1 are targeted to the cyclin E promoter (Nielsen, 2001).

Collectively, these results implicate each of the components of the SUV39H1-HP1 complex in the repression functions of the Rb protein. In this model Rb brings to the promoter the SUV39H1 enzyme (and possibly other members of this family) to methylate Lys 9 of histone H3 and provides a binding site for HP1. Methylation by SUV39H1 cannot take place on an already acetylated lysine. Thus the deacetylase activity associated with Rb may be a necessary preceding step to SUV39H1-mediated methylation. The precise function of HP1 in repression is unclear. HP1 may protect the methyl group on Lys 9 from attack from potential demethylases; it may bring in other repressive functions, or it may enhance the stability of the Rb-associated repressor complex (Nielsen, 2001).

HP1 is found associated with a number of transcriptional repressors, suggesting that it may have a role in repressing many other promoters. Thus, the results presented here extend the role of SUV39H1 and HP1 beyond heterochromatic gene silencing to a more general, genome-wide function in repressing gene transcription (Nielsen, 2001).

Inositide signaling at the plasma membrane has been implicated in the regulation of numerous cellular processes including cytoskeletal dynamics, vesicle trafficking, and gene transcription. Studies have also shown that a distinct inositide pathway exists in nuclei, where it may regulate nuclear processes such as mRNA export, cell cycle progression, gene transcription, and DNA repair. Nuclear PtdIns(4,5)P2 synthesis is stimulated during progression from G1 through S phase, although mechanistic details of how cell cycle progression impinges on the regulation of nuclear inositides is unknown. pRB, which regulates progression of cells from G1 through S phase interacts both in vitro and in vivo with Type Ialpha PIPkinases, the enzymes responsible for nuclear PtdIns(4,5)P2 synthesis. Moreover, this interaction stimulates the activity of Type I PIPkinase in an in vitro assay. Using murine erythroleukamia (MEL) cells, expressing a temperature-sensitive mutant of large T antigen (LTA), in vivo changes have been demonstrated in nuclear PtdIns(4,5)P2 levels that are consistent with the ability of LTA to disrupt pRB/Type I interactions. This study, for the first time, provides a potential mechanism for how cell cycle progression could regulate the levels of nuclear inositides (Divecha, 2002).

The mammalian DNA (cytosine-5) methyltransferase (Dnmt1) is involved in the maintenance of methylation patterns in the genome during DNA replication and development. The retinoblastoma gene product, Rb, is a cell cycle regulator protein that represses transcription by recruiting histone deacetylase (HDAC1). In vivo, histone deacetylase associates with Dnmt1. Rb itself associates with human Dnmt1 (hDnmt1) independently of its own phosphorylation status. Methyltransferase activity co-purifies with Rb. The regulatory domain of hDnmt1 binds strongly to the B and C pockets of Rb (amino acids 701-872) and inhibits methyltransferase activity by disruption of the hDnmt1-DNA binary complex. Weak interaction of Rb pockets A and B with Dnmt1 was also observed. Overexpression of Rb leads to hypomethylation of the cellular DNA, suggesting that Rb may modulate Dnmt1 activity during DNA replication in the cell cycle (Pradhan, 2002).

The therapeutic value of DNA-damaging antineoplastic agents is dependent upon their ability to induce tumor cell apoptosis while sparing most normal tissues. A component of the apoptotic response to these agents in several different types of tumor cells is the deamidation of two asparagines in the unstructured loop of Bcl-x_L. Deamidation of these asparagines imports susceptibility to apoptosis by disrupting the ability of Bcl-x_L to block the proapoptotic activity of BH3 domain-only proteins. Conversely, Bcl-x_L deamidation is actively suppressed in fibroblasts, and suppression of deamidation is an essential component of their resistance to DNA damage-induced apoptosis. These results suggest that the regulation of Bcl-x_L deamidation has a critical role in the tumor-specific activity of DNA-damaging antineoplastic agents (Deverman, 2002).

In addition to its tumor-suppressor activity, Rb is a potent antiapoptotic protein -- loss of Rb in normal fibroblasts confers sensitivity to DNA-damaging agents, and reintroduction of Rb into Rb null tumors confers resistance to these agents. Hence, it was reasoned that Rb must suppress proapoptotic signals. Indeed, Rb suppresses the inactivating deamidation of Bcl-xL, and these findings indicate that the antiapoptotic activity of Rb is dependent upon the ability of Rb to suppress Bcl-xL deamidation. Finally, the data suggest that the inactivation of Rb increases the susceptibility of tumor cells to DNA-damaging agents in part because inactivation of Rb is permissive for Bcl-xL deamidation (Deverman, 2002).

The CCAAT displacement protein (CDP-cut/CUTL1/cux) performs a key proliferation-related function as the DNA binding subunit of the cell cycle controlled HiNF-D complex. HiNF-D interacts with all five classes (H1, H2A, H2B, H3, and H4) of the cell-cycle dependent histone genes, which are transcriptionally and coordinately activated at the G(1)/S phase transition independent of E2F. The tumor suppressor pRB/p105 is an intrinsic component of the HiNF-D complex. However, the molecular interactions that enable CDP and pRB to form a complex and thus convey cell growth regulatory information onto histone gene promoters must be further defined. Using transient transfections, it has been shown that CDP represses the H4 gene promoter and that pRB functions with CDP as a co-repressor. Direct physical interaction between CDP and pRB was observed in glutathione-S-transferase (GST) pull-down assays. Furthermore, interactions between these proteins were established by yeast and mammalian two-hybrid experiments and co-immunoprecipitation assays. Confocal microscopy shows that subsets of each protein are co-localized in situ. Using a series of pRB mutants, it has been found that the CDP/pRB interaction, similar to the E2F/pRB interaction, utilizes the A/B large pocket (LP) of pRB. Thus, several converging lines of evidence indicate that complexes between CDP and pRB repress cell cycle regulated histone gene promoters (Gupta, 2003).

The liver is capable of completely regenerating itself in response to injury and after partial hepatectomy. In liver of old animals, the proliferative response is dramatically reduced, the mechanism for which is unknown. The liver specific protein, C/EBPalpha (see Drosophila Slbo), normally arrests proliferation of hepatocytes through inhibiting cyclin dependent kinases (cdks). Evidence that aging switches the liver-specific pathway of C/EBPalpha growth arrest to repression of E2F transcription. An age-specific C/EBPalpha-Rb-E2F4 complex has been identified that binds to E2F-dependent promoters and represses these genes. The C/EBPalpha-Rb-E2F4 complex occupies the c-myc promoter and blocks induction of c-myc in livers of old animals after partial hepatectomy. These results show that the age-dependent switch from cdk inhibition to repression of E2F transcription causes a loss of proliferative response in the liver because of an inability to induce E2F target genes after partial hepatectomy, providing a possible mechanism for the age-dependent loss of liver regenerative capacity (Iakova, 2003).

The retinoblastoma protein (RB) facilitates adipocyte differentiation by inducing cell cycle arrest and enhancing the transactivation by the adipogenic CCAAT/enhancer binding proteins (C/EBP). The peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear receptor pivotal for adipogenesis, promotes adipocyte differentiation more efficiently in the absence of RB. PPARgamma and RB coimmunoprecipitate, and this PPARgamma-RB complex also contains the histone deacetylase HDAC3, thereby attenuating PPARgamma's capacity to drive gene expression and adipocyte differentiation. Dissociation of the PPARgamma-RB-HDAC3 complex by RB phosphorylation or by inhibition of HDAC activity stimulates adipocyte differentiation. These observations underscore an important function of both RB and HDAC3 in fine-tuning PPARgamma activity and adipocyte differentiation (Fajas, 2002b).

The detection of a PPARgamma-RB-HDAC3 protein complex on the promoter of well-established PPARgamma targets in vivo, as well as the fact that the proadipogenic effects of PPARgamma are blunted in one case by the presence of RB and in the other, stimulated by HDAC inhibitors, underscores the relevance of the repression of PPARgamma by RB and HDAC3. Complexes involved in nuclear receptor-mediated transcriptional repression often contain HDACs. The thyroid receptor, as does PPARgamma, specifically recruits HDAC3. Participation of HDACs in the repressive activity of nuclear receptor is facilitated by the presence of other corepressors, such as the silencing mediator of retinoic and thyroid receptors (SMRT) or the nuclear receptor corepressor (N-CoR). Furthermore, corepressors such as SMRT and N-CoR are required for the activation of HDAC3 in such complexes. It is therefore tempting to speculate that RB fulfills such a corepressor role for PPARgamma (Fijas, 2002b).

YY1 regulates the expression of genes with important functions in DNA replication, protein synthesis, and cellular response to external stimuli during cell growth and differentiation. How YY1 accomplishes such a variety of functions is unknown. A subset of the nuclear YY1 appears to be O-GlcNAcylated regardless of the differentiation status of the cells. Glucose strongly stimulates O-linked N-acetylglucosaminylation (O-GlcNAcylation) on YY1. Glycosylated YY1 no longer binds the retinoblastoma protein (Rb). Upon dissociation from Rb, the glycosylated YY1 is free to bind DNA. The ability of the O-glycosylation on YY1 to disrupt the complex with Rb leads to a proposal that O-glycosylation might have a profound effect on cell cycle transitions that regulate the YY1-Rb heterodimerization and promote the activity of YY1. These observations provide strong evidence that YY1-regulated transcription is very likely connected to the pathway of glucose metabolism that culminates in the O-GlcNAcylation on YY1, changing its function in transcription (Hiromura, 2003).

Retinoblastoma gene product (pRB) plays critical roles in regulation of the cell cycle and tumor suppression. It is known that downregulation of pRB can stimulate carcinogenesis via abrogation of the pRB pathway, although the mechanism has not been elucidated. In this study, it was found that Mdm2, a ubiquitin ligase for p53, promotes ubiquitin-dependent degradation of pRB. pRB is efficiently ubiquitinated by wild-type Mdm2 in vivo as well as in vitro, but other RB family proteins are not. Mutant Mdm2 with a substitution in the RING finger domain shows dominant-negative stabilization of pRB. Both knockout and knockdown of Mdm2 causes accumulation of pRB. Moreover, Mdm2 inhibits pRB-mediated flat formation of Saos-2 cells. Downregulation of pRB expression is correlated with a high level of expression of Mdm2 in human lung cancers. These results suggest that Mdm2 regulates function of pRB via ubiquitin-dependent degradation of pRB (Uchida, 2005).

Fer (see Drosophila Fps oncogene analog) is a nuclear and cytoplasmic intracellular tyrosine kinase. This study shows that Fer is required for cell-cycle progression in malignant cells. Decreasing the level of Fer using the RNA interference (RNAi) approach impedes the proliferation of prostate and breast carcinoma cells and leads to their arrest at the G0/G1 phase. At the molecular level, knockdown of Fer results in the activation of the retinoblastoma protein (pRB), and this is reflected by profound hypo-phosphorylation of pRB on both cyclin-dependent kinase CDK4 and CDK2 phosphorylation sites. Dephosphorylation of pRB is not seen upon the direct targeting of either CDK4 or CDK2 expression, and is only partially achieved by the simultaneous depletion of these two kinases. Amino-acid sequence analysis revealed two protein phosphatase 1 (PP1) binding motifs in the kinase domain of Fer and the association of Fer with the pRB phosphatase PP1alpha was verified using co-immunoprecipitation analysis. Downregulation of Fer potentiates the activation of PP1alpha and overexpression of Fer decreases the enzymatic activity of that phosphatase. These findings portray Fer as a regulator of cell-cycle progression in malignant cells and as a potential target for cancer intervention (Pasder, 2006).

Retinoblastoma protein (Rb) is a multifunctional tumor suppressor, frequently inactivated in certain types of human cancer. Nucleolin (see Drosophila Modulo) is an abundant multifunctional phosphoprotein of proliferating and cancerous cells, recently identified as cell cycle-regulated transcription activator, controlling expression of human papillomavirus type 18 (HPV18) oncogenes in cervical cancer. Here it was found that nucleolin is associated with Rb in intact cells in the G1 phase of the cell cycle, and the complex formation is mediated by the growth-inhibitory domain of Rb. Association with Rb inhibits the DNA binding function of nucleolin and in consequence the interaction of nucleolin with the HPV18 enhancer, resulting in Rb-mediated repression of the HPV18 oncogenes. The intracellular distribution of nucleolin in epithelial cells is Rb-dependent, and an altered nucleolin localization in human cancerous tissues results from a loss of Rb. These findings suggest that deregulated nucleolin activity due to a loss of Rb contributes to tumor development in malignant diseases, thus providing further insights into the molecular network for the Rb-mediated tumor suppression (Grinstein, 2006).

Genetic studies in C. elegans identified lin-9 to function together with the retinoblastoma homologue lin-35 in vulva differentiation. A human homologue of Lin-9 (hLin-9) functions in the mammalian pRB pathway. hLin-9 binds to pRB and cooperates with pRB in flat cell formation in Saos-2 cells. In addition, hLin-9 synergizes with pRB and Cbfal to transactivate an osteoblast-specific reporter gene. In contrast, hLin-9 is not involved in pRB-mediated inhibition of cell cycle progression or repression of E2F-dependent transactivation. Consistent with these data, hLin-9 is able to associate with partially penetrant pRB mutants that do not bind to E2F, but retain the ability to activate transcription and to promote differentiation. hLin-9 can also inhibit oncogenic transformation, dependent on the presence of a functional pRB protein. RNAi-mediated knockdown of Lin-9 can substitute for the loss of pRB in transformation of human primary fibroblasts. These data suggest that hLin-9 has tumor-suppressing activities and that the ability of hLin-9 to inhibit transformation is mediated through its association with pRB (Gagrica, 2004).

Changes in histone methylation status regulate chromatin structure and DNA-dependent processes such as transcription. Recent studies indicate that, analogous to other histone modifications, histone methylation is reversible. Retinoblastoma binding protein 2 (RBP2), a nuclear protein implicated in the regulation of transcription and differentiation by the retinoblastoma tumor suppressor protein, contains a JmjC domain recently defined as a histone demethylase signature motif. This study reports that RBP2 is a demethylase that specifically catalyzes demethylation on H3K4, whose methylation is normally associated with transcriptionally active genes. RBP2−/− mouse cells displayed enhanced transcription of certain cytokine genes, which, in the case of SDF1, was associated with increased H3K4 trimethylation. Furthermore, RBP2 specifically demethylates H3K4 in biochemical and cell-based assays. These studies provide mechanistic insights into transcriptional regulation by RBP2 and provide the first example of a mammalian enzyme capable of erasing trimethylated H3K4 (Klose, 2007).

The adenovirus (Ad) E1A (Ad-E1A) oncoprotein mediates cell transformation, in part, by displacing E2F transcription factors from the retinoblastoma protein (pRb) tumor suppressor. This study determined the crystal structure of the pRb pocket domain in complex with conserved region 1 (CR1) of Ad5-E1A. The structure and accompanying biochemical studies reveal that E1A-CR1 binds at the interface of the A and B cyclin folds of the pRb pocket domain, and that both E1A-CR1 and the E2F transactivation domain use similar conserved nonpolar residues to engage overlapping sites on pRb, implicating a novel molecular mechanism for pRb inactivation by a viral oncoprotein (Liu, 2007).

AMP-activated protein kinase (AMPK) is an evolutionarily conserved metabolic sensor that responds to alterations in cellular energy levels to maintain energy balance. While its role in metabolic homeostasis is well documented, its role in mammalian development is less clear. This study demonstrates that mutant mice lacking the regulatory AMPK β1 subunit have profound brain abnormalities. The β1^−/− mice show atrophy of the dentate gyrus and cerebellum, and severe loss of neurons, oligodendrocytes, and myelination throughout the central nervous system. These abnormalities stem from reduced AMPK activity, with ensuing cell cycle defects in neural stem and progenitor cells (NPCs). The β1^−/− NPC deficits result from hypophosphorylation of the retinoblastoma protein (Rb), which is directly phosphorylated by AMPK at Ser⁸⁰⁴. The AMPK-Rb axis is utilized by both growth factors and energy restriction to increase NPC growth. These results reveal that AMPK integrates growth factor signaling with cell cycle control to regulate brain development (Dasgupta, 2009).

pRB-mediated inhibition of cell proliferation is a complex process that depends on the action of many proteins. However, little is known about the specific pathways that cooperate with the Retinoblastoma protein (pRB) and the variables that influence pRB's ability to arrest tumor cells. Here two short hairpin RNA (shRNA) screens are described that identify kinases that are important for pRB to suppress cell proliferation and pRB-mediated induction of senescence markers. The results reveal an unexpected effect of LATS2, a component of the Hippo pathway, on pRB-induced phenotypes. Partial knockdown of LATS2 strongly suppresses some pRB-induced senescence markers. Further analysis shows that LATS2 cooperates with pRB to promote the silencing of E2F target genes, and that reduced levels of LATS2 lead to defects in the assembly of DREAM (DP, RB [retinoblastoma], E2F, and MuvB) repressor complexes at E2F-regulated promoters. Kinase assays show that LATS2 can phosphorylate DYRK1A, and that it enhances the ability of DYRK1A to phosphorylate the DREAM subunit LIN52. Intriguingly, the LATS2 locus is physically linked with RB1 on 13q, and this region frequently displays loss of heterozygosity in human cancers. Thee results reveal a functional connection between the pRB and Hippo tumor suppressor pathways, and suggest that low levels of LATS2 may undermine the ability of pRB to induce a permanent cell cycle arrest in tumor cells (Tschöp, 2011).

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