Retinoblastoma-family protein
Retinoblastoma interactions with Brahma family proteins and SWI/SNF The retinoblastoma tumor suppressor protein (RB) binds several cellular proteins involved in cell cycle progression. Using the yeast two-hybrid system, RB was shown to bind specifically to the protein BRG1. BRG1 shares extensive sequence similarity to Drosophila Brahma, an activator of homeotic gene expression, and the
yeast transcriptional activator SNF2/SW12. BRG1 contains an RB-binding motif found in viral oncoproteins and binds to the A/B pocket and the
hypophosphorylated form of RB. BRG1 does not bind RB in viral oncoprotein-transformed cells. Coimmunoprecipitation experiments suggested BRG1 associates
with the RB family in vivo. In the human carcinoma cell line SW13, BRG1 exhibits tumor suppressor activity by inducing formation of flat, growth-arrested cells.
This activity depends on the ability of BRG1 to cooperate and complex with RB, since both an RB-nonbinding mutant of BRG1 and the sequestration of RB by
adenovirus E1A protein abolished flat cell formation (Dunaief, 1994).
hBRG1 and hBRM are mammalian homologs of the SNF2/SW12 yeast transcriptional activator and of Drosophila Brahma. These proteins
exist in a large multisubunit complex that likely serves to remodel chromatin: in so doing, this complex facilitates the
function of specific transcription factors. The retinoblastoma protein inhibits cell cycle progression by
repressing transcription of specific growth-related genes. The members of the hBRG1/hBRM family of proteins interact with the pRB family of proteins, which
includes pRB, p107, and p130. Interaction between the hBRG1/hBRM family with the pRB family likely
influences cellular proliferation, since both hBRG1 and hBRM (but not mutants of these proteins unable to bind to
pRB family members) inhibit the formation of drug-resistant colonies when transfected into a SW13 human
adenocarcinoma cell line, lacking endogenous hBRG1 or hBRM. hBRM and two isoforms of
hBRG1 induce the formation of flat, growth-arrested cells in a pRB family-dependent manner when introduced
into SW13 cells. This flat-cell inducing activity is severely reduced by cotransfection of the wild-type E1A
protein and variably reduced by the cotransfection of mutants of E1A that lack the ability to bind to some or all
members of the pRB family (Strober, 1996).
The mammalian SWI-SNF complex is a chromatin-remodelling machine
involved in the modulation of gene expression. Its activity relies on
two closely related ATPases known as brm/SNF2alpha; these two proteins can cooperate with nuclear receptors for
transcriptional activation. In addition, they are involved in the
control of cell proliferation, most probably by facilitating
p105Rb repression of E2F transcriptional activity. In the
present study, the ability of various brm/SNF2alpha
deletion mutants to reverse the transformed phenotype of
ras-transformed fibroblasts has been studied. Deletions within the
p105Rb LXCXE binding motif or the conserved bromodomain have
only a moderate effect. On the other hand, a 49-amino-acid segment,
rich in lysines and arginines and located immediately downstream of the
p105Rb interaction domain, appears to be essential in this
assay. This region is also required for cooperation of brm/SNF2alpha
with the glucocorticoid receptor in transfection experiments, but only in the context of a reporter construct integrated in the cellular genome. The region has homology to the AT hooks present in
high-mobility-group protein I/Y DNA binding domains and is required for
the tethering of brm/SNF2alpha to chromatin (Bourachot, 1999).
Yeast and mammalian SWI-SNF complexes regulate transcription through active modification of chromatin structure. Human SW-13
adenocarcinoma cells lack BRG1 protein, a component of SWI-SNF that has a DNA-dependent ATPase activity essential for
SWI-SNF function. BRG1 specifically represses transcription from a transfected c-fos promoter and
correspondingly blocks transcriptional activation of the endogenous c-fos gene. Mutation of lysine residue 798 in the DNA-dependent
ATPase domain of BRG1 significantly reduces its ability to repress c-fos transcription. Repression by BRG1 requires the cyclic AMP response element of the c-fos
promoter but not nearby binding sites for Sp1, YY1, or TFII-I. Repression of c-fos by BRG1, a known Rb binding protein, depends on Rb. Using human C33A cervical carcinoma cells, which lack BRG1 and also express a nonfunctional Rb
protein, transcriptional repression by BRG1 is weak unless wild-type Rb is also supplied. Interestingly, Rb-dependent repression by BRG1 is found to take
place through a pathway that is independent of transcription factor E2F (Murphy, 1999).
Considering the -76 to +10 region of the c-fos promoter in isolation, mutation of the cyclic AMP response element (CRE) results in complete loss of sensitivity to BRG1 in these
assays. A CRE is also located in the corresponding location of the human promoter. Thus, a possible model for the effect of BRG1 on c-fos could involve
nucleosomal rearrangement of the c-fos promoter (already occupied by ATF/CREB) and repression of ATF/CREB-dependent transcription by an Rb-associated
histone deacetylase. Interestingly, the CRE is known to bind transcription factor ATF-2, which was reported to interact directly with Rb (in that
case, the ATF-2-Rb interaction correlated with transcriptional activation, not repression). ATF-2 has been detected in extracts of SW-13 cells by Western blot
analysis using a specific antibody against ATF-2, and experiments are now in progress to examine its role in BRG1-mediated repression (Murphy, 1999).
Rb forms a repressor complex containing histone deacetylase (HDAC) and the hSWI/SNF nucleosome remodeling complex, which inhibits
transcription of genes for cyclins E and A and arrests cells in the G1 phase of the cell cycle. Phosphorylation of Rb by cyclin D/cdk4 disrupts association with
HDAC, relieving repression of the cyclin E gene and G1 arrest. However, the Rb-hSWI/SNF complex persists and is sufficient to maintain repression of the cyclin A
and cdc2 genes, inhibiting exit from S phase. HDAC-Rb-hSWI/SNF and Rb-hSWI/SNF then appear to maintain the order of cyclin E and A expression during the
cell cycle, which in turn regulates exit from G1 and from S phase, respectively (Zhang, 2000).
Mutation of the yeast SWI/SNF ATPases, SWI2/SNF2, has been shown to lead to deregulation of a subset of genes, with more genes being activated by the mutations than repressed. This finding was surprising because SWI/SNF activity had previously been associated predominantly with transcriptional activation. Recent findings indicate that the corepressor HDAC can
be found in the hSWI/SNF-like Mi2b nucleosome remodeling complex. These studies provide evidence that SWI/SNF-like complexes can also participate in transcriptional repression. A recent study has also shown that the c-fos
promoter can be repressed by Rb and that BRG1 is required for this repression. However, the lack of E2F sites in the c-fos promoter
raises the possibility that this effect of Rb and BRG1 on the c-fos promoter may be indirect. Indeed, the c-fos promoter is sensitive to growth suppression: C33a cells are growth arrested by the combination of Rb and BRG1. Thus, inhibition of c-fos expression by Rb and
BRG1 in these studies may simply reflect the growth arrest that is imposed by the combination of Rb and BRG1. The results of the current study, however, demonstrate that Rb and
BRG1 cooperate to inhibit genes such as cyclin E, cyclin A, and cdc2, which are known targets of Rb-E2F and participate in cell cycle progression (Zhang, 2000 and references therein).
Recent results suggest that SWI/SNF-like nucleosome remodeling complexes and related remodeling complexes can regulate both the formation and positioning of
nucleosomes along a promoter.
In genes such as HO in yeast, where SWI/SNF is important for transcriptional activation, it is associated with transcriptional activators and HAT activity. In contrast, when recruited by Rb into a complex with HDAC, hSWI/SNF appears to be important for repression. Thus, it is possible that SWI/SNF
facilitates the action of both HAT or HDACs on nucleosomes in vivo. Alternatively, SWI/SNF may be important for both assembling or disassembling chromatin
after histones in nucleosomes are modified by HAT or HDACs. It is interesting to note that in some situations Rb is associated with transcriptional activation. For
example, Rb can enhance BRG1-dependent transcriptional activation by the glucocorticoid receptor. In this situation, SWI/SNF and
Rb are recruited to a promoter via a transcriptional activator associated with HAT activity (the glucocorticoid receptor) (Zhang, 2000 and references therein).
Evidence is presented that HDAC and BRG1 interact with different sites on Rb, allowing formation of an HDAC-Rb-hSWI/SNF complex. It appears that
phosphorylation of Rb by cyclin D/cdk4 during G1 disrupts interaction of HDAC with Rb-hSWI/SNF, allowing expression of cyclin E and progression into S phase. These results imply that cyclin E expression is a downstream target of cyclin D/cdk4. And indeed, recent studies using a 'knock-in' of the cyclin E gene
into the cyclin D1 gene locus in mice have provided evidence that cyclin E is genetically downstream of cyclin D1. In contrast to Rb-HDAC, the Rb-hSWI/SNF complex is not disrupted by cyclin D/cdk4 phosphorylation of Rb, allowing it to persist into S phase where it continues
to repress the cyclin A and cdc2 genes, thereby inhibiting (or delaying) movement into M phase. After cyclin E accumulates and cyclin E/cdk2 becomes
active, it appears to block Rb-hSWI/SNF activity at least in part by phosphorylation of BRG1. This allows cyclin A and cdc2 expression and progression of cells into M phase. In the absence of p16ink4a, HDAC-Rb-hSWI/SNF repression of cyclin E
expression is not sufficient to completely block cyclin E/cdk2 activity (because p21waf1/cip1 and p27kip1 are sequestered away from cyclin E/cdk2 by cyclin
D/cdk4). Since cyclin E/cdk2 activity is not completely blocked in the p16ink4a(-) cells, they move into S phase. However, they have difficulty exiting S phase
because Rb-hSWI/SNF inhibits expression of cyclin A and cdc2. This leads to additional DNA synthesis (endoreduplication). The cells eventually manage to exit S
phase and undergo mitosis, but there is no cytokinesis and they arrest with multiple nuclei. It is unclear whether Rb-hSWI/SNF also has a function in regulating a step
late in mitosis that leads to formation of multiple nuclei or whether the multiple nuclei are simply the result of the additional DNA synthesis that occurs before the cells
precede into mitosis (Zhang, 2000 and references therein).
A recent study has provided evidence of a role for Rb and E2F in regulation of the level of cyclin B1 and thus entry into mitosis. Cyclin A/cdk2 phosphorylates the Cdh1 subunit of anaphase promoting complex (APC), blocking activity of this ubiquitin ligase,
which normally degrades cyclin B1. Rb/E2F-mediated inhibition of cyclin A expression leads to activation of APC and destruction of cyclin B1. Thus,
expression of cyclin A in S phase (following cyclin E/cdk2-mediated inactivation of Rb-hSWI/SNF) would lead to cyclin A/cdk2-dependent inhibition
of APC, thereby triggering accumulation of cyclin B1. In support of this model, it has been demonstrated that APC remains active following mitosis, and this activity
persists through G1 and is not lost until S phase (perhaps coinciding with inhibition of Rb-hSWI/SNF and the appearance of cyclin A).
Loss of Rb-hSWI/SNF also results in increased expression of cdc2. Together, the increase in cyclin B1 and cdc2 would lead to accumulation of cyclin B1/cdc2,
which is required for entry into mitosis. Forcibly maintaining Rb-hSWI/SNF would then be expected to inhibit or delay movement of cells from S phase to M phase
by inhibiting formation of cyclin B1/cdc2 (Zhang, 2000).
Rb is linked to Polycomb repressive pathways Association between Rb and PcG proteins forms a repressor complex that blocks entry of cells into mitosis. Also, evidence is provided that Rb colocalizes with nuclear PcG complexes and is important for association of PcG complexes with nuclear targets. The Rb-PcG complex may provide a means to link cell cycle arrest to differentiation events leading to embryonic pattern formation (Dahiya, 2001).
Loss of cell cycle control is a hallmark of tumorigenic transformation. A critical regulator of the cell cycle is the Rb family. One target of Rb family members is E2F transcription factors. E2F binding sites are found, for example, in the promoter region of genes for the cyclin A and E regulatory subunits of cyclin-dependent kinase 2 (cdk2), whose activity is essential for cell cycle progression. Binding of Rb to E2F not only masks the transactivation domain of E2F; the Rb-E2F complex that forms at promoters of cell cycle control genes recruits chromatin-remodeling enzymes such as histone deacetylase (HDAC) to actively repress transcription. The ability of Rb to bind HDAC and to arrest cells in G1 is blocked when Rb is phosphorylated by cyclin D/cdk4 or cdk6 (Dahiya, 2001 and reference therein).
The cdk inhibitor p16 blocks cdk4/6 activity, leading directly to accumulation of hypophosphorylated Rb. The participation of p16 and Rb in a common pathway is illustrated by the fact that p16 cannot arrest Rb(-) cells, and by the finding that only one or the other of these tumor suppressors is mutated in tumors (because mutation of p16 leads to activation of cdk4/6 and hyperphosphorylation [inactivation] of Rb). Recent studies have found that embryo fibroblasts from mice lacking both E2F4 and E2F5 cannot be arrested by either p16 or dominant-negative Ras, both of which are dependent upon accumulation of hypophosphorylated Rb for growth arrest. These results suggest that E2F4/5 is required for targeting Rb family-corepressor complexes to cell cycle control genes, and that in the absence of E2F4/5, these genes are not repressed when hypophosphorylated Rb accumulates. In support of these findings, it has also been found that displacement of Rb-E2F complexes from promoters with a dominant-negative E2F (DN-E2F) containing a DNA binding domain but lacking an Rb binding site, or titration of Rb-E2F complexes away from promoters by transfection of a plasmid containing multiple E2F binding site repeats, prevents Rb-mediated repression of cyclin E and A expression and growth arrest by p16. Taken together, these results suggest that E2F4/5 recruits Rb family repressor complexes to promoters of cell cycle genes, leading to a block in cell proliferation. In contrast to E2F4/5, E2F1-3 are thought to contribute an important transactivation function to cell cycle progression and apoptosis. Accordingly, these E2F family members are thought to regulate a distinct set of genes from E2F4/5 (Dahiya, 2001 and references therein).
Rb can interact with HDAC-1-3, and histone deacetylase has been demonstrated to be required genetically for Rb function in C. elegans. HDAC activity is reportedly required for Rb family repression of the cyclin E gene. Also, it was proposed that this repression of cyclin E expression is relieved when growth factors signal through the Ras/MAP kinase pathway to induce expression of D cyclins, which in turn combine with cdk4/6 to form active kinases that phosphorylate Rb. This phosphorylation induces a conformational change in Rb that displaces HDAC, thereby relieving repression of cyclin E expression. Cyclin E then combines with cdk2 to form a kinase that is essential for progression of cells into S phase, at least in part because it is required for assembly of origins of DNA replication (Dahiya, 2001).
Rb repressor activity and growth suppression also require interaction with BRG1 or Brm, which are central components of SWI/SNF chromatin-remodeling complexes. Mice heterozygous for BRG1 or the SNF5 component of the SWI/SNF complex are prone to tumor formation, and BRG1 has been found to be mutated in multiple human tumor cell lines. Furthermore, genetic screens in Drosophila have identified Brm and two other SWI/SNF components as enhancers of E2F activity. These results are consistent with a role for the SWI/SNF complex as a tumor suppressor critical for the Rb family pathway. Accordingly, BRG1 has been found to be required for Rb to repress expression of cyclins E and A and cdc2. However, while this repression of cyclin E expression requires HDAC, repression of cyclin A and cdc2 expression does not. Cyclin A/cdk2 phosphorylation of the cdh1 subunit of the anaphase-promoting complex reportedly blocks its ubiquitin ligase-mediated turnover of cyclin B. Repression of cyclin A and cdc2 expression by the Rb family then prevents assembly of cyclin B/cdc2, which is required for entry into mitosis. Therefore, in addition to blocking the G1/S transition, this inhibition of cyclin B/cdc2 activity allows Rb to also block entry of cells into mitosis. The finding that repression of cyclin A and cdc2 expression is HDAC independent suggests that distinct Rb repressor complexes may block the cell cycle at G1/S (e.g., repression of cyclin E expression) and G2/M (e.g., repression of cyclin A and cdc2 expression) (Dahiya, 2001).
Evidence is presented that a polycomb group (PcG) protein, HPC2, in association with its RING finger protein binding partner, Ring1, serves as an HDAC-independent corepressor for Rb, which specifically represses expression of cyclin A and cdc2 and arrests cells in G2. PcG proteins classically repress Hox genes, and this repression coupled with transcriptional activation by trithorax proteins imposes the patterns of Hox expression required for proper embryonic development. However, in addition to their role in repression of Hox genes, PcG proteins are critical regulators of the cell cycle. Mice lacking one of the PcG family members, Bmi-1 (BMI-1 in humans), have severe defects in lymphoid cell proliferation. And, overexpression of Bmi-1 as a result of viral insertion stimulates cell proliferation and is oncogenic. The molecular basis for this has recently been uncovered. Bmi-1 represses expression of the INK4a/ARF locus encoding the cdk4/6 inhibitor p16 and the ARF protein. Also, crossing the Bmi-1-/- mice into an INK4a/ARF-/- background reverses much of the proliferative defect in the Bmi-1-/- mice. p16 is a central component of the Rb pathway in human cells, and in the absence of Bmi-1, p16 accumulates, blocking cyclin D/cdk4 activity and leading to accumulation of hypophosphorylated (active) Rb and growth arrest.
As with Bmi-1, another PcG family member, M33 (CBX2/HPC1 in humans), is required for normal proliferation of fibroblasts and lymphocytes. However, it is not yet clear how M33 is involved in regulation of cell proliferation. In contrast to Bmi-1 and M33, other PcG family members, such as eed (EED in humans and Esc in Drosophila) and HPC2 (Mpc2 in mice), are negative regulators of the cell cycle. Expression of eed blocks cell proliferation, and expression of a dominant-negative hpc2 leads to transformed morphology and growth of cells in suspension. Eed+/- mice have a myelo- and lympho-proliferative syndrome associated with lymphoid tumor formation. A cross of the eed+/- mice into a Bmi-1-/- background largely reverses the lymphoproliferative defect in the Bmi-1-/- mice, indicating that the family members have opposing activities on cell proliferation and leading the authors to conclude that Bmi-1 is epistatic to eed (Dahiya, 2001 and references therein).
Both HPC2 and Bmi-1 interact with the RING finger protein Ring1, and the Ring1
binding domain is required for Bmi-1 to repress the INK4a/ARF locus and for HPC2 to arrest cells. Ring1 associates with Rb in vivo, and Ring1 coimmunoprecipitates in a complex with E2F in an Rb-dependent fashion. These results provide evidence that Ring1 is present in the E2F-Rb-CtBP-HPC2
complex. When Ring1 is expressed in HPC2(-) cells, there is little detectable effect on the cell cycle profile; however, when a subthreshold level (a level where little or no increase in cells in S
or G2/M is evident) of HPC2 (or Rb and HPC2) is expressed, Ring1 synergizes with HPC2 to trigger accumulation of cells in G2 (Dahiya, 2001).
Ring1 binds to the C-terminal end of HPC2. Therefore, an HPC2 mutant was used
with the Ring1 binding site deleted (deletion of the C-terminal 30 amino acids). CtBP binds to the PIDLRS sequence N-terminal to this deletion, such that this C-terminal deletion of HPC2 does not affect its binding to CtBP, nor does it affect the ability of HPC2 to interact with Rb in coimmunoprecipitation assays. Therefore, this C-terminal truncation mutant of HPC2 competes with wild-type HPC2 for binding to CtBP in the E2F-Rb-CtBP-HPC2 complex, but it should act as a dominant negative because it is unable to recruit Ring1, which is essential for PcG function. Indeed, expression of this HPC2 mutant has been shown to produce a transformed morphology in cells, and to allow growth in suspension; these phenotypes were reversed by coexpression of wild-type HPC2. Rb- and HPC2-dependent repression is blocked by this DN-HPC2 in transfection assays (Dahiya, 2001).
PcG proteins such as Bmi and HPC2 are present in a single large complex. It is then unclear how Bmi and HPC2 might target distinct sets of genes and have opposing effects on the cell cycle if they are in a common complex. And further, it is unclear why there are substantial differences in homeotic transformations seen in mice lacking different PcG proteins. It is suggested that the relative concentration of a given PcG protein in the complex may direct the targeting of the complex. Such a model may allow for an 'all or none' targeting effect. For example, an excess of Bmi would lead to the complex being directed toward the p16/ARF locus and away from cyclin A and cdc2 genes. And vice versa, excess HPC2 would target the complex toward cyclin A and cdc2 genes and away from the INK4a/ARF locus (Dahiya, 2001).
Genetic studies have demonstrated that Bmi1 promotes cell proliferation and stem cell self-renewal with a correlative decrease of p16INK4a expression. Polycomb genes EZH2 and BMI1 repress p16 expression in human and mouse primary cells, but not in cells deficient for pRB protein function. The p16 locus is H3K27-methylated and bound by BMI1, RING2, and SUZ12. Inactivation of pRB family proteins abolishes H3K27 methylation and disrupts BMI1, RING2, and SUZ12 binding to the p16 locus. These results suggest a model in which pRB proteins recruit PRC2 to trimethylate p16, priming the BMI1-containing PRC1L ubiquitin ligase complex to silence p16 (Kotake, 2007).
The mammalian pRB family proteins, pRB, p107, and p130 (also known as pocket proteins), play a key role in controlling the G1-to-S transition of the cell cycle and maintaining differentiated cells in a reversible quiescent or permanent senescent arrest state. The pocket proteins are hypophosphorylated in cells exiting mitosis as well as in quiescent cells, where they bind to and negatively regulate the function of the E2F family transcription factors. In cells entering the cell cycle, extracellular mitogens first induce the expression of D-type cyclins, which bind to and activate CDK4 and CDK6, leading to the phosphorylation of pRB family proteins, causing functional inactivation by E2F dissociation, thereby promoting a G1-to-S transition. Inhibition of CDK4 and CDK6 by the INK4 family of CDK inhibitors (p16INK4a, p15INK4b, p18INK4c, and p19INK4d) retains pRB family proteins in their hypophosphorylated, growth-suppressive states and prevents G1-to-S progression. Disruption of the INK4-RB pathway, consisting of INK4-cyclinDs-CDK4/6-RB-E2Fs, deregulates G1-to-S control and represents a common event in the development of most, if not all, types of cancer (Kotake, 2007).
Among the major challenges toward a better understanding of G1 control by the INK4-pRB pathway is how different INK4 genes are regulated, thereby linking G1 control to different cellular pathways. INK4 proteins are relatively stable, and the primary regulation of INK4 is through transcriptional control. The expression of each of the INK4 genes is distinctly different during development, in different adult tissues, and in response to different cellular conditions. There have been only a few reports wherein a transcriptional regulator has been demonstrated to bind to an INK4 promoter by either gel shift or chromatin immunoprecipitation (ChIP) assay. Identification of factors that directly bind to INK4 promoters holds the key to linking different cellular pathways to G1 control by the INK4-pRB pathway, but these links remain disproportionately poorly understood in comparison with knowledge of the function of the INK4-pRB pathway (Kotake, 2007).
To elucidate the molecular mechanisms regulating p16 expression, whether p16 gene expression is directly regulated by BMI1, an oncogene that encodes a transcriptional repressor of the Polycomb group (PcG) of proteins, was directly tested. Deletion of Bmi1 retards cell proliferation, causes premature senescence in mouse embryonic fibroblasts (MEFs), and reduces the number of hematopoietic stem cells, with an associated up-regulation of p16 (and to a lesser extent of p19Arf). Codeletion of p16 (or p16-Arf) partially rescues the proliferative defects of Bmi1-null cells, providing genetic evidence supporting a functional interaction between the Bmi1 and p16 genes. However, whether BMI1 directly binds to and regulates the transcription of the p16 gene has not been demonstrated. A notable feature of p16 is its high level of expression in virally transformed cells and its inverse correlation with pRB function, suggesting a negative regulation of p16 gene expression by pRB. Therefore whether pRB and BMI1 collaboratively repress p16 expression was also examined (Kotake, 2007).
These results provide the first biochemical evidence supporting a direct regulation of p16 transcription by the PRC2 histone methyltransferase complex and the BMI1-RING2-containing PRC1 histone ubiquitin ligase complex. Both H3K27 methylation at and BMI1/RING2 binding to the p16 locus require the function of the pRB family proteins, linking for the first time H3K27 methylation and the function of BMI1 with the pRB proteins. The detailed biochemical mechanism by which pRB family proteins collaborate with BMI1 to repress p16 transcription is yet to be determined. In repeated attempts, binding of pRB to the p16 locus could not be detected. The simplest model suggested by these results is that the pRB family proteins are either involved in regulating the enzymatic activity or the recruitment of PRC2 to the p16 locus. H3K27 methylation by PRC2 would then facilitate recruitment of the BMI1-containing PRC1L complex to ubiquitinate H2A, leading to p16 silencing (Kotake, 2007).
The results also suggest a regulatory loop between p16 and the pocket proteins, with p16 acting as an upstream activator of the pocket proteins and the pocket proteins repressing p16 transcription as negative feedback. INK4 proteins are intrinsically stable and, once synthesized, stably bind to and inhibit the activity of CDK4/6 by both interfering with ATP binding and by reducing the cyclin-CDK4/6 surface. Without a mechanism for repressing INK4 expression, mitogen-induced cyclin D synthesis would not be able to compete off INK4 from CDK4/6, and displaced, monomeric cyclin D proteins would be rapidly degraded, leaving a constitutive activation of RB function and locking cells in a permanent G1-arrested state. Repression of p16 expression by pRB family proteins thus also constitutes a feedback loop to set up a balance between INK4-mediated inhibition and cyclin D-mediated activation of G1 progression. This function of p16, however, must be repressed in stem cells, which undergo continuous proliferation and self-renewal in vivo. It is speculated that one mechanism to achieve this is through expression of BMI1 in the stem cell compartment (Kotake, 2007).
Retinoblastoma interactions with the transcriptional apparatus The retinoblastoma susceptibility gene product (Rb) generally represses RNA polymerase III (Pol
III)-directed transcription. This implies that Rb interacts with essential transcription factors. Mutations
in either the A or B subdomains in the Rb pocket interfere with Rb-mediated repression of Pol
III-directed transcription, which indicates that both subdomains are directly involved in this activity.
Addition of either purified TFIIIB or purified TFIIIC2 partially relieves Rb-mediated repression and
restores activity to nuclear extracts that had been depleted of essential factors by binding to Rb. Pull
down and coimmunoprecipitation experiments as well as functional assays indicate that Rb interacts
with both TFIIIB and TFIIIC2 and that the A subdomain is primarily required for binding TFIIIB and
the B subdomain for binding TFIIIC2. While Rb interacts with both factors, the A subdomain is more
important than the B subdomain in directing Rb-mediated repression; TFIIIB is the principal target
of that activity (Chu, 1997).
Rb binds directly to the largest TFIID subunit,
TATA-binding protein associated factor TAF(II)250 (Drosophila homolog: TBP-associated factor 250kD), first identified as the cell cycle regulatory protein
CCG1. The domains in Rb and TAF(II)250 important for their interaction in vitro and in
vivo have been mapped. Both the amino terminus and the large pocket of Rb are able to associate independently with
TAF(II)250. The binding domain(s) within the large pocket is distinct from the viral oncoprotein and
E2F binding region since certain pocket mutations (which abolish E1A binding) do not abolish
TAF(II)250 binding. Consistent with the large pocket of Rb binding to TAF(II)250, the large pocket
domains of both p107 and p130 are able to bind to TAF(II)250 in vivo. At
least two regions of TAF(II)250 are able to bind to the large pocket of Rb independently, whereas the
amino terminus of Rb binds to a distinct domain in TAF(II)250. Rb can
bind to TFIID in vitro, presumably in part through an interaction with TAF(II)250. These results suggest
a complex interaction between Rb and TAF(II)250 and imply that TAF(II)250, TFIID, and potentially
other basal transcription factors are targets for regulation by Rb and Rb-related proteins (Shao, 1997).
The retinoblastoma tumor suppressor protein, Rb, interacts directly with the largest TATA-binding
protein-associated factor, TAFII250, through multiple regions in each protein. To define the potential role(s)
of this interaction, it was necessary to examine whether Rb could regulate the intrinsic, bipartite kinase activity of TAFII250. Rb is able to inhibit the kinase activity of immunopurified and gel-purified recombinant
TAFII250. Rb inhibits the autophosphorylation of TAFII250 as well as its phosphorylation of the RAP74
subunit of TFIIF in a dose-responsive manner. Inhibition of TAFII250 kinase activity involves the Rb pocket
(amino acids 379 to 928) but not its amino terminus. In addition, Rb appears to specifically inhibit the
amino-terminal kinase domain of TAFII250 through a direct protein-protein interaction. Two different tumor-derived Rb pocket mutants, C706F and Deltaex22, are functionally
defective for kinase inhibition, even though they are able to bind the amino terminus of TAFII250. These results
suggest a novel mechanism of transcriptional regulation by Rb, involving direct interaction with TAFII250
and inhibition of its ability to phosphorylate itself, RAP74, and possibly other targets (Siegert, 1999).
The retinoblastoma susceptibility gene product 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 has been 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).
The level of ribosomal gene transcription has been shown to be finely regulated in response to changes in cell growth rate and the state of differentiation. This
regulation is believed, at least in part, to be due to a change in the number of actively transcribed genes.
Transcription of the ribosomal genes by RNA polymerase I (PolI) is activated both in vitro and in vivo by UBF. The recruitment of this protein to the PolI promoter is in fact the first step in ribosomal gene
activation, permitting the subsequent association of the TATA-binding protein (TBP)-containing complex SL-1, and hence of the polymerase. UBF contains multiple tandem homologies to the DNA binding domain of high mobility group 1 (HMG-1), the HMG box, and loops approximately 140 bp of ribosomal DNA into a single turn, a structure that has been called the ribosomal enhancesome. Data on the promotion of PolI transcription in vertebrates are compatible with the formation of two precisely juxtaposed
enhancesomes on the PolI promoter as a prerequisite to promoter recognition by SL-1. Mammalian and Xenopus UBFs are functionally interchangeable for this task in vivo. However, enhancesome formation is clearly incompatible with the nucleosomal
chromatin structure of the inactive genes. Therefore, the transition from the inactive to active ribosomal gene state requires the replacement of one or more nucleosomes with enhancesomes. Chromatin remodeling has been shown to be facilitated by the recruitment of
co-activators with acetyltransferase activity. Further, the HMG box of Drosophila
TCF/LEF functionally recruits the histone acetyltransferase (HAT) CREB-binding protein (CBP). The potential of CBP to activate ribosomal transcription by PolI has been investigated (Pelletier, 2000 and references therein).
RNA polymerase I (PolI) transcription is activated by the HMG box architectural upstream binding factor (UBF), which loops approximately 140 bp of DNA into the enhancesome,
necessitating major chromatin remodeling. The acetyltransferase CBP is recruited to and acetylates UBF both in vitro and in vivo. CBP activates
PolI transcription in vivo through its acetyltransferase domain and acetylation of UBF facilitates transcription derepression and activation in vitro. CBP activation and
Rb suppression of ribosomal transcription by recruitment to UBF are mutually exclusive, regulating in vivo PolI transcription through an acetylation-deacetylation
'flip-flop.' Thus, PolI transcription is regulated by protein acetylation, and the competitive recruitment of CBP and Rb (Pelletier, 2000).
Rb suppresses ribosomal transcription in vitro via a direct interaction with UBF. An interaction has been identified in vitro between Rb aa 379-928 and the HMG boxes 1 and/or 2 of mUBF. Other data further
suggest that the Rb 'pocket' (aa 379-792) is sufficient for transcriptional suppression in vitro and for the Rb-UBF interaction in vivo. Since HMG boxes 1 and 2 also bind CBP, it is possible that Rb and CBP binding to UBF are exclusive events. If this is the case,
suppression by Rb and activation by CBP would also be exclusive (Pelletier, 2000).
Rb suppresses ribosomal transcription in vivo. Consistent with a role for the Rb pocket in this suppression, it has also been found that the
Rb-related pocket protein p107 is equally effective in this suppression. It was next determined if the pocket domain of Rb (aa 379-792) could
bind HMG boxes 1 and/or 2 of xUBF. Rb(379-792) binds a polypeptide containing HMG boxes 1 and 2, but does not bind to
sequences C-terminal of HMG box 2. Thus the pocket region of Rb is sufficient to bind xUBF. It was also found that the individual HMG boxes 1 and 2 bind the Rb pocket region less efficiently than does the box12 combination.
Rb and CBP were then placed in competition for xUBF. Preincubation of full-length xUBF with the interaction domain CBP2 is sufficient to inhibit subsequent
binding to Rb(379-792). Conversely, pre-incubation of xUBF with Rb(379-792) inhibits its subsequent binding to CBP2. These
data strongly suggest that suppression of PolI transcription by Rb results, at least in part, from its capacity to interfere with the recruitment of CBP to UBF. However, in other systems, Rb has also been shown to suppress transcription by the recruitment of histone deacetylase 1 (HDAC1). Hence, Rb could also potentially reverse the catalytic effects of recruiting CBP to UBF. It was therefore asked if HDAC1
deacetylates xUBF acetylated with the CBP-HAT domain and whether the presence of Rb enhances this deacetylation. The rate of deacetylation of
xUBF by HDAC1 in the presence of Rb(379-792) is nearly two times more rapid than in its absence. Suppression of PolI transcription in vivo by Rb
(and p107) is at least partly relieved by inhibiting deacetylation with TSA. The fact that Rb suppression can not be reversed with TSA alone is consistent with Rb also preventing CBP recruitment to UBF. In fact, the Rb-induced
suppression of PolI transcription can be completely relieved, and indeed reversed, by the coexpression of CBP in combination with TSA treatment (Pelletier, 2000).
The recruitment of CBP to UBF could activate transcription (1) by the acetylation of UBF, (2) by the acetylation of local chromatin, (3) by displacing Rb, or (4) by a
combination of these effects. The fact that Rb can cooperate in the deacetylation of UBF suggests that acetylated UBF can effectively bind Rb and
this was confirmed in pulldown experiments. The role of CBP, Rb, and acetylation
in DNA binding by UBF was also investigated. Neither an excess of CBP2 nor saturation acetylation of UBF with the HAT domain of CBP has any detectable effect on UBF's capacity to
bind the ribosomal promoter DNA. Rb(379-928), [or the pocket domain Rb(379-792) or
GST-Rb(379-928)], has no effect on DNA binding by UBF (Pelletier, 2000).
Since the DNA binding of UBF is unaffected by CBP or Rb binding or by acetylation, it was asked if acetylated UBF is necessary for transcription activation
in vitro. Bacterially produced UBF, which is necessarily unacetylated, has been found in many laboratories to be refractory for in vitro transcription. However, UBF produced in mammalian and insect cells or by in vitro translation has been found to be functional. This suggests that post-transcriptional modification of UBF may be important, and indeed this has been shown to be the
case for UBF phosphorylation. Rat and mouse nuclear extracts were therefore depleted of endogenous UBF and used to study the capacity of bacterially expressed (i.e.,
unacetylated) UBF to activate transcription from the rat or mouse PolI promoters. Bacterially produced rUBF and
xUBF were either acetylated with matrix-immobilized active CBP HAT domain or mock acetylated (unacetylated) with the immobilized inactive HAT
domain and then the HAT protein was removed by centrifugation. UBF has been shown both to derepress PolI transcription in vitro as well as to activate it. The derepression properties of rUBF in the rat extract was investigated in competition with added histone H1. As expected, addition of H1 to the rat extract represses transcription of the rat promoter, and this repression is even
more pronounced after UBF depletion. Addition of unacetylated rUBF does not relieve H1 repression, and even increases it somewhat. However, the
acetylated rUBF relieves H1 repression and gives about a 2-fold increase in transcription (or more than 4-fold the level observed in the presence of the same amount
of unacetylated UBF). The capacity of UBF to activate transcription from the mouse promoter was also tested in a UBF-depleted mouse nuclear extract. Here the unacetylated rUBF gives a small degree of transcription activation (1.7 times), but the acetylated rUBF activates much more effectively (3.5 times).
Xenopus UBF has been shown to activate the mouse promoter in vivo, although it has also been shown to be ineffective in activating the rat or human promoters in
vitro. Bacterial unacetylated xUBF has a clear repressive (0.5 times) effect on the mouse
promoter in vitro, but after acetylation, this repression is completely relieved and transcription is somewhat activated (Pelletier, 2000).
These data strongly support a 'flip-flop' model for the regulation of ribosomal transcription by CBP and Rb-HDAC1. (The term 'flip-flop' is used to
describe a system with two alternative semistable states, here CBP-bound or Rb-bound UBF.) The formation of a UBF-CBP complex activates transcription by
acetylation of UBF itself, and perhaps also by opening up the adjacent ribosomal chromatin, allowing further UBF ingression and gene activation. Excess Rb prevents
formation of a UBF-CBP complex and, by recruiting HDAC1, catalyses UBF deacetylation and hence its inactivation. Acetylation of UBF significantly
enhances its ability to activate PolI transcription in vitro. Although changes in DNA binding and the ability of UBF to bind Rb have been excluded, the mechanism by
which UBF acetylation functions remains unknown. One possible explanation being actively pursued is that acetylation induces a structural change in UBF.
Quite possibly Rb recruitment to UBF has roles other than just to promote UBF deacetylation. Rb can inhibit SL-1 recruitment to
UBF. Rb may also cooperate in deacetylation of adjacent histones. Enhancesome
structure, with its single 140 bp loop of DNA, could accommodate the core histones in a weak association with the DNA. Yet, xUBF can also
associate stably with nucleosomes. Thus, the CBP/Rb flip-flop could catalyze the transition between a predominantly nucleosomal and a
predominantly enhancesomal gene state, the transition not necessarily requiring complete displacement of either core histones or UBF. It has in fact been observed
that the core histones remain associated with the active ribosomal genes, but only via their N-terminal domains. Whether a
nucleosome-enhancesome transition is facilitated by UBF acetylation, histone acetylation, or a combination of the two must now be determined (Pelletier, 2000).
Recent data suggest that both acetylation and phosphorylation can cooperate to activate transcription in vivo. UBF is known to be multiply phosphorylated, mainly within the C-terminal acidic domain but also in HMG box 5. Each of these modifications has been shown to
activate transcription in vitro and, in the case of the acidic domain, phosphorylation has been shown to enhance recruitment of SL-1. Here acetylation is also important for UBF function. In future work attempts will be made to test whether a functional link exists between the
phosphorylation and the acetylation of UBF (Pelletier, 2000).
The C. elegans synthetic multivulva (synMuv) genes act redundantly to antagonize the specification of vulval cell fates, which are promoted by an RTK/Ras pathway. At least 26 synMuv genes have been genetically identified, several of which encode proteins with homologs that act in chromatin remodeling or transcriptional repression. This study reports the molecular characterization of two synMuv genes, lin-37 and lin-54.lin-37 and lin-54 encode proteins in a complex with at least seven synMuv proteins, including LIN-35, the only C. elegans homolog of the mammalian tumor suppressor Rb. Biochemical analyses of mutants suggest that LIN-9, LIN-53, and LIN-54 are required for the stable formation of this complex. This complex is distinct from a second complex of synMuv proteins with a composition similar to that of the mammalian Nucleosome Remodeling and Deacetylase complex. The class B synMuv complex identified in this study is evolutionarily conserved and likely functions in transcriptional repression and developmental regulation (Harrison, 2006; full text of article).
LIN-37 and LIN-54 form a multisubunit protein complex together with at least five other class B synMuv proteins: LIN-9, LIN-35 Rb, LIN-52, LIN-53 RbAp48, and DPL-1 DP. This DP, Rb, and MuvB (DRM) complex is biochemically and genetically distinct from a NuRD-like complex that includes HDA-1 HDAC1, LET-418 Mi2, and LIN-53 RbAp48. These findings suggest that LIN-35 Rb and DPL-1 DP likely have a function in vulval development distinct from recruitment of the NuRD complex (Harrison, 2006).
The DRM complex is similar to two recently described and highly similar complexes that contain several Drosophila homologs of class B synMuv proteins (Korenjak, 2004; Lewis, 2004). The Myb–MuvB complex was purified by immunoprecipitation of the LIN-54 homolog Mip120 or the LIN-9 homolog Mip130 from Drosophila tissue-culture cells and coimmunoprecipitating proteins were identified by mass spectrometry. The Myb–MuvB complex contains stoichiometric levels of Mip130, RBF, Mip40, Mip120, p55, dDP, dE2F2, and dLin52, which are homologs of LIN-9, LIN-35, LIN-37, LIN-54, LIN-53, DPL-1, EFL-1, and LIN-52, respectively. This complex also contains substoichiometric amounts of Rpd3, the fly homolog of HDA-1, and L(3)MBT, a protein similar to the class B synMuv protein LIN-61. The dREAM complex was identified by biochemical purification of Drosophila Rb-containing complexes from embryo extracts followed by mass spectrometry and Western blot analyses. The dREAM complex contains all of the proteins identified in the Myb–MuvB complex at stoichiometric levels except for dLin52 (Korenjak, 2004). The differences between the dREAM and Myb–MuvB complexes might be a consequence of the methods used for purification or might reflect the existence in different tissues or during different developmental stages of multiple subcomplexes with overlapping components. Both the dREAM and Myb–MuvB complexes can mediate transcriptional repression of many E2F-target genes (Harrison, 2006).
The similarity between the C. elegans DRM complex and the Drosophila dREAM and Myb–MuvB complexes indicates that the DRM complex likely also acts in transcriptional repression. Given the broad expression patterns of the synMuv genes and the multiple phenotypic abnormalities caused by the loss of individual synMuv proteins, it is proposed that, similar to their Drosophila counterparts, the DRM complex proteins are involved in the repression of many targets important for diverse biological functions (Harrison, 2006).
The DRM complex differs slightly from both the dREAM and the Myb–MuvB complexes. Unlike the dREAM complex, the DRM complex contains a LIN-52 dLin52-like protein. Unlike the Myb–MuvB complex, the DRM complex does not contain HDA-1 Rpd3 or LIN-61 L(3)MBT. The similarities of the DRM, dREAM, and Myb–MuvB complexes suggest that there is a core complex consisting of LIN-35 RBF, EFL-1 E2F2, DPL-1 DP, LIN-9 Mip130, LIN-37 Mip40, LIN-52 dLin-52, LIN-53 p55, and LIN-54 Mip120 and that this complex might associate with other proteins during specific stages of development or in certain cell types (Harrison, 2006).
The dREAM and Myb–MuvB complexes both contain the DNA-binding protein Myb. There is no clear Myb homolog in C. elegans. It is possible that the C. elegans DRM complex does not contain a Myb ortholog or that the functional ortholog of the Drosophila Myb protein found in the dREAM and Myb–MuvB complexes might not be readily identifiable by comparisons of primary sequence (Harrison, 2006).
It is proposed that the DRM complex could be recruited to DNA by multiple DNA-binding factors, including LIN-54 and the heterodimeric transcription factor formed by EFL-1 and DPL-1. The DRM complex could then act with the NuRD-like complex to repress transcription. Alternatively, the DRM complex and the NuRD-like complex could act sequentially. The NURD-like complex could deacetylate the N-terminal tails of histones, and the DRM subsequently could bind these unmodified histone tails, preventing their acetylation. The dREAM complex previously has been shown to bind unmodified histone H4 tails, supporting this model. This binding might be mediated by LIN-53, because the mammalian homolog RbAp48 binds histone H4. Deacetylated histones are associated with transcriptionally repressed areas of the genome. Thus, by protecting histone tails from future acetylation, the DRM complex could act to maintain transcriptional repression of nearby genes (Harrison, 2006).
Although neither the DRM nor the dREAM complexes contains known chromatin-modifying or chromatin-remodeling enzymes, these complexes might require the activity of a histone deacetylase to mediate transcriptional repression. Mutations in genes encoding components of either the DRM or the NuRD-like complex require an additional class A or class C synMuv mutation to produce a highly penetrant Muv phenotype. However, mutations affecting two of the NuRD-like complex components, HDA-1 and LET-418, alone can cause low penetrance Muv phenotypes, suggesting that the chromatin-remodeling and chromatin-modifying activities of this complex might be required more broadly for the transcriptional repression of genes necessary for proper vulval development than is the activity of the DRM complex. Perhaps other class B synMuv proteins not associated with the DRM complex, for example, HPL-2, LIN-36, or LIN-61, act with the DRM complex to maintain the repressed state formed by the activity of the NuRD-like complex (Harrison, 2006).
The high degree of conservation shared by the DRM/Myb-MuvB/dREAM complexes in C. elegans and Drosophila and the important roles that the components of DRM complex play in C. elegans development suggest that a similar complex exists in other organisms, including humans. The core components of these complexes have homologs in humans, and the human homolog of LIN-9, hLin-9, can associate with Rb to specifically promote differentiation (but not to inhibit cell-cycle progression). Perhaps Rb or other Rb-family proteins act within the context of a DRM-like complex to control differentiation. Rb could act as a tumor suppressor through such DRM-mediated regulation of differentiation in addition to its role in cell-cycle regulation. Further biochemical and genetic studies of nematodes, insects, and mammals should elucidate the role that this conserved protein complex plays in development and in carcinogenesis (Harrison, 2006).
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