Retinoblastoma-family protein
Retinoblastoma proteins act as transcriptional activators and repressors Forced expression of the retinoblastoma (RB) gene product inhibits the proliferation of cells in culture.
A major target of the RB protein is the S-phase-inducing transcription factor E2F1. RB binds directly
to the activation domain of E2F1 and silences it, thereby preventing cells from entering S phase. To
induce complete G1 arrest, RB requires the presence of the hbrm/BRG-1 proteins (see Drosophila Brahma), which are
components of the coactivator SWI/SNF complex. This cooperation is mediated through a physical
interaction between RB and hbrm/BRG-1. In transfected cells RB can simultaneously contact both
E2F1 and hbrm, thereby targeting hbrm to E2F1. E2F1 and hbrm are indeed found
within the same complex in vivo. RB and hbrm cooperate to repress E2F1 activity in
transient transfection assays. The ability of hbrm to cooperate with RB to repress E2F1 is dependent
upon several distinct domains of hbrm, including the RB binding domain and the NTP binding site.
However, the bromodomain seems dispensable for this activity. Taken together, these results point out an
unexpected role as corepressor for the hbrm protein. The ability of hbrm and RB to cooperate in
repressing E2F1 activity could be an underlying mechanism for the observed cooperation between
hbrm and RB to induce G1 arrest. The domain of hbrm that binds RB has
transcriptional activation potential which RB can repress. This suggest that RB not only targets hbrm
but also regulates its activity (Trouche, 1997).
The retinoblastoma tumour-suppressor protein Rb inhibits cell proliferation by repressing a subset of
genes that are controlled by the E2F family of transcription factors and which are involved in
progression from the G1 to the S phase of the cell cycle. Rb, which is recruited to target promoters by
E2F1, represses transcription by masking the E2F1 transactivation domain and by inhibiting surrounding
enhancer elements, an active repression that could be crucial for the proper control of progression
through the cell cycle. Some transcriptional regulators act by acetylating or deacetylating the tails
protruding from the core histones, thereby modulating the local structure of chromatin: for example,
some transcriptional repressors function through the recruitment of histone deacetylases. The histone deacetylase HDAC1 physically interacts and cooperates with Rb. In HDAC1, the
sequence involved is an LXCXE motif, similar to that used by viral transforming proteins to contact Rb.
These results strongly suggest that the Rb/HDAC1 complex is a key element in the control of cell
proliferation and differentiation and that it is a likely target for transforming viruses. These results suggest that Rb represses transcription by recruiting a histone deacetylase (Magnaghi-Jaulin, 1998).
The retinoblastoma protein (Rb) silences specific genes that are active in the S phase of the cell cycle
and which are regulated by E2F transcription factors. Rb binds to the activation domain of E2F and
then actively represses the promoter by a mechanism that is poorly understood. Rb
associates with a histone deacetylase, HDAC1, through the Rb 'pocket' domain. Association with the
deacetylase is reduced by naturally occurring mutations in the pocket and by binding of the human
papilloma virus oncoprotein E7. Rb can recruit histone deacetylase to E2F: Rb
cooperates with HDAC1 to repress the E2F-regulated promoter of the gene encoding the cell-cycle
protein cyclin E. Inhibition of histone deacetylase activity by trichostatin A (TSA) inhibits Rb-mediated
repression of a chromosomally integrated E2F-regulated promoter. These results indicate that histone
deacetylases are important for regulating the cell cycle and that active transcriptional repression by Rb
may involve the modification of chromatin structure (Brehn, 1998).
p107 is a member of the pocket family of proteins that includes the retinoblastoma tumor suppressor.
Overexpression of p107 arrests cells in G1, suggesting that it is important for cell cycle control. This growth
suppression is mediated at least in part through the interaction of p107 with a member of the E2F family of cell
cycle transcription factors; this interaction can be disrupted by oncoproteins from DNA tumor viruses
(such as adenovirus E1a) that bind p107. Not only does the binding of p107 to E2F inactivate E2F, but when p107 is tethered to the promoter through binding to E2F it functions as a general
transcriptional repressor. This general repressor activity is also evident when p107 is fused to the DNA
binding domain of Gal4, it could be directly targeted to the promoter in an E2F-independent fashion.
Using p107 mutants, the regions of the protein required for transcriptional repression and cell
growth suppression have been compared. The pocket domain is sufficient for inactivation of E2F, general repressor
activity, and most of the growth suppressor activity. Binding of conserved region 1 from Ela to p107 blocks
interaction with E2F, but it does not affect general repressor activity, demonstrating that binding and
inactivation of E2F and general repressor activity are distinguishable properties of p107. Within the pocket,
two conserved domains, A and B, are sufficient for growth suppression and transcriptional repressor
activity. These two domains are fully functional when they are coexpressed as
separate proteins; it is suggested that the domains may interact at the promoter to form an
active pocket (Starostik, 1996).
Cyclin D1 controls the timing of S phase onset in mammalian cells, acting as a positive regulator
of the transcription factor E2F. Cyclin D1 overexpression leads to the activation of the
dihydrofolate reductase gene promoter, acting through the E2F binding site in the promoter.
P16INK4 represses this interaction; this repression can be released by overexpression of
cdk4. Thus cyclin D1 and its associated kinase have a direct role in cell cycle regulation of E2F
activity and consequently of S phase-specific gene expression. E2F binding sites bind complexes
containing the retinoblastoma protein, while in Rb-deficient cell lines overexpression of cyclin D1
fails to activate E2F-dependent transcription, suggesting that Rb may be involved in promoter
activation (Schulze, 1994).
The retinoblastoma protein family has been implicated in growth control and modulation of the activity of
genes involved in cell proliferation, such as B-myb (see Drosophila Myb oncogene-like). Recent evidence indicates that the product of the B-myb
gene is necessary for the growth and survival of several human and murine cell lines. Upon overexpression,
B-myb induces deregulated cell growth of certain cell lines. B-myb overexpression is able to
induce DNA synthesis in p107 growth-arrested human osteosarcoma cells (SAOS2). p107 might exert its
growth-suppressive activity by regulating B-myb gene transcription. Indeed, p107 down-modulates B-myb
promoter activity and drastically decreases E2F-mediated transactivation. B-myb is able to stimulate the
DNA synthesis of both stably and transiently transfected human glioblastoma cells (T98G). Altogether, these
data provide definitive evidence that the human B-myb protein is involved in growth control of human cells,
and that p107 has a significant role in regulating B-myb gene activity (Sala, 1996).
Endochondral ossification is the process of skeletal bone growth via the formation of a cartilage template that subsequently undergoes mineralization to form trabecular bone. Genetic mutations affecting the proliferation or differentiation of chondrocytes result in skeletal abnormalities. Activating transcription factor-2 (ATF-2) modulates expression of cell cycle regulatory genes in chondrocytes, and mutation of ATF-2 results in a dwarfed phenotype. This study investigated the regulatory role that ATF-2 plays in expression of the pocket proteins, cell cycle regulators important in cellular proliferation and differentiation. The spatial and temporal pattern of pocket protein expression was identified in wild type and mutant growth plates. Expression of retinoblastoma (pRb) mRNA and protein were decreased in ATF-2 mutant primary chondrocytes. pRb mRNA expression was coordinated with chondrogenic differentiation and cell cycle exit in ATDC5 cells. Type X collagen immunohistochemistry was performed to visualize a delay in differentiation in response to loss of ATF-2 signaling. Chondrocyte proliferation was also affected by loss of ATF-2. These studies suggest pRb plays a role in chondrocyte proliferation, differentiation and growth plate development by modulating cell cycle progression. ATF-2 regulates expression of pRb within the developing growth plate, contributing to the skeletal phenotype of ATF-2 mutant mice through the regulation of chondrocyte proliferation and differentiation (Vale-Cruz, 2008).
Targets of Retinoblastoma proteins as regulators of transcription Transcription by RNA polymerase (pol) III is under cell-cycle control, being higher in S and G2 than in G0 and
early G1 phases. Many transformed cell types have elevated pol III activity, presumably to sustain sufficient
protein synthesis for unrestrained growth. The retinoblastoma tumour-suppressor protein restricts cellular
proliferation, and is often found mutated in transformed cells. Rb can repress the
level of transcription from pol III templates both in vitro and vivo. Analysis of Rb-deficient SAOS2 cells and
primary fibroblasts from Rb-/- mice demonstrates elevated levels of pol III activity in the absence of functional
Rb protein. Rb-induced repression of pol III activity is alleviated by mutations in the Rb pocket domain that
occur naturally in tumours, and by viral transforming proteins that bind and inactivate Rb. These results
implicate repression of pol III transcription as a mechanism for Rb-induced growth arrest, and suggest that
restraining protein biosynthesis may be important in the prevention of tumour development (White, 1996).
The histone H1(0)-encoding gene is expressed in vertebrates in differentiating cells during the arrest of proliferation. In the H1(0) promoter, a specific regulatory element, named the H4 box, exhibits features that implicate a role in mediating H1(0) gene expression in response to both differentiation and cell cycle control signals. For instance, within the linker histone gene family, the H4 box is found only in the promoters of differentiation-associated subtypes, suggesting that it is specifically involved in differentiation-dependent expression of these genes. In addition, an element nearly identical to the H4 box is conserved in the promoters of histone H4-encoding genes and is known to be involved in their cell cycle-dependent expression. The transcription factors interacting with the H1(0) H4 box were therefore expected to link differentiation-dependent expression of H1(0) to the cell cycle control machinery. The aim of this work has been to identify such transcription factors and to obtain information concerning the regulatory pathway involved. Interestingly, the cloning strategy led to the isolation of a retinoblastoma protein (RB) partner known as HBP1. HBP1, a high-mobility group box transcription factor, interacts specifically with the H1(0) H4 box and moreover is expressed in a differentiation-dependent manner. HBP1-encoding gene is able to produce different forms of HBP1. Both HBP1 and RB are involved in the activation of H1(0) gene expression. It is therefore proposed that HBP1 mediates a link between the cell cycle control machinery and cell differentiation signals. Through modulating the expression of specific chromatin-associated proteins such as histone H1(0), HBP1 plays a vital role in chromatin remodeling events during the arrest of cell proliferation in differentiating cells (Lemercier, 2000).
The activity of the E2F transcription factor is controlled by physical association with the
retinoblastoma protein (pRB) and two related proteins, p107 and p130. The pRB family
members are thought to control different aspects of E2F activity, but it has been unclear what
the respective functions of these proteins might be. To dissect the specific functions of pRB,
p107, and p130, a study was carried out to determine how the expression of E2F-regulated
genes changes in cultures of primary cells lacking each of these family members. Whereas no
changes were found in the expression of E2F-target genes in cells lacking either p107 or p130,
deregulated expression of E2F targets is seen in cells lacking pRB and in cells lacking both
p107 and p130. Surprisingly, the genes that are deregulated in these two settings are completely
different. cyclin E and p107 are derepressed in pRb mutants. B-myb, cdc2, E2F-1, TS, RRM2
and cyclin A2 are derepressed in p107mutant/p130mutant cells. These findings show that pRB
and p107/p130 indeed provide different functions in E2F regulation and identify target genes that
are dependent on pRB family proteins for their normal expression. Because deregulation of E2F
activity is thought to play an important role in promoting the proliferation of Rb mutant tumor
cells, it is surprising that far more extensive changes in E2F activity are found in p107/p130
double mutants than in Rb mutants. Perhaps, however, the set of E2F targets genes that are misexpressed
in Rb mutants might be more important for cell proliferation than the E2F targets that are
misexpressed in p107/p130 double mutants (Hurford, 1997).
Using the method of differential display (DDRT-PCR) in
combination with nuclear run-on analyses, a number of genes could be detected that are upregulated by
ectopic expression of the Rb gene in Rb-deficient mammary carcinoma cells. Not only could
stimulation of the endogenous mutant Rb gene be detected but also positive regulation of genes coding for diverse classes
of proteins, including the endothelial growth regulator endothelin-1 and two proteoglycans: versican and PG40.
Using a second approach, gene expression was investigated in cell lines established from Rb deficient
heterozygous and homozygous knockout mouse embryos and normal mice. Several genes were detected whose
expression correlates either positively or negatively with the presence of Rb (Rohde, 1996).
A prominent feature of cell differentiation is the initiation and maintenance of an
irreversible cell cycle arrest with the complex involvement of the retinoblastoma (RB)
family (RB, p130, p107). The HBP1 transcriptional repressor has been isolated as a
potential target of the RB family in differentiated cells. By homology, HBP1 is a
sequence-specific HMG transcription factor, of which LEF-1 (Drosophila homolog: Pangolin) is the best-characterized
family member. Several features of HBP1 suggest an intriguing role as a
transcriptional and cell cycle regulator in differentiated cells:
Taken together, the results suggest that HBP1 may represent a unique transcriptional
repressor with a role in initiation and establishment of cell cycle arrest during
differentiation (Tevosian. 1997).
The E2F transcription factors are essential regulators of cell growth in multicellular organisms, controlling the expression of a number of genes whose products are involved in DNA replication and cell proliferation. In Saccharomyces cerevisiae, the MBF and SBF transcription complexes have functions similar to those of E2F proteins in higher eukaryotes, by regulating the timed expression of genes implicated in cell cycle progression and DNA synthesis. The CDC6 gene is a target for MBF and SBF-regulated transcription. S. cerevisiae Cdc6p induces the formation of the prereplication complex and is essential for initiation of DNA replication. Interestingly, the Cdc6p homolog in Schizosaccharomyces pombe, Cdc18p, is regulated by DSC1, the S. pombe homolog of MBF. By cloning the promoter for the human homolog of Cdc6p and Cdc18p, it has been demonstrated that the cell cycle-regulated transcription of this gene is dependent on E2F. In vivo footprinting data demonstrate that the identified E2F sites are occupied in resting cells and in exponentially growing cells, suggesting that E2F is responsible for downregulating the promoter in early phases of the cell cycle and the subsequent upregulation when cells enter S phase. These data also demonstrate that the human CDC6 protein (hCDC6) is essential and limiting for DNA synthesis, since microinjection of an anti-CDC6 rabbit antiserum blocks DNA synthesis and CDC6 cooperates with cyclin E to induce entry into S phase in cotransfection experiments. Furthermore, E2F is sufficient to induce expression of the endogenous CDC6 gene even in the absence of de novo protein synthesis. In conclusion, these results provide a direct link between regulated progression through G1 controlled by the pRB pathway and the expression of proteins essential for the initiation of DNA replication (Hateboer 1998).
The tumor suppressor RB regulates diverse cellular processes such as G1/S transition, cell
differentiation, and cell survival. Indeed, Rb-knockout mice exhibit phenotypes including ectopic mitosis,
defective differentiation, and extensive apoptosis in the neurons. Using differential display, a novel
gene, Rig-1, has been isolated based on its elevated expression in the hindbrain and spinal cord of
Rb-knockout embryos. The longest open reading frame of Rig-1 encodes a polypeptide that consists of
a putative extracellular segment with five immunoglobulin-like domains and three fibronectin III-like
domains, a putative transmembrane domain, and a distinct intracellular segment. The Rig-1 sequence
is 40% identical to the recently identified Roundabout protein. Consistent with the predicted
transmembrane nature of the protein, Rig-1 protein is present in the membranous fraction. Antisera
raised against the putative extracellular and intracellular segments of Rig-1 react with an
approximately 210-kDa protein in mouse embryonic CNS. Rig-1 mRNA is transiently expressed in
the embryonic hindbrain and spinal cord. Elevated levels of Rig-1 mRNA and protein were found in
Rb-/- embryos. Ectopic expression of a transmembrane form of Rig-1, but not the secreted form,
promotes neuronal cell entrance to S phase and represses the expression of a marker of differentiated
neuron, Talpha1 tubulin. Thus Rig-1, a possible distant relative of Roundabout, may mediate some of the
pleiotropic roles of RB in the developing neurons (Yuan, 1999).
During endochondral bone development, both the chondrogenic differentiation of mesenchyme and the hypertrophic differentiation of chondrocytes coincide with the proliferative arrest of the differentiating cells. However, the mechanisms by which differentiation is coordinated with cell cycle withdrawal, and the importance of this coordination for skeletal development, have not been defined. Through analysis of mice lacking the pRB-related p107 and p130 proteins, it was found that p107 is required in prechondrogenic condensations for cell cycle withdrawal and for quantitatively normal alpha1(II) collagen expression. Remarkably, the p107-dependent proliferative arrest of mesenchymal cells is not needed for qualitative changes that are associated with chondrogenic differentiation, including production of Alcian blue-staining matrix and expression of the collagen IIB isoform. In chondrocytes, both p107 and p130 contribute to cell cycle exit, and p107 and p130 loss is accompanied by deregulated proliferation, reduced expression of Cbfa1, and reduced expression of Cbfa1-dependent genes that are associated with hypertrophic differentiation. Moreover, Cbfa1 is detected, and hypertrophic differentiation occurs, only in chondrocytes that have undergone or are undergoing a proliferative arrest. The results suggest that Cbfa1 links a p107- and p130-mediated cell cycle arrest to chondrocyte terminal differentiation (Rossi, 2002).
Differentiation is a coordinated process of irreversible cell cycle exit and tissue-specific gene expression. To probe the functions of the retinoblastoma protein (RB) family in cell differentiation, HBP1 was isolated as a specific target of RB and p130. HBP1 was a transcriptional repressor and a cell cycle inhibitor. The induction of HBP1, RB, and p130 upon differentiation in the muscle C2C12 cells suggested a coordinated role. The expression of HBP1 is shown to unexpectedly block muscle cell differentiation without interfering with cell cycle exit. Moreover, the expression of MyoD and myogenin, but not Myf5, is inhibited in HBP1-expressing cells. HBP1 inhibits transcriptional activation by the MyoD family members. The inhibition of MyoD family function by HBP1 requires binding to RB and/or p130. Since Myf5 might function upstream of MyoD, these data suggested that HBP1 probably blocks differentiation by disrupting Myf5 function, thus preventing expression of MyoD and myogenin. Consistent with this, the expression of each MyoD family member reverses the inhibition of differentiation by HBP1. Further investigation implicated the relative ratio of RB to HBP1 as a determinant of whether cell cycle exit or full differentiation occurred. At a low RB/HBP1 ratio, cell cycle exit occurs but there is no tissue-specific gene expression. At elevated RB/HBP1 ratios full differentiation occurs. Similar changes in the RB/HBP1 ratio have been observed in normal C2 differentiation. Thus, it is postulated that the relative ratio of RB to HBP1 may be one signal for activation of the MyoD family. A model is proposed in which a checkpoint of positive and negative regulation may coordinate cell cycle exit with MyoD family activation to give fidelity and progression in differentiation (Shih, 1998).
Retinoblastoma (Rb)/E2F complexes repress expression of many genes
important for G1-to-S transition, but also appear to regulate
gene expression at other stages of the cell cycle. In C.
elegans, lin-35/Rb and other synthetic Multivulva (SynMuv)
group B genes function redundantly with other sets of genes to
regulate G1/S progression, vulval and pharyngeal
differentiation, and other unknown processes required for viabilty.
lin-35/Rb, efl-1/E2F, and other
SynMuv B genes negatively regulate a component of the
anaphase-promoting complex or cyclosome (APC/C). The APC/C is a
multisubunit complex that promotes metaphase-to-anaphase progression
and G1 arrest by targeting different substrates for
ubiquitination and proteasome-mediated destruction. The C.
elegans APC/C gene mat-3/APC8 has been defined by
temperature-sensitive embryonic lethal alleles that strongly affect
germline meiosis and mitosis but only weakly affect somatic
development. Severe nonconditional mat-3 alleles
and a hypomorphic viable allele (ku233) are described, all of which affect
postembryonic cell divisions including those of the vulval lineage.
The ku233 lesion is located outside of the mat-3 coding
region and reduces mat-3 mRNA expression. Loss-of-function
alleles of lin-35/Rb and other SynMuv B genes suppress
mat-3(ku233) defects by restoring mat-3 mRNA to wild-type
levels. Therefore, Rb/E2F complexes appear to repress mat-3 expression (Garbe, 2004).
Using genome-wide analysis of transcription factor occupancy, this study investigated the mechanisms underlying three mammalian growth arrest pathways that require the pRB tumor suppressor family. It was found that p130 and E2F4 cooperatively repress a common set of genes under each growth arrest condition and showed that growth arrest is achieved through repression of a core set of genes involved not only in cell cycle control but also mitochondrial biogenesis and metabolism. Motif-finding algorithms predicted the existence of nuclear respiratory factor-1 (NRF1) binding sites in E2F target promoters, and genome-wide factor binding analysis confirmed these predictions. NRF1 (Drosophila homolog: Erect wing), a factor known to regulate expression of genes involved in mitochondrial function, is a coregulator of a large number of E2F target genes. These studies provide insights into E2F regulatory circuitry, suggest how factor occupancy can predict the expression signature of a given target gene, and reveal pathways deregulated in human tumors (Cam, 2003).
To understand cell cycle control mechanisms in early development and how they change during differentiation, embryonic stem cells were used to model embryonic events. The results demonstrate that as pluripotent cells differentiate, the length of G(1) phase increases substantially. At the molecular level, this is associated with a significant change in the size of active cyclin-dependent kinase (Cdk) complexes, the establishment of cell cycle-regulated Cdk2 activity and the activation of a functional Rb-E2F pathway. The switch from constitutive to cell cycle-dependent Cdk2 activity coincides with temporal changes in cyclin A2 and E1 protein levels during the cell cycle. Transcriptional mechanisms underpin the down-regulation of cyclin levels and the establishment of their periodicity during differentiation. As pluripotent cells differentiate and pRb/p107 kinase activities become cell cycle dependent, the E2F-pRb pathway is activated and imposes cell cycle-regulated transcriptional control on E2F target genes, such as cyclin E1. These results suggest the existence of a feedback loop where Cdk2 controls its own activity through regulation of cyclin E1 transcription. Changes in rates of cell division, cell cycle structure and the establishment of cell cycle-regulated Cdk2 activity can therefore be explained by activation of the E2F-pRb pathway (White, 2005).
Retinoblastoma proteins: development and differentiation The tumor suppressor Rb and the NuRD (nucleosome remodeling and histone deacetylation) complex have been implicated in transcriptional repression during cell cycle progression and cell fate specification. The Rb/E2F complex physically interacts with and thus recruits the NuRD complex to actively repress transcription. C. elegans counterparts of Rb, E2F/DP, and some NuRD complex components appear to function in a common class B synthetic Multivulva (synMuv) pathway to antagonize RTK/Ras signaling during vulval fate specification. Therefore, it has been suggested that they function together in a single complex to repress vulva-specific gene transcription. However, little is known about the in vivo interactions between these class B synMuv genes and their relationships with other pathways in specific cellular processes during vulval development. It has been shown that C. elegans Rb/E2F and NuRD complexes antagonize Ras activity by controlling a lin-39 Hox-mediated cell fusion event that regulates the competence of vulval cells. Interestingly, Rb/E2F and NuRD complexes exhibit very different genetic properties. While the NuRD complex negatively regulates lin-39 Hox activity, likely by downregulating its expression, RB/E2F appears to play dual roles in regulating lin-39: a negative role in controlling its activity and a previously uncharacterized positive role in regulating its expression (Chen, 2001).
The retinoblastoma gene product has been implicated in the regulation of
multiple cellular and developmental processes, including a well-defined role
in the control of cell cycle progression. The Caenorhabditis elegans retinoblastoma protein homolog, LIN-35, is also a key regulator of cell cycle
entry and, as shown by studies of synthetic multivulval genes, plays an
important role in the determination of vulval cell fates. An
additional and unexpected function has been demonstrated for lin-35 in organ morphogenesis.
Using a genetic approach to isolate lin-35 synthetic-lethal
mutations, redundant roles have been identified for lin-35 and
ubc-18, a gene that encodes an E2 ubiquitin-conjugating enzyme
closely related to human UBCH7. lin-35 and ubc-18 cooperate
to control one or more steps during pharyngeal morphogenesis. Based on genetic
and phenotypic analyses, this role for lin-35 in pharyngeal
morphogenesis appears to be distinct from its cell cycle-related functions. lin-35 and ubc-18 may act in concert to regulate the levels of one or more critical targets during C. elegans development (Fay, 2003).
Studies have shown that a synthetic multivulva phenotype results from mutations in genes that antagonize the ras-mediated intercellular signaling system responsible for vulval induction in Caenorhabditis elegans. Synthetic multivulva mutations define two classes of genes, A and B, and a mutation in a gene of each class is required to produce the multivulva phenotype. The ectopic vulval tissue in multivulva animals is generated by vulval precursor cells that in the wild type do not generate vulval tissue. One of the class B synthetic multivulva genes, lin-35, encodes a protein similar to the retinoblastoma (Rb) protein. This paper describes the isolation and characterization of 50 synthetic multivulva mutations, the identification of new components of both the class A and class B lin-35 Rb pathways, and the cloning of lin-52, a class B gene that may have a conserved role in Rb-mediated signaling (Thomas, 2003).
lin-35, a member of the class B synMuv pathway, encodes a protein similar to the mammalian tumor suppressor pRb. Other genes with class B synMuv activity encode DP (dpl-1), E2F (efl-1), RbAp48 (lin-53), histone deacetylase (hda-1), and HP1 family proteins (hpl-2). In addition to their role in vulval development, many class B genes have been shown to regulate G1-to-S phase progression in the cell cycle. These genes include dpl-1, efl-1, lin-9, lin-15B, lin-35, and lin-36; other class B genes, hda-1, let-418, lin-37, lin-53, and tam-1, do not appear to be involved in cell cycle control. Even among the subgroup of class B genes that are involved in cell cycle control, lin-35 and lin-15B have been shown to have partially nonoverlapping functions. These results suggest that either the class B synMuv genes act differently in vulval development and cell cycle control or more subtle differences in their roles in vulval development have not been detected (Thomas, 2003).
Mammalian homologs of some of these class B synMuv proteins are known to functionally, and in some cases physically, interact with pRb. These and other parallels indicate that the class B synMuv pathway is an analog of Rb pathways in other organisms, particularly those pathways in which Rb is involved in chromatin remodeling. Consequently, additional class B synMuv genes may have homologs with analogous functions in other organisms. One such gene is lin-52. lin-52 encodes a small protein, portions of which are conserved in similarly small proteins predicted by the human, mouse, and Drosophila genome sequences. The further analysis of lin-52 and other synMuv genes should help elucidate the mechanisms of action of Rb-like proteins and their regulators and effectors. The determination of how the class B synMuv genes negatively regulate the vulval induction process should provide insight concerning the antagonistic actions of Rb-mediated and Ras-mediated pathways (Thomas, 2003).
In screens for genetic modifiers of lin-35/Rb, the C. elegans retinoblastoma protein homolog, a mutation in xnp-1 (Drosophila homolog: XNP)) was identified. Mutations in xnp-1, including a presumed null allele, are viable and, in general, appear indistinguishable from the wild type. In contrast, xnp-1 lin-35 double mutants are typically sterile and exhibit severe defects in gonadal development. Analyses of the abnormal gonads indicate a defect in the lineages that generate cells of the sheath and spermatheca. xnp-1 encodes the C. elegans homolog of ATR-X, a human disease gene associated with severe forms of mental retardation and urogenital developmental defects. xnp-1/ATR-X is a member of the Swi2/Snf2 family of ATP-dependent DEAD/DEAH box helicases, which function in nucleosome remodeling and transcriptional regulation. Expression of an xnp-1::GFP promoter fusion is detected throughout C. elegans development in several cell types including neurons and cells of the somatic gonad. These findings demonstrate a new biological role for Rb family members in somatic gonad development and implicate lin-35 in the execution of multiple cell fates in C. elegans. In addition, these results suggest a possible conserved function for xnp-1/ATR-X in gonadal development across species (Bender, 2004).
xnp-1 encodes the C. elegans homolog of the human ATR-X gene, a member of the Swi/Snf superfamily of ATP-dependent chromatin remodeling helicases. Mutations in human ATR-X lead to severe mental retardation as well as many secondary anomalies including urogenital defects in approximately 80% of ATR-X patients. The mutation identified in xnp-1(fd2) mutants leads to a substitution (R → K) of a highly conserved arginine at amino acid position 1130 (corresponding to human ATR-X position 2197) in the C terminus of the peptide. Interestingly, an analysis of molecular lesions from ATR-X patients indicates that mutations affecting the C-terminal region of the ATR-X protein are often associated with the most severe forms of urogenital defects. In contrast to humans, however, expression of the gonadal defect in C. elegans is dependent upon the coordinate inactivation of class B SynMuv genes such as lin-35. Thus, in C. elegans, lin-35 and xnp-1 function redundantly in the control of gonadal development (Bender, 2004).
Studies on Swi/Snf family members have indicated their importance in many diverse biological processes, most of which can be linked mechanistically to the control of nucleosome remodeling and gene expression. The precise level of control exerted by Swi/Snf members has been reported to range from gene-specific to global and appears to depend on several factors including the particular Swi/Snf complex involved, associations with various binding partners, genetic background, and cell cycle phase. Moreover, the effects exerted by Swi/Snf complexes on individual target genes can be either repressive or activating; the outcome most likely depends on the influence of other bound regulators such as histone modifying enzymes (Bender, 2004).
An obvious model to account for the functional redundancy of LIN-35 and XNP-1 is that both proteins share in common one or more transcriptional targets. Thus, in single-mutant backgrounds, sufficient regulation of the target can be brought about through the intact pathway acting alone. However, in double mutants, two means of regulation are missing and the shared target (or targets) may become grossly deregulated. Based on precedent from studies on the transcriptional effects of Rb family members, as well as other lin-35 synthetic mutants, the actions of both LIN-35 and XNP-1 on the shared target(s) are found to be repressive in nature (Bender, 2004).
As to how many common targets might be affected in the double mutants is an open question. Many studies analyzing the transcriptional targets of individual Swi/Snf complexes have been carried out, and they suggest that Swi/Snf proteins may regulate the expression of sizeable numbers (on the order of several hundred to several thousand) of physically disparate target genes. Likewise, many recent reports seeking to determine the transcriptional targets of Rb family members suggest that Rb family members may collectively regulate the expression of up to several hundred genes. Although such studies may be significantly compromised by issues such as cell- and tissue-type specific differences, genetic redundancy, and indirect effects, they provide at least some basis for estimating the number of genes that may be co-regulated by LIN-35 and XNP-1 in C. elegans. Namely, assuming a nonbiased set of 250 independent targets for both LIN-35 and XNP-1, as well as a genome consisting of 17,000 genes, it would be predicted that on average, 3.7 genes would be regulated by both factors. Although such calculations are highly speculative, they do suggest that the observed phenotype of xnp-1 lin-35 mutants could be due to the missexpression of a relatively small number of genes, perhaps even a single common target. Identification of such targets, either by genetics or other means, will await further studies (Bender, 2004).
Mutations in the XNP/ATR-X gene cause several X-linked mental retardation syndromes in humans (see Drosophila Mei-41). The XNP/ATR-X gene encodes a DNA-helicase belonging to the SNF2 family. It has been proposed that XNP/ATR-X might be involved in chromatin remodelling. The lack of a mouse model for the ATR-X syndrome has, however, hampered functional studies of XNP/ATR-X. C. elegans possesses one homolog of the XNP/ATR-X gene, named xnp-1. By analysing a deletion mutant, it has been shown that xnp-1 is required for the development of the embryo and the somatic gonad. Moreover, abrogation of xnp-1 function in combination with inactivation of genes of the NuRD complex, as well as lin-35/Rb and hpl-2/HP1 leads to a stereotyped block of larval development with a cessation of growth but not of cell division. A specific function for xnp-1 together with lin-35 or hpl-2 has been demonstrated in the control of transgene expression, a process known to be dependent on chromatin remodelling. This study thus demonstrates that in vivo XNP-1 acts in association with RB, HP1 and the NuRD complex during development (Cardoso, 2005).
The class A, B and C synthetic multivulva (synMuv) genes act redundantly to
negatively regulate the expression of vulval cell fates in Caenorhabditis
elegans. The class B and C synMuv proteins include homologs of proteins
that modulate chromatin and influence transcription in other organisms similar
to members of the Myb-MuvB/dREAM, NuRD and Tip60/NuA4 complexes. To determine
how these chromatin-remodeling activities negatively regulate the vulval
cell-fate decision, a suppressor of the synMuv phenotype was isolated and it was found that the suppressor gene encodes the C. elegans homolog of
Drosophila melanogaster ISWI. The C. elegans ISW-1 protein
likely acts as part of a Nucleosome Remodeling Factor (NURF) complex with
NURF-1, a nematode ortholog of NURF301, to promote the synMuv phenotype.
isw-1 and nurf-1 mutations suppress both the synMuv
phenotype and the multivulva phenotype caused by overactivation of the Ras
pathway. These data suggest that a NURF-like complex promotes the expression of
vulval cell fates by antagonizing the transcriptional and chromatin-remodeling
activities of complexes similar to Myb-MuvB/dREAM, NuRD and Tip60/NuA4.
Because the phenotypes caused by a null mutation in the tumor-suppressor and
class B synMuv gene lin-35 Rb and a gain-of-function mutation in
let-60 Ras are suppressed by reduction of isw-1 function,
NURF complex proteins might be effective targets for cancer therapy (Andersen, 2006; full text of article).
To determine the roles of the retinoblastoma gene (Rb-1) in skeletal muscle differentiation in vitro, C2 myoblasts have been isolated; they stably express an antisense RNA directed to the 3'-untranslated region (3'UTR) of Rb-1 mRNA. The levels of Rb-1 mRNA and its product (pRb) in the clones transfected with antisense Rb are markedly decreased to 25%-35% of those in the control clones. Cell growth of the clones is accelerated, especially in medium containing low concentrations of fetal calf serum. Even in differentiation medium with a low mitogen level, the antisense Rb clones proliferate as single-nucleated myoblast-like cells without expressing the sarcometric myosin heavy chain protein, whereas the control clones form highly multinucleated myotubes after 4 days of culture under the same conditions. Under this condition, the levels of Rb-1 mRNA and pRb in the antisense Rb clones are 30%-50% of those in the control clone, and no divergent increase in the Rb-family protein p107 expression is observed. This inhibited differentiation is abrogated by reintroducing to the clones transfected with antisense Rb, with expression vectors for the sense 3'UTR of Rb-1 mRNA, or with Rb-1 mRNA lacking its 3'UTR. In the antisense Rb clone cultured in differentiation medium, the amounts of MyoD and myogenin mRNA are markedly decreased on the 2nd day of culture in the differentiation medium. The expression of cell cycle-promoting genes including E2F-1 and cyclin D1 are up-regulated throughout the experiment. These results demonstrate that pRb is essential for the completion of terminal differentiation in C2 cells (Kobayashi, 1998).
To define a mechanism by which retinoblastoma protein (Rb) functions in cellular differentiation, primary fibroblasts from the lung buds of wild-type (RB+/+) and null-mutant (RB-/-) mouse embryos have been examined. In culture, the RB+/+ fibroblasts differentiate into fat-storing cells, either
spontaneously or in response to hormonal induction; otherwise syngenic RB-/- fibroblasts cultured in identical conditions do not. Ectopic expression of normal Rb, but not Rb with a single point mutation, enables RB-/- fibroblasts to differentiate into adipocytes. Rb appears in murine fibroblasts
to activate CCAAT/enhancer-binding proteins (C/EBPs), a family of transcription factors crucial for adipocyte differentiation. Physical interaction between Rb and C/EBPs, demonstrated by reciprocal coimmunoprecipitation, occurs only in differentiating cells. Wild-type Rb also enhances the binding of C/EBP to cognate DNA sequences in vitro and the transactivation of a C/EBPß-responsive promoter in cells. Taken together, these observations establish a direct and
positive role for Rb in terminal differentiation. Such a role contrasts with the function of Rb in arresting cell cycle progression in G1 by negative regulation of other transcription factors, like E2F-1 (Chen, 1996).
Prior to death at embryonic day 14.5, mice deficient for the RB gene (RB-/-), show increased cell death in
all tissues that normally express RB1: the nervous system, liver, lens, and skeletal muscle precursor cells.
Transgenic mice (RBlox) were generated that express low levels of pRb, driven by an RB1 minigene.
RBlox/RB-/- mutant fetuses die at birth with specific skeletal muscle defects, including increased cell
death prior to myoblast fusion, shorter myotubes with fewer myofibrils, reduced muscle fibers,
accumulation of elongated nuclei that actively synthesize DNA within the myotubes, and reduction in
expression of the late muscle-specific genes MCK and MRF4. Thus, insufficient pRb results in failure of
myogenesis in vivo, and is manifest in two ways: (1) the massive apoptosis of myoblasts implicates a positive role for
pRb in cell survival, and (2) surviving myotubes fail to develop normally and accumulate large
polyploid nuclei, implicating pRb in permanent withdrawal from the cell cycle. These results demonstrate
a role for pRb during terminal differentiation of skeletal muscles in vivo and place pRb at a nodal point
that controls cell proliferation, differentiation, and death (Zacksenhaus, 1996).
The severe neurological deficit in embryos carrying null mutations for the retinoblastoma (Rb) gene
suggests that Rb plays a crucial role in neurogenesis. While developing neurons undergo apoptosis in
vivo neural precursor cells cultured from Rb-deficient embryos appear to differentiate and survive. To
determine whether Rb is an essential regulator of the intrinsic pathway modulating terminal mitosis, the terminal differentiation of primary cortical progenitor cells and bFGF-dependent neural
stem cells derived from Rb-deficient mice was examined. Although Rb -/- neural precursor cells are able to
differentiate in vitro, these cells exhibit a significant delay in terminal mitosis relative to
wild-type cells. Furthermore, Rb -/- cells surviving in vitro exhibit an upregulation of p107 that is found
in complexes with E2F3. This suggests that p107 may partially compensate for the loss of Rb in neural
precursor cells. Functional ablation of Rb family proteins by adenovirus-mediated delivery of an E1A
N-terminal mutant results in apoptosis in Rb-deficient cells, consistent with the interpretation that other
Rb family proteins may facilitate differentiation and survival. While p107 is upregulated and interacts
with the putative Rb target E2F3 in neural precursor cells, these results indicate that it clearly cannot
restore normal E2F regulation. Rb-deficient cells exhibit a significant enhancement of E2F 1 and 3
activity throughout differentiation concomitant with the aberrant expression of E2F-inducible genes. In
these studies it is shown that Rb is essential for the regulation of E2F 1 and 3 activity as well as the
onset of terminal mitosis in neural precursor cells (Callaghan, 1999).
During skeletal muscle differentiation, a subset of
myoblasts remains quiescent and undifferentiated but
retains the capacity to self-renew and give rise to
differentiating myoblasts: this sub-population of
muscle cells has been termed 'reserve cells'. In
order to characterise genes that can regulate the ratio
between reserve cells and differentiating myoblasts, members of the retinoblastoma tumor
suppressor family have been examined. Although pRb and
p107 positively regulate muscle cell differentiation, the role of p130 in muscle cells remains unknown. p130 (protein and mRNA), but
neither pRb nor p107, preferentially accumulates during
muscle differentiation in reserve cells. Also, p130 is the
major Rb-family protein present in E2F complexes in
this sub-population of cells. Although forced expression
of either p130 or pRb in mouse C2 myoblasts efficiently
blocks cell cycle progression, only p130 inhibits the
differentiation program. Furthermore, muscle cells
overexpressing p130 have reduced levels of the muscle-promoting
factor MyoD. In addition, p130 represses the
transactivation capacity of MyoD, an effect abolished by
co-transfection of pRb. Thus, it is proposed that p130, by
blocking cell cycle progression and differentiation,
could be part of a specific pathway that defines a pool
of reserve cells during terminal differentiation (Carnac, 2000).
The p53 oncosuppressor protein regulates cell cycle checkpoints and apoptosis, but increasing evidence also indicates its involvement in differentiation and development. In the presence of differentiation-promoting stimuli, p53-defective myoblasts exit from the cell cycle but do not differentiate into myocytes and myotubes. To identify the pathways through which p53 contributes to skeletal muscle differentiation, the expression was examined of a series of genes regulated during myogenesis in parental and dominant-negative p53 (dnp53)-expressing C2C12 myoblasts. In dnp53-expressing C2C12 cells, as well as in p53 minus primary myoblasts, pRb is hypophosphorylated and proliferation stops. However, these cells do not upregulate pRb and have reduced MyoD activity. The transduction of exogenous p53 or Rb genes in p53-defective myoblasts rescues MyoD activity and differentiation potential. Additionally, in vivo studies on the Rb promoter demonstrate that p53 regulates the
Rb gene expression at transcriptional level through a p53-binding site. Therefore, p53 regulates myoblast differentiation by means of pRb without affecting its cell cycle-related functions (Porrello, 2000).
In physiological proliferating conditions, p53-impaired myoblasts did not show any modification of the Rb gene expression. These observations are consistent with the notion that p53 is not involved in cell cycle control in normal proliferating conditions. In contrast, it is well known that different types of stressing stimuli promote p53 activation. In this type of situation, p53 is known to promote pRb hypophosphorylation and inhibition of DNA synthesis through the transcriptional induction of p21Waf1/Cip1. Indeed, compared with the parental cells, C2-dnp53 cells do not arrest in the G1 phase of the cell cycle in response to doxorubicin-induced DNA damage. Together with the findings obtained in differentiating conditions, these results indicate the presence of two different types of p53-dependent regulation of pRb. One operates through p21Waf1/Cip1 transcription, and the other through direct Rb transcription. These observations are consistent with the emerging idea that p53 regulates transcription of different genes, depending on the type of stimuli that provoked its activation. Interestingly, the existence of a positively regulated p53-binding site on the Rb promoter has been known for several years, but no transcriptional induction of the Rb gene was found in apoptotic or growth-arresting situations, so far. These results reveal the existence of a physiological condition in which p53 directly transactivates the Rb gene (Porrello, 2000)
The retinoblastoma tumor suxzppressor (Rb) plays an important role in the regulation of cell cycle progression and terminal differentiation of
many cell types. Rb-/- mouse embryos die at midgestation with defects in cell cycle regulation, control of apoptosis and terminal
differentiation. However, chimeric mice composed of wild-type and Rb-deficient cells are viable and show minor abnormalities. To
determine the role of Rb in development more precisely, chimeric embryos and adults made with marked Rb-/- cells were analyzed. Like
their germline Rb-/- counterparts, brains of midgestation chimeric embryos exhibit extensive ectopic S-phase entry. In Rb-mutants, this is accompanied by widespread apoptosis. However, in chimeras, the majority of Rb-deficient cells survive and differentiate into neuronal fates. Rescue of Rb-/- neurons in the presence of wild-type cells occurs after induction of the p53 pathway and leads to accumulation of cells with 4n DNA content. Therefore, the role of Rb during development can be divided into a cell-autonomous function in exit from the cell cycle and a non-cell-autonomous role in the suppression of apoptosis and induction of differentiation (Lipinski, 2001).
In addition to its role in negative regulation of cell cycle entry and apoptosis, pRB has been proposed to promote terminal differentiation of many cell types, including myocytes, adipocytes and neurons. In the CNS, increased levels of pRB activity in wild type and onset of the cell cycle defect in germline Rb-/- and chimeric embryos correlate with the initiation of cell cycle exit of neuronal precursor cells, suggesting that this is the cell population affected by the loss of Rb. In addition, although expression of early neuronal differentiation markers is correctly initiated in Rb-/- embryos, it quickly declines and fully differentiated neurons are never observed. In vitro, functional ablation of the pRB-family proteins by expression of viral oncoproteins in differentiating cortical progenitor cells prevents exit from the cell cycle and leads to death.
Nevertheless, Rb-/- chimeric adults have normal overall brain architecture, and Rb-deficient cells in chimeric CNS have normal neuronal or glial morphology.
Therefore, any requirement for pRB function in neuronal differentiation and survival does not appear to be cell autonomous. Given the increased G2 cell population present in chimeric brains, it is possible that at least some of the differentiated Rb-/- neurons are arrested in G2 instead of the G1-phase of the cell cycle. This would suggest that G2 arrest, with 4n DNA content, is compatible with neuronal differentiation. In addition, pRB has been proposed to coordinate the initiation of the differentiation program with terminal cell cycle withdrawal. In chimeric embryos, initiation of expression of neuronal differentiation markers concurrent with S-phase progression of Rb-/- cells is observed, suggesting that in Rb-deficient neuronal precursors, induction of the neuronal differentiation program might occur without prior exit from the cell cycle (Lipinski, 2001).
Within the chimeric CNS, the only exception to the differentiation rescue seemed to be the Rb-/- Purkinje neurons, which were abnormally enlarged and showed
nuclear pleomorphism. It has been reported that inactivation of the Rb-family proteins by expression of viral oncoproteins leads to cell cycle re-entry and death of mature Purkinje cells, but not of cortical neurons. The findings support the notion that Purkinje neurons are uniquely dependent on the presence of functional pRB for normal development in a cell-autonomous fashion. Another tissue that appears to require the presence of functional pRB in a cell-autonomous manner is the ocular lens. No suppression of either cell cycle defect or cell death is seen in the lens, and adult Rb-/- chimeras develop bilateral cataracts. These findings suggest that pRB is required in a cell-autonomous manner for cell cycle regulation in all tissue types, while the requirement for suppression of apoptosis and induction of differentiation is either cell-autonomous or non-cell-autonomous, depending on the tissue type involved (Lipinski, 2001).
Although the underlying molecular mechanisms remain to be elucidated, in the CNS of Rb-/- chimeras, cells expressing wild-type pRB are able to rescue their Rb-deficient neighbors from apoptosis and possibly cause them to arrest in the G2-phase of the cell cycle. This suggests that cell-cell interactions might play a much larger role in the regulation of aberrant cell cycle and cell death than previously recognized. Although the data are restricted to the study of pRB in a developmental setting, similar interactions between wild-type and Rb-deficient cells might be expected to occur during tumorigenesis, where a mixture of cells with different genotypes is also present. Thus, tumor development and progression could be affected by the responsiveness of tumor cells mutant in the pRB pathway to factors produced by genetically normal cells (Lipinski, 2001).
The incidence of osteosarcoma is increased 500-fold in patients who inherit mutations in the RB gene. To understand why the retinoblastoma protein (pRb) is specifically targeted in osteosarcoma, its function in osteogenesis was studied. Loss of pRb but not p107 or p130 blocks late osteoblast differentiation. pRb physically interacts with the osteoblast transcription factor, CBFA1, and associates with osteoblast-specific promoters in vivo in a CBFA1-dependent fashion. Association of pRb with CBFA1 and promoter sequences results in synergistic transactivation of an osteoblast-specific reporter. This transactivation function is lost in tumor-derived pRb mutants, underscoring a potential role in tumor suppression. Thus, pRb functions as a direct transcriptional coactivator promoting osteoblast differentiation, which may contribute to the targeting of pRb in osteosarcoma (Thomas, 2001).
When preadipocytes reenter the cell cycle, Peroxisome proliferator-activated receptor gamma (PPARgamma) expression is induced, coincident with an increase in DNA synthesis, suggesting the involvement of the E2F family of cell cycle regulators. E2F1 induces PPARgamma transcription during clonal expansion, whereas E2F4 represses PPARgamma expression during terminal adipocyte differentiation. Using a combination of in vivo experiments with knockout and chimeric animals and in vitro experiments, it has been demonstrated that the absence of E2F1 impairs,
whereas depletion of E2F4 stimulates, adipogenesis. E2Fs hence represent the link between proliferative signaling pathways, triggering clonal expansion, and terminal adipocyte differentiation through regulation of PPARgamma expression. This underscores the complex role of the E2F protein family in the control of both cell proliferation and differentiation (Fajas, 2002a).
The E2F and pRB family members appear to participate in the regulation of cell cycle events that are required for adipogenesis. In growth-arrested preadipocytes, E2F4 and E2F5 are complexed with p130, leading to repression of its target genes. Upon reentry into cell cycle of these growth-arrested preadipocytes, p130, as well as the other members of the retinoblastoma family, is phosphorylated by the cyclin/cdk holoenzymes, releasing the E2F complex, resulting in the activation of the E2F target genes. After several rounds of DNA synthesis, the cyclin-dependent kinase inhibitors, such as p21, p27, and p18, are induced, and they mediate cell cycle exit and maintain the irreversible growth arrest characteristic of terminal adipocyte differentiation. PPARgamma and C/EBPalpha have been shown to contribute to this permanent cell cycle exit by inhibiting the E2F DNA binding activity and upregulating the levels of p21, respectively. There is also evidence that pRB plays a positive role in adipocyte differentiation through association and activation of C/EBPalpha. In this study, it is shown that the E2F proteins play a direct role in the regulation of early adipocyte differentiation. E2F1 and 3 trigger the expression of PPARgamma during the early stages of adipogenesis, whereas E2F4 represses expression of PPARgamma at the terminal stage of adipocyte differentiation (Fajas, 2002a).
Correct cell cycle regulation and terminal mitosis are critical for nervous system development. The retinoblastoma (Rb) protein is a key regulator of these processes: Rb-/- embryos die by E15.5, exhibiting gross hematopoietic and neurological defects. The extensive apoptosis in Rb-/- embryos has been attributed to aberrant S phase entry resulting in conflicting growth control signals in differentiating cells. To assess the role of Rb in cortical development in the absence of other embryonic defects, mice with telencephalon-specific Rb deletions were examined. Animals carrying a floxed Rb allele were interbred with mice with cre recombinase expressed by virtue of cre having been knocked into the Foxg1 locus. This results in a deletion of Rb from cre expressing cells. Unlike germline knockouts, mice specifically deleted for Rb in the developing telencephalon survive until birth. In these mutants, Rb-/- progenitor cells divide ectopically, but are able to survive and differentiate. Mutant brains exhibit enhanced cellularity due to increased proliferation of neuroblasts. These studies demonstrate that: (1) cell cycle deregulation during differentiation does not necessitate apoptosis; (2) Rb-deficient mutants exhibit enhanced neuroblast proliferation, and (3) terminal mitosis may not be required to initiate differentiation (Ferguson, 2002).
The involvement of the retinoblastoma gene product (Rb) and its family members (p107 and p130) in cell cycle exit and terminal differentiation of neural precursor cells has been demonstrated in vitro. To investigate the roles of Rb and p107 in growth, differentiation and apoptosis in the developing and mature cerebellum, Rb was selectively inactivated either alone or in combination with p107 in cerebellar precursor cells or in Purkinje cells. In mouse models, it is shown that (1) Rb is required for differentiation, cell cycle exit and survival of granule cell precursors; (2) p107 can not fully compensate for the loss of Rb function in granule cells; (3) Rb and p107 are not required for differentiation and survival of Purkinje cells during embryonic and early postnatal development; (4) Rb function in
Purkinje cells is cell autonomous, and (5) loss of Rb deficient CNS precursor cells is mediated by p53-independent apoptosis (Marino, 2003).
pRb, p107 and p130 are important regulators of cell cycle and have extensive overlapping functions; however, only Rb has been shown to be a bone fide tumor suppressor. Defining the overlapping versus distinct pocket protein functions is therefore an important step to understanding the unique role of Rb. Using lung as a model, the present studies demonstrate that pocket proteins are important not only in regulating cell cycle and survival but also in cell lineage specification. An inducible lung-specific Rb knockout strategy was used to demonstrate that Rb is specifically required for restricting neuroendocrine cell fate despite functional compensation for Rb deficiency in other cell types. Ablation of total Rb family function results in opposing effects in specification along distinct cell lineages, providing evidence that pocket proteins inhibit neuroendocrine cell fate while being required for differentiation in other cell types. These findings identify a novel role for pocket proteins in cell fate determination, and establish a unique cell lineage-specific function for Rb that explains, at least in part, why Rb and p16 are inactivated in phenotypically distinct carcinomas (Wikenheiser-Brokamp, 2004).
Lung carcinomas are divided into small cell lung cancer (SCLC) and non-small cell lung cancers (NSCLC), based upon distinct clinical and pathologic features. Rb gene mutations occur in nearly all SCLC, whereas p16 is the preferential target for inactivation in NSCLC. The p16 protein inhibits cyclin D/cdk4,6 kinase activity thus maintaining Rb in its active, hypophosphorylated state. Inactivation of p16 occurs in many human cancers and results in constitutive hyperphosphorylation and thus inactivation of Rb. The remarkably tight inverse correlation between mutational inactivation of Rb and loss of p16 expression suggests that these proteins function in a common regulatory pathway. Why then are different components of the Rb pathway selectively mutated in distinct carcinomas? One hypothesis is that Rb gene mutations are seen in SCLC because neuroendocrine cells are exquisitely sensitive to Rb loss because of a lack of functional compensation by p107 and/or p130 in this cell lineage. By contrast, Rb mutations are not detected in NSCLC because these tumors arise from nonneuroendocrine cell lineages that show functional compensation for Rb deficiency. In support of this hypothesis, Rb function was demonstrated to be specifically required for regulation of neuroendocrine but not other epithelial cell lineages in the current studies. Total pocket protein inactivation results in marked epithelial abnormalities throughout the epithelium, implying that p107 and/or p130 provide a redundant or compensatory function in other cell lineages. Inactivation of p16 alters total pocket protein function, thereby eliminating family member compensation that occurs with Rb loss alone. Moreover, p107 or p130 is required along with Rb for p16-mediated growth arrest in mouse embryo fibroblasts. Thus, the data support the hypothesis that Rb gene mutation is sufficient to generate SCLC but that p16 inactivation is required to generate NSCLC because of differing degrees of functional redundancy among pocket proteins in distinct cell types. In human cancers, p16 inactivation occurs with much greater frequency than Rb gene mutations suggesting that, in contrast to lung neuroendocrine cells and retinoblasts, most cells require loss of total pocket protein function rather than simply Rb to progress to cancer (Wikenheiser-Brokamp, 2004).
Certain cells of the human retina are extremely sensitive to loss of function of the retinoblastoma tumor suppressor gene RB. Retinoblastomas develop early in life and at high frequency in individuals heterozygous for a germ-line RB mutation, and sporadic retinoblastomas invariably have somatic mutation in the RB gene. In contrast, retinoblastomas do not develop in Rb+/- mice. Although retinoblastoma is thought to have developmental origins, the function of Rb in retinal development has not been fully characterized. The role of Rb in normal retinal development and in retinoblastoma was examined using conditional Rb mutations in the mouse. In late embryogenesis, Rb-deficient retinas exhibited ectopic S-phase and high levels of p53-independent apoptosis, particularly in the differentiating retinal ganglion cell layer. During postnatal retinal development, loss of Rb leads to more widespread retinal apoptosis, and adults show loss of photoreceptors and bipolar cells. Conditional Rb mutation in the retina does not result in retinoblastoma formation even in a p53-mutant background. However, on a p107- or p130-deficient background, Rb mutation in the retina causes retinal dysplasia or retinoblastoma (MacPherson, 2004).
The molecular mechanisms governing early cardiogenesis are still largely
unknown. Interestingly, the retinoblastoma protein (Rb), a regulator of cell
cycle, has recently emerged as a new candidate regulating cell differentiation.
Rb-/- mice die at midgestation and mice lacking E2f1/E2f3, downstream components
of the Rb-dependent transcriptional pathway, die of heart failure. To gain
insight into the function of Rb pathway in early cardiogenesis, Rb-/-
embryonic stem (ES) cells differentiating into cardiomyocytes were used. Rb-/- cells
display a dramatic delay in expression of cardiac-specific transcription
factors and in turn, the whole process of cardiac differentiation. The
phenotype of Rb-/- ES cell-derived cardiomyocytes is rescued by reintroducing
Rb in cardiac progenitors, by stimulating the BMP-dependent cardiogenic pathway
or by overexpression of Nkx2.5. ES cells deficient in the recently identified
factor LEK1, a murine homolog of the cardiomyogenic factor 1, or specific
disruption of Rb-LEK1 interaction into the nucleus of differentiating ES cells
recapitulates the delay in cardiac differentiation of Rb-/- ES cells. Thus, evidence is provided
for a novel Rb/LEK1-dependent and BMP-independent
transcriptional program, which plays a pivotal role in priming ES cells toward a
cardiac fate (Papadimou, 2005).
The inactivation of the retinoblastoma (Rb) tumor suppressor gene in mice results in ectopic proliferation, apoptosis, and impaired differentiation in extraembryonic, neural, and erythroid lineages, culminating in fetal death by embryonic day 15.5 (E15.5). The specific loss of Rb in trophoblast stem (TS) cells, but not in trophoblast derivatives, leads to an overexpansion of trophoblasts, a disruption of placental architecture, and fetal death by E15.5. Despite profound placental abnormalities, fetal tissues appeared remarkably normal, suggesting that the full manifestation of fetal phenotypes requires the loss of Rb in both extraembryonic and fetal tissues. Loss of Rb results in an increase of E2f3 expression, and the combined ablation of Rb and E2f3 significantly suppresses Rb mutant phenotypes. This rescue appears to be cell autonomous since the inactivation of Rb and E2f3 in TS cells restores placental development and extends the life of embryos to E17.5. Taken together, these results demonstrate that loss of Rb in TS cells is the defining event causing lethality of Rb–/– embryos and reveal the convergence of extraembryonic and fetal functions of Rb in neural and erythroid development. It is concluded that the Rb pathway plays a critical role in the maintenance of a mammalian stem cell population (Wenzel, 2007).
Hematopoiesis is maintained by stem cells (HSCs) that undergo fate decisions by integrating intrinsic and extrinsic signals, with the latter derived from the bone marrow (BM) microenvironment. Cell-cycle regulation can modulate stem cell fate, but it is unknown whether this represents an intrinsic or extrinsic effector of fate decisions. This study investigated the role of the retinoblastoma protein (RB), a central regulator of the cell cycle, in hematopoiesis. Widespread inactivation of RB in the murine hematopoietic system resulted in profound myeloproliferation. HSCs were lost from the BM due to mobilization to extramedullary sites and differentiation. This phenotype was not intrinsic to HSCs, but, rather, was the consequence of an RB-dependent interaction between myeloid-derived cells and the microenvironment. These findings demonstrate that myeloproliferation may result from perturbed interactions between hematopoietic cells and the niche. Therefore, RB extrinsically regulates HSCs by maintaining the capacity of the BM to support normal hematopoiesis and HSCs (Walkley, 2007).
Pocket proteins (pRb, p107 and p130) are well studied in their role of regulating cell cycle progression. Increasing evidence suggests that these proteins also control early differentiation and even later stages of cell maturation, such as migration. However, pocket proteins also regulate apoptosis, and many of the developmental defects in knock out models have been attributed to increased cell death. This study eliminated ectopic apoptosis in the developing brain through the deletion of Bax, and showed that pocket proteins are required for radial migration independent of their role in cell death regulation. Following loss of pRb and p107, a population of cortical neurons fails to pass through the intermediate zone into the cortical plate. Importantly, these neurons are born at the appropriate time and this migration defect cannot be rescued by eliminating ectopic cell death. In addition, it was shown that pRb and p107 regulate radial migration through a cell autonomous mechanism since pRb/p107 deficient neurons fail to migrate to the correct cortical layer within a wild type brain. These results define a novel role of pocket proteins in regulating cortical lamination through a cell autonomous mechanism independent of their role in apoptosis (Svoboda, 2013).
Retinoblastoma (RB; see Drosophila Rb) is a tumor suppressor gene which regulates cell cycle entry to S phase via E2F transcription factors (see Drosophila E2F). Using knockout (KO) mice, it has been described that Rb plays a role in cell migration and differentiation in developing and adult brain as well as apoptosis. In addition, the RB family is required for the self-renewal and survival of human embryonic stem cells (ESCs). However, little is known about the role of this gene in human brain development. This study investigated the role of RB in cerebral organoids from human ESCs deficient for RB. RB was shown to be expressed abundantly in neural stem/progenitor cells in organoids at 15 and 28 days in culture. The results revealed that the loss of RB promotes S phase entry of DCX+ cells and increases apoptosis of Sox2+ neural stem/progenitor cells, Doublecortin+ and Tuj1+ (Neuron-specific class III β-tubulin) neurons, which was associated with the upregulation of the CYCLIN A2 and of the apoptosis-regulating BAX genes. Moreover, aberrant Tuj1+ neuronal migration was observed in RB-KO organoids, and upregulation of the VLDLR gene, a receptor important in Reelin signaling. Interestingly, ectopically localized Tuj1+ cells were also found in teratomas from RB-KO human ESCs. These results suggest that RB gene has critical roles in human brain development (Matsui, 2017).
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