Retinoblastoma protein meets chromatin

doi:10.1016/S0968-0004(99)01368-7

Trends in Biochemical Sciences

Volume 24, Issue 4, 1 April 1999, Pages 142-145

https://doi.org/10.1016/S0968-0004(99)01368-7 Get rights and content

Abstract

The retinoblastoma (RB) protein exerts its tumour-suppressor function by repressing the transcription of cellular genes required for DNA replication and cell division. Recent investigations into the mechanism of RB repression have revealed that RB can regulate transcription by effecting changes in chromatin structure. These findings point towards a link between chromatin regulation and cancer.

Section snippets

Mechanisms of transcriptional repression by retinoblastoma protein

How does RB repress transcription once it is recruited to the promoter by binding to E2F? The binding site for RB resides within the activation domain of E2F (Ref. 7). This suggests that RB could operate by masking the activation domain and thereby preventing the latter from interacting functionally with components of the general transcription machinery, such as the TATA-box-binding protein (TBP). The fact that the E2F residues contacted by RB are required for binding of TBP by E2F and

Retinoblastoma protein and histone acetylation

Several groups have recently reported that RB is associated with histone deacetylase activity and can bind to at least two members of the HDAC family, HDAC1 and HDAC230, 31, 32. HDAC1 preferentially binds to the active, hypophosphorylated form of RB (Ref. 31). Furthermore, E2F1, RB and HDAC1 can form a trimeric complex, and RB can recruit histone deacetylase activity to E2F in vitro30, 32. If the interaction between histone deacetylase and RB is relevant to transcriptional repression, we can

Retinoblastoma protein and chromatin remodelling

Can RB affect chromatin structure by other mechanisms? Chromatin structure appears to be different in RB^+/+ and RB^−/− MEFs: chromatin from RB^−/− cells is more accessible to micrococcal-nuclease digestion, a property that is indicative of an open chromatin conformation⁴³. This correlates with increased histone-H1 phosphorylation in RB^−/− cells, which in turn has been attributed to higher levels of cyclin-E–CDK2 activity in these cells³⁷. RB, therefore, could indirectly promote a closed,

Acknowledgements

We apologize to colleagues whose work could not be cited owing to space limitations. We thank R. E. Herrera for communicating results prior to publication. Research in the laboratory is funded by the Cancer Research Campaign. A. B. is supported by the Association for International Cancer Research.

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The mammalian cell cycle has been extensively studied regarding cancer etiology, progression, and therapeutic intervention. The canonical cell cycle framework is supported by a plethora of data pointing to a relatively simple linear pathway in which mitogenic signals are integrated in a stepwise fashion to allow progression through G1/S with coordinate actions of cyclin-dependent kinases (CDK)4/6 and CDK2 on the RB tumor suppressor. Recent work on adaptive mechanisms and intrinsic heterogeneous dependencies indicates that G1/S control of the cell cycle is a variable signaling pathway rather than an invariant engine that drives cell division. These alterations can limit the effectiveness of pharmaceutical agents but provide new avenues for therapeutic interventions. These findings support a dystopian view of the cell cycle in cancer where the canonical utopian cell cycle is often not observed. However, recognizing the extent of cell cycle heterogeneity likely creates new opportunities for precision therapeutic approaches specifically targeting these states.
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The pocket domain contains a cleft that binds an “LxCxE” motif originally identified in viral oncoproteins [18]. Histone deacetylases, chromatin remodeling complexes, Cdh1, and a number of other cellular proteins bind Rb in an “LxCxE” cleft-dependent manner [19–21], although detailed structural analysis of these associations is limited [22]. The best-characterized pocket protein interactions are with transcription factors.
The human pocket proteins retinoblastoma (Rb), p107, and p130 are critical negative regulators of the cell cycle and contribute to tumor suppression. While strong structural conservation within the pocket protein family provides for some functional redundancy, important differences have been observed and may underlie the reason that Rb is a uniquely potent tumor suppressor. It has been proposed that distinct pocket protein activities are mediated by their different E2F transcription factor binding partners. In humans, Rb binds E2F1–E2F5, whereas p107 and p130 almost exclusively associate with E2F4 and E2F5. To identify the molecular determinants of this specificity, we compared the crystal structures of Rb and p107 pocket domains and identified several key residues that contribute to E2F selectivity in the pocket family. Mutation of these residues in p107 to match the analogous residue in Rb results in an increase in affinity for E2F1 and E2F2 and an increase in the ability of p107 to inhibit E2F2 transactivation. Additionally, we investigated how phosphorylation by Cyclin-dependent kinase on distinct residues regulates p107 affinity for the E2F4 transactivation domain. We found that phosphorylation of residues S650 and S975 weakens the E2F4 transactivation domain binding. Our data reveal molecular features of pocket proteins that are responsible for their similarities and differences in function and regulation.
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Finally, new Rb tumor suppressor activities have been characterized that might utilize independent regulation, and clear predictions can now be made for how the code could separately turn off these different activities. Rb was initially characterized as a regulator of transcription [8,9]. It inhibits expression of genes under the control of E2F transcription factors by binding E2Fs and recruiting co-repressor proteins that modify histones and chromatin.
Multisite phosphorylation modulates the function of regulatory proteins with complex signaling properties and outputs. The retinoblastoma tumor suppressor protein (Rb) is inactivated by cyclin-dependent kinase (Cdk) phosphorylation in normal and cancer cell cycles, so understanding the molecular mechanisms and effects of Rb phosphorylation is imperative. Rb functions in diverse processes regulating proliferation, and it has been speculated that multisite phosphorylation might act as a code in which discrete phosphorylations control specific activities. The idea of an Rb phosphorylation code is evaluated here in light of recent studies of Rb structure and function. Rb inactivation is discussed with an emphasis on how multisite phosphorylation changes Rb structure and associations with protein partners.
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The aim of this work was to define the role of pRb depletion in the proliferation and differentiation of avian retinoblasts in vitro. For this purpose vectors expressing pRb short hairpin RNA were used to deplete pRb in cultures of avian neuroretinal cells. Down regulation of pRb was observed by Western blot and quantification of nuclear pRb. Cell proliferation and differentiation were studied following BrdU labeling and immunostaining. Transfection significantly down-regulated pRb in neuroretinal cells. Long-term effect of pRb depletion mainly induced proliferation of epithelial-like cells that expressed markers of reactive Müller glial cells. A minority of these cells that survived passaging could be maintained as neurosphere-like aggregates with low pRb, not observed in control cultures. BrdU labeling followed by a two week chase showed the presence of cells still remained labelled, indicating low cell cycling. Under appropriate conditions, these aggregates differentiate in precursors of amacrine interneurons shown by the expression of AP2, in absence of the photoreceptors marker visinin and the late neuronal marker MAP2. Taken together these data show that decrease pRb level in cultures of avian neuroretinal cells promotes the emergence and proliferation of stem cell/progenitors from reactive-like Muller cells.
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Transcription Factors Recruit the Phosphorylated HDAC2 Forms—We had previously reported that Sp1 and Sp3 preferentially associated with phosphorylated HDAC2 (9). Rb, p53, YY1, and NF-κB (p50, p65 subunits) are transcription factors known to achieve transcription silencing or dynamic acetylation/deacetylation by recruiting HDAC1 and 2 to specific promoters via Sin3 and/or NuRD complexes (23–26). To determine whether these transcription factors preferentially recruited phosphorylated HDAC2, we carried out immunoprecipitations of MCF-7 cell lysates with antibodies to these transcription factors.
Histone deacetylase 2 (HDAC2) is one of the histone-modifying enzymes that regulate gene expression by remodeling chromatin structure. Along with HDAC1, HDAC2 is found in the Sin3 and NuRD multiprotein complexes, which are recruited to promoters by DNA-binding proteins. In this study, we show that the majority of HDAC2 in human breast cancer cells is not phosphorylated. However, the minor population of HDAC2, preferentially cross-linked to DNA by cisplatin, is mono-, di-, or tri-phosphorylated. Furthermore, HDAC2 phosphorylation is required for formation of Sin3 and NuRD complexes and recruitment to promoters by transcription factors including p53, Rb, YY1, NF-κB, Sp1, and Sp3. Unmodified HDAC2 requires linker DNA to associate with chromatin but is not cross-linked to DNA by formaldehyde. We provide evidence that unmodified HDAC2 is associated with the coding region of transcribed genes, whereas phosphorylated HDAC2 is primarily recruited to promoters.
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Trends in Biochemical Sciences

ReviewRetinoblastoma protein meets chromatin

Abstract

Section snippets

Mechanisms of transcriptional repression by retinoblastoma protein

Retinoblastoma protein and histone acetylation

Retinoblastoma protein and chromatin remodelling

Acknowledgements

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Review
Retinoblastoma protein meets chromatin