Mol Cell Biol 20(18): 6799-6805

Role of the LXCXE Binding Site in Rb Function

Division of Molecular Oncology, Departments of Medicine and Cell Biology, Washington University School of Medicine, St. Louis, Missouri 63110

^{Corresponding author. Mailing address: Campus Box 8069, Division of Molecular Oncology, Washington University School of Medicine, 660 South Euclid Ave., St. Louis, MO 63110. Phone: (314) 362-8989. Fax: (314) 747-2797. E-mail:}ude.ltsuw.mi@naedd.

Received 2000 Mar 8; Revisions requested 2000 Apr 10; Accepted 2000 Jun 7.

Abstract

Oncoproteins from DNA tumor viruses such as adenovirus E1a, simian virus 40 T antigen, and human papillomavirus E7 contain an LXCXE sequence, which they use to bind the retinoblastoma protein (Rb) and inhibit its function. Cellular proteins such as histone deacetylases 1 and 2 (HDAC1 and -2) also contain an LXCXE-like sequence, which they use to interact with Rb. The LXCXE binding site in Rb was mutated to assess its role in Rb function. These mutations inhibited binding to HDAC1 and -2, which each contain an LXCXE-like sequence, but had no effect on binding to HDAC3, which lacks an LXCXE-like sequence. Mutation of the LXCXE binding site inhibited active transcriptional repression by Rb and prevented it from effectively repressing the cyclin E and A gene promoters. In contrast, mutations in the LXCXE binding site did not prevent Rb from binding and inactivating E2F. Thus, the LXCXE mutations appear to separate Rb's ability to bind and inactivate E2F from its ability to efficiently recruit HDAC1 and -2 and actively repress transcription. In transient assays, several of the LXCXE binding site mutants caused an increase in the percentage of cells in G₁ by flow cytometry, suggesting that they can arrest cells. However, this effect was transient, as none of the mutants affected cell proliferation in longer-term assays examining bromodeoxyuridine incorporation or colony formation. Our results then suggest that the LXCXE binding site is important for full Rb function. Mutation of the LXCXE binding site does not inhibit binding of the BRG1 ATPase component of the SWI/SNF nucleosome remodeling complex, which has been shown previously to be important for Rb function. Indeed, overexpression of BRG1 and Rb in cells deficient for the proteins led to stable growth inhibition, suggesting a cooperative role for SWI/SNF and the LXCXE binding site in efficient Rb function.

Abstract

The retinoblastoma protein (Rb) is an important regulator of the cell cycle (42). One target of Rb is the E2F family of cell cycle transcription factors, and binding of Rb blocks transcriptional activation by E2F (1, 11, 25, 32, 34). There are conflicting reports as to whether this inactivation results simply from the binding of Rb to the transactivation domain of E2F, or whether recruitment of chromatin remodeling enzymes is required. In in vitro transcription assays Rb blocks transcriptional activation by E2F-1 in the apparent absence of chromatin remodeling complexes, suggesting that Rb may function simply by binding and masking the transactivation domain of E2F-1 (33). However, other studies have demonstrated that Rb can interact with chromatin remodeling enzymes to repress E2F activity (3, 29). One of these enzymes is histone deacetylase (HDAC), a family of at least seven different enzymes that removes acetyl groups from the tails of histone octamers. This removal of acetyl groups appears to facilitate condensation of nucleosomes into chromatin, which in turn blocks access of transcription factors, leading to gene repression (23, 24, 45). In contrast to in vitro assays, transfection assays in vivo have suggested that interaction of Rb with HDAC is required for Rb to inhibit E2F-1 (3, 29). Furthermore, the active repression by the Rb-E2F complex at the promoters of cell cycle genes is thought to be mediated at least in part by recruitment of HDAC, and HDAC activity appears to be required for Rb to repress several cellular genes (28). An IXCXE site in the C terminus of HDAC1 seems to be important in mediating association with Rb (29).

In addition to HDACs, Rb also interacts with two other chromatin remodeling enzymes, BRG1 and BRM (8, 35, 38). These proteins are ATPases which are central components of the human SWI-SNF nucleosome remodeling complex. SWI-SNF was first identified in yeast where the ATPase SWI2-SNF2 appears to be a homologue of mammalian BRG1 and BRM (reviewed in reference 23). SWI/SNF seems to function by regulating nucleosome formation and positioning around genes. Several different SWI-SNF-related remodeling complexes have now been identified, and these complexes appear to have similar activities in in vitro assays. While SWI-SNF has been thought to be involved primarily in transcriptional activation, mutation of SWI2-SNF2 led to both activation and repression of genes in yeast (more genes were activated than repressed), suggesting that SWI-SNF may also be involved in transcriptional repression (19). Additionally, SWI-SNF-related complexes have been shown more directly to be involved in transcriptional repression. For example, the Mi2β complex is associated with repression, and it is thought that the presence of HDAC1 in the complex is required for this activity (22, 40, 48).

It has been demonstrated that expression of BRG1 in SW13 cells, which are deficient for both BRG1 and BRM (30) but are Rb^{leads to growth arrest (}8). Inhibition of Rb function by expression of adenovirus E1a prevented this arrest, and mutation of E1a to selectively block its interaction with Rb significantly reduced this effect of E1a. Additionally, a dominant-negative form of BRM, containing a mutant ATPase domain but an intact Rb binding site, was able to inhibit growth suppression by Rb (8). Two additional studies also point to a role for BRG1 in Rb function: recently, it was shown that SWI-SNF activity is important for Rb repression of the c-fos gene (31), and earlier studies provided evidence that expression of BRM in BRG1/BRM-deficient cells was required for Rb to efficiently inhibit transcriptional activation by E2F-1 (37). Taken together, the above studies point to potentially important roles for HDAC and SWI-SNF in Rb activity.

Like HDAC1, BRG1 contains an LXCXE site, and deletion of a BRG1 region containing the LXCXE site results in loss of binding to Rb (8). An LXCXE sequence is also found in adenovirus E1a, human papillomavirus (HPV) E7, and simian virus 40 T antigen (7, 10, 13, 20). These DNA tumor virus oncogene products use the LXCXE motif for high-affinity binding and inhibition of Rb. Without the LXCXE site, these oncoproteins cannot transform cells. The fact that viruses target the LXCXE binding site of Rb and that this is necessary for transformation points to the importance of this site in Rb function. The Rb pocket has been cocrystallized with an LXCXE peptide, allowing localization of the LXCXE binding site (26). A hydrophobic groove in Rb pocket domain B forms the binding site, where the four conserved amino acids Tyr 709, Lys 713, Tyr 756, and Asn 757 are involved in contacting the backbone of the LXCXE peptide. We found that mutation of these contact amino acids inhibited binding of Rb to LXCXE-like proteins such as adenovirus E1a and HDAC1 and -2 but not HDAC3, which lacks an LXCXE-like motif. The LXCXE binding site mutations inhibited HDAC-dependent active repression and efficient growth suppression by Rb, providing evidence that the LXCXE binding site is important for efficient Rb function. However, the mutations did not affect either binding of Rb to E2F or the ability of Rb to inhibit transcriptional activation by E2F. These results suggest that although the LXCXE binding site has a critical role in active transcriptional repression by Rb and is important for full Rb function, this site is not required for Rb binding and inhibition of E2F. The LXCXE binding site mutations then separate Rb functions of binding and inactivation of E2F from recruitment of LXCXE proteins. The LXCXE binding site mutations in Rb did not affect binding to the SWI-SNF ATPase, BRG1, and we found that overexpression of BRG1 with the Rb mutants led to growth arrest. These results suggest a level of cooperation between LXCXE proteins and SWI-SNF in efficient Rb function.

ACKNOWLEDGMENTS

We thank N. Dyson and J. Wang for communicating results prior to publication, S. Schreiber for the HDAC1 to HDAC6 expression vectors, K. Helin for the E2F-1 expression vector, D. Ayer for MLPCAT, S. Goff for the BRG1 expression vector, R. Weinberg for the cycE-luc reporter, C. Brechot for the cycA-luc reporter, S. Cotter for the Flag-tagged BRG1 expression vector, and A. Postigo for the LexA-tagged HDAC3.

This study was supported by grants from the NIH to D.C.D. A.D. was supported by training grant HL07317-22 from the National Heart Lung and Blood Institute.

ACKNOWLEDGMENTS

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