HPV 16E7 and 48E7 proteins use different mechanisms to target p130 to overcome cell cycle block.

BACKGROUND: Retinoblastoma like protein 2 (RBL2) or p130 is a member of the pocket protein family, which is infrequently mutated in human tumours. Its expression is posttranscriptionally regulated and largely G0 restricted. We have previously shown that E6/E7 oncoproteins encoded by human papillomavirus (HPV) type 16, which is a high-risk type for cervical cancer development, must target p130 to promote the host cell to exit from quiescence (G0) state and enter S phase of the cell cycle. P130 is associated with the DREAM (DP, RB-like, E2F and MuvB) complex in G0/G1, which prevents S phase progression by repressing transcription of E2F-regulated genes. E7 proteins could potentially disrupt the p130-DREAM complex through two known mechanisms: direct interaction with p130 or induction of cyclin dependent kinase 2 (CDK2) phosphorylation by interacting with its inhibitor, p21(CIP1). METHODS: In this study we have used p130 mutants deficient in binding the E7 LXCXE domain (p130mE7), unphosphorylatable by CDK2 (p130PM22) or a combination of both (p130PM22/mE7) to investigate these mechanisms used by E7 proteins to disrupt the p130-DREAM complex and promote cell cycle progression. RESULTS: We found that HPV16 E7 binding to p130 through its LXCXE domain was absolutely required to disrupt p130-DREAM to promote S phase of the cell cycle, as HPV16 E7 was unable to suppress p130mE7 but could suppress p130PM22. In contrast, the E7 protein encoded by a cutaneous HPV type that lacks a functional LXCXE domain, HPV 48 E7, was also able to disrupt p130-DREAM to promote cell cycling, but through the alternative mechanism. Thus, HPV48 E7 could suppress a cell cycle block imposed by p130mE7, but was unable to suppress p130PM22. CONCLUSIONS: Overall, these results indicate that suppression of p130 is required for HPV-induced cell cycling, and that different HPV E7 proteins can use alternative mechanisms to achieve this.

Binding of FoxM1 to G2/M gene promoters is dependent upon B-Myb.

The promoters of genes which regulate entry into and progress through mitosis are typically induced maximally in G2 by transcription factors that include B-Myb and FoxM1. As FoxM1 gene transcription is a target of B-Myb, we investigated in this study how these transcription factors functionally interact to regulate these G2/M genes. Using a 3T3 cell line containing floxed B-myb alleles (B-myb(F/F)) that could be conditionally deleted by Cre recombinase, we confirmed that B-myb knockout caused both decreased mRNA expression of several G2/M genes, including FoxM1, and delayed entry into mitosis. Although FoxM1 protein expression was actually unaffected by B-myb knockout when quiescent B-myb(F/F) 3T3 cells re-entered the cell cycle upon serum-stimulation, chromatin immunoprecipitation revealed that FoxM1 binding to G2/M promoters was substantially reduced. FoxM1 transcriptional activity requires sequential phosphorylation by Cyclin-dependent kinases and Plk1, which are B-Myb target genes, and we found that phosphorylation at Plk1-specific sites was somewhat reduced upon B-myb knockout. Neither this effect nor nuclear accumulation of FoxM1, which was unaffected by B-myb knockout, was sufficient to account for the dependence on B-Myb for FoxM1 promoter binding, however. More significantly, assays using paired Birc5 (survivin) promoter-luciferase reporters with either wild-type or mutated Myb binding sites showed that FoxM1 was unable to bind and activate the promoter in the absence of B-Myb binding. Our data suggest that B-Myb is required as a pioneer factor to enable FoxM1 binding to G2/M gene promoters and explains how these transcription factors may collaborate to induce mitosis.

Disruption of repressive p130-DREAM complexes by human papillomavirus 16 E6/E7 oncoproteins is required for cell-cycle progression in cervical cancer cells.

Human papillomaviruses (HPVs) with tropism for mucosal epithelia are the major aetiological factors in cervical cancer. Most cancers are associated with so-called high-risk HPV types, in particular HPV16, and constitutive expression of the HPV16 E6 and E7 oncoproteins is critical for malignant transformation in infected keratinocytes. E6 and E7 bind to and inactivate the cellular tumour suppressors p53 and Rb, respectively, thus delaying differentiation and inducing proliferation in suprabasal keratinocytes to enable HPV replication. One member of the Rb family, p130, appears to be a particularly important target for E7 in promoting S-phase entry. Recent evidence indicates that p130 regulates cell-cycle progression as part of a large protein complex termed DREAM. The composition of DREAM is cell cycle-regulated, associating with E2F4 and p130 in G0/G1 and with the B-myb transcription factor in S/G2. In this study, we addressed whether p130-DREAM is disrupted in HPV16-transformed cervical cancer cells and whether this is a critical function for E6/E7. We found that p130-DREAM was greatly diminished in HPV16-transformed cervical carcinoma cells (CaSki and SiHa) compared with control cell lines; however, when E6/E7 expression was targeted by specific small hairpin RNAs, p130-DREAM was reformed and the cell cycle was arrested. We further demonstrated that the profound G1 arrest in E7-depleted CaSki cells was dependent on p130-DREAM reformation by also targeting the expression of the DREAM component Lin-54 and p130. The results show that continued HPV16 E6/E7 expression is necessary in cervical cancer cells to prevent cell-cycle arrest by a repressive p130-DREAM complex.

p27Kip1 and p130 cooperate to regulate hematopoietic cell proliferation in vivo.

To investigate the potential functional cooperation between p27Kip1 and p130 in vivo, we generated mice deficient for both p27Kip1 and p130. In p27Kip1-/-; p130-/- mice, the cellularity of the spleens but not the thymi is significantly increased compared with that of their p27Kip1-/- counterparts, affecting the lymphoid, erythroid, and myeloid compartments. In vivo cell proliferation is significantly augmented in the B and T cells, monocytes, macrophages, and erythroid progenitors in the spleens of p27Kip1-/-; p130-/- animals. Immunoprecipitation and immunodepletion studies indicate that p130 can compensate for the absence of p27Kip1 in binding to and repressing CDK2 and is the predominant CDK-inhibitor associated with the inactive CDK2 in the p27Kip1-/- splenocytes. The finding that the p27Kip1-/-; p130-/- splenic B cells are hypersensitive to mitogenic stimulations in vitro lends support to the concept that the hyperproliferation of splenocytes is not a result of the influence of their microenvironment. In summary, our findings provide genetic and molecular evidence to show that p130 is a bona fide cyclin-dependent kinase inhibitor and cooperates with p27Kip1 to regulate hematopoietic cell proliferation in vivo.

B-Myb overcomes a p107-mediated cell proliferation block by interacting with an N-terminal domain of p107.

B-Myb is a cell-cycle regulated transcription factor which is implicated in cell proliferation and has an essential role in early embryonic development. In this study we examined the functions of B-Myb required to overcome G1 arrest in Saos-2 cells induced by the retinoblastoma-related p107 protein. Our results demonstrated that this activity was independent of B-Myb transactivation function, but correlated with its capacity to form an in vivo complex with p107. A large proportion of B-Myb formed complexes with p107 in cotransfected cells, however, B-Myb bound weakly to the related p130 protein and not at all to pRb. In contrast to the E2F transcription factors, which bind the p107 C-terminal pocket domain, B-Myb recognizes an N-terminal p107 region which overlaps the larger cyclin-binding domain. B-Myb and cyclin A2 formed mutually exclusive complexes with p107, and B-Myb enhanced the activity of co-transfected cyclin E kinase activity, implying that B-Myb affects the cell cycle by preventing sequestration of active cyclin/cdk2 complexes. This study defines a novel function of B-Myb and further suggests that the p107 N-terminus provides an interaction domain for transcription factors involved in cell cycle control.

A B-myb--DREAM complex is not critical to regulate the G2/M genes in HPV-transformed cell lines.

BACKGROUND/AIM: It is well-established that HPV E7 proteins, encoded by human papillomavirus (HPV) genes, frequently associated with cervical cancers bind avidly to the retinoblastoma (RB) family of pocket proteins and disrupt their association with members of the E2F transcription factor family. Our previous study showed that the repressive p130-dimerization partner, RB-like, E2F and multi-vulval class (DREAM) complex was disrupted by HPV16 E7 proteins in order to maintain the viral replication in CaSki cells. However, we would like to address whether the activator B-myb-DREAM complex is critical in regulating the replication and mitosis phase since our previous study showed increased B-myb-DREAM expression in HPV-transformed cell lines when compared to control cells. RESULTS: The association of B-myb with both LIN-54 and LIN-9 was equally decreased by depleting LIN-54 in CaSki cells. Flow cytometry analysis showed that LIN-54 depletion caused an increased proportion of G2/M cells in T98G, SiHa and CaSki cells. The mRNA levels of certain S/G2 genes such as cyclin B, aurora kinase A and Polo-like kinase 1 have demonstrated a marginal increased in CaSki-Lin-54-depleted cells when compared to SiHa- and T98G-Lin-54-depleted cells. We further confirmed this experiment by depleting the B-myb itself in CaSki cells and the results showed the same pattern of cell cycle and mRNA levels for S/G2 genes when compared to LIN-54- and LIN-9-depleted cells. CONCLUSION: The B-myb-DREAM complex might not be vital for progression through mitosis in cells lacking a G1/S checkpoint and not as crucial as the p130-DREAM complex for the survival of the HPV virus.

The cell cycle-regulated B-Myb transcription factor overcomes cyclin-dependent kinase inhibitory activity of p57(KIP2) by interacting with its cyclin-binding domain.

The cell cycle-regulated B-Myb transcription factor is required for early embryonic development and is implicated in regulating cell growth and differentiation. In addition to its transcriptional regulatory properties, recent data indicate that B-Myb can release active cyclin/Cdk2 activity from the retinoblastoma-related p107 protein by directly interacting with the p107 N terminus. As this p107 domain has homology to the cyclin-binding domains of the p21(Waf1/Cip1) family of cyclin-dependent kinase inhibitors (CKIs), we investigated in this study whether B-Myb could also interact with these CKIs. No in vivo interaction was found with either p21(Waf1/Cip1) or p27(KIP1), however, binding to p57(KIP2) was readily detectable in both in vivo and in vitro assays. The B-Myb-interacting region of p57(KIP2) mapped to the cyclin-binding domain. Consistent with this, B-Myb competed with cyclin A2 for binding to p57(KIP2), resulting in release of active cyclin/Cdk2 kinase. Moreover, B-Myb partially overcame the ability of p57(KIP2) to induce G1 arrest in Saos-2 cells. Despite similarities with previous p107 studies, the B-Myb domains required for interaction with p57(KIP2) were quite different from those implicated for p107. Thus, it is evident that B-Myb may promote cell proliferation by a non-transcriptional mechanism that involves release of active cyclin/Cdk2 from p57(KIP2) as well as p107.

A B-myb promoter corepressor site facilitates in vivo occupation of the adjacent E2F site by p107 x E2F and p130 x E2F complexes.

Transcription from the B-myb (MybL2 gene) promoter is strictly cell cycle-regulated by repression mediated through an E2F site during G(0)/early G(1). We report here the characterization of a corepressor site (downstream repression site (DRS)) required for this activity that is closely linked to the E2F site. Systematic mutagenesis of the DRS enabled a consensus to be derived, and it is notable that this sequence is compatible with cell cycle gene homology region sequences associated with cell cycle-dependent elements in the cyclin A, cdc2, and CDC25C promoters. The B-myb promoter is inappropriately active during G(0) in mouse embryo fibroblasts lacking the p107 and p130 pocket proteins, and we show that the ability of transfected p107 and p130 to re-impose repression on the promoter is dependent on the DRS. In contrast, transfected Rb was unable to repress the B-myb promoter. Consistent with the notion that Rb.E2F complexes are unable to bind the B-myb promoter E2F site in vivo, footprinting showed that this site is unoccupied in cells lacking p107 and p130. Chromatin immunoprecipitation assays showed a requirement for the DRS in recruiting p107 and p130 complexes to the B-myb promoter, indicating that in vivo the DRS governs the occupancy of the adjacent E2F site by transcriptional repressors.

E2F mediates cell cycle-dependent transcriptional repression in vivo by recruitment of an HDAC1/mSin3B corepressor complex.

Despite biochemical and genetic data suggesting that E2F and pRB (pocket protein) families regulate transcription via chromatin-modifying factors, the precise mechanisms underlying gene regulation by these protein families have not yet been defined in a physiological setting. In this study, we have investigated promoter occupancy in wild-type and pocket protein-deficient primary cells. We show that corepressor complexes consisting of histone deacetylase (HDAC1) and mSin3B were specifically recruited to endogenous E2F-regulated promoters in quiescent cells. These complexes dissociated from promoters once cells reached late G1, coincident with gene activation. Interestingly, recruitment of HDAC1 complexes to promoters depended absolutely on p107 and p130, and required an intact E2F-binding site. In contrast, mSin3B recruitment to certain promoters did not require p107 or p130, suggesting that recruitment of this corepressor can occur via E2F-dependent and -independent mechanisms. Remarkably, loss of pRB had no effect on HDAC1 or mSin3B recruitment. p107/p130 deficiency triggered a dramatic loss of E2F4 nuclear localization as well as transcriptional derepression, which is suggested by nucleosome mapping studies to be the result of localized hyperacetylation of nucleosomes proximal to E2F-binding sites. Taken together, these findings show that p130 escorts E2F4 into the nucleus and, together with corepressor complexes that contain mSin3B and/or HDAC1, directly represses transcription from target genes as cells withdraw from the cell cycle.