The transcription factor MYC binds specific DNA sites in cellular chromatin and induces the acetylation of histones H3 and H4. However, the histone acetyltransferases (HATs) that are responsible for these modifications have not yet been identified. MYC associates with TRRAP, a subunit of distinct macromolecular complexes that contain the HATs GCN5/PCAF or TIP60. Although the association of MYC with GCN5 has been shown, its interaction with TIP60 has never been analysed. Here, we show that MYC associates with TIP60 and recruits it to chromatin in vivo with four other components of the TIP60 complex: TRRAP, p400, TIP48 and TIP49. Overexpression of enzymatically inactive TIP60 delays the MYC-induced acetylation of histone H4, and also reduces the level of MYC binding to chromatin. Thus, the TIP60 HAT complex is recruited to MYC-target genes and, probably with other other HATs, contributes to histone acetylation in response to mitogenic signals.

In mammalian cells, cyclin E±CDK2 complexes are activated in the late G 1 phase of the cell cycle and are believed to have an essential role in promoting S-phase entry. We have targeted the murine genes CCNE1 and CCNE2, encoding cyclins E1 and E2. Whereas single knockout mice were viable, double knockout embryos died around midgestation. Strikingly, however, these embryos showed no overt defects in cell proliferation. Instead, we observed developmental phenotypes consistent with placental dysfunction. Mutant placentas had an overall normal structure, but the nuclei of trophoblast giant cells, which normally undergo endoreplication and reach elevated ploidies, showed a marked reduction in DNA content. We derived trophoblast stem cells from double knockout E3.5 blastocysts. These cells retained the ability to differentiate into giant cells in vitro, but were unable to undergo multiple rounds of DNA synthesis, demonstrating that the lack of endoreplication was a cell-autonomous defect. Thus, during embryonic development, the needs for E-type cyclins can be overcome in mitotic cycles but not in endoreplicating cells.

E2F proteins can either activate or repress transcription. Following mitogenic stimulation, repressive E2F4-p130-histone deacetylase complexes dissociate from, while activating species (E2F1, -2, and -3) associate with, target promoters. Histones H3 and H4 simultaneously become hyperacetylated, but it remains unclear whether this is a prerequisite or a consequence of E2F binding. Here, we show that activating E2F species are required for hyperacetylation of target chromatin in human cells. Overexpression of a dominant-negative (DN) E2F1 mutant in serum-stimulated T98G cells blocked all E2F binding, H4 acetylation, and, albeit partially, H3 acetylation. Target gene activation and S-phase entry were also blocked by DN E2F1. Conversely, ectopic activation of E2F1 rapidly induced H3 and H4 acetylation, demonstrating a direct role for E2F in these events. E2F1 was previously shown to bind the histone acetyltransferases (HATs) p300/CBP and PCAF/GCN5. In our hands, ectopically expressed E2F1 also bound the unrelated HAT Tip60 and induced recruitment of five subunits of the Tip60 complex (Tip60, TRRAP, p400, Tip48, and Tip49) to target promoters in vivo. Moreover, E2F-dependent recruitment of Tip60 to chromatin occurred in late G 1 following serum stimulation. We speculate that the activities of multiple HAT complexes account for E2F-dependent acetylation, transcription, and S-phase entry.

Somatic activation of Ras occurs frequently in human cancers, including one-third of lung cancers. Activating Ras mutations also occur in the germline, leading to complex developmental syndromes. The precise mechanism by which Ras activation results in human disease is uncertain. Here we describe the phenotype of a mouse engineered to harbor a germline oncogenic K-ras G12D mutation. This mouse exhibits early embryonic lethality due to a placental trophoblast defect. Reconstitution with a wild-type placenta rescues the early lethality, but mutant embryos still succumb to cardiovascular and hematopoietic defects. In addition, mutant embryos demonstrate a profound defect in lung branching morphogenesis associated with striking up-regulation of the Ras/mitogen-activated protein kinase (MAPK) antagonist Sprouty-2 and abnormal localization of MAPK activity within the lung epithelium. This defect can be significantly suppressed by lentiviral short hairpin RNA (shRNA)-mediated knockdown of Sprouty-2 in vivo. Furthermore, in the context of K-ras G12D -mediated lung tumorigenesis, Sprouty-2 is also up-regulated and functions as a tumor suppressor to limit tumor number and overall tumor burden. These findings indicate that in the lung, Sprouty-2 plays a critical role in the regulation of oncogenic K-ras, and implicate counter-regulatory mechanisms in the pathogenesis of Ras-based disease.[Keywords: K-ras; mouse models; lung development; lung cancer; Sprouty] Supplemental material is available at http://www.genesdev.org.

Genetic alteration of the p53 tumor suppressor gene, which monitors DNA damage and operates cell cycle checkpoints, is a major factor in the development of human malignancies. The p53 protein belongs to a family that also includes two structurally related proteins, p63 and p73. Although all three proteins share similar transcriptional functions and antiproliferative effects, each of them appears to play a distinct role in development and tumor suppression. One of the principal regulators of p53 activity is the MDM2 protein. The interaction of MDM2 with p53 inhibits p53 transcriptional activity and targets p53 for ubiquitin-dependent degradation. The ability of MDM2 to inhibit p53 functions is antagonized by the ARF oncosuppressor protein. We show here that like p53, the p63␣ and p63␥ isoforms are able to associate with human MDM2 (HDM2). Overexpression of HDM2 increased the steady-state level of intracellular p63 and enhanced its transcriptional activity. Both effects appeared to be counteracted by ARF coexpression. These data indicate that p63 can be activated by HDM2 under conditions in which p53 is inhibited. Therefore, HDM2 expression could support p63-specific transcriptional functions on a common set of genes, keeping interference by p53 at a minimum.The p63 gene, which maps on the 3q27-28 region, is one of the members of the p53 gene family. Unlike p53, it shows a complex pattern of expression due to alternative splicing and promoter usage that results in multiple isoforms with different biological activities (1, 2). Initiation of transcription in exon 1 produces the TA isotypes, containing the evolutionarily conserved transactivation, DNA-binding, and oligomerization domains, whereas initiation in exon 3Ј gives rise to the ⌬N isotypes that lack the TA domain (3). p63 shows a remarkable structural similarity to p53 and to the related p73 protein: ϳ60% of the amino acids of the human p63 and p73 proteins in the region corresponding to the DNA-binding domain are identical to those of p53 (4).In contrast with the ubiquitous expression of p53, p63 exhibits a rather tissue-specific distribution in that it is most detectable in the basal layer of stratified epithelia, including the epidermis, where the ⌬Np63␣ isotype is predominantly expressed (3). However, it is still not known how the expression of different isoforms of p63 is regulated in different tissues and during development.All three members of the p53 family share similar transcriptional functions, as p63 and p73 can also activate many of the p53 target genes, although with varying efficiency (5). Moreover, like p73, p63 is able to induce apoptosis and growth suppression in a manner similar to p53 (4).Molecular alterations of p63 or p73 in human cancers appear to be rare; unlike p53-deficient mice, those lacking p73 or p63 show no increased susceptibility to spontaneous tumorigenesis (6, 7). Viral oncoproteins such as SV40 large T antigen, adenovirus E1B, and human papilloma virus E6, which bind and inactivate p53, do not target p73 and p63 (2, 8). Thu...