E type cyclins (E1 and E2) are believed to drive cell entry into the S phase. It is widely assumed that the two E type cyclins are critically required for proliferation of all cell types. Here, we demonstrate that E type cyclins are largely dispensable for mouse development. However, endoreplication of trophoblast giant cells and megakaryocytes is severely impaired in the absence of cyclin E. Cyclin E-deficient cells proliferate actively under conditions of continuous cell cycling but are unable to reenter the cell cycle from the quiescent G(0) state. Molecular analyses revealed that cells lacking cyclin E fail to normally incorporate MCM proteins into DNA replication origins during G(0)-->S progression. We also found that cyclin E-deficient cells are relatively resistant to oncogenic transformation. These findings define a molecular function for E type cyclins in cell cycle reentry and reveal a differential requirement for cyclin E in normal versus oncogenic proliferation.

Activity of the hypoxia-inducible factor (HIF) complex is controlled by oxygen-dependent hydroxylation of prolyl and asparaginyl residues. Hydroxylation of specific prolyl residues by 2-oxoglutarate (2-OG)-dependent oxygenases mediates ubiquitinylation and proteasomal destruction of HIF-␣. Hydroxylation of an asparagine residue in the C-terminal transactivation domain (CAD) of HIF-␣ abrogates interaction with p300, preventing transcriptional activation. Yeast two-hybrid assays recently identified factor inhibiting HIF (FIH) as a protein that associates with the CAD region of HIF-␣. Since FIH contains certain motifs present in iron-and 2-OG-dependent oxygenases we investigated whether FIH was the HIF asparaginyl hydroxylase. Assays using recombinant FIH and HIF-␣ fragments revealed that FIH is the enzyme that hydroxylates the CAD asparagine residue, that the activity is directly inhibited by cobalt(II) and limited by hypoxia, and that the oxygen in the alcohol of the hydroxyasparagine residue is directly derived from dioxygen. Sequence analyses involving FIH link the 2-OG oxygenases with members of the cupin superfamily, including Zn(II)-utilizing phosphomannose isomerase, revealing structural and evolutionary links between these metal-binding proteins that share common motifs.Hypoxia in animals activates a broad range of homeostatic responses via induction of a transcriptional complex termed hypoxia-inducible factor (HIF) 1 (1, 2). HIF is a heterodimer of ␣-and ␤-subunits with regulation by dioxygen availability being mediated by post-translational modification of the ␣-subunits (1, 2). In mammalian cells, two HIF-␣ subunit isoforms (HIF-1␣ and HIF-2␣) are regulated by dioxygen levels. Each HIF-␣ protein contains an internal oxygen-dependent degradation domain possessing targeting motifs for proteolytic regulation and a C-terminal transactivation domain (CAD) independently regulated by dioxygen, irrespective of changes in protein abundance, through interaction with the CH1 domain of the co-activator p300 (for review, see Ref.3).The oxygen-dependent degradation of HIF-␣ by proteolysis is regulated by the hydroxylation of specific prolyl residues (Pro-402 and Pro-564 in human HIF-1␣) that mediate recognition of HIF-␣ by the von Hippel-Lindau (VHL) ubiquitinylation complex and consequent proteasomal destruction (4 -7). Combined structural analysis and genetic approaches led to the identification of three isoforms of human HIF prolyl hydroxylase (PHD1-3, prolyl hydroxylase domain) together with homologues in a range of organisms (8,9). In vitro analyses together with sequence and mutational analyses identified these as belonging to a subfamily of the Fe(II)-and 2-oxoglutarate (2-OG)-dependent oxygenases (4,5,8,9). Limiting oxygen availability in hypoxia, or direct inhibition of the PHD enzymes by cobaltous ions and iron chelators, allows HIF-␣ to escape hydroxylation and recognition by pVHL, providing insights into the mechanism by which these stimuli suppress HIF-␣ degradation and activate the transcriptional casca...

D-type cyclins (cyclins D1, D2, and D3) are regarded as essential links between cell environment and the core cell cycle machinery. We tested the requirement for D-cyclins in mouse development and in proliferation by generating mice lacking all D-cyclins. We found that these cyclin D1(-/-)D2(-/-)D3(-/-) mice develop until mid/late gestation and die due to heart abnormalities combined with a severe anemia. Our analyses revealed that the D-cyclins are critically required for the expansion of hematopoietic stem cells. In contrast, cyclin D-deficient fibroblasts proliferate nearly normally but show increased requirement for mitogenic stimulation in cell cycle re-entry. We found that the proliferation of cyclin D1(-/-)D2(-/-)D3(-/-) cells is resistant to the inhibition by p16(INK4a), but it critically depends on CDK2. Lastly, we found that cells lacking D-cyclins display reduced susceptibility to the oncogenic transformation. Our results reveal the presence of alternative mechanisms that allow cell cycle progression in a cyclin D-independent fashion.

ABSTRACTp300 and CBP are homologous transcription adapters targeted by the E1A oncoprotein. They participate in numerous biological processes, including cell cycle arrest, differentiation, and transcription activation. p300 and͞or CBP (p300͞CBP) also coactivate CREB. How they participate in these processes is not yet known. In a search for specific p300 binding proteins, we have cloned the intact cDNA for HIF-1␣. This transcription factor mediates hypoxic induction of genes encoding certain glycolytic enzymes, erythropoietin (Epo), and vascular endothelial growth factor. Hypoxic conditions lead to the formation of a DNA binding complex containing both HIF-1␣ and p300͞CBP. Hypoxia-induced transcription from the Epo promoter was specifically enhanced by ectopic p300 and inhibited by E1A binding to p300͞CBP. Hypoxia-induced VEGF and Epo mRNA synthesis were similarly inhibited by E1A. Hence, p300͞CBP-HIF complexes participate in the induction of hypoxia-responsive genes, including one (vascular endothelial growth factor) that plays a major role in tumor angiogenesis. Paradoxically, these data, to our knowledge for the first time, suggest that p300͞ CBP are active in both transformation suppression and tumor development.

Recruitment of p300/CBP by the hypoxia-inducible factor, HIF-1, is essential for the transcriptional response to hypoxia and requires an interaction between the p300/CBP CH1 region and HIF-1␣. A new p300-CH1 interacting protein, p35srj, has been identified and cloned. p35srj is an alternatively spliced isoform of MRG1, a human protein of unknown function. Virtually all endogenous p35srj is bound to p300/CBP in vivo, and it inhibits HIF-1 transactivation by blocking the HIF-1␣/p300 CH1 interaction. p35srj did not affect transactivation by transcription factors that bind p300/CBP outside the CH1 region. Endogenous p35srj is up-regulated markedly by the HIF-1 activators hypoxia or deferoxamine, suggesting that it could operate in a negative-feedback loop. In keeping with this notion, a p300 CH1 mutant domain, defective in HIF-1 but not p35srj binding, enhanced endogenous HIF-1 function. In hypoxic cells, p35srj may regulate HIF-1 transactivation by controlling access of HIF-1␣ to p300/CBP, and may keep a significant portion of p300/CBP available for interaction with other transcription factors by partially sequestering and functionally compartmentalizing cellular p300/CBP.[Key Words: Hypoxia; HIF-1; transcription; p35srj; p300; CBP; regulation]Received August 14, 1998; revised version accepted November 17, 1998.Localized tissue hypoxia is a major factor in the pathogenesis of tumor vascularization, myocardial ischemia, and stroke. An hypoxic signal is transduced through a hemoprotein oxygen sensor and results in the induction of the transcription factor, hypoxia inducible factor-1 (HIF-1). HIF-1 consists of HIF-1␣ and ARNT (aryl hydrocarbon nuclear translocator) subunits for review, see Guillemin and Krasnow 1997). Hypoxia results in the stabilization of the HIF-1␣ protein, a marked increase in its transactivation potential, and heterodimerization of HIF-1␣ with ARNT to form active HIF-1 (Li et al. 1996;Jiang et al. 1997;Kallio et al. 1997;Pugh 1997;Huang et al. 1998). The active HIF-1 heterodimer binds to its cognate response element and activates transcription of several hypoxia-induced genes, including those encoding erythropoietin, vascular endothelial growth factor (VEGF), and key glycolytic enzymes. HIF-1␣ deficiency results in embryonic lethality, with severe neurological and cardiovascular developmental abnormalities and impaired expression of HIF-1 target genes. This suggests that HIF-1␣ is a global regulator of developmental and cellular O 2 homeostasis (Iyer et al. 1998).HIF-1␣ activates transcription by recruiting the transcriptional adapter/histone acetyltransferase proteins, p300 and CBP (CREB-binding protein), to a transcription complex. This is essential for the normal cellular response to hypoxia . p300 and CBP (Fig.1A) are homologous, ubiquitously expressed, nuclear phosphoproteins that contain three highly conserved cysteine-histidine-rich domains (CH1, -2, and -3). p300/ CBP function as coactivators, linking signal-responsive DNA-bound transcriptional activators to the basal transcription mac...

Congenital Heart Defects (CHD) have a neonatal incidence of 0.8-1%1,2. Despite abundant examples of monogenic CHD in humans and mice, CHD has a low absolute sibling recurrence risk (~2.7%)3, suggesting a considerable role for de novo mutations (DNM), and/or incomplete penetrance4,5. De novo protein-truncating variants (PTVs) have been shown to be enriched among the 10% of ‘syndromic’ patients with extra-cardiac manifestations6,7. We exome sequenced 1,891 probands, including both syndromic (S-CHD, n=610) and non-syndromic cases (NS-CHD, n=1,281). In S-CHD, we confirmed a significant enrichment of de novo PTVs, but not inherited PTVs, in known CHD-associated genes, consistent with recent findings8. Conversely, in NS-CHD we observed significant enrichment of PTVs inherited from unaffected parents in CHD-associated genes. We identified three novel genome-wide significant S-CHD disorders caused by DNMs in CHD4, CDK13 and PRKD1. Our study reveals distinct genetic architectures underlying the low sibling recurrence risk in S-CHD and NS-CHD.

The protein EP300 and its paralog CREBBP (CREB-binding protein) are ubiquitously expressed transcriptional co-activators and histone acetyl transferases. The gene EP300 is essential for normal cardiac and neural development, whereas CREBBP is essential for neurulation, hematopoietic differentiation, angiogenesis and skeletal and cardiac development. Mutations in CREBBP cause Rubinstein-Taybi syndrome, which is characterized by mental retardation, skeletal abnormalities and congenital cardiac defects. The CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2) binds EP300 and CREBBP with high affinity and regulates gene transcription. Here we show that Cited2-/- embryos die with cardiac malformations, adrenal agenesis, abnormal cranial ganglia and exencephaly. The cardiac defects include atrial and ventricular septal defects, overriding aorta, double-outlet right ventricle, persistent truncus arteriosus and right-sided aortic arches. We find increased apoptosis in the midbrain region and a marked reduction in ErbB3-expressing neural crest cells in mid-embryogenesis. We show that CITED2 interacts with and co-activates all isoforms of transcription factor AP-2 (TFAP2). Transactivation by TFAP2 isoforms is defective in Cited2-/- embryonic fibroblasts and is rescued by ectopically expressed CITED2. As certain Tfap2 isoforms are essential in neural crest, neural tube and cardiac development, we propose that abnormal embryogenesis in mice lacking Cited2 results, at least in part, from its role as a Tfap2 co-activator.

The transcription factor ISGF3 transduces interferon (IFN)-alpha signals and activates the transcription of cellular antiviral defence genes. Adenovirus E1A blocks the IFN-alpha response, allowing unhindered viral replication. ISGF3 consists of Stat1, Stat2 and p48. Here we show that p300 and/or CBP (CREB-binding protein), which are transcription adaptors targeted by E1A, interact specifically with Stat2. Binding occurs between the first cysteine-histidine-rich region of p300/CBP and the carboxy-terminal segment of Stat2, a domain essential for ISGF3 function. We find that this domain of Stat2 has transactivation potential, which correlates with its binding to p300/CBP. Moreover, E1A represses Stat2 transactivation and IFN-alpha-activated transcription by inhibiting p300/CBP function. This provides a new mechanism for inhibition of the IFN-alpha-activated antiviral response by E1A, and supports the view that E1A binding to p300/CBP has functional significance for adenovirus replication in its natural host.