The F-box protein Skp2 mediates c-Myc ubiquitylation by binding to the MB2 domain. However, the turnover of cMyc is largely dependent on phosphorylation of threonine-58 and serine-62 in MB1, residues that are often mutated in cancer. We now show that the F-box protein Fbw7 interacts with and thereby destabilizes c-Myc in a manner dependent on phosphorylation of MB1. Whereas wild-type Fbw7 promoted c-Myc turnover in cells, an Fbw7 mutant lacking the F-box domain delayed it. Furthermore, depletion of Fbw7 by RNA interference increased both the abundance and transactivation activity of c-Myc. Accumulation of cMyc was also apparent in mouse Fbw7 À/À embryonic stem cells. These observations suggest that two F-box proteins, Fbw7 and Skp2, differentially regulate c-Myc stability by targeting MB1 and MB2, respectively.

Autism spectrum disorder (ASD) comprises a range of neurodevelopmental disorders characterized by deficits in social interaction and communication as well as by restricted and repetitive behaviours. ASD has a strong genetic component with high heritability. Exome sequencing analysis has recently identified many de novo mutations in a variety of genes in individuals with ASD, with CHD8, a gene encoding a chromatin remodeller, being most frequently affected. Whether CHD8 mutations are causative for ASD and how they might establish ASD traits have remained unknown. Here we show that mice heterozygous for Chd8 mutations manifest ASD-like behavioural characteristics including increased anxiety, repetitive behaviour, and altered social behaviour. CHD8 haploinsufficiency did not result in prominent changes in the expression of a few specific genes but instead gave rise to small but global changes in gene expression in the mouse brain, reminiscent of those in the brains of patients with ASD. Gene set enrichment analysis revealed that neurodevelopment was delayed in the mutant mouse embryos. Furthermore, reduced expression of CHD8 was associated with abnormal activation of RE-1 silencing transcription factor (REST), which suppresses the transcription of many neuronal genes. REST activation was also observed in the brains of humans with ASD, and CHD8 was found to interact physically with REST in the mouse brain. Our results are thus consistent with the notion that CHD8 haploinsufficiency is a highly penetrant risk factor for ASD, with disease pathogenesis probably resulting from a delay in neurodevelopment.

Mammalian Fbw7 (also known as Sel-10, hCdc4, or hAgo) is the F-box protein component of an SCF (Skp1-Cul1-F-box protein-Rbx1)-type ubiquitin ligase, and the mouse Fbw7 is expressed prominently in the endothelial cell lineage of embryos. We generated mice deficient in Fbw7 and found that the embryos died in utero at embryonic day 10.5-11.5, manifesting marked abnormalities in vascular development. Vascular remodeling was impaired in the brain and yolk sac, and the major trunk veins were not formed. In vitro para-aortic splanchnopleural explant cultures from Fbw7 ؊/؊ embryos also manifested an impairment of vascular network formation. Notch4, which is the product of the proto-oncogene Int3 and an endothelial cell-specific mammalian isoform of Notch, accumulated in Fbw7 ؊/؊ embryos, resulting in an increased expression of Hey1, which encodes a transcriptional repressor that acts downstream of Notch signaling and is implicated in vascular development. Expression of Notch1, -2, or -3 or of cyclin E was unaffected in Fbw7 ؊/؊ embryos. Mammalian Fbw7 thus appears to play an indispensable role in negative regulation of the Notch4-Hey1 pathway and is required for vascular development.

The chromodomain helicase DNA-binding (CHD) family of enzymes is thought to regulate gene expression, but their role in the regulation of specific genes has been unclear. Here we show that CHD8 is expressed at a high level during early embryogenesis and prevents apoptosis mediated by the tumour suppressor protein p53. CHD8 was found to bind to p53 and to suppress its transactivation activity. CHD8 promoted the association of p53 and histone H1, forming a trimeric complex on chromatin that was required for inhibition of p53-dependent transactivation and apoptosis. Depletion of CHD8 or histone H1 resulted in p53 activation and apoptosis. Furthermore, Chd8−/− mice died early during embryogenesis, manifesting widespread apoptosis, whereas deletion of p53 ameliorated this developmental arrest. These observations reveal a mode of p53 regulation mediated by CHD8, which may set a threshold for induction of apoptosis during early embryogenesis by counteracting p53 function through recruitment of histone H1.

Iron-dependent degradation of iron-regulatory protein 2 (IRP2) is a key event for maintenance of an appropriate intracellular concentration of iron. Although FBXL5 (F box and leucine-rich repeat protein 5) is thought to mediate this degradation, the role of FBXL5 in the control of iron homeostasis in vivo has been poorly understood. We have now found that mice deficient in FBXL5 died in utero, associated with excessive iron accumulation. This embryonic mortality was prevented by additional ablation of IRP2, suggesting that impaired IRP2 degradation is primarily responsible for the death of Fbxl5(-)(/-) mice. We also found that liver-specific deletion of Fbxl5 resulted in deregulation of both hepatic and systemic iron homeostasis, leading to the development of steatohepatitis. The liver-specific mutant mice died with acute liver failure when fed a high-iron diet. Thus, our results uncover a major role for FBXL5 in ensuring an appropriate supply of iron to cells.

Members of the chromodomain helicase DNA-binding (CHD) family of proteins are thought to regulate gene expression. Among mammalian CHD proteins, CHD8 was originally isolated as a negative regulator of the Wnt-␤-catenin signaling pathway that binds directly to ␤-catenin and suppresses its transactivation activity. The mechanism by which CHD8 inhibits ␤-catenin-dependent transcription has been unclear, however. Here we show that CHD8 promotes the association of ␤-catenin and histone H1, with formation of the trimeric complex on chromatin being required for inhibition of ␤-catenin-dependent transactivation. A CHD8 mutant that lacks the histone H1 binding domain did not show such inhibitory activity, indicating that histone H1 recruitment is essential for the inhibitory effect of CHD8. Furthermore, either depletion of histone H1 or expression of a dominant negative mutant of this protein resulted in enhancement of the response to Wnt signaling. These observations reveal a new mode of regulation of the Wnt signaling pathway by CHD8, which counteracts ␤-catenin function through recruitment of histone H1 to Wnt target genes. Given that CHD8 is expressed predominantly during embryogenesis, it may thus contribute to setting a threshold for responsiveness to Wnt signaling that operates in a development-dependent manner.

The Wnt signaling pathway plays a pivotal role in vertebrate early development and morphogenesis. Duplin (axis duplication inhibitor) interacts with ␤-catenin and prevents its binding to Tcf, thereby inhibiting downstream Wnt signaling. Here we show that Duplin is expressed predominantly from early-to mid-stage mouse embryogenesis, and we describe the generation of mice deficient in Duplin. Duplin ؊/؊ embryos manifest growth retardation from embryonic day 5.5 (E5.5) and developmental arrest accompanied by massive apoptosis at E7.5. The mutant embryos develop into an egg cylinder but do not form a primitive streak or mesoderm. Expression of ␤-catenin target genes, including those for T (brachyury), Axin2, and cyclin D1, was not increased in Duplin ؊/؊ embryos, suggesting that the developmental defect is not simply attributable to upregulation of Wnt signaling caused by the lack of this inhibitor. These results suggest that Duplin plays an indispensable role, likely by a mechanism independent of inhibition of Wnt signaling, in mouse embryonic growth and differentiation at an early developmental stage.

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