The PIK3CA gene encoding the p110α subunit of Class IA phosphatidylinositol 3-kinases (PI3Ks) is frequently mutated in human tumors. Mutations in the PIK3CB gene encoding p110β, the only other widely expressed Class IA PI3K, have not been reported. We compared the biochemical activity and transforming potential of mutant forms of p110α and p110β in a human mammary epithelial cell system. The two most common tumor-derived alleles of p110α, H1047R and E545K , potently activated PI3K signaling. Human mammary epithelial cells expressing these alleles grew efficiently in soft agar and as orthotopic tumors in nude mice. We also examined a third class of mutations in p110α, those in the p85-binding domain. A representative tumor-derived p85-binding-domain mutant R38H showed modestly reduced p85 binding and weakly activated PI3K/Akt signaling. In contrast, a deletion mutant lacking the entire p85-binding domain efficiently activated PI3K signaling. When we constructed in p110β a mutation homologous to the E545K allele of p110α, the resulting p110β mutant was only weakly activated and allowed minimal soft-agar growth. However, a gene fusion of p110β with the membrane anchor from c-Src was highly active and transforming in both soft-agar and orthotopic nude mouse assays. Thus, although introduction of activating mutations from p110α at the corresponding sites in p110β failed to render the enzyme oncogenic in human cells, the possibility remains that other mutations might activate the β isoform.

Mutations in the PHF8 gene, which encodes the plant homeo domain (PHD) finger protein 8, are connected to X-linked mental retardation associated with cleft lip and cleft palate. Two groups now report that the PHF8 protein is a histone demethylase with activity against H4K20me1 (histone H4 lysine 20). Qi et al. report a role for PHF8 in regulating gene expression, as well as in neuronal cell survival and craniofacial development in zebrafish. The results suggest there may be a link between histone methylation dynamics and X-linked mental retardation. Liu et al. show that PHF8 is linked to two distinct events during cell-cycle progression. PHF8 is recruited to the promoters of G1/S-phase genes where it removes H4K20me1 and contributes to gene activation, whereas dissociation of PHF8 from chromatin in prophase allows H4K20me1 to accumulate during mitosis. PHF8 is a JmjC domain-containing protein, the gene for which has been linked to X-linked mental retardation (XLMR). These authors demonstrate PHF8 to be a histone demethylase with activity against H4K20me1. It has a role in regulating gene expression as well as in neuronal cell survival and craniofacial development in zebrafish. The results suggest there may be a link between histone methylation dynamics and XLMR. X-linked mental retardation (XLMR) is a complex human disease that causes intellectual disability1. Causal mutations have been found in approximately 90 X-linked genes2; however, molecular and biological functions of many of these genetically defined XLMR genes remain unknown. PHF8 (PHD (plant homeo domain) finger protein 8) is a JmjC domain-containing protein and its mutations have been found in patients with XLMR and craniofacial deformities. Here we provide multiple lines of evidence establishing PHF8 as the first mono-methyl histone H4 lysine 20 (H4K20me1) demethylase, with additional activities towards histone H3K9me1 and me2. PHF8 is located around the transcription start sites (TSS) of ∼7,000 RefSeq genes and in gene bodies and intergenic regions (non-TSS). PHF8 depletion resulted in upregulation of H4K20me1 and H3K9me1 at the TSS and H3K9me2 in the non-TSS sites, respectively, demonstrating differential substrate specificities at different target locations. PHF8 positively regulates gene expression, which is dependent on its H3K4me3-binding PHD and catalytic domains. Importantly, patient mutations significantly compromised PHF8 catalytic function. PHF8 regulates cell survival in the zebrafish brain and jaw development, thus providing a potentially relevant biological context for understanding the clinical symptoms associated with PHF8 patients. Lastly, genetic and molecular evidence supports a model whereby PHF8 regulates zebrafish neuronal cell survival and jaw development in part by directly regulating the expression of the homeodomain transcription factor MSX1/MSXB, which functions downstream of multiple signalling and developmental pathways3. Our findings indicate that an imbalance of histone methylation dynamics has a critical role in XLMR.

The PI3K pathway is frequently activated in cancer; therefore, considerable effort is focused on identifying compounds that can inhibit specific pathway components, particularly the hallmark oncogene PIK3CA. Although targeted inhibition of a cancer survival gene holds significant promise, there are concerns that drug resistance may emerge within the cancerous cells, thus limiting clinical efficacy. Using genetically defined human mammary epithelial cells, we evolved resistance to the PI3K/mammalian target of rapamycin (mTOR) inhibitor BEZ235, and by genome-wide copy number analyses, we identified MYC and eIF4E amplification within the resistant cells. Importantly, either MYC or eukaryotic translation initiation factor 4E (eIF4E) was required to bypass pharmacological PI3K/mTOR inhibition in resistant cells. Furthermore, these cells displayed elevated 5′ cap-dependent protein translation. Collectively, these findings suggest that analysis of drivers of protein translation could facilitate the identification of cancer lesions that confer resistance to PI3K pathway-targeted drugs.

There is an interesting overlap of function in a wide range of organisms between genes that modulate the stress responses and those that regulate aging phenotypes and, in some cases, lifespan. We have therefore screened mutagenized zebrafish embryos for the altered expression of a stress biomarker, senescence-associated β-galactosidase (SA-β-gal) in our current study. We validated the use of embryonic SA-β-gal production as a screening tool by analyzing a collection of retrovirus-insertional mutants. From a pool of 306 such mutants, we identified 11 candidates that showed higher embryonic SA-β-gal activity, two of which were selected for further study. One of these mutants is null for a homologue of Drosophila spinster, a gene known to regulate lifespan in flies, whereas the other harbors a mutation in a homologue of the human telomeric repeat binding factor 2 (terf2) gene, which plays roles in telomere protection and telomere-length regulation. Although the homozygous spinster and terf2 mutants are embryonic lethal, heterozygous adult fish are viable and show an accelerated appearance of aging symptoms including lipofuscin accumulation, which is another biomarker, and shorter lifespan. We next used the same SA-β-gal assay to screen chemically mutagenized zebrafish, each of which was heterozygous for lesions in multiple genes, under the sensitizing conditions of oxidative stress. We obtained eight additional mutants from this screen that, when bred to homozygosity, showed enhanced SA-β-gal activity even in the absence of stress, and further displayed embryonic neural and muscular degenerative phenotypes. Adult fish that are heterozygous for these mutations also showed the premature expression of aging biomarkers and the accelerated onset of aging phenotypes. Our current strategy of mutant screening for a senescence-associated biomarker in zebrafish embryos may thus prove to be a useful new tool for the genetic dissection of vertebrate stress response and senescence mechanisms. By performing genetic mutant screens using senescence-associated biomarkers, we show that the zebrafish is a tractable model system for the study of aging. In vertebrate organisms, it has not previously been possible to carry out systematic screens for genes that are important for stress responses and aging in an unbiased way. However, such vertebrate models are of considerable importance, given the provocative evidence of common biochemical and functional pathways modulating stress responses and lifespan as well as aging in a wide range of organisms. Our present study has successfully employed a colorimetric high-throughput method using a senescence-associated β-galactosidase-based assay to screen for mutations that alter the stress responses in zebrafish embryos, in the hope that these might represent potential aging mutants. Subsequently, the mutations identified by embryonic senescence have indeed displayed adult aging-related phenotypes in zebrafish. Hence, our method for the identification of mutant zebrafish has the immediate potential to accelerate the discovery of novel genes and new functions relevant for our understanding of aging processes in vertebrates. Such knowledge will be essential for the ultimate development of pharmacological, nutritional, and behavioral interventions for the amelioration of oxidative stress- and age-associated diseases and disabilities in humans.