The Klotho gene encodes a 130-kDa single-pass transmembrane protein with a short cytoplasmic domain (10 amino acids) and is expressed predominantly in the kidney. Mice carrying a loss-of-function mutation in the Klotho gene develop a syndrome resembling human aging, including shortened life span, skin atrophy, muscle atrophy, osteoporosis, arteriosclerosis, and pulmonary emphysema (1). Conversely, overexpression of the Klotho gene extends the life span and increases resistance to oxidative stress in mice (2-4). These observations suggest that the Klotho gene functions as an aging suppressor gene. The extracellular domain of Klotho protein is shed and secreted in the blood (2, 5), potentially functioning as a humoral factor that signals suppression of intracellular insulin/IGF1 signaling, which partly contributes to its anti-aging properties (2). However, a signaling pathway(s) directly activated by Klotho protein, including the identity of the Klotho receptor, has not been determined. The function of the transmembrane form of Klotho protein also remains to be determined.Fibroblast growth factor-23 (FGF23) 2 was originally identified as a gene mutated in patients with autosomal dominant hypophosphatemic rickets (6), where mutations in the FGF23 gene conferred resistance to inactivation by protease cleavage, resulting in elevated serum levels of FGF23 (7-12). FGF23 inhibits phosphate transport in renal proximal tubular cells and in proximal tubules perfused in vitro (13). Consistent with these findings, mice defective in FGF23 expression show increased renal phosphate reabsorption and hyperphosphatemia (14). Although FGF23 binds to multiple FGF receptors (FGFRs) (15), it has modest receptor affinity (K D ϭ 200 -700 nM) and often requires cofactors such as heparin or glycosaminoglycan (15, 16) to activate FGF signaling in cultured cells and to inhibit phosphate transport in proximal tubules perfused in vitro (13).Klotho-deficient mice (Klotho Ϫ/Ϫ mice) and FGF23 deficient mice (Fgf23 Ϫ/Ϫ mice) develop many common phenotypes, including shortened life span, growth retardation, infertility, muscle atrophy, hypoglycemia, and vascular calcification in the kidneys. Notably, they both have increased serum levels of phosphate (14, 17). These observations have led us to the hypothesis that Klotho and FGF23 may function via a common signal transduction pathway. In this report we show that Klotho binds to multiple FGFRs and functions as a cofactor necessary for FGF signaling activation by FGF23. MATERIALS AND METHODSExpression Vectors-Complementary DNA containing the mouse FGFRs coding region (IMAGE Clone, Invitrogen, supplemental Fig. 1) were cloned into pcDNA3.1(ϩ) expression vector (Invitrogen). Before subcloning, a V5-epitope tag was added to the C terminus and appropriate restriction enzyme sites to the both ends using synthetic oligonucleotides and polymerase chain reaction. Expression vectors for the mouse FGF23 resistant to proteolytic inactivation (R179Q) (18), the transmembrane form of mouse Klotho, and the extracel...

SUMMARY Polycomb proteins play essential roles in stem cell renewal and human disease. Recent studies of HOX genes and X-inactivation have provided evidence for RNA cofactors in Polycomb repressive complex 2 (PRC2). Here, we develop a RIP-seq method to capture the PRC2 transcriptome and identify a genome-wide pool of >9,000 PRC2-interacting RNAs in embryonic stem cells. The transcriptome includes antisense, intergenic, and promoter-associated transcripts, as well as many unannotated RNAs. A large number of transcripts occur within imprinted regions, oncogene and tumor suppressor loci, and stem-cell-related bivalent domains. We provide evidence for direct RNA-protein interactions, most likely via the Ezh2 subunit. We also identify Gtl2 RNA as a PRC2 cofactor that directs PRC2 to the reciprocally imprinted Dlk1 coding gene. Thus, Polycomb proteins interact with a genome-wide family of RNAs, some of which may be used as biomarkers and therapeutic targets for human disease.

The fibroblast growth factor (FGF) 19 subfamily of ligands, FGF19, FGF21, and FGF23, function as hormones that regulate bile acid, fatty acid, glucose, and phosphate metabolism in target organs through activating FGF receptors (FGFR1-4). We demonstrated that Klotho and ␤Klotho, homologous single-pass transmembrane proteins that bind to FGFRs, are required for metabolic activity of FGF23 and FGF21, respectively. Here we show that, like FGF21, FGF19 also requires ␤Klotho. Both FGF19 and FGF21 can signal through FGFR1-3 bound by ␤Klotho and increase glucose uptake in adipocytes expressing FGFR1. Additionally, both FGF19 and FGF21 bind to the ␤Klotho-FGFR4 complex; however, only FGF19 signals efficiently through FGFR4. Accordingly, FGF19, but not FGF21, activates FGF signaling in hepatocytes that primarily express FGFR4 and reduces transcription of CYP7A1 that encodes the rate-limiting enzyme for bile acid synthesis. We conclude that the expression of ␤Klotho, in combination with particular FGFR isoforms, determines the tissue-specific metabolic activities of FGF19 and FGF21.