Two Krebs cycle genes, fumarate hydratase (FH) and succinate dehydrogenase (SDH), are mutated in a subset of human cancers, leading to accumulation of their substrates, fumarate and succinate, respectively. Here we demonstrate that fumarate and succinate are competitive inhibitors of multiple a-ketoglutarate (a-KG)-dependent dioxygenases, including histone demethylases, prolyl hydroxylases, collagen prolyl-4-hydroxylases, and the TET (ten-eleven translocation) family of 5-methlycytosine (5mC) hydroxylases. Knockdown of FH and SDH results in elevated intracellular levels of fumarate and succinate, respectively, which act as competitors of a-KG to broadly inhibit the activity of a-KG-dependent dioxygenases. In addition, ectopic expression of tumor-derived FH and SDH mutants inhibits histone demethylation and hydroxylation of 5mC. Our study suggests that tumor-derived FH and SDH mutations accumulate fumarate and succinate, leading to enzymatic inhibition of multiple a-KG-dependent dioxygenases and consequent alterations of genome-wide histone and DNA methylation. These epigenetic alterations associated with mutations of FH and SDH likely contribute to tumorigenesis.[Keywords: FH; SDH; metabolites; a-KG-dependent dioxygenases; DNA methylation; histone methylation] Supplemental material is available for this article. Received March 7, 2012; revised version accepted May 9, 2012. Several lines of evidence, including the recent identification of mutations affecting isocitrate dehydrogenase (IDH), fumarate hydratase (FH), and succinate dehydrogenase (SDH), have demonstrated that mutations in certain metabolic enzymes may play a causal role in tumorigenesis. The NADP + -dependent IDH genes IDH1 and IDH2 are frequently mutated in >75% of glioma (Parsons et al. 2008),

The Yes-associated protein (YAP) transcriptional coactivator is a key regulator of organ size and a candidate human oncogene inhibited by the Hippo tumor suppressor pathway. The TEAD family of transcription factors binds directly to and mediates YAP-induced gene expression. Here we report the three-dimensional structure of the YAP (residues 50-171)-TEAD1 (residues 194-411) complex, in which YAP wraps around the globular structure of TEAD1 and forms extensive interactions via three highly conserved interfaces. Interface 3, including YAP residues 86-100, is most critical for complex formation. Our study reveals the biochemical nature of the YAP-TEAD interaction, and provides a basis for pharmacological intervention of YAP-TEAD hyperactivation in human diseases. The Yes-associated protein (YAP) is a transcriptional coactivator (Yagi et al. 1999). YAP knockout in mice causes embryonic lethality (Morin-Kensicki et al. 2006), indicating its critical role in development. In contrast, transgenic expression of YAP dramatically increases mouse liver size in a reversible fashion (Camargo et al. 2007;Dong et al. 2007), suggesting a key role of YAP in organ size regulation. Consistent with its growth-promoting function, yap genomic amplification and elevated protein levels have been observed in several human cancers (Overholtzer et al. 2006;Zender et al. 2006;Dong et al. 2007;Zhao et al. 2007;Steinhardt et al. 2008). Furthermore, expression of active YAP potently induces transformation in both NIH3T3 and MCF10A cells (Overholtzer et al. 2006;Zhao et al. 2009), and liver-specific transgenic expression of YAP causes tumor formation in vivo (Camargo et al. 2007;Dong et al. 2007). These observations support the function of YAP as a human oncogene.As a transcriptional coactivator, YAP needs to bind transcription factors to stimulate gene expression. Reported YAP target transcription factors include TEAD, p73, Runx2, and the ErbB4 cytoplasmic domain (Yagi et al. 1999;Strano et al. 2001;Vassilev et al. 2001;Komuro et al. 2003). However, only TEAD has been demonstrated to be important for the growth-promoting function of YAP Zhao et al. 2008). In humans, the TEAD family has four highly homologous proteins sharing a conserved DNA-binding TEA domain. YAP and TEAD1 bind to a common set of promoters in MCF10A cells (Zhao et al. 2008). Knockdown of TEAD aborts expression of the majority of YAP-inducible genes and largely attenuates YAP-induced overgrowth, epithelial-mesenchymal transition (EMT), and oncogenic transformation (Zhao et al. 2008). Furthermore, the phenotype of TEAD1/TEAD2 double-knockout mice resembles YAP knockout mice . Notably, the role of the YAP and TEAD complex in growth promotion is implicated in Sveinsson's chorioretinal atrophy caused by the TEAD1 Y406H mutation, which abolishes its interaction with and activation by YAP (Kitagawa 2007;Zhao et al. 2008). Consistently, Scalloped (Sd), the Drosophila homolog of TEAD, directly mediates Yorkie (Yki)-induced gene expression and overgrowth phenotypes (Zhao et al. 2007;Gou...

Protein Phosphatase 2A (PP2A) plays an essential role in many aspects of cellular physiology. The PP2A holoenzyme consists of a heterodimeric core enzyme, which comprises a scaffolding subunit and a catalytic subunit, and a variable regulatory subunit. Here we report the crystal structure of the heterotrimeric PP2A holoenzyme involving the regulatory subunit B'/B56/PR61. Surprisingly, the B'/PR61 subunit has a HEAT-like (huntingtin-elongation-A subunit-TOR-like) repeat structure, similar to that of the scaffolding subunit. The regulatory B'/B56/PR61 subunit simultaneously interacts with the catalytic subunit as well as the conserved ridge of the scaffolding subunit. The carboxyterminus of the catalytic subunit recognizes a surface groove at the interface between the B'/B56/PR61 subunit and the scaffolding subunit. Compared to the scaffolding subunit in the PP2A core enzyme, formation of the holoenzyme forces the scaffolding subunit to undergo pronounced conformational rearrangements. This structure reveals significant ramifications for understanding the function and regulation of PP2A.

TET proteins oxidize 5-methylcytosine (5mC) on DNA and play important roles in various biological processes. Mutations of TET2 are frequently observed in myeloid malignance. Here, we present the crystal structure of human TET2 bound to methylated DNA at 2.02 Å resolution. The structure shows that two zinc fingers bring the Cys-rich and DSBH domains together to form a compact catalytic domain. The Cys-rich domain stabilizes the DNA above the DSBH core. TET2 specifically recognizes CpG dinucleotide and shows substrate preference for 5mC in a CpG context. 5mC is inserted into the catalytic cavity with the methyl group orientated to catalytic Fe(II) for reaction. The methyl group is not involved in TET2-DNA contacts so that the catalytic cavity allows TET2 to accommodate 5mC derivatives for further oxidation. Mutations of Fe(II)/NOG-chelating, DNA-interacting, and zinc-chelating residues are frequently observed in human cancers. Our studies provide a structural basis for understanding the mechanisms of TET-mediated 5mC oxidation.