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Mitochondrial DNA (mtDNA) has been reported to contain 5-methylcytosine (5mC) at CpG dinucleotides, as in the nuclear genome, but neither the mechanism generating mtDNA methylation nor its functional significance is known. We now report the presence of 5-hydroxymethylcytosine (5hmC) as well as 5mC in mammalian mtDNA, suggesting that previous studies underestimated the level of cytosine modification in this genome. DNA methyltransferase 1 (DNMT1) translocates to the mitochondria, driven by a mitochondrial targeting sequence located immediately upstream of the commonly accepted translational start site. This targeting sequence is conserved across mammals, and the encoded peptide directs a heterologous protein to the mitochondria. DNMT1 is the only member of the three known catalytically active DNA methyltransferases targeted to the mitochondrion. Mitochondrial DNMT1 (mtDNMT1) binds to mtDNA, proving the presence of mtDNMT1 in the mitochondrial matrix. mtDNMT1 expression is up-regulated by NRF1 and PGC1α, transcription factors that activate expression of nuclear-encoded mitochondrial genes in response to hypoxia, and by loss of p53, a tumor suppressor known to regulate mitochondrial metabolism. Altered mtDNMT1 expression asymmetrically affects expression of transcripts from the heavy and light strands of mtDNA. Hence, mtDNMT1 appears to be responsible for mtDNA cytosine methylation, from which 5hmC is presumed to be derived, and its expression is controlled by factors that regulate mitochondrial function. mitochondrial epigenetics | epigenetics | 5-hydroxymethylation

Careful patient selection combined with skilled cannulation minimizes complications. Higher-risk procedures should be performed in specialist centers.

SUMMARYMaternal IgG is transferred to the suckling mouse and rat through a major histocompatibility complex (MHC ) class I-related Fc receptor (FcRn) on the brush border of the proximal small intestine. We have previously described a site on the epithelial surface of the human fetal intestine with IgG binding characteristics similar to FcRn. We report here the identification by reverse transcriptase polymerase chain reaction amplification and sequencing of the human orthologue of rat and mouse FcRn in tissue obtained from human fetal and adult intestine. FcRn protein was detected in adult human intestine by western blot. Immunohistochemical studies of sections of human intestine show that the FcRn is localized mostly to the epithelial cells, where it is in the apical region. These data suggest that the binding of IgG previously seen in the fetal intestine is due to the presence of FcRn. Potential roles for this MHC class I-like Fc receptor in the human intestine include the transfer of passive immunity, induction of oral tolerance, and immunosurveillance.

SummaryFewer than half of children with high-risk neuroblastoma survive. Many of these tumors harbor high-level amplification of MYCN, which correlates with poor disease outcome. Using data from our large drug screen we predicted, and subsequently demonstrated, that MYCN-amplified neuroblastomas are sensitive to the BCL-2 inhibitor ABT-199. This sensitivity occurs in part through low anti-apoptotic BCL-xL expression, high pro-apoptotic NOXA expression, and paradoxical, MYCN-driven upregulation of NOXA. Screening for enhancers of ABT-199 sensitivity in MYCN-amplified neuroblastomas, we demonstrate that the Aurora Kinase A inhibitor MLN8237 combines with ABT-199 to induce widespread apoptosis. In diverse models of MYCN-amplified neuroblastoma, including a patient-derived xenograft model, this combination uniformly induced tumor shrinkage, and in multiple instances led to complete tumor regression.

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Folylpoly-gamma-glutamate synthetase (FPGS) is essential for the survival of proliferating mammalian cells and central to the action of all "classical" folate antimetabolites. We report the isolation of cDNAs corresponding to the 5' ends of FPGS mRNA from both human and hamster cells which include a start codon upstream of and in-frame with the AUG in the previously reported FPGS open reading frame. The predicted hamster and human amino-terminal extension peptides have features consistent with a mitochondrial targeting sequence. Ribonuclease protection and 5'-rapid amplification of cDNA ends assays indicated multiple transcriptional start sites consistent with the sequence of the promoter region of this gene, which was highly GC-rich and did not contain TATA or CCAAT elements. These start sites would generate two classes of transcripts, one including the upstream AUG and one in which only the downstream AUG would be available for translation initiation. Transfection of the full length human cDNA into cells lacking FPGS restored their ability to grow in the absence of glycine, a product of mitochondrial folate metabolism, as well as of thymidine and purines. Therefore, we propose that the mitochondrial and cytosolic forms of FPGS are derived from the same gene, arising from the use of the two different translation initiation codons, and that the translation products differ by the presence of a 42-residue amino-terminal mitochondrial leader peptide.