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Differential DNA methylation of the paternal and maternal alleles regulates the parental origin-specific expression of imprinted genes in mammals (1, 2). The methylation imprints are established in male and female germ cells during gametogenesis, and the de novo DNA methyltransferase DNMT3A and its cofactor DNMT3L are required in this process (3-5). However, the mechanisms underlying locus- and parental-specific targeting of the de novo DNA methylation machinery in germline imprinting are poorly understood. Here we show that amine oxidase (flavin-containing) domain 1 (AOFl), a protein related to the lysine demethylase KDMl (also known as LSDl) (6), functions as a histone H3 lysine 4 (H3K4) demethylase and is required for de novo DNA methylation of some imprinted genes in oocytes. AOFl, now renamed lysine demethylase 1B (KDM1B) following a new nomenclature (7), is highly expressed in growing oocytes where genomic imprints are established. Targeted disruption of the gene encoding KDM1B had no effect on mouse development and oogenesis. However, oocytes from KDM1B-deficient females showed a substantial increase in H3K4 methylation and failed to set up the DNA methylation marks at four out of seven imprinted genes examined. Embryos derived from these oocytes showed biallelic expression or biallelic suppression of the affected genes and died before mid-gestation. Our results suggest that demethylation of H3K4 is critical for establishing the DNA methylation imprints during oogenesis., KDM1B is related to KDMl in that it possesses a flavin adenine dinucleotide-dependent amine oxidase domain and a SWIRM domain (6). In KDMl, the amine oxidase domain contains the demethylase [...]

Thioredoxin (Trx) is a cytosolic, redox-active protein that is secreted from many cells and has several extracellular functions. In activated lymphocytes, the pathway of secretion does not involve the Golgi apparatus. Levels of extracellular Trx are decreased by the antioxidant N-acetylcysteine. Hence, the secretion of Trx could be altered by the redox status of the cell or the protein. To study Trx mutants, we characterized the secretion of human Trx from Chinese hamster ovary cells. Secretion of human Trx is unaffected by brefeldin A, slow but efficient, and sensitive to low temperature and factors in serum. We demonstrate that N-acetylcysteine reduces the cellular level of Trx but not the proportion secreted; thus this chemical does not block the nonclassic pathway for Trx secretion. Furthermore, we find that mutations in either the active site or the dimerization site of Trx do not alter its secretion. Thus the nonclassic secretion of Trx is not dependent on the redox status of either the cell or the protein. protein trafficking; protein secretion

Histone methylation and DNA methylation cooperatively regulate chromatin structure and gene activity. How these two systems coordinate with each other remains unclear. Here we study the biological function of lysine-specific demethylase 1 (LSD1, also known as KDM1 and AOF2), which has been shown to demethylate histone H3 on lysine 4 (H3K4) and lysine 9 (H3K9). We show that LSD1 is required for gastrulation during mouse embryogenesis. Notably, targeted deletion of the gene encoding LSD1 (namely, Aof2) in embryonic stem (ES) cells induces progressive loss of DNA methylation. This loss correlates with a decrease in DNA methyltransferase 1 (Dnmt1) protein, as a result of reduced Dnmt1 stability. Dnmt1 protein is methylated in vivo, and its methylation is enhanced in the absence of LSD1. Furthermore, Dnmt1 can be methylated by Set7/9 (also known as KMT7) and demethylated by LSD1 in vitro. Our findings suggest that LSD1 demethylates and stabilizes Dnmt1, thus providing a previously unknown mechanistic link between the histone and DNA methylation systems. [ABSTRACT FROM AUTHOR]

Studies have shown that DNA (cytosine-5-)-methyltransferase 1 (DNMT1) is the principal enzyme responsible for maintaining CpG methylation and is required for embryonic development and survival of somatic cells in mice. The role of DNMT1 in human cancer cells, however, remains highly controversial. Using homologous recombination, here we have generated a DNMT1 conditional allele in the human colorectal carcinoma cell line HCT116 in which several exons encoding the catalytic domain are flanked by loxP sites. Cre recombinase–mediated disruption of this allele results in hemimethylation of ∼20% of CpG-CpG dyads in the genome, coupled with activation of the G2/M checkpoint, leading to arrest in the G2 phase of the cell cycle. Although cells gradually escape from this arrest, they show severe mitotic defects and undergo cell death either during mitosis or after arresting in a tetraploid G1 state. Our results thus show that DNMT1 is required for faithfully maintaining DNA methylation patterns in human cancer cells and is essential for their proliferation and survival. [ABSTRACT FROM AUTHOR]

Histone methylation and DNA methylation cooperatively regulate chromatin structure and gene activity. How these two systems coordinate with each other remains unclear. Here we study the biological function of lysine-specific demethylase 1 (LSD1, also known as KDM1 and AOF2), which has been shown to demethylate histone H3 on lysine 4 (H3K4) and lysine 9 (H3K9). We show that LSD1 is required for gastrulation during mouse embryogenesis. Notably, targeted deletion of the gene encoding LSD1 (namely, Aof2) in embryonic stem (ES) cells induces progressive loss of DNA methylation. This loss correlates with a decrease in DNA methyltransferase 1 (Dnmt1) protein, as a result of reduced Dnmt1 stability. Dnmt1 protein is methylated in vivo, and its methylation is enhanced in the absence of LSD1. Furthermore, Dnmt1 can be methylated by Set7/9 (also known as KMT7) and demethylated by LSD1 in vitro. Our findings suggest that LSD1 demethylates and stabilizes Dnmt1, thus providing a previously unknown mechanistic link between the histone and DNA methylation systems.