Epigenetic instability in ES cells and cloned mice.

Cloning by nuclear transfer (NT) is an inefficient process in which most clones die before birth and survivors often display growth abnormalities. In an effort to correlate gene expression with survival and fetal overgrowth, we have examined imprinted gene expression in both mice cloned by nuclear transfer and in the embryonic stem (ES) cell donor populations from which they were derived. The epigenetic state of the ES cell genome was found to be extremely unstable. Similarly, variation in imprinted gene expression was observed in most cloned mice, even in those derived from ES cells of the same subclone. Many of the animals survived to adulthood despite widespread gene dysregulation, indicating that mammalian development may be rather tolerant to epigenetic aberrations of the genome. These data imply that even apparently normal cloned animals may have subtle abnormalities in gene expression.

Retroviral expression in embryonic stem cells and hematopoietic stem cells.

Achieving long-term retroviral expression in primary cells has been problematic. De novo DNA methylation of infecting proviruses has been proposed as a major cause of this transcriptional repression. Here we report the development of a mouse stem cell virus (MSCV) long terminal repeat-based retroviral vector that is expressed in both embryonic stem (ES) cells and hematopoietic stem (HS) cells. Infected HS cells and their differentiated descendants maintained long-term and stable retroviral expression after serial adoptive transfers. In addition, retrovirally infected ES cells showed detectable expression level of the green fluorescent protein (GFP). Moreover, GFP expression of integrated proviruses was maintained after in vitro differentiation of infected ES cells. Long-term passage of infected ES cells resulted in methylation-mediated silencing, while short-term expression was methylation independent. Tissues of transgenic animals, which we derived from ES cells carrying the MSCV-based provirus, did not express GFP. However, treatment with the demethylating agent 5-azadeoxycytidine reactivated the silent provirus, demonstrating that DNA methylation is involved in the maintenance of retroviral repression. Our results indicate that retroviral expression in ES cells is repressed by methylation-dependent as well as methylation-independent mechanisms.

DNA methyltransferase contributes to delayed ischemic brain injury.

DNA methylation is important for controlling the profile of gene expression and is catalyzed by DNA methyltransferase (MTase), an enzyme that is abundant in brain. Because significant DNA damage and alterations in gene expression develop as a consequence of cerebral ischemia, we measured MTase activity in vitro and DNA methylation in vivo after mild focal brain ischemia. After 30 min middle cerebral artery occlusion (MCAo) and reperfusion, MTase catalytic activity and the 190 kDa band on immunoblot did not change over time. However, [(3)H]methyl-group incorporation into DNA increased significantly in wild-type mice after reperfusion, but not in mutant mice heterozygous for a DNA methyltransferase gene deletion (Dnmt(S/+)). Dnmt(S/+) mice were resistant to mild ischemic damage, suggesting that increased DNA methylation is associated with augmented brain injury after MCA occlusion. Consistent with this formulation, treatment with the MTase inhibitor 5-aza-2'-deoxycytidine and the deacetylation inhibitor trichostatin A conferred stroke protection in wild-type mice. In contrast to mild stroke, however, DNA methylation was not enhanced, and reduced dnmt gene expression was not protective in an ischemia model of excitotoxic/necrotic cell death. In conclusion, our results demonstrate that MTase activity contributes to poor tissue outcome after mild ischemic brain injury.

DNA hypomethylation perturbs the function and survival of CNS neurons in postnatal animals.

DNA methyltransferase I (Dnmt1), the maintenance enzyme for DNA cytosine methylation, is expressed at high levels in the CNS during embryogenesis and after birth. Because embryos deficient for Dnmt1 die at gastrulation, the role of Dnmt1 in the development and function of the nervous system could not be studied by using this mutation. We therefore used the cre/loxP system to produce conditional mutants that lack Dnmt1 in neuroblasts of embryonic day 12 embryos or in postmitotic neurons of the postnatal animal. Conditional deletion of the Dnmt1 gene resulted in rapid depletion of Dnmt1 proteins, indicating that the enzyme in postmitotic neurons turns over quickly. Dnmt1 deficiency in postmitotic neurons neither affected levels of global DNA methylation nor influenced cell survival during postnatal life. In contrast, Dnmt1 deficiency in mitotic CNS precursor cells resulted in DNA hypomethylation in daughter cells. Whereas mutant embryos carrying 95% hypomethylated cells in the brain died immediately after birth because of respiratory distress, mosaic animals with 30% hypomethylated CNS cells were viable into adulthood. However, these mutant cells were eliminated quickly from the brain within 3 weeks of postnatal life. Thus, hypomethylated CNS neurons were impaired functionally and were selected against at postnatal stages.

[Evaluation of selected parameters of connective tissue metabolism in patients with primary open angle glaucoma]. / Ocena wybranych parametrów metabolizmu tkanki lacznej u chorych na jaskre prosta.

PURPOSE: To evaluate the plasma concentration of P I CP and P III NP as markers of the connective tissue metabolism in human with and without POAG. The mechanical properties of the collagen could be one of the common factors involved in the pathogenesis of chronic open angle glaucoma. MATERIAL AND METHODS: P I CP and P III NP were studied in serum in 20 patients with POAG and in 10 healthy subjects using RIA method. RESULTS: The concentration of P III NP in the blood serum was markedly increased (3.99 +/- 1.5 micrograms/l) compared with control group (2.84 +/- 0.77 micrograms/l), p < 0.05. There was not significant difference in concentration of P I CP in both groups.

Self-splicing group I intron in cyanobacterial initiator methionine tRNA: evidence for lateral transfer of introns in bacteria.

A group I self-splicing intron has been found in the anticodon loop of tRNA(fMet) genes in three cyanobacterial genera: Dermocarpa, Scytonema and Synechocystis; it is absent in nine others. The Synechocystis intron is also interrupted by an open reading frame (ORF) of 150 codons. Of these three bacteria, only Scytonema also contains the group I intron that has previously been reported in tRNA(Leu) (UAA) genes in both cyanobacteria and chloroplasts. The presence of an ORF in the tRNA(fMet) intron, the sporadic distribution of the intron among cyanobacteria and the lack of correlation between relatedness of the intron sequences and the bacteria in which they reside, are all consistent with recent introduction of this intron by lateral transfer.

Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a.

Current evidence indicates that methylation of cytosine in mammalian DNA is restricted to both strands of the symmetrical sequence CpG, although there have been sporadic reports that sequences other than CpG may also be methylated. We have used a dual-labeling nearest neighbor technique and bisulphite genomic sequencing methods to investigate the nearest neighbors of 5-methylcytosine residues in mammalian DNA. We find that embryonic stem cells, but not somatic tissues, have significant cytosine-5 methylation at CpA and, to a lesser extent, at CpT. As the expression of the de novo methyltransferase Dnmt3a correlates well with the presence of non-CpG methylation, we asked whether Dnmt3a might be responsible for this modification. Analysis of genomic methylation in transgenic Drosophila expressing Dnmt3a reveals that Dnmt3a is predominantly a CpG methylase but also is able to induce methylation at CpA and at CpT.

Sequence-specific methylation of the mouse H19 gene in embryonic cells deficient in the Dnmt-1 gene.

We have used Dnmtc/c ES cells that are homozygous for disruption of the DNA methyltransferase gene to address how de novo methylation is propagated and whether it is directed to specific sites in the early embryo. We examined the imprinted H19 gene and the specific-sequence region implicated as an "imprinting mark" to determine whether de novo methylation was occurring at a restricted set of sites. Since the "imprinting mark" was found to be methylated differentially at all stages of development, we reasoned that the sequence may still be a target for the de novo methylation activity found in the Dnmtc/c cells, even though the loss of maintenance the methylase activity renders the H19 promoter active. We used bisulfite genomic sequencing to determine the methylation state of the imprinted region of the H19 gene and found a low level of DNA methylation at specific single CpG sites in the upstream region of the imprinted H19 sequence in the Dnmtc/c mutant ES cells. Moreover, these CpG sites appeared to be favoured targets for further de novo methylation of neighbouring CpG sites in rescued ES cells, which possess apparently normal maintenance activity. Our data provide further evidence for a separate methylating activity in ES cells and indicate that this activity displays sequence specificity.