Chromatin structure and methylation of rat rRNA genes studied by formaldehyde fixation and psoralen cross-linking

By using formaldehyde cross-linking of histones to DNA and gel retardation assays we show that formaldehyde fixation, similar to previously established psoralen photocross-linking, discriminates between nucleosome-packed (inactive) and nucleosome-free (active) fractions of ribosomal RNA genes. By both cross-linking techniques we were able to purify fragments from agarose gels, corresponding to coding, enhancer and promoter sequences of rRNA genes, which were further investigated with respect to DNA methylation. This approach allows us to analyse independently and in detail methylation patterns of active and inactive rRNA gene copies by the combination of HpaII and MspI restriction enzymes. We found CpG methylation mainly present in enhancer and promoter regions of inactive rRNA gene copies. The methylation of one single HpaII site, located in the promoter region, showed particularly strong correlation with the transcriptional activit

Melatonin inhibits the protein c anticoagulant Pathway in rats

This study examines the influence of melatonin on the PC anticoagulant pathway in rats. The experiment was performed on 52 male white Wistar rats weighing 200-220 g. Animals were equally divided into 4 groups. They were treated in three consecutive days, every 12 hours, subcutaneously: 1st group - with saline solution (solvent for melatonin and luzindole); 2nd group - with melatonin, daily dose 0.2 mg/kg body weight; 3rd group - with luzindole, nonstop dose of 0.4 mg/kg body weight; 4th group - melatonin, one hour after pretreatment with luzindole. The required amount of blood was taken under urethane narcosis via direct cardiac puncture. After three days of administration of melatonin, a significant decrease in the antigen concentration of protein C, protein C activity, activated protein C and thrombomodulin was observed. The soluble form of the endothelial receptor for protein C, activity of protein S and free protein S were significantly elevated. The competitive melatonin receptor antagonist - luzindole, when administered alone and in pretreatment, effectively removes the observed effects of melatonin by blocking exogenous, as well as endogenous melatonin. In conclusion, our data give us reason to assume that melatonin significantly reduces the activity of the protein C anticoagulant pathway in rats

Effects of Melatonin and Luzindole on plasma levels of tissue factor, tissue factor Pathway inhibitor and Von Willebrand factor in rats

Introduction: The role of melatonin in hemostasis is still poorly studied.Purpose: The purpose of this study was to investigate the effects of melatonin and luzindole, inhibitor of the melatonin receptors - MT1 and MT2, on plasma levels of tissue factor, tissue factor pathway inhibitor and von Willebrand factor.Material and methods: The study was performed on 52 male Wistar rats, kept at 12/12 h natural regimen dark/light. The daily doses of melatonin 0.2 mg/kg b.m. and luzindole 0.4 mg/kg b.m. were applied subcutaneously twice daily at intervals of 12h for three consecutive days. The rats were distributed into four equal groups (n = 13) and were treated as follows: the first group (control group) - by saline; the second group - by melatonin; the third group - by luzindole; and the fourth group - by luzindole and an hour later by melatonin.Results: The results show that melatonin significantly increases the plasma levels of TF, reduces the values of free TFPI antigen and free TFPI activity, and increases the values of vWF antigen and vWF activity. Applied alone, luzindole lowers the plasma levels of TF, increases the values of free TFPI antigen and free TFPI activity; decreases the values of vWF antigen and vWF activity. Pretreatment with luzindole repeats the effect of self-administration. The received data show that melatonin induces a pronounced tendency to hypercoagulability in rats by a significant increase in TF, a decrease of free TFPI and free TFPI activity, as well as an increase in vWF antigen and vWF activity.Conclusions: Lizindole self-administration and pretreatment show a decisive involvement of MT1 and MT2 receptors for accomplishing the effects of the hormone

Targeting of De Novo DNA Methylation Throughout the Oct-4 Gene Regulatory Region in Differentiating Embryonic Stem Cells

Differentiation of embryonic stem (ES) cells is accompanied by silencing of the Oct-4 gene and de novo DNA methylation of its regulatory region. Previous studies have focused on the requirements for promoter region methylation. We therefore undertook to analyse the progression of DNA methylation of the ∼2000 base pair regulatory region of Oct-4 in ES cells that are wildtype or deficient for key proteins. We find that de novo methylation is initially seeded at two discrete sites, the proximal enhancer and distal promoter, spreading later to neighboring regions, including the remainder of the promoter. De novo methyltransferases Dnmt3a and Dnmt3b cooperate in the initial targeted stage of de novo methylation. Efficient completion of the pattern requires Dnmt3a and Dnmt1, but not Dnmt3b. Methylation of the Oct-4 promoter depends on the histone H3 lysine 9 methyltransferase G9a, as shown previously, but CpG methylation throughout most of the regulatory region accumulates even in the absence of G9a. Analysis of the Oct-4 regulatory domain as a whole has allowed us to detect targeted de novo methylation and to refine our understanding the roles of key protein components in this process

The SNF2 family ATPase LSH promotes cell-autonomous de novo DNA methylation in somatic cells

Methylation of DNA at carbon 5 of cytosine is essential for mammalian development and implicated in transcriptional repression of genes and transposons. New patterns of DNA methylation characteristic of lineage-committed cells are established at the exit from pluripotency by de novo DNA methyltransferases enzymes, DNMT3A and DNMT3B, which are regulated by developmental signaling and require access to chromatin-organized DNA. Whether or not the capacity for de novo DNA methylation of developmentally regulated loci is preserved in differentiated somatic cells and can occur in the absence of exogenous signals is currently unknown. Here, we demonstrate that fibroblasts derived from chromatin remodeling ATPase LSH (HELLS)-null mouse embryos, which lack DNA methylation from centromeric repeats, transposons and a number of gene promoters, are capable of reestablishing DNA methylation and silencing of misregulated genes upon re-expression of LSH. We also show that the ability of LSH to bind ATP and the cellular concentration of DNMT3B are critical for cell-autonomous de novo DNA methylation in somatic cells. These data suggest the existence of cellular memory that persists in differentiated cells through many cell generations and changes in transcriptional state

Immortality, but not oncogenic transformation, of primary human cells leads to epigenetic reprogramming of DNA methylation and gene expression

Tumourigenic transformation of normal cells into cancer typically involves several steps resulting in acquisition of unlimited growth potential, evasion of apoptosis and non-responsiveness to growth inhibitory signals. Both genetic and epigenetic changes can contribute to cancer development and progression. Given the vast genetic heterogeneity of human cancers and difficulty to monitor cancer-initiating events in vivo, the precise relationship between acquisition of genetic mutations and the temporal progression of epigenetic alterations in transformed cells is largely unclear. Here, we use an in vitro model system to investigate the contribution of cellular immortality and oncogenic transformation of primary human cells to epigenetic reprogramming of DNA methylation and gene expression. Our data demonstrate that extension of replicative life span of the cells is sufficient to induce accumulation of DNA methylation at gene promoters and large-scale changes in gene expression in a time-dependent manner. In contrast, continuous expression of cooperating oncogenes in immortalized cells, although essential for anchorage-independent growth and evasion of apoptosis, does not affect de novo DNA methylation at promoters and induces subtle expression changes. Taken together, these observations imply that cellular immortality promotes epigenetic adaptation to highly proliferative state, whereas transforming oncogenes confer additional properties to transformed human cells

Recruitment of MBD1 to target genes requires sequence-specific interaction of the MBD domain with methylated DNA

MBD1, a member of the methyl-CpG-binding domain family of proteins, has been reported to repress transcription of methylated and unmethylated promoters. As some MBD1 isoforms contain two DNA-binding domains—an MBD, which recognizes methylated DNA; and a CXXC3 zinc finger, which binds unmethylated CpG—it is unclear whether these two domains function independently of each other or if they cooperate in facilitating recruitment of MBD1 to particular genomic loci. In this report we investigate DNA-binding specificity of MBD and CXXC3 domains in vitro and in vivo. We find that the methyl-CpG-binding domain of MBD1 binds more efficiently to methylated DNA within a specific sequence context. We identify genes that are targeted by MBD1 in human cells and demonstrate that a functional MBD domain is necessary and sufficient for recruitment of MBD1 to specific sites at these loci, while DNA binding by the CXXC3 motif is largely dispensable. In summary, the binding preferences of MBD1, although dependent upon the presence of methylated DNA, are clearly distinct from those of other methyl-CpG-binding proteins, MBD2 and MeCP2

Smchd1-Dependent and -Independent Pathways Determine Developmental Dynamics of CpG Island Methylation on the Inactive X Chromosome

X chromosome inactivation involves multiple levels of chromatin modification, established progressively and in a stepwise manner during early development. The chromosomal protein Smchd1 was recently shown to play an important role in DNA methylation of CpG islands (CGIs), a late step in the X inactivation pathway that is required for long-term maintenance of gene silencing. Here we show that inactive X chromosome (Xi) CGI methylation can occur via either Smchd1-dependent or -independent pathways. Smchd1-dependent CGI methylation, the primary pathway, is acquired gradually over an extended period, whereas Smchd1-independent CGI methylation occurs rapidly after the onset of X inactivation. The de novo methyltransferase Dnmt3b is required for methylation of both classes of CGI, whereas Dnmt3a and Dnmt3L are dispensable. Xi CGIs methylated by these distinct pathways differ with respect to their sequence characteristics and immediate chromosomal environment. We discuss the implications of these results for understanding CGI methylation during development