CXXC domain of human DNMT1 is essential for enzymatic activity

DNA cytosine methylation is one of the major epigenetic gene silencing marks in the human genome facilitated by DNA methyltransferases. DNA cytosine-5 methyltransferase 1 (DNMT1) performs maintenance methylation in somatic cells. In cancer cells, DNMT1 is responsible for the aberrant hypermethylation of CpG islands and the silencing of tumor suppressor genes. Here we show that the catalytically active recombinant DNMT1, lacking 580 amino acids from the amino terminus, binds to unmethylated DNA with higher affinity than hemimethylated or methylated DNA. To further understand the binding domain of enzyme, we have used gel shift assay. We have demonstrated that the CXXC region (C is cysteine; X is any amino acid) of DNMT1 bound specifically to unmethylated CpG dinucleotides. Furthermore, mutation of the conserved cysteines abolished CXXC mediated DNA binding. In transfected COS-7 cells, CXXC deleted DNMT1 (DNMT1 (DeltaCXXC)) localized on replication foci. Both point mutant and DNMT1 (DeltaCXXC) enzyme displayed significant reduction in catalytic activity, confirming that this domain is crucial for enzymatic activity. A permanent cell line with DNMT1 (DeltaCXXC) displayed partial loss of genomic methylation on rDNA loci, despite the presence of endogenous wild-type enzyme. Thus, the CXXC domain encompassing the amino terminus region of DNMT1 cooperates with the catalytic domain for DNA methyltransferase activity.     

Early Life Stress Inhibits Expression of Ribosomal RNA in the Developing Hippocampus

Children that are exposed to abuse or neglect show abnormal hippocampal function. However, the developmental mechanisms by which early life stress (ELS) impairs normal hippocampal development have not been elucidated. Here we propose that exposure to ELS blunts normal hippocampal growth by inhibiting the availability of ribosomal RNA (rRNA). In support of this hypothesis, we show that the normal mouse hippocampus undergoes a growth-spurt during the second week of life, followed by a gradual decrease in DNA and RNA content that persists into adulthood. This developmental pattern is associated with accelerated ribosomal RNA (rRNA) synthesis during the second week of life, followed by a gradual decline in rRNA levels that continue into adulthood. Levels of DNA methylation at the ribosomal DNA (rDNA) promoter are lower during the second week of life compared to earlier development or adulthood. Exposure to brief daily separation (BDS), a mouse model of early life stress, increased DNA methylation at the ribosomal DNA promoter, decreased rRNA levels, and blunted hippocampal growth during the second week of life. Exposure to acute (3 hrs) maternal separation decreased rRNA and increased DNA methylation at the rDNA proximal promoter, suggesting that exposure to stress early in life can rapidly regulate the availability of rRNA levels in the developing hippocampus. Given the critical role that rRNA plays in supporting normal growth and development, these findings suggest a novel molecular mechanism to explain how stress early in life impairs hippocampus development in the mouse.     

Critical period of nonpromoter DNA methylation acquisition during prenatal male germ cell development

The prenatal period of germ cell development is a key time of epigenetic programming in the male, a window of development that has been shown to be influenced by maternal factors such as dietary methyl donor supply. DNA methylation occurring outside of promoter regions differs significantly between sperm and somatic tissues and has recently been linked with the regulation of gene expression during development as well as successful germline development. We examined DNA methylation at nonpromoter, intergenic sequences in purified prenatal and postnatal germ cells isolated from wildtype mice and mice deficient in the DNA methyltransferase cofactor DNMT3L. Erasure of the parental DNA methylation pattern occurred by 13.5 days post coitum (dpc) with the exception of approximately 8% of loci demonstrating incomplete erasure. For most loci, DNA methylation acquisition occurred between embryonic day 13.5 to 16.5 indicating that the key phase of epigenetic pattern establishment for intergenic sequences in male germ cells occurs prior to birth. In DNMT3L-deficient germ cells at 16.5 dpc, average DNA methylation levels were low, about 30% of wildtype levels; however, by postnatal day 6, about half of the DNMT3L deficiency-specific hypomethylated loci had acquired normal methylation levels. Those loci normally methylated earliest in the prenatal period were the least affected in the DNMT3L-deficient mice, suggesting that some loci may be more susceptible than others to perturbations occurring prenatally. These results indicate that the critical period of DNA methylation programming of nonpromoter, intergenic sequences occurs in male germline progenitor cells in the prenatal period, a time when external perturbations of epigenetic patterns could result in diminished fertility.     

AUF1 cell cycle variations define genomic DNA methylation by regulation of DNMT1 mRNA stability

DNA methylation is a major determinant of epigenetic inheritance. DNA methyltransferase 1 (DNMT1) is the enzyme responsible for the maintenance of DNA methylation patterns during cell division, and deregulated expression of DNMT1 leads to cellular transformation. We show herein that AU-rich element/poly(U)-binding/degradation factor 1 (AUF1)/heterogeneous nuclear ribonucleoprotein D interacts with an AU-rich conserved element in the 3' untranslated region of the DNMT1 mRNA and targets it for destabilization by the exosome. AUF1 protein levels are regulated by the cell cycle by the proteasome, resulting in cell cycle-specific destabilization of DNMT1 mRNA. AUF1 knock down leads to increased DNMT1 expression and modifications of cell cycle kinetics, increased DNA methyltransferase activity, and genome hypermethylation. Concurrent AUF1 and DNMT1 knock down abolishes this effect, suggesting that the effects of AUF1 knock down on the cell cycle are mediated at least in part by DNMT1. In this study, we demonstrate a link between AUF1, the RNA degradation machinery, and maintenance of the epigenetic integrity of the cell.     

Absence of ribosomal DNA amplification in the meroistic (telotrophic) ovary of the large milkweed bug Oncopeltus fasciatus (Dallas) (Hemiptera: Lygaeidae)

In the typical meroistic insect ovary, the oocyte nucleus synthesizes little if any RNA. Nurse cells or trophocytes actively synthesize ribosomes which are transported to and accumulated by the oocyte. In the telotrophic ovary a morphological separation exists, the nurse cells being localized at the apical end of each ovariole and communicating with the ooocytes via nutritive cords. In order to determine whether the genes coding for ribosomal RNA (rRNA) are amplified in the telotrophic ovary of the milkweed bug Oncopeltus fasciatus, the percentages of the genome coding for ribosomal RNA in somatic cells, spermatogenic cells, ovarian follicles, and nurse cells were compared. The oocytes and most of the nurse cells of O. fasciatus are uninucleolate. DNA hybridizing with ribosomal RNA is localized in a satellite DNA, the density of which is 1.712 g/cm(-3). The density of main-band DNA is 1.694 g/cm(-3). The ribosomal DNA satellite accounts for approximately 0.2% of the DNA in somatic and gametogenic tissues of both males and females. RNA-DNA hybridization analysis demonstrates that approximately 0.03% of the DNA in somatic tissues, testis, ovarian follicles, and isolated nurse cells hybridizes with ribosomal RNA. The fact that the percentage of DNA hybridizing with rRNA is the same in somatic and in male and female gametogenic tissues indicates that amplification of ribosomal DNA does not occur in nurse cells and that if it occurs in oocytes, it represents less than a 50-fold increase in ribosomal DNA. An increase in total genome DNA accounted by polyploidization appears to provide for increasing the amount of ribosomal DNA in the nurse cells.     

Cellular content of ribosomal RNA in relation to the progression and competence signals governing proliferation of 3T3 and SV40-3T3 cells

The method for differential fluorescence staining of cellular RNA and DNA by acridine orange (AO) was optimized for 3T3 and SV40-3T3 cells. Cellular contents of DNA and of ribosomal RNA (rRNA) were determined by dual-channel flow cytometry during cell-density-dependent proliferation and after stimulation of quiescent cells. With increasing density of 3T3 cells, cellular content of rRNA decreases by about 60%, whereas SV40-3T3 cells do not exhibit a significant dependence of rRNA content on cell density. 3T3 cells stimulated early after becoming quiescent resume reaccumulation of rRNA after a delay of only 4 h, whereas cells maintained at quiescence for several days exhibit a delay of about 12 h before a significant rise of rRNA is observed. The extent of rise of cellular rRNA content after different regimens of stimulation of quiescent 3T3 cells does not correlate well with the fraction of cells entering the cell cycle. These and other reported instances of discordance between rRNA content and stimulation into the cell cycle are resolved by showing that of the two signals governing entry into the cell cycle only the progression signal, but not the competence signal is associated with reaccumulation of cellular rRNA. The present results are consistent with the progression function being in essence the achievement of a threshold number of ribosomes per cell, which in conjunction with the competence signal is sufficient for initiation of the cell cycle.