The Communication Factor EDF and the Toxin–Antitoxin Module mazEF Determine the Mode of Action of Antibiotics

It was recently reported that the production of Reactive Oxygen Species (ROS) is a common mechanism of cell death induced by bactericidal antibiotics. Here we show that triggering the Escherichia coli chromosomal toxin–antitoxin system mazEF is an additional determinant in the mode of action of some antibiotics. We treated E. coli cultures by antibiotics belonging to one of two groups: (i) Inhibitors of transcription and/or translation, and (ii) DNA damaging. We found that antibiotics of both groups caused: (i) mazEF-mediated cell death, and (ii) the production of ROS through MazF action. However, only antibiotics of the first group caused mazEF-mediated cell death that is ROS-dependent, whereas those of the second group caused mazEF-mediated cell death by an ROS-independent pathway. Furthermore, our results showed that the mode of action of antibiotics was determined by the ability of E. coli cells to communicate through the signaling molecule Extracellular Death Factor (EDF) participating in mazEF induction

CTCF Elements Direct Allele-Specific Undermethylation at the Imprinted H19 Locus

AbstractThe H19 imprinted gene locus is regulated by an upstream 2 kb imprinting control region (ICR) that influences allele-specific expression, DNA methylation [1], and replication timing [2]. This ICR becomes de novo methylated during late spermatogenesis in the male [3] but emerges from oogenesis in an unmethylated form, and this allele-specific pattern is then maintained throughout early development [4] and in all tissues of the mouse [5]. We have used a genetic approach involving transfection into embryonic stem (ES) cells in order to decipher how the maternal allele is protected from de novo methylation at the time of implantation [6]. Our studies show that CCCTC binding factor (CTCF) boundary elements within the ICR [7, 8] have the ability to prevent de novo methylation on the maternal allele. Since CTCF does not recognize its binding sequence when methylated [9, 10], this reaction does not occur on the paternal allele, thus preserving the gamete-derived, allele-specific pattern. These results suggest that CTCF may play a general role in the maintenance of differential methylation patterns in vivo

Role of transcription complexes in the formation of the basal methylation pattern in early development

Following erasure in the blastocyst, the entire genome undergoes de novo methylation at the time of implantation, with CpG islands being protected from this process. This bimodal pattern is then preserved throughout development and the lifetime of the organism. Using mouse embryonic stem cells as a model system, we demonstrate that the binding of an RNA polymerase complex on DNA before de novo methylation is predictive of it being protected from this modification, and tethering experiments demonstrate that the presence of this complex is, in fact, sufficient to prevent methylation at these sites. This protection is most likely mediated by the recruitment of enzyme complexes that methylate histone H3K4 over a local region and, in this way, prevent access to the de novo methylation complex. The topological pattern of H3K4me3 that is formed while the DNA is as yet unmethylated provides a strikingly accurate template for modeling the genome-wide basal methylation pattern of the organism. These results have far-reaching consequences for understanding the relationship between RNA transcription and DNA methylation

An upstream repressor element plays a role in Igf2 imprinting

The imprinted Igf2 gene is associated with a small upstream region that is differentially methylated on the active paternal allele. We have identified a repressor element within this sequence and shown that repression is probably mediated through a trans- acting factor, GCF2. DNA methylation of this site abrogates both protein binding and repressor activity. Targeting experiments demonstrate that this element plays a role in the repression of the maternal Igf2 gene in vivo