SummaryEpidemiological and clinical studies have shown that double infection with herpes simplex virus type 2 (HSV-2) and Chlamydia trachomatis occurs in vivo . We hypothesized that co-infection would alter replication of these agents. To test this hypothesis, HeLa cells were infected with C. trachomatis serovar E, followed 24 h later by HSV-2 strain 333. Transmission electron microscopic (TEM) analyses indicated that, by 10 h after HSV addition, reticulate bodies (RBs) in coinfected cells were swollen, aberrantly shaped and electron-lucent. In infectious titre assays, HSV-2 coinfection abrogated production of infectious chlamydial progeny. Western blot analyses indicated that accumulation of chlamydial major outer membrane protein (MOMP) was decreased by HSV co-infection while accumulation of chlamydial heat-shock protein 60-1 (HSP60-1) was increased. Polymerase chain reaction (PCR) experiments indicated that chlamydial genome copy number was unaltered by HSV-2 superinfection. Semi-quantitative, reverse transcription PCR (RT-PCR) experiments demonstrated that levels of chlamydial groEL , ftsK , ftsW , dnaA and unprocessed 16S rRNA transcripts were not changed by HSV-2 super-infection. These data indicate that HSV-2 superinfection drives chlamydia into a viable but noncultivable state, which is the hallmark of persistence. Because chlamydial HSP60-1 has been associated with immunopathology in vivo , these results also suggest that disease severity might be increased in coinfected individuals.

At the end of mammalian spermatogenesis, chromatin in differentiating germ cells is extensively remodeled, with the majority of nucleosomes being removed and ultimately exchanged by highly basic proteins named protamines. Residual nucleosomes are, to various degrees, retained at regulatory sequences in human and mouse sperm. Moreover, certain histone variants and modifications remain present in regulatory sequences of subsets of genes in spermatozoa, providing opportunities for paternal inheritance of chromatin states and epigenetic control of gene expression in the subsequent generation. Here we describe in detail a method that enables the generation of soluble chromatin samples from mouse and human spermatozoa within 1 d. These samples are amendable to chromatin immunoprecipitation and high-throughput sequencing of nucleosome-associated genomic DNA, which require several additional days. We also provide computational scripts that allow straightforward analysis of large genome-wide data sets by biologists with limited computational experience. This protocol will facilitate studies of mechanisms of chromatin remodeling and epigenetic reprogramming during spermatogenesis and of paternal epigenetic inheritance. Similarly, it will help in the study of the causes of human male infertility.

The majority of mammalian promoters are CpG islands; regions of high CG density that require protection from DNA methylation to be functional. Importantly, how sequence architecture mediates this unmethylated state remains unclear. To address this question in a comprehensive manner, we developed a method to interrogate methylation states of hundreds of sequence variants inserted at the same genomic site in mouse embryonic stem cells. Using this assay, we were able to quantify the contribution of various sequence motifs towards the resulting DNA methylation state. Modeling of this comprehensive dataset revealed that CG density alone is a minor determinant of their unmethylated state. Instead, these data argue for a principal role for transcription factor binding sites, a prediction confirmed by testing synthetic mutant libraries. Taken together, these findings establish the hierarchy between the two cis-encoded mechanisms that define the DNA methylation state and thus the transcriptional competence of CpG islands.DOI: http://dx.doi.org/10.7554/eLife.04094.001

For the most part metazoan genomes are highly methylated and harbor only small regions with low or absent methylation. In contrast, partially methylated domains (PMDs), recently discovered in a variety of cell lines and tissues, do not fit this paradigm as they show partial methylation for large portions (20%–40%) of the genome. While in PMDs methylation levels are reduced on average, we found that at single CpG resolution, they show extensive variability along the genome outside of CpG islands and DNase I hypersensitive sites (DHS). Methylation levels range from 0% to 100% in a roughly uniform fashion with only little similarity between neighboring CpGs. A comparison of various PMD-containing methylomes showed that these seemingly disordered states of methylation are strongly conserved across cell types for virtually every PMD. Comparative sequence analysis suggests that DNA sequence is a major determinant of these methylation states. This is further substantiated by a purely sequence based model which can predict 31% (R2) of the variation in methylation. The model revealed CpG density as the main driving feature promoting methylation, opposite to what has been shown for CpG islands, followed by various dinucleotides immediately flanking the CpG and a minor contribution from sequence preferences reflecting nucleosome positioning. Taken together we provide a reinterpretation for the nucleotide-specific methylation levels observed in PMDs, demonstrate their conservation across tissues and suggest that they are mainly determined by specific DNA sequence features.

Staphylococcus aureus undergoes a density-dependent conversion in phenotype from tissue-adhering to tissue-damaging and phagocyte-evading that is mediated in part by the quorum-sensing operon, agr, and its effector, RNAIII. Contributions of host factors to this mechanism for regulating virulence have not been studied. We hypothesized that fibrinogen, as a component of the inflammatory response, could create spatially constrained microenvironments around bacteria that increase density independently of bacterial numbers and thus potentiate quorum-sensing-dependent virulence gene expression. Here we show that transient fibrinogen depletion significantly reduces the bacterial burden and the consequential morbidity and mortality during experimental infection with wild-type S. aureus, but not with bacteria that lack expression of the quorum-sensing operon, agr. In addition, it inhibits in vivo activation of the promoter for the agr effector, RNAIII, and downstream targets of RNAIII, including α hemolysin and capsule production. Moreover, both in vitro and in vivo, the mechanism for promoting this phenotypic switch in virulence involves clumping of the bacteria, demonstrating that S. aureus responds to fibrinogen-mediated bacterial clumping by enhancing density-dependent virulence gene expression. These data demonstrate that down-modulation of specific inflammatory components of the host that augment bacterial quorum sensing can be a strategy for enhancing host defense against infection.