Differential viral accessibility (DIVA) identifies alterations in chromatin architecture through large-scale mapping of lentiviral integration sites.

Alterations in chromatin structure play a major role in the epigenetic regulation of gene expression. Here, we describe a step-by-step protocol for differential viral accessibility (DIVA), a method for identifying changes in chromatin accessibility genome-wide. Commonly used methods for mapping accessible genomic loci have strong preferences toward detecting 'open' chromatin found at regulatory regions but are not well suited to studying chromatin accessibility in gene bodies and intergenic regions. DIVA overcomes this limitation, enabling a broader range of sites to be interrogated. Conceptually, DIVA is similar to ATAC-seq in that it relies on the integration of exogenous DNA into the genome to map accessible chromatin, except that chromatin architecture is probed through mapping integration sites of exogenous lentiviruses. An isogenic pair of cell lines are transduced with a lentiviral vector, followed by PCR amplification and Illumina sequencing of virus-genome junctions; the resulting sequences define a set of unique lentiviral integration sites, which are compared to determine whether genomic loci exhibit significantly altered accessibility between experimental and control cells. Experienced researchers will take 6 d to generate lentiviral stocks and transduce the target cells, a further 5 d to prepare the Illumina sequencing libraries and a few hours to perform the bioinformatic analysis

CATaDa reveals global remodelling of chromatin accessibility during stem cell differentiation in vivo

During development eukaryotic gene expression is coordinated by dynamic changes in chromatin structure. Measurements of accessible chromatin are used extensively to identify genomic regulatory elements. Whilst chromatin landscapes of pluripotent stem cells are well characterised, chromatin accessibility changes in the development of somatic lineages are not well defined. Here we show that cell-specific chromatin accessibility data can be produced via ectopic expression of E. coli Dam methylase in vivo, without the requirement for cell-sorting (CATaDa). We have profiled chromatin accessibility in individual cell-types of Drosophila neural and midgut lineages. Functional cell-type specific enhancers were identified, as well as novel motifs enriched at different stages of development. Finally, we show global changes in the accessibility of chromatin between stem-cells and their differentiated progeny. Our results demonstrate the dynamic nature of chromatin accessibility in somatic tissues during stem cell differentiation and provide a novel approach to understanding gene regulatory mechanisms underlying development

Composition and diversity of the subgingival microbiome and its relationship with age in postmenopausal women: an epidemiologic investigation

Abstract Background The extent to which the composition and diversity of the oral microbiome varies with age is not clearly understood. Methods The 16S rRNA gene of subgingival plaque in 1219 women, aged 53–81 years, was sequenced and its taxonomy annotated against the Human Oral Microbiome Database (v.14.5). Composition of the subgingival microbiome was described in terms of centered log(2)-ratio (CLR) transformed OTU values, relative abundance, and prevalence. Correlations between microbiota abundance and age were evelauted using Pearson Product Moment correlations. P-values were corrected for multiple testing using the Bonferroni method. Results Of the 267 species identified overall, Veillonella dispar was the most abundant bacteria when described by CLR OTU (mean 8.3) or relative abundance (mean 8.9%); whereas Streptococcus oralis, Veillonella dispar and Veillonella parvula were most prevalent (100%, all) when described as being present at any amount. Linear correlations between age and several CLR OTUs (Pearson r = − 0.18 to 0.18), of which 82 (31%) achieved statistical significance (P < 0.05). The correlations lost significance following Bonferroni correction. Twelve species that differed across age groups (each corrected P < 0.05); 5 (42%) were higher in women ages 50–59 compared to ≥70 (corrected P < 0.05), and 7 (48%) were higher in women 70 years and older. Conclusions We identified associations between several bacterial species and age across the age range of postmenopausal women studied. Understanding the functions of these bacteria could identify intervention targets to enhance oral health in later life.https://deepblue.lib.umich.edu/bitstream/2027.42/152202/1/12903_2019_Article_906.pd

Intercalation of small molecules into DNA in chromatin is primarily controlled by superhelical constraint

The restricted access of regulatory factors to their binding sites on DNA wrapped around the nucleosomes is generally interpreted in terms of molecular shielding exerted by nucleosomal structure and internucleosomal interactions. Binding of proteins to DNA often includes intercalation of hydrophobic amino acids into the DNA. To assess the role of constrained superhelicity in limiting these interactions, we studied the binding of small molecule intercalators to chromatin in close to native conditions by laser scanning cytometry. We demonstrate that the nucleosome-constrained superhelical configuration of DNA is the main barrier to intercalation. As a result, intercalating compounds are virtually excluded from the nucleosome-occupied regions of the chromatin. Binding of intercalators to extranucleosomal regions is limited to a smaller degree, in line with the existence of net supercoiling in the regions comprising linker and nucleosome free DNA. Its relaxation by inducing as few as a single nick per ~50 kb increases intercalation in the entire chromatin loop, demonstrating the possibility for long-distance effects of regulatory potential

Molecular Evolution and Population Genetics of Tomato spotted wilt virus (TSWV).

The overall goal of this dissertation research was to elucidate the molecular evolution and population genetics of Tomato Spotted Wilt virus (TSWV), at the species level and within individual isolates, and to develop a standardized diagnostic system that can be used to assign attribution to initial TSWV infections. Initially, using consensus sequence data from genes encoding five viral proteins we applied a multilocus molecular population genetic framework to characterize the genetic status and recent evolutionary history of the TSWV species. Our analysis provided the first demonstration of population structuring and species-wide population expansions for TSWV, attributed possibly to founder effects. Also, we identified positive selection favoring divergence between Tospovirus species and purifying selection acting at the species level to preserve protein function. In addition, we were able to discover specific amino acid sites subject to positive selection within Bunyaviridae and to estimate the level of genetic heterogeneity of the TSWV species. Subsequently, in order to characterize the population history and genetic structure of individual wild-type TSWV isolates, thirteen geographically and host-diverse isolates were amplified, cloned and 516 clones were sequenced. Estimation of levels of genetic diversity and haplotype analysis revealed that natural TSWV isolates are highly heterogeneous viral populations that consist of one or more haplotypes with high frequency and an array of closely related rare haplotypes, some of which are defective. These viral populations exhibit a high transitional bias, attributed to the function of RNA-dependent RNA polymerase or an editing enzyme such as dsRAD. Also high levels of among-population differentiation were observed induced by geographic and/or host related factors. Demographic analysis based on tests of neutrality, gene genealogies and the coalescent revealed an excess of rare polymorphism and a shallow population genetic architecture consistent with a model of population growth for all analyzed TSWV isolates. Finally, data from genes encoding two viral proteins (NSm and L) were used for analysis of optimal informational content and for phylogenetic analysis. Our research has identified partial sequence regions that contain similar phylogenetic information and perform as well as the complete NSm and RdRp genes, for branching points statistically supported (bootstrap value>50%). We propose a new advanced diagnostic system, which will use the NSm and RdRp local regions together with the N gene of TSWV to assign attribution to initial TSWV infections and prevent their spread to an epidemic form

Abstract A50: The identification of an Ets1-driven gene signature in head and neck squamous cell carcinoma

Abstract Head and Neck Squamous Cell Carcinoma (HNSCC) is a heterogeneous disease of significant mortality and morbidity and with limited treatment options. This highlights the urgent need for a better understanding of the underlying biology of HNSCC. Recent genomic analysis of HNSCC tumors has identified four distinct molecular classes, of which the mesenchymal subtype is particularly aggressive in nature and associated with poor survival. We have discovered that Ets1, a prototypic oncogenic transcription factor, is selectively enriched in the mesenchymal HNSCC tumors along with other key drivers of Epithelial to Mesenchymal transition (EMT). Further analysis of gene expression datasets reveals that Ets1 expression, while low in normal epithelium, increases in pre-malignant and malignant tissues, suggesting a possible role for Ets1 during early tumorigenesis. Strikingly, Kaplan Meier analysis shows that Ets1 expression is associated with both poor survival in HNSCC and high incidence of tumor recurrence. We have performed hierarchical clustering of 33 HNSCC cell lines to show that these cell lines exhibit gene expression profiles that are representative of the four tumor subtypes. To better understand the Ets1-driven molecular processes in EMT high tumors, we have utilized SCC25, a mesenchymal HNSCC cell line to perform global transcriptomic analysis of effects of Ets1 knockdown. Additionally, we have also performed ChIP-seq experiments to identify genome wide transcriptional targets of Ets1 in SCC25. Taken together, these studies have uncovered an Ets1 intrinsic core gene signature that can serve as a valuable tool for further tumor classification and cancer-specific pathway discovery. Interestingly, by cross-referencing with the Cancer Genome Atlas HNSCC RNA-Seq dataset, we demonstrate that Ets1 expression is highly correlated with the majority of the Ets1 signature target genes thus reinforcing a functional relationship. Collectively, our findings not only identify several Ets1 dependent genes that are key to initiating and maintaining EMT in HNSCC, but also unearth novel targets that can be exploited for future therapeutics against this deadly cancer. Citation Format: Christian Gluck, Isha Sethi, Maria Tsompana, Satrajit Sinha. The identification of an Ets1-driven gene signature in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A50.</jats:p