No abstract

Exposure to physiological estrogens or xenoestrogens (e.g., zearalenone or bisphenol A) increases the risk for cancer. However, little information is available on their significance in ovarian cancer. We present a comprehensive study on the effect of estradiol, zearalenone and bisphenol A on the phenotype, mRNA, intracellular and cell-free miRNA expression of human epithelial ovarian cell lines. Estrogens induced a comparable effect on the rate of cell proliferation and migration as well as on the expression of estrogen-responsive genes (GREB1, CA12, DEPTOR, RBBP8) in the estrogen receptor α (ERα)-expressing PEO1 cell line, which was not observable in the absence of this receptor (in A2780 cells). The basal intracellular and cell-free expression of miR200s and miR203a was higher in PEO1, which was accompanied with low ZEB1 and high E-cadherin expression. These miRNAs showed a rapid but intermittent upregulation in response to estrogens that was diminished by an ERα-specific antagonist. The role of ERα in the regulation of the MIR200B-MIR200A-MIR429 locus was further supported by publicly available ChIP-seq data. MiRNA expression of cell lysates correlated well with cell-free miRNA expression. We conclude that cell-free miR200s might be promising biomarkers to assess estrogen sensitivity of ovarian cells.

IRF3, IRF5 and IRF9 are transcription factors, which play distinct roles in the regulation of antiviral and inflammatory responses. The determinants that mediate IRF-specific enhancer selection are not fully understood. To uncover regions occupied predominantly by IRF3, IRF5 or IRF9, we performed ChIP-seq experiments in activated murine dendritic cells. The identified regions were analysed with respect to the enrichment of DNA motifs, the interferon-stimulated response element (ISRE) and ISRE half-site variants, and chromatin accessibility. Using a machine learning method, we investigated the predictability of IRF-dominance. We found that IRF5-dominant regions differed fundamentally from the IRF3- and IRF9-dominant regions: ISREs were rare, while the NFKB motif and special ISRE half-sites, such as 5′-GAGA-3′ and 5′-GACA-3′, were enriched. IRF3- and IRF9-dominant regions were characterized by the enriched ISRE motif and lower frequency of accessible chromatin. Enrichment analysis and the machine learning method uncovered the features that favour IRF3 or IRF9 dominancy (e.g. a tripartite form of ISRE and motifs for NF-κB for IRF3, and the GAS motif and certain ISRE variants for IRF9). This study contributes to our understanding of how IRF members, which bind overlapping sets of DNA sequences, can initiate signal-dependent responses without activating superfluous or harmful programmes.

We have previously observed phenotypic and developmental changes upon the ectopic expression of the RUNX3 or the ZBTB46 transcription factors in mouse embryonic stem cell (ESC) derived progenitors. In this study we evaluated the gene expression profiles of the RUNX3- and the ZBTB46-instructed murine ESCs with RNA-Seq testing two next generation sequencing (NGS) technologies. We compared the DNA nanoball (DNB) based MGI DNBSEQ G400 sequencer with the bridge-PCR based Illumina NextSeq 500 instrument. Moreover, we also compared two types of MGI sequencing reagents (Standard- versus Hot-MPS) with the DNBSEQ G400. Importantly, very similar gene expression profile and greatly overlapping RUNX3 and ZBTB46 regulated gene sets were detected with both platforms. Moreover, almost identical gene expression pattern was obtained with the Hot-MPS reagent compared to the Standard-MPS chemistry. This transcriptomic analysis also facilitated the identification of RUNX3 and ZBTB46 regulated genes. For example, we found that Gzmd, Gdf6 and Ccr7 genes were robustly upregulated upon the forced expression of Runx3, on the other hand, Gpx2, Tdpoz4 and Arg2 were induced upon the ectopic expression of Zbtb46. Together these findings demonstrate that the DNBSEQ G400 system is also suitable for global transcript profiling and target gene selection with lower cost.

The antiviral state, an initial line of defense against viral infection, is established by a set of IFN-stimulated genes (ISGs) encoding antiviral effector proteins. The effector ISGs are transcriptionally regulated by type I IFNs mainly via activation of IFN-stimulated gene factor 3 (ISGF3). In this study, the regulatory elements of effector ISGs were characterized to determine the (epi)genetic features that enable their robust induction by type I IFNs in multiple cell types. We determined the location of regulatory elements, the DNA motifs, the occupancy of ISGF3 subunits (IRF9, STAT1, and STAT2) and other transcription factors, and the chromatin accessibility of 37 effector ISGs in murine dendritic cells. The IFN-stimulated response element (ISRE) and its tripartite version occurred most frequently in the regulatory elements of effector ISGs than in any other tested ISG subsets. Chromatin accessibility at their promoter regions was similar to most other ISGs but higher than at the promoters of inflammation-related cytokines, which were used as a reference gene set. Most effector ISGs (81.1%) had at least one ISGF3 binding region proximal to the transcription start site (TSS), and only a subset of effector ISGs (24.3%) was associated with three or more ISGF3 binding regions. The IRF9 signals were typically higher, and ISRE motifs were “stronger” (more similar to the canonical sequence) in TSS-proximal versus TSS-distal regulatory regions. Moreover, most TSS-proximal regulatory regions were accessible before stimulation in multiple cell types. Our results indicate that “strong” ISRE motifs and universally accessible promoter regions that permit robust, widespread induction are characteristic features of effector ISGs.