Using a recently developed chemical approach, we have generated a genome-wide map of nucleosomes in vivo in Schizosaccharomyces pombe (S. pombe) at base pair resolution. The shorter linker length previously identified in S. pombe is due to a preponderance of nucleosomes separated by ∼4/5 bp, placing nucleosomes on opposite faces of the DNA. The periodic dinucleotide feature thought to position nucleosomes is equally strong in exons as in introns, demonstrating that nucleosome positioning information can be superimposed on coding information. Unlike the case in Saccharomyces cerevisiae, A/T-rich sequences are enriched in S. pombe nucleosomes, particularly at ±20 bp around the dyad. This difference in nucleosome binding preference gives rise to a major distinction downstream of the transcription start site, where nucleosome phasing is highly predictable by A/T frequency in S. pombe but not in S. cerevisiae, suggesting that the genomes and DNA binding preferences of nucleosomes have coevolved in different species. The poly (dA-dT) tracts affect but do not deplete nucleosomes in S. pombe, and they prefer special rotational positions within the nucleosome, with longer tracts enriched in the 10-to 30-bp region from the dyad. S. pombe does not have a welldefined nucleosome-depleted region immediately upstream of most transcription start sites; instead, the −1 nucleosome is positioned with the expected spacing relative to the +1 nucleosome, and its occupancy is negatively correlated with gene expression. Although there is generally very good agreement between nucleosome maps generated by chemical cleavage and micrococcal nuclease digestion, the chemical map shows consistently higher nucleosome occupancy on DNA with high A/T content.chromatin structure | heterochromatin | gene regulation E ukaryotic DNA is organized into nucleosome arrays that facilitate proper genome compaction and regulation of the genetic information. Genome-wide nucleosome maps from different organisms have provided important insights into how nucleosome positioning regulates chromosome functions (1-6). It is commonly accepted that the DNA sequence itself plays a major role in the exact positions where histone octamers bind DNA to form nucleosomes (7-9). Most remarkably, nucleosomes prefer special dinucleotide motifs at specific rotational angles within the nucleosome core (10, 11) and disfavor poly (dA-dT) tracts (12-15). Although the ability of DNA to interact with the histone core is important, nucleosome occupancy is influenced by additional extrinsic factors, including species-specific DNA binding proteins (16), ATP-dependent remodeling complexes (17,18), and the transcription machinery (19)(20)(21)(22).The underlying mechanisms for the species-or cell typespecific nucleosome positioning features have not been well understood. The fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae are two highly diverged model organisms widely used to study basic biological processes in eukaryotes (23). Although similar in siz...

Ovarian cancer is the most lethal gynecological malignancy, primarily because its origin and initiation factors are unknown. A secretory murine oviductal epithelial (MOE) model was generated to address the hypothesis that the fallopian tube is an origin for high-grade serous cancer. MOE cells were stably altered to express mutation in p53, silence PTEN, activate AKT, and amplify KRAS alone and in combination, to define if this cell type gives rise to tumors and what genetic alterations are required to drive malignancy. Cell lines were characterized in vitro and allografted into mice. Silencing PTEN formed high-grade carcinoma with wide spread tumor explants including metastasis into the ovary. Addition of p53 mutation to PTEN silencing did not enhance this phenotype, whereas addition of KRAS mutation reduced survival. Interestingly, PTEN silencing and KRAS mutation originating from ovarian surface epithelium generated endometrioid carcinoma, suggesting that different cellular origins with identical genetic manipulations can give rise to distinct cancer histotypes. Defining the roles of specific signaling modifications in tumorigenesis from the fallopian tube/oviduct is essential for early detection and development of targeted therapeutics. Further, syngeneic MOE allografts provide an ideal model for pre-clinical testing in an in vivo environment with an intact immune system.

Cooperativity in transcription factor (TF) binding is essential in eukaryotic gene regulation, and arises through diverse mechanisms. Here we focus on one mechanism, collaborative competition, which is of interest because it arises both automatically, with no requirement for TF co-evolution, and spontaneously, with no requirement for ATP-dependent nucleosome remodeling factors. Previous experimental studies of collaborative competition analyzed cases in which target sites for pairs of cooperating TFs were contained within the same side of the nucleosome. Here we utilize new assays to measure cooperativity in protein binding to pairs of nucleosomal DNA target sites. We focus on the cases that are of greatest in vivo relevance, in which one binding site is located close to the end of a nucleosome, and the other binding site is located at diverse positions throughout the nucleosome. Our results reveal energetically significant positive (favorable) cooperativity for pairs of sites on the same side of the nucleosome, but, for the cases examined, energetically insignificant cooperativity between sites on opposite sides of the nucleosome. These findings imply a special significance for TF binding sites that are spaced within one half nucleosome length (74 bp) or less along the genome, and may prove useful for prediction of cooperatively acting TFs genome-wide.

High-grade serous carcinoma (HGSC) is the most lethal ovarian cancer histotype. The fallopian tube secretory epithelial cells (FTSECs) are a proposed progenitor cell type. Genetically altered FTSECs form tumors in mice; however, a spontaneous HGSC model has not been described. Apart from a subpopulation of genetically predisposed women, most women develop ovarian cancer spontaneously, which is associated with aging and lifetime ovulations. A murine oviductal cell line (MOELOW) was developed and continuously passaged in culture to mimic cellular aging (MOEHIGH). The MOEHIGH cellular model exhibited a loss of acetylated tubulin consistent with an outgrowth of secretory epithelial cells in culture. MOEHIGH cells proliferated significantly faster than MOELOW, and the MOEHIGH cells produced more 2D foci and 3D soft agar colonies as compared to MOELOW MOEHIGH were xenografted into athymic female nude mice both in the subcutaneous and the intraperitoneal compartments. Only the subcutaneous grafts formed tumors that were negative for cytokeratin, but positive for oviductal markers, such as oviductal glycoprotein 1 and Pax8. These tumors were considered to be poorly differentiated carcinoma. The differential molecular profiles between MOEHIGH and MOELOW were determined using RNA-Seq and confirmed by protein expression to uncover pathways important in transformation, like the p53 pathway, the FOXM1 pathway, WNT signaling, and splicing. MOEHIGH had enhanced protein expression of c-myc, Cyclin E, p53, and FOXM1 with reduced expression of p21. MOEHIGH were also less sensitive to cisplatin and DMBA, which induce lesions typically repaired by base-excision repair. A model of spontaneous tumorogenesis was generated starting with normal oviductal cells. Their transition to cancer involved alterations in pathways associated with high-grade serous cancer in humans.

BackgroundThe fallopian tube epithelium is one of the potential sources of high-grade serous ovarian cancer (HGSC). The use of estrogen only hormone replacement therapy increases ovarian cancer (OVCA) risk. Despite estrogen’s influence in OVCA, selective estrogen receptor modulators (SERMs) typically demonstrate only a 20 % response rate. This low response could be due to a variety of factors including the loss of estrogen receptor signaling or the role of estrogen in different potential cell types of origin. The response of fallopian tube epithelium to SERMs is not known, and would be useful when determining therapeutic options for tumors arising from this cell type, such as HGSC.ResultsUsing normal murine derived oviductal epithelial cells (mouse equivalent to the fallopian tube) estrogen receptor expression was confirmed and interaction with its ligand, estradiol, triggered mRNA and protein induction of progesterone receptor (PR). The SERMs 4-hydroxytamoxifen, raloxifene and desmethylarzoxifene, functioned as estrogen receptor antagonists in oviductal cells. Cellular proliferation and migration assays suggested that estradiol does not significantly impact cellular migration and increased proliferation. Further, using RNAseq, the oviduct specific transcriptional genes targets of ER when stimulated by estradiol and 4-hydroxytamoxifen signaling were determined and validated. The RNA-seq revealed enrichment in proliferation, anti-apoptosis, calcium signaling and steroid signaling processes. Finally, the ER and PR receptor status of a panel of HGSC cell lines was investigated including Kuramochi, OVSAHO, OVKATE, OVCAR3, and OVCAR4. OVSAHO demonstrated receptor expression and response, which highlights the need for additional models of ovarian cancer that are estrogen responsive.ConclusionsOverall, the fallopian tube has specific gene targets of estrogen receptor and demonstrates a tissue specific response to SERMs consistent with antagonistic action.Electronic supplementary materialThe online version of this article (doi:10.1186/s13048-016-0213-3) contains supplementary material, which is available to authorized users.