No abstract

Ewing sarcoma usually expresses the EWS/FLI fusion transcription factor oncoprotein. EWS/FLI regulates myriad genes required for Ewing sarcoma development. EWS/FLI binds GGAA-microsatellite sequences in vivo and in vitro. These sequences provide EWS/FLI-mediated activation to reporter constructs, suggesting that they function as EWS/FLI-response elements. We now demonstrate the critical role of an EWS/FLI-bound GGAA-microsatellite in regulation of the gene as well as for Ewing sarcoma proliferation and anchorage-independent growth. Clinically, genomic GGAA-microsatellites are highly variable and polymorphic. Current data suggest that there is an optimal "sweet-spot" GGAA-microsatellite length (of 18-26 GGAA repeats) that confers maximal EWS/FLI-responsiveness to target genes, but the mechanistic basis for this remains unknown. Our biochemical studies, using recombinant Δ22 (a version of EWS/FLI containing only the FLI portion), demonstrate a stoichiometry of one Δ22-monomer binding to every two consecutive GGAA-repeats on shorter microsatellite sequences. Surprisingly, the affinity for Δ22 binding to GGAA-microsatellites significantly decreased, and ultimately became unmeasureable, when the size of the microsatellite was increased to the sweet-spot length. In contrast, a fully functional EWS/FLI mutant (Mut9, which retains approximately half of the EWS portion of the fusion) showed low affinity for smaller GGAA-microsatellites but instead significantly increased its affinity at sweet-spot microsatellite lengths. Single-gene ChIP and genome-wide ChIP-sequencing (ChIP-seq) and RNA-seq studies extended these findings to the in vivo setting. Together, these data demonstrate the critical requirement of GGAA-microsatellites as EWS/FLI activating response elements in vivo and reveal an unexpected role for the EWS portion of the EWS/FLI fusion in binding to sweet-spot GGAA-microsatellites.

The Fallopian tube has been reported to undergo cyclical changes. However, many studies of tubal ultrastructure have either examined one segment of the tube only or studied animal oviducts. The aim of this study was to document in detail the combined morphological and ultrastructural features of the epithelial lining along the length of the tube in women at different stages of the menstrual cycle. We report an increase in the proportion of ciliated cells along the tube, being highest in the fimbriae, but no substantial difference between the follicular and luteal phases of the menstrual cycle. In the late follicular phase, fragments of cytoplasmic and cellular material were seen in the isthmic lumen but not in the outer tubal segments. Similarly, surface domes and secretory granules were more prominent in the mid-tube and ampullary sections than in the fimbriae. This surface activity was followed by relative quiescence in the early/mid luteal phase with reversion to a more active surface but with little secretory activity in the late luteal phase. These findings along the Fallopian tube substantiate the concept of functional differentiation between the different segments and necessitate further studies to determine its clinical relevance.

Oestrogen and progesterone are known to require their corresponding steroid receptors to manifest structural and functional effects in the Fallopian tube, uterus and other target organs. This study compares cyclical variations of these receptors in the uterus and in different segments of the Fallopian tube in the same subjects using an immunocytochemical technique. The results show that in the Fallopian tube, isthmic and ampullary epithelial and stromal oestrogen receptors increased in the follicular phase to a peak at mid cycle and then declined in the late luteal phase. The intensity of immunostaining of oestrogen receptors was less in the Fallopian tube than in endometrial glandular epithelium. The fimbrial end demonstrated an opposite pattern of staining to other segments of the tube. Progesterone receptor immunostaining was more intense than that for oestrogen receptors in the follicular phase, and, whereas it disappeared completely from the endometrial glandular epithelium in the late luteal phase, positive staining was clearly visualized in the tubal epithelium and stroma and endometrial stroma at this stage of the menstrual cycle. These differences in the steroid receptor content may reflect the changing and different functional roles of these regions and may have important implications on human reproduction.

The association between PCOS and EC appears confined to premenopausal women. The tendency for cyclin D1-expressing endometrial tumours to be more prevalent in women with PCO challenges the assumption that EC prognosis is improved in women with PCOS.