CTCF is a master regulator that plays important roles in genome architecture and gene expression. How CTCF is recruited in a locus-specific manner is not fully understood. Evidence from epigenetic processes, such as X-chromosome inactivation (XCI), indicates that CTCF associates functionally with RNA. Using genome-wide approaches to investigate the relationship between its RNA interactome and epigenomic landscape, here we report that CTCF binds thousands of transcripts in mouse embryonic stem cells, many in close proximity to CTCF’s genomic binding sites. CTCF is a specific and high-affinity RNA-binding protein (Kd <1 nM). During XCI, CTCF differentially binds the active and inactive X-chromosomes and interacts directly with Tsix, Xite, and Xist RNAs. Tsix and Xite RNAs target CTCF to the X-inactivation center, thereby inducing homologous X-chromosome pairing. Our work elucidates one mechanism by which CTCF is recruited in a locus-specific manner and implicates CTCF-RNA interactions in long-range chromosomal interactions.

Flk1 is the major receptor for VEGF on endothelial cells. During embryogenesis, flk1 is required for both vasculogenesis and angiogenesis and abnormally elevated flk1 expression is often associated with pathological conditions in adults. While the biological function of flk1 has been studied extensively, very little is known about how the flk1 gene is regulated at the transcriptional level. Our transgenic study led to the identification of a flk1 endothelial enhancer positioned approximately 5 kb upstream of the flk1 translation initiation site. Binding sites for FoxH1, scl, ets and gata factors are found in the zebrafish flk1 endothelial enhancer, as well as in upstream sequences of mouse flk1 and human kdr genes, suggesting that the regulatory machinery for flk1/kdr is conserved from fish to mammals. The roles of scl, ets and gata factors in hemangioblasts have been well defined, but the significance of FoxH1 in vessel formation has not been explored previously. Here we show that FoxH1 binds to the flk1 endothelial enhancer in vitro and functions as a repressor for flk1 transcription in cultured cells. Consistent with these findings, the expression level of flk1 is elevated in embryos lacking both maternal and zygotic FoxH1. We further show that overexpression of FoxH1 has a negative effect on vascular formation that can be counteracted by the down-regulation of smad2 activity in zebrafish embryos. Taken together, our data provide the first evidence that flk1 is a direct target of FoxH1 and that FoxH1 is involved in vessel formation in zebrafish.

Telomeric regions are known to be transcribed in several organisms. Although originally reported to be transcribed from all chromosomes with enrichment near the inactive X of female cells, we show that telomeric RNAs in fact are enriched on both sex chromosomes of the mouse in a developmentally specific manner. In female stem cells, both active Xs are marked by the RNAs. In male stem cells, both the X and the Y accumulate telomeric RNA. Distribution of telomeric RNAs changes during cell differentiation, after which they associate only with the heterochromatic sex chromosomes of each sex. FISH mapping suggests that accumulated telomeric RNAs localize at the distal telomeric end. Interestingly, telomeric expression changes in cancer and during cellular stress. Furthermore, RNA accumulation increases in Dicer-deficient stem cells, suggesting direct or indirect links to RNAi. We propose that telomeric RNAs are tied to cell differentiation and may be used to mark pluripotency and disease.

BackgroundImbalances in X-linked gene dosage between the sexes are resolved by transcriptionally silencing one of two X-chromosomes in female cells of the early mammalian embryo. X-inactivation is triggered by expression of the non-coding Xist gene. In turn, Xist is dually regulated by the antisense Tsix RNA and by the Oct4 pluripotency factor. Although there is general agreement that Tsix is an inhibitor of Xist, some laboratories have observed ectopic Xist induction in differentiating male ES cells when Tsix is mutated, whereas we have not observed significant changes in Xist. These observational differences have led to fundamentally diverse models of X-chromosome counting. Here, we investigate if different methods of cell differentiation and use of all -trans retinoic acid (RA) could be causative factors and how they might impact Xist expression.ResultsWe compared suspension and cell-adhesion cultures in the presence or absence of RA and find that RA significantly impacts Xist expression in Tsix-mutant male cells. Whereas the standard embryoid body method infrequently leads to ectopic Xist expression, adding RA generates a significant number of Xist-positive male cells. However, while normal Xist clouds in wild-type female cells are robust and well-circumscribed, those found in the RA-treated mutant males are loosely dispersed. Furthermore, ectopic Xist expression does not generally lead to complete gene silencing. We attribute the effect of RA on Xist to RA's repressive influence on Oct4, a pluripotency factor recently shown to regulate Tsix and Xist. RA-treated ES cells exhibit accelerated decreases in Oct4 RNA levels and also display accelerated loss of binding to Xist intron 1. When Tsix is deficient, the faster kinetics of Oct4 loss tip the equilibrium towards Xist expression. However, the aberrant Xist clusters are unlikely to explain elevated cell death, as X-linked silencing does not necessarily correlate with the qualitatively aberrant Xist clusters.ConclusionsWe conclude that RA treatment leads to premature downregulation of Oct4 and partial derepression of Xist irrespective of X-chromosome counting. RA-induced Xist clusters in male cells do not result in global or stable silencing, and excess cell death is not observed. These data and RA's known pleiotropic effects on ES transcription networks suggest that RA differentation bypasses normal X-inactivation controls and should be used judiciously. We propose that the likelihood of Xist expression is determined by a balance of multiple Xist activators and repressors, and that levels of Oct4 and Tsix are crucial toward achieving this balance.