Characterization of CTCF and CTCFL binding. A Examples of CTCF- and CTCFL-binding site location. The genomic location of CTCF (upper part) and CTCFL (middle and bottom parts) binding sites in the absence (−CTCFL, middle) or presence (+CTCFL, bottom) of CTCFL, within the Stra8, Prss50 and Gal3st1 genes. The vertical axes show the number of unique sequence reads. B CTCFL binds to Stra8 and Prss50. Band shift analyses of GFP-CTCFL on Stra8 and Prss50 fragments. GFP-CTCFL binding can be super shifted (marked with asterisks) with anti-GFP, but not with an Actin antibody. Band shifts were performed under excess probe conditions. C In vitro effect of CTCFL on CTCF binding. Band shift analyses with GFP-CTCF and/or GFP-CTCFL on Prss50- and Stra8-bindings sites. GFP-CTCFL is added in increasing amounts (1-, 2-, 5- and 10-fold compared to GFP-CTCF). To allow competition, the band shift was performed under probe-limiting concentrations. D Cellular effect of CTCFL on CTCF binding. ChIP analyses with CTCFL (blue), CTCF (red) and pre-immune (yellow) antisera in ES cells that were either non-transfected (−) or transiently transfected CTCFL-V5-GFP (+). According to ChIP-sequencing data, Prss50, Stra8 and Vps18 bind both CTCF and CTCFL, whereas Gal3st1 only binds CTCFL, and Chr10 only binds CTCF. A CTCF- and CTCFL-negative site within the Amylase gene is used as reference and set to 1. Error bars represent standard deviations of biological replicates. E Competition between CTCF and CTCFL in ES cells. Genome-wide binding of CTCF was compared to that of CTCFL by ChIP-Seq using non-transfected ES cells and ES cells transiently transfected with GFP-CTCFL. The left hand panel shows the effect of CTCFL binding on shared CTCFL/CTCF sites that showed >1.5 fold difference in CTCF binding. The effect is categorized into sites with increased (up) or decreased (down) CTCF binding. The right hand panel shows a more general effect of CTCFL binding on CTCF binding. Here, we examined the change in CTCF binding in all shared CTCF(L)-binding sites (all) compared to those shared sites that were significantly changed in CTCF binding (changed). The effect on CTCF binding is plotted as log2-fold difference. F In vivo CTCF(L) binding. ChIP was performed using anti-CTCF (red) or anti-CTCFL (blue) antibodies, or pre-immune serum, on the indicated sites (A: Amylase, S: Stra8, P: Prss50, V: Vps18) in nuclei from dissociated seminiferous tubules, partly purified by elutriation. Relative enrichment is shown compared to Amylase.