Systematic accumulation of Tsix -associated RNA Polymerase II (RNAPII) at the limit of the H3K27 tri-methylation domain of the Xic in undifferentiated embryonic stem (ES) cells. (A) Profile of RNAPII distribution in wild-type male ES cells (CK35 cell line). A schematic representation of the Xist/Tsix locus is shown (top). Red and green arrows indicate the orientation of Tsix and Xist transcription units, respectively. The orange box represents the Xite locus. The purple box corresponds to the DXPas34 enhancer. (B) Distribution of RNAPII at the Tsix 3' region in wild-type (CK35, in black) and three different Tsix-mutant male ES cell lines (Ma2L in red, ΔPas34 in yellow and AV in orange). The RNAPII profile is also shown for the corresponding Ma2L control, Ma1L (in grey). The AV cell line, derived from CK35 wild-type cells, carries a 15-kb-long deletion encompassing the major Tsix promoter. (C-E) RNAPII accumulation in three independent Tsix mutant male ES cells: Ma2L (C), ΔPas34 (D) and AV (E). A schematic representation of the region analyzed in each ChIP assay is shown (top). The colour code of the genetic elements is the same as in (A). In (C), the position of the transcriptional STOP signal introduced in the Ma2L cell line is indicated. In (D, E), arrows indicate position of the deletions introduced to generate ΔPas34 and AV, respectively. (F) ChIP analysis of H3K27 tri-methylation in wild-type (CK35, in black) and AV male ES cells (in red). Dotted lines indicate the position of the 15-kb region deleted in the AV cell line.