Tsix transcription affects the expression of genes located in the distal part of the Xic. RT-PCR analysis of several genes lying within the Xic: Xpct, Cnbp2, Ftx, Jpx, Tsx and Chic1 in different embryonic stem cells, after normalization to Arpo PO transcript levels.

Loss of H3K27 tri-methylation at the Xic during embryonic stem (ES) cell differentiation. (A) Map of Xic (see Figure 1A). (B-D) Extensive ChIP analysis of H3K27 trimethylation in (B) wild-type female ES cells (LF2), (C) wild-type male revertant cells (Ma1L), or (D) Tsix-truncated ES cells (Ma2L). ChIP experiments were performed on undifferentiated ES cells (black line) and after 2 and 4 days (orange and red lines, respectively) of differentiation with retinoic acid.

H3K4 methylation is not involved in the H3K27me3 boundary formation at the Xic. (A) Map of Xic (see Figure 1A). (B) ChIP analysis showing the distribution of histone H3 modifications at the Xic in female embryonic stem (ES) cells (LF2) using antibodies against H3K27 trimethylation (H3K27me3, black line), H3K4 dimethylation (H3K4me2, red line) and H3K4 trimethylation (H3K4me3, blue line). (C) Extensive analysis of H3K4me2 and (D) H3K4me3 in wild-type (Ma1L, black line) and Tsix-truncated (Ma2L, red line) male ES cells. Insets focus on the percentage of immunoprecipitation observed at the Xist 5' region.

H3K27 tri-methylation spreads from the hotspot into Xist in the absence of Tsix transcription. (A) Map of Xic (see Figure 1A). (B) ChIP analysis of H3K27 trimethylation in male Tsix-truncated embryonic stem (ES) cells (Ma2L cell line, red line) and in the corresponding revertant (Ma1L, black line). In the Ma2L cell line, Tsix transcription has been truncated through the insertion of a loxP-flanked transcriptional STOP signal downstream of the Tsix promoter (represented in the graph by the STOP symbol). The Ma1L revertant was obtained from Ma2L after deletion of the STOP signal. (C) Similar analysis in male wild-type (CK35 cell line, black line) and mutated (ΔPas34, red line) ES cells in which Tsix transcription is drastically reduced. The ΔPas34 cell line was generated by deleting DXPas34, an enhancer of Tsix located near the Tsix major promoter. Dotted lines in the graph indicate the location of the 1.2-kb deletion carried by ΔPas34 ES cells.

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.

Distribution of H3K27 tri-methylation within a 300-kb region of the Xic in undifferentiated male and female embryonic stem (ES) cells. (A) Schematic diagram of the 300-kb region analyzed in our ChIP experiments showing the location of the different transcription units of the Xic. Arrows indicate the direction of each transcription unit. Coding genes are indicated in black, non-coding genes with coloured arrows. The orange box represents the Xite locus, an enhancer region that displays transcriptional activity on the major Tsix promoter. The purple box represents the so-called region B, a complex transcriptional unit that produces both sense and antisense transcripts [40]. We used in each ChIP experiment a set of 383 primer pairs that were designed automatically to cover the 300-kb region (except repeat regions: see Materials and methods). (B) ChIP analysis of H3K27 trimethylation in undifferentiated female (LF2) ES cells and (C) in male (CK35 cell line) ES cells. Both graphs show the percentage of immunoprecipitation (%IP) obtained after normalization to the input. All values are mean ± standard deviation. The average percentage of immunoprecipitation calculated for each position was plotted against the genomic location (bp). The +1 coordinate corresponds to position 1,005,322,247 in NCBI build 37.