Spreading of Xist RNA in T37H is attenuated at the onset of X inactivation. (A) Metaphase chromosomes from a 3-day differentiated TMA-2/S8 cell after RNA FISH with Xist probe (Left, green), followed by DNA FISH with brown (b) and diabetes (db) probes (Center, red). In the example shown, Xist RNA does not spread beyond the translocation breakpoint. (B) Representative examples of Xist RNA localization on 4^Xi chromosomes in 3-day differentiating TMA-2/S8 ES cells. Cells are classified according to the same criteria as used in Fig. 2. Schematic on the left illustrates the extent of Xist RNA spread (green box), for each of the three categories. (C) Summary of Xist RNA analysis in 3-day differentiating TMA-2/S8 ES cells. Each category of cells is shown as a proportion of the total number of cells with a 4^Xi chromosome. Data obtained for T37H fibroblasts published in ref. 19 are shown for comparison.

A large gene-rich/LINE-1-poor domain at the translocation breakpoint correlates with restricted spreading of X inactivation. (A) Fine-mapping of the chromosome 4 breakpoint. Schematic shows an expanded view of the region on chromosome 4 between 128 and 150 Mb, with the location of BAC probes assigned to the 4^X (red) or X⁴ (green) translocation product by using DNA FISH analysis. Example shows DNA FISH with BAC clones RP23–3D14 (red) and RP23–468M24 (green), spaced 1 Mb apart at 142 and 143 Mb on chromosome 4. (B) The figure illustrates LINE-1 (L1) homology as the percentage of total sequence (black bars) and the number of genes (red bars) in 1-Mb intervals along the entire length of chromosome X (Upper) and chromosome 4 (Lower). For the X chromosome, the location of the T37H breakpoint (cytogenetic band XA2, approximately Mb 15–32) is indicated with a thick black line. For chromosome 4, the precisely mapped position of the T37H breakpoint (142–143 Mb) is marked with the large arrow. The positions of the b and db loci are also shown. A 20-Mb L1-poor/gene-rich domain occurring at the breakpoint is highlighted with a dotted line. Data are extracted from Ensembl mouse genome sequence release m34.

Characteristics of TMA-2 ES cells carrying the T37H translocation. (A) Ideogram of the mouse chromosomes X and 4 and the X⁴ and 4^X translocation products in the T37H translocation. Approximate location of the T37H translocation breakpoint, Xist, brown (b) and diabetes (db) loci as well as X-specific 141 probe are shown. (B) Localization of db and b loci (green) and X-specific 141 repeat (red) on 4^X product in T37H translocation. The chromosome is counterstained with DAPI (blue). Computer-generated, G-like banding of the same chromosome based on DAPI staining is shown alongside. Brackets indicate chromosome X and 4 parts. (C) An example of a metaphase spread illustrating X⁴ (arrowhead) and 4^X (arrow) T37H translocation products in comparison with the normal X chromosome (arrow). X-specific 141 probe (red) is used for chromosome identification. (D) Analysis of karyotype stability of TMA-2/S8 clone. Preservation of both normal X and 4^X chromosomes was analyzed by DNA FISH with X chromosome paint at passages 3, 5, 6, 10, and 27 (P3, P5, P6, P10, and P27, respectively). (E) Dynamics of X inactivation in TMA-2/S8 ES cell differentiation analyzed by Xist RNA accumulation in differentiating cells at days 1, 2, 3, and 5.

Attenuated spread of chromatin modifications in T37H at the onset of X inactivation. (A) Metaphase chromosomes from a 5-day differentiated TMA-2/S8 cell, illustrating lack of H3K27 histone methylation in the autosomal part of 4^Xi chromosome (Left). Arrows indicate chromosomes 4^X and 4 as identified by DNA FISH with chromosome 4 markers brown (b) and diabetes (db) (Right). (B) Representative examples of H3K27me3 staining of the 4^Xi chromosome in differentiating TMA-2/S8 ES cells. Metaphases with H3K27me3 on the 4^X chromosome were classified into four categories depending on the extent of spread into chromosome 4 material: up to translocation (B1), up to db/b (B2), beyond db/b (B3), and up to the centromere (B4). Schematic shows the extent of spread for each category. The chromosome 4 constituent is indicated with a bracket, the red box illustrates the degree of H3K27me27 spread, and green circles show the position of db/b markers on chromosome 4. Each example is presented, illustrating H3K27me3 (shown in red on one panel and, to improve contrast, uncolored in another panel), position of db/b markers (green), and secondary constriction close to the translocation breakpoint (DAPI) (C) Summary showing percentage of cells for each category illustrated in B. Data are shown for TMA-2/S8 cells differentiated for 3 days (dark blue bar) and 5 days (light blue bar) and adult T37H fibroblasts (white bar). Scoring data and the number of metaphases analyzed are shown in the table (Lower). (D) Representative example of acH4 and H3K27me3 staining of the 4^Xi chromosome in 5-day differentiated TMA-2/S8 ES cells. Schematic on the right shows the extent of 3meH3K27 spreading and schematic on the left illustrates the extent of H4 hypoacetylation (white box). Note that there is a region of overlap between 3meH3K27 and acH4 corresponding approximately to the region between translocation breakpoint and db/b markers. (E) Summary of H4 hypoacetylation analysis in 5-day differentiated TMA-2/S8 ES cells. Metaphases with a 4^Xi chromosome were categorized as in B and C. Scoring data and the number of metaphases analyzed are shown in the table (Lower). Data for T37H somatic cells are from Duthie et al. (19) and are included for comparison.