Nuclear topology of D4Z4 units in control and FSHD myoblasts. 4q subtelomere architecture in 1.4 μm midprojections of 3D-preserved interphase nuclei from healthy (i, iii, iv and v) and FSHD myoblasts (ii, vi and vii) immunofluorescence in situ hybridization (immuno-FISH) using anti-H3K27me3 antibody (scale bar = 5 μm). The chromosome 4q territories are shown in blue, the bacterial artificial chromosome (BAC) upstream of the FRG1 gene in red, and the BAC containing a D4Z4 array in green. The arrows highlight the 4q subtelomeres identified by the cohybridization of both BACs and the chromosome 4 painting. The arrowhead in (ii) identifies a 4q subtelomere showing reduced hybridization with the green BAC and probably corresponding to the contracted D4Z4 allele. (iii) and (vi) The same nuclei as those respectively shown in (i) and (ii) were immunostained with anti-H3K27me3 antibody (red); the green spots correspond to hybridization with the D4Z4-containing BAC; the arrows and arrowhead identify the 4q alleles. (iv) and (v) Representative confocal sections of a nucleus from healthy myoblasts consisting of 4q D4Z4 alleles (green) that were negative ((iv), only green) or positive ((v), green and yellow) for colocalization with anti-H3K27me3 immunofluorescence (red). (vii) A 3 × enlargement of the confocal section in (vi) showing a nucleus from FSHD myoblast consisting of 4q D4Z4 alleles (green) that were negative (arrowhead, only green) or positive (arrow, green and yellow) for colocalization with anti-H3K27me3 immunofluorescence (red).