FIG. 7.

Repeat silencing in the early preimplantation embryo does not require Xist. Cot-1 and Xist RNA FISH with subsequent DNA FISH using the Xic probe, Sx7, in the wild-type two-cell embryo (A), and Cot-1 RNA FISH with subsequent DNA FISH in the Xist mutant two-cell embryo using a combination of Sx7 and πXE9 probes to distinguish X^M (wild-type) from X^P (Xist deficient) (B). (C) Pictorial representation of the DAPI staining pattern and corresponding Cot-1 RNA FISH pattern at the two-cell stage. Because the nucleolus and perinucleolar heterochromatin are devoid of Cot-1 signal, the Cot-1 hole is always larger than the DAPI hole left by the nucleolus itself. When the DNA FISH signal in question localizes to the perinucleolar Cot-1 hole, the signal is scored as nucleolar association inactive. On the other hand, when the signal localizes in Cot-1⁺ regions, it is scored as no association active. (D) Xic nucleolar association of X^P versus X^M. Cot-1, LINE, B1, and B2 RNA FISH were performed on wild-type and Xist-deficient two-cell embryos in combination with an Xic probe (Sx7). DNA FISH was conducted subsequently to compare the frequency with which X^P and X^M come in direct contact with the nucleolus. The combination of Sx7 and πXE9 probes was used to distinguish X^M (wild-type) from X^P (Xist deficient). When the Sx7 signal was directly adjacent to the nucleolus or located in the perinucleolar heterochromatic ring, the chromosome was judged to be nucleolus associated and Cot-1⁻. For the two-cell embryo, there was 100% correlation between nucleolus-associated and Cot-1⁻, LINE⁻, and SINE B2⁻ states of X^P; there was a 95% correlation between the nucleolus-associated and the SINE B1⁻ state. In contrast, chromosomes that were not nucleolus associated were Cot-1⁺. P values were calculated using the student t test. WT, wild-type. Xist−, X^MX^P;Xist−.