Genetic analysis of the mouse X inactivation center defines an 80-kb multifunction domain - PubMed (original) (raw)

Genetic analysis of the mouse X inactivation center defines an 80-kb multifunction domain

J T Lee et al. Proc Natl Acad Sci U S A. 1999.

Abstract

Dosage compensation in mammals occurs by X inactivation, a silencing mechanism regulated in cis by the X inactivation center (Xic). In response to developmental cues, the Xic orchestrates events of X inactivation, including chromosome counting and choice, initiation, spread, and establishment of silencing. It remains unclear what elements make up the Xic. We previously showed that the Xic is contained within a 450-kb sequence that includes Xist, an RNA-encoding gene required for X inactivation. To characterize the Xic further, we performed deletional analysis across the 450-kb region by yeast-artificial-chromosome fragmentation and phage P1 cloning. We tested Xic deletions for cis inactivation potential by using a transgene (Tg)-based approach and found that an 80-kb subregion also enacted somatic X inactivation on autosomes. Xist RNA coated the autosome but skipped the Xic Tg, raising the possibility that X chromosome domains escape inactivation by excluding Xist RNA binding. The autosomes became late-replicating and hypoacetylated on histone H4. A deletion of the Xist 5' sequence resulted in the loss of somatic X inactivation without abolishing Xist expression in undifferentiated cells. Thus, Xist expression in undifferentiated cells can be separated genetically from somatic silencing. Analysis of multiple Xic constructs and insertion sites indicated that long-range Xic effects can be generalized to different autosomes, thereby supporting the feasibility of a Tg-based approach for studying X inactivation.

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Figures

Figure 1

Figure 1

YAC fragmentation scheme (A) and vector (B). (C) Xic deletion panel. E, _Eag_I; N, _Not_I; B, _Bss_HII; S, _Sal_I; Sf, _Sfi_I. (D) Map of P1 clones.

Figure 2

Figure 2

Xist expression in Tg ES and EB cells. (A) RNA FISH of undifferentiated ES cells with a rhodamine-labeled Xist probe (red). X, endogenous; T, Tg; Blue, 4′,6-diamidino-2-phenylindole (dapi) staining. (B) RNA FISH of day 9 EB cells (Xist RNA, red). Double Xist expressers are indicated by asterisks; the arrow indicates Xist RNA coat on a πJL2.5 metaphase chromosome. (C) RNA/DNA FISH for Xist RNA (red) and Zfx or vector DNA (FITC, green) was performed for all ES lines to determine Xist RNA origin. πJL2.5 EB is shown. Coincident red and green resulted in white.

Figure 3

Figure 3

Xist is expressed in class I somatic cells, and the RNA coats the autosome. (A) RNA/DNA FISH of normal female and Tg fibroblasts. Xist RNA is shown in red; Zfx genomic DNA is shown in green. (B) πJL1.4.1 chromosomes were hybridized to Xist probes (red) without chromosome denaturation, then stripped, denatured, and hybridized to chromosome 3 (Ch3)-specific paints (Ch.paint; red) and Tg probes (green). Arrows indicate normal homologues of Ch3. (C) Xist RNA skips the Xic Tg as indicated by the arrows. This experiment was performed as described for B.

Figure 4

Figure 4

Delayed replication timing of the Tg autosome in class I fibroblasts. πJL2.5.5 is shown. Arrows indicate labeling by Ch8-specific paints (Ch.paint) and Tg probes; asterisks indicate Y. BrdU, BrdUrd.

Figure 5

Figure 5

H4 hypoacetylation of Tg autosomes in class I somatic cells. (A) Immunofluorescence of πJL2.5.5 chromosome spreads with antiacetylated H4 antibodies (green). The Ch8 Tg (arrows) and the Y (asterisk) were hypoacetylated. The Tg is shown in red. (B) In πJL1.4.1, some Tg autosomes showed partial deacetylation (arrows). The Tg is pseudocolored yellow. (C) A control female cell showing one hypoacetylated Xi (arrow).

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