Disruption of Ini1 leads to peri-implantation lethality and tumorigenesis in mice

Abstract

SNF5/INI1 is a component of the ATP-dependent chromatin remodeling enzyme family SWI/SNF. Germ line mutations of INI1 have been identified in children with brain and renal rhabdoid tumors, indicating that INI1 is a tumor suppressor. Here we report that disruption of Ini1 expression in mice results in early embryonic lethality. Ini1-null embryos die between 3.5 and 5.5 days postcoitum, and Ini1-null blastocysts fail to hatch, form the trophectoderm, or expand the inner cell mass when cultured in vitro. Furthermore, we report that approximately 15% of Ini1-heterozygous mice present with tumors, mostly undifferentiated or poorly differentiated sarcomas. Tumor formation is associated with a loss of heterozygocity at the Ini1 locus, characterizing Ini1 as a tumor suppressor in mice. Thus, Ini1 is essential for embryo viability and for repression of oncogenesis in the adult organism.

FIG. 1

Disruption of Ini1. (A) Targeting strategy for Ini1. A retroviral promoter trap vector was inserted in intron 3 of Ini1. sa and sd, the splice acceptor site in the β-geo gene cassette and the splice donator site in the puromycin selection marker, respectively. Carets represent the site of alternate splicing. Positions of the primers used for PCR analysis are shown. LTR (del), long terminal repeat deleted; Aⁿ, polyadenylation signal; Puro, puromycin. (B) Genotyping of Ini1-targeted mice by PCR. Genomic DNA was harvested from tails and genotyped as described in Materials and Methods. The size and position of wild-type (WT) and targeted bands are indicated. (C) Genotyping of Ini1-targeted embryos. Embryos 6.5 d.p.c. and younger were genotyped by nested PCR. The size and position of wild-type and targeted bands are indicated.

FIG. 2

Ini1 is expressed in ES cells and ubiquitously throughout development. (A) β-galactosidase staining of targeted ES cells showing expression of Ini1. Wild-type (WT) AB2.2 ES cells were used as a control. (B) Whole mount staining of Ini1^in3/+ embryos showing ubiquitous expression of Ini1 at indicated time points. Wild-type embryos at 6.5 and 10.5 d.p.c. are shown as controls.

FIG. 3

Ini1-null mice are early embryonic lethal and fail to hatch in vitro. Blastocysts were harvested from C57BL/6 Ini1^in3/+ females and plated in culture for 96 h, at which time outgrowths were processed for PCR. Blastocysts are shown before and after culturing. TE, trophectoderm; ICM, inner cell mass.

FIG. 4

Ini1-heterozygous mice present with various tumors. Microscopic features of different tumors from Ini1^in3/+ mice. Parafin-embedded tumors were sectioned, stained with hematoxylin and eosin, and examined under a microscope at magnification ×75. Mouse numbers corresponding to those presented in Table 2 are indicated in each panel.

FIG. 5

Loss of heterozygosity at the Ini1 locus results in tumor formation. Tumor samples and control tissue (wild-type [WT] male brain) were processed for Western analysis as detailed in Materials and Methods. The band corresponding to the Ini1 protein is indicated. NS represents a nonspecific band.