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. 2006 Oct;26(19):7201-10.
doi: 10.1128/MCB.00459-06.

Disruption of Ledgf/Psip1 results in perinatal mortality and homeotic skeletal transformations

Heidi G Sutherland  1 , Kathryn Newton, David G Brownstein, Megan C Holmes, Clémence Kress, Colin A Semple, Wendy A Bickmore
Affiliations

Disruption of Ledgf/Psip1 results in perinatal mortality and homeotic skeletal transformations

Heidi G Sutherland et al. Mol Cell Biol. 2006 Oct.

Abstract

PC4- and SF2-interacting protein 1 (Psip1)-also known as lens epithelium-derived growth factor (Ledgf)-is a chromatin-associated protein that has been implicated in transcriptional regulation, mRNA splicing, and cell survival in vitro, but its biological function in vivo is unknown. We identified an embryonic stem cell clone with disrupted Psip1 in a gene trap screen. The resulting Psip1-betageo fusion protein retains chromatin-binding activity and the PWWP and AT hook domains of the wild-type protein but is missing the highly conserved C terminus. The majority of mice homozygous for the disrupted Psip1 gene died perinatally, but some survived to adulthood and displayed a range of phenotypic abnormalities, including low fertility, an absence of epididymal fat pads, and a tendency to develop blepharitis. However, contrary to expectations, the lens epithelium was normal. The mutant mice also exhibited motor and/or behavioral defects such as hind limb clenching, reduced grip strength, and reduced locomotor activity. Finally, both Psip1(-/-) neonates and surviving adults had craniofacial and skeletal abnormalities. They had brachycephaly, small rib cages, and homeotic skeletal transformations with incomplete penetrance. The latter phenotypes suggest a role for Psip1 in the control of Hox expression and may also explain why PSIP1 (LEDGF) is found as a fusion partner with NUP98 in myeloid leukemias.

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Figures

FIG. 1.
FIG. 1.
Gene trap disruption of the Psip1 locus. (A) Predicted intron-exon structure of mouse Psip1 and antisense Snapc3 with integration sites of the F9/3G5 and ES149 gene trap lines indicated. (B) Diagram of the Psip1 p52 and p75 protein isoforms and the predicted gene trap fusion proteins in F9/3G5 and ES149 gene trap lines. Known protein motifs predicted by SMART/PFAM and the experimentally determined human immunodeficiency virus IBD are depicted.
FIG. 2.
FIG. 2.
Subcellular localization of Psip1-βgeo fusion proteins. Shown is immunostaining of F9/3G5 and ES149 cells with an anti-β-gal antibody showing localization of the Psip1-β-geo fusion proteins in the nuclei and on chromosomes in mitotic cells (arrows). In some ES149 nuclei, the fusion protein is concentrated at the DAPI-bright heterochromatic foci (arrowheads).
FIG. 3.
FIG. 3.
Genotyping of ES149 mice for gene trap disruption of Psip1. (A) PCR analysis of WT (+/+) and heterozygous (+/−) and homozygous (−/−) Psip1 mutant mice with primers for lacZ (L) (432-bp band) and endogenous Psip1 (P) (860-bp band). (B) Southern blot analysis of HindIII-digested genomic DNA from +/+, +/−, and −/− mice probed with a PCR product for exon 9 of Psip1. This results in a 4-kb band for the endogenous Psip1 locus and an 8-kb band for the gene-trapped locus.
FIG. 4.
FIG. 4.
Psip1 expression in WT and mutant mice. (A) X-Gal staining of WT and Psip1+/− embryos at 11.5 dpc. (B) RT-PCR analysis of Psip1 and Snapc3 mRNAs in Psip1+/+, Psip1+/−, and Psip1−/− mice. Gapdh was used as a control. (C) Western blot analysis of p75/Psip1 in protein extracts form +/+, +/−, and −/− embryos (top). The Coomassie-stained polyacrylamide gel at the bottom shows protein loading.
FIG. 5.
FIG. 5.
Phenotypic analysis of surviving adult Psip1−/− mutant mice in the MF1 background. (A) Psip1−/− mutant mice have a reduction of intraabdominal fat, in particular, an absence of epididymal fat pads (fp), compared to their +/+ littermates. (B) H&E staining of seminiferous tubules from heterozygous (+/−) and Psip1−/− mutant mice. Both show normal-appearing basilar spermatogonia, several layers of differentiating spermatocytes, and maturing spermatids at the lumen. Bars, 50 μm. (C) H&E staining of eyes from WT (+/+) and Psip1−/− mutant mice showing a normal lens epithelium (le). (D) Chronic ulcerative blepharitis in Psip1−/− mutant mice. The mouse on the left is 2 months old; the right eye is asymptomatic, and the left eye shows mild symptoms. The mouse on the right is 4 months old; symptoms in the right eye are mild, and those in the left eye are severe. (E) H&E staining of the eyelid of a Psip1−/− mutant mouse showing ulcerated epidermis covered with adherent fibrin plaque. The adjacent intact epidermis is hyperplastic, and chronic mixed inflammatory cells have infiltrated the dermis. Bar, 100 μm. (F) Psip1−/− mutant mice clench their hind limbs, in contrast to the hind limb extension of +/+ mice. (G) Psip1−/− mutant mice have brachycephaly with broad, shortened faces and jaws. (H) Alizarin red-alcian blue staining of skulls shows that the cranial bones and nasal process are broader and shorter in Psip1−/− mutant mice compared to those of +/− (and +/+) mice.
FIG. 6.
FIG. 6.
Alizarin red-alcian blue staining reveals craniofacial and skeletal abnormalities in Psip1−/− mutant mice. (A) Staining of +/+ and Psip1−/− mutant MF1 newborn skeletons. −/− pups have domed skulls (arrowheads) and reduced interparietal, exoccipital, and supraoccipital bones (double arrowheads). (B) Psip1−/− mutant pups lack physiological curvature of the spine and have a small rib cage and a short sternum, compared to +/+ and +/− littermates. (C) Dissected rib cages from C57BL/6 +/+, +/−, and −/− newborn pups. (D) Cervical region, showing misshapen C1 and C2 and an incomplete ectopic rib (arrow) from C7 that is fused with the cartilage of T1. (E) Adult skeletons showing an incomplete ectopic rib (arrowed) from C7 unilaterally fused with T1 and a prominent spinous process, characteristic for T2, incorrectly associated with T3 in a Psip1−/− mutant animal. (F) +/+ and −/− MF1 newborn skeletons showing a reduced number of lumbar vertebrae and transformation of L6 into S1.
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