Coactivator PRIP, the Peroxisome Proliferator-activated Receptor-interacting Protein, Is a Modulator of Placental, Cardiac, Hepatic, and Embryonic Development*

Nuclear receptor coactivator PRIP (peroxisome proliferator-activated receptor (PPARγ)-interacting protein) and PRIP-interacting protein with methyltransferase activity, designated PIMT, appear to serve as linkers between cAMP response element-binding protein-binding protein (CBP)/p300-anchored and PBP (PPARγ-binding protein)-anchored coactivator complexes involved in the transcriptional activity of nuclear receptors. To assess the biological significance of PRIP, we disrupted the PRIP gene in mice by homologous recombination. Mice nullizygous for PRIP died between embryonic day 11.5 and 12.5 (postcoitum) due in most part to defects in the development of placenta, heart, liver, nervous system, and retardation of embryonic growth. Transient transfection assays using fibroblasts isolated from PRIP−/− embryos revealed a significant decrease in the capacity for ligand-dependent transcriptional activation of retinoid X receptor α and to a lesser effect on PPARγ transcriptional activity. These observations indicate that PRIP like PBP, CBP, and p300 is an essential and nonredundant coactivator.

Nuclear receptor coactivator PRIP (peroxisome proliferator-activated receptor (PPAR␥)-interacting protein) and PRIP-interacting protein with methyltransferase activity, designated PIMT, appear to serve as linkers between cAMP response element-binding protein-binding protein (CBP)/p300-anchored and PBP (PPAR␥-binding protein)-anchored coactivator complexes involved in the transcriptional activity of nuclear receptors. To assess the biological significance of PRIP, we disrupted the PRIP gene in mice by homologous recombination. Mice nullizygous for PRIP died between embryonic day 11.5 and 12.5 (postcoitum) due in most part to defects in the development of placenta, heart, liver, nervous system, and retardation of embryonic growth. Transient transfection assays using fibroblasts isolated from PRIP ؊/؊ embryos revealed a significant decrease in the capacity for ligand-dependent transcriptional activation of retinoid X receptor ␣ and to a lesser effect on PPAR␥ transcriptional activity. These observations indicate that PRIP like PBP, CBP, and p300 is an essential and nonredundant coactivator.
Of interest is that the recently identified coactivator designated PRIP/ASC2/RAP250/NRC/TRBP has also been shown to interact with several nuclear receptors and with CBP/p300 and TRAP130 of the TRAP/DRIP/ARC complex (10 -14). Thus, PRIP appears to serve as a bridge between the first complex anchored by CBP/p300 and the downstream TRAP/DRIP/ARC mediator complex anchored by PBP. Furthermore, the recently isolated PRIP-interacting protein with RNA methyltransferase activity, designated PIMT (25), forms a complex with CBP, p300, and PBP (26), further attesting to the possibility that two major multiprotein cofactor complexes anchored by CBP/p300 and PBP, respectively, merge into one megacomplex on DNA template (28). Since PRIP and PRIP-binding protein PIMT appear to link the two cofactor complexes under in vitro conditions, we have found it necessary to explore the biological function of PRIP by generating mice with PRIP null phenotype. We now demonstrate that PRIP is critical for the embryonic development, since disruption of the PRIP gene in the mouse leads to embryonic lethality around E11.5 to E12.5 days, implying that PRIP (like CBP/p300 and PBP) is also critical for embryonic development and survival.

MATERIALS AND METHODS
Construction of Targeting Vector-The genomic DNA fragment containing the full-length PRIP gene was isolated from a mouse 129/Sv P1 bacteriophage library (Genome Systems, St. Louis, MO), using polymerase chain reaction with primers 5Ј-CAATGCAGCCTGTTCCTGT-3Ј and 5Ј-GCTGCTGCATCACCATGAAA-3Ј designed from mouse PRIP cDNA sequence (10). A Cre-LoxP system was employed to delete exon 7 from the PRIP gene. For this purpose, we constructed a triple-LoxP PRIP targeting vector to generate a "floxed" mouse PRIP-targeted locus.
Generation of PRIP Null Mice-The targeting vector was linearized and electroporated into HM1 embryonic stem (ES) cells (28). Transfected ES cells were selected in the medium with G418 (200 g/ml), and the surviving colonies were screened for homologous recombination by PCR with primers P1 5Ј-CCTACAGCTGCAAGCAAATC-3Ј and P2 5Ј-TATACGAAGTTATGCGGCCG-3Ј. ES cells with the appropriate PRIP floxed targeted locus were further confirmed by Southern blot analysis (Fig. 1B), and the euploid selected ES cells were used for injection into 3.5-day-old blastocysts derived from C57/B6 mouse by the Northwestern University Targeted Mutagenesis Facility to generate chimeric mice. Chimeric male mice were bred with wild type C57/B6 female to produce heterozygous mice, which were then crossed with EIIa-Cre transgenic mice (29) to delete the DNA fragment between LoxP1 and LoxP3. EIIa-cre-mediated recombination occurs early in development (2-8 cells), and mice carrying the allele with deletion were crossed with wild type C57/B6 to achieve germ line transmission. The heterozygous mice with expected deletion were interbred to generate homozygous mutants.
Genotyping of Mice and Embryos-DNA extracted from the tail tips of mice and from the yolk sac of embryos was used for genotyping by PCR. The mice carrying the recombination between LoxP1 and LoxP3 on one allele were identified by PCR with primers P1/P2 and P3 (5Ј-C-GGCCGCATAACTTCGTATA-3Ј)/P4 (5Ј-TTCTTCTTCCGAGGCGGTT-T-3Ј). Presence of P1/P2 product, while lacking P3/P4 amplification, indicated the deletion of PRIP gene on one allele. The homozygosity for the deletion was detected by the absence of exon 7 as ascertained by PCR with primers P5 5Ј-ACGGGCCACCAAATATGATG-3Ј and primer P4 (see above) ( Fig. 1C).
RT-PCR and Western Blots-For RT-PCR, total RNA was extracted from embryos with TRIzol reagent (Invitrogen). Primers 5Ј-CCTA-CAGCTGCAAGCAAATC-3Ј and 5Ј-CGAACATGCTGCATGAGCTGA-3Ј were used to amplify the region between exon 6 and exon 8 from the PRIP homozygous mutant. To detect PRIP protein in embryos, whole cell lysates were prepared from the embryos by homogenization and probed with anti-mouse PRIP antiserum. The signal was detected by ECL detection system.
Histological Analysis and Immunohistochemistry-Age-matched embryos were fixed in paraformaldehyde or 10% of buffered formalin, embedded in paraffin, serially sectioned at 5-M thickness in sagittal or transverse planes, and stained with hematoxylin and eosin. Immunohistochemical staining for the localization of proliferating cell nuclear antigen (PCNA) was performed using a standard avidin-biotin-peroxidase complex protocol as described previously (24). Giemsa stain was done using the standard protocol.
Isolation of Fibroblasts from Embryos and Transfection of Primary Fibroblasts-Mouse embryonic fibroblasts (MEF) were isolated from E11.5 embryos and cultured in Dulbecco's modified Eagle's medium with 10% of fetal bovine serum as described (22). For transfection, 2 ϫ 10 5 of MEFs were plated in six-well plates for overnight culture. The transfections were carried out with LipofectAMINE-2000 Plus reagent according to manufacturer's instruction (Invitrogen). Plasmids pCMV-PPAR␥, PPRE-TK-LUC, pCMV-RXR, RXRE-TK-LUC, pCMV-RAR, and RARE-TK-LUC were as described previously (10,25,26). ␤-Galactosidase expression vector pCMV␤ was used as a cotransfectant, which served as control for transfection efficiency. Cell extracts were prepared 36 h after transfection and were assayed for luciferase and ␤-galactosidase activities.

Disruption of PRIP Gene in Mice-
We constructed a conditional knock-out allele of PRIP by using the Cre/loxP recombination system according to the strategy in Fig. 1A. To generate conventional PRIP knock-out mice (in which the gene is permanently inactivated at the germ cell stage), it was necessary to induce recombination between loxP sites. To achieve heterozygosity, PRIP-targeted mice were bred with homozygous Cre transgenic mouse line, EII-cre (29). The EII-cre mice carry the Cre transgene under the control of the adenovirus EIIa promoter and express Cre recombinase only in early mouse embryos (2-8 cell stage), and it induces the recombination between the two loxP sites with the same orientation (29). We detected the expected, all types of recombinants among the offspring (29). The recombination between loxP1 and loxP3 resulted in the deletion of PRIP exon 7 and a reading frameshift to generate a stop codon right after the fusion between exon 6 and exon 8. The chimeras were crossed to wild type mice to produce heterozygous mice carrying one recombinant PRIP allele, and the homozygous mice were obtained from heterozygous mating. By sequencing the RT-PCR products, exon 7 was not found in mRNA transcribed from the recombinant PRIP allele, and the reading frameshift was introduced by the deletion leading to a premature stop codon (data not shown). As the result, only 488 amino acids at the N terminus containing no LXXLL motif can possibly be translated from the mRNA, but no PRIP protein was detected by Western blot analysis (data not shown).
Embryonic Lethality and Growth Retardation of PRIP Null Mice-Among 26 new-born pups, and 54 mice that were 3 weeks old generated from intercrosses between heterozygous PRIP mutant mice, no homozygous mutants were detected. Genotyping the embryos at different stages of gestation showed that no PRIP null embryos survived beyond E13.5 (13.5 days postcoitum). However, heart beating was observed among the majority of viable PRIP null embryos recovered between E11.5 and E12.5 and few were moribund or dead. These observations indicate lethality occurred in a relatively narrow window of time as no viable PRIP null embryos were seen at E13.5. The viable PRIP Ϫ/Ϫ embryos recovered at E11.5 and E12.5 exhibited clear evidence of growth retardation compared with the wild type and heterozygous littermates. PRIP Ϫ/Ϫ embryos appeared strikingly different at the gross level at E12.5, they were pale and smaller in size than their PRIP ϩ/ϩ and PRIP ϩ/Ϫ littermates (Fig. 2, A and B). Normally, extraembryonic mesoderm of the yolk sac gives rise to blood and endothelial cells, which form blood islands. The extraembryonic membrane covering PRIP null embryos contained fewer vessels in contrast to wild type yolk sac with its well developed blood vessels (Fig.  2B). In addition, superficial vasculature was less obvious in PRIP null mutants (Fig. 2D). While the liver of wild type embryos was easily visualized by its rich vasculature through the skin, only a pale primitive liver bud was discerned in PRIP Ϫ/Ϫ embryos (Fig. 2D). Failure of palatal shelf to fuse, abnormal finder separations, and developmental abnormalities in brain were detected in PRIP Ϫ/Ϫ mutants (not illustrated).
Lack of Organized Spongiotrophoblast Layer in PRIP Mutant Placenta-In wild type placenta at E12.5 contains three distinct trophoblast cell structures: the innermost labyrinthine layer, the intermediate spongiotrophoblast layer, and the outermost trophoblast giant cell layer. The labyrinthine zone formed by the fusion of chorion with allantois is composed of extensively branched fetal blood vessels and maternal blood sinuses among strands of diploid trophoblast cells that separate the maternal blood sinuses from fetal blood vessels. In PRIP null placenta, no compact layer of spongiotrophoblast cells was observed between labyrinth zone and trophoblast giant cell layer. Instead, islands of spongiotrophoblast-like cells dissociated from trophoblast giant cell layer and migrating into the labyrinth zone were common occurrence in PRIP Ϫ/Ϫ placenta (Fig. 3B). In wild type placenta blood sinuses are filled with maternal blood cells throughout the labyrinthine layer (Fig. 3, A, C, and F), whereas most of the tortuous vessels in the PRIP Ϫ/Ϫ placenta were enlarged, ruptured, and generally empty (Fig. 3, B, D, and F). While the chorioallantoic fusion appeared to occur in PRIP mutant placenta, chorionic trophoblast cells clustered in labyrinth had multiple nuclei, and these clusters showed insufficient blood vessel branching. These changes are reminiscent of some of the placental defects observed in PBP null mutants (22,24). However, changes in PRIP Ϫ/Ϫ placenta appeared less profound when compared with PBP Ϫ/Ϫ placenta (24). Nucleated fetal erythroblasts in PRIP Ϫ/Ϫ placenta had irregular shaped nuclei with very little cytoplasm (Fig. 3F). Trophoblast cell proliferation in PRIP null placenta as assessed by PCNA immunostaining was significantly lower than that observed in PRIP ϩ/ϩ placenta (data not shown).
PRIP Ϫ/Ϫ Embryos Manifest Cardiac Defects-Inefficient pumping by the heart leading to circulation failure is one of the major causes of embryonic lethality during middle gestation. The development of heart requires coordinated differentiation of several embryonic lineages, including the myocytes of myocardium, the endothelial cells of the endocardium, and the cells of the neural crest that form the outflow tract. At E12.5, the heart of PRIP Ϫ/Ϫ embryos exhibited defects involving all three lineages (Fig. 4, A and B). In PRIP Ϫ/Ϫ heart, the epicardium, consisting of a single layer of mesothelial cells lining against the compact layer of myocardium, appeared to separate from underlying myocardium and this space is filled with blood cells (Fig. 4B). Pericardial space surrounding the heart in mutant was much smaller than their wild type littermate. Unlike the ventricles in wild type heart, which consisted of multicell thick compact layer with well developed trabeculae, the ventricles in PRIP Ϫ/Ϫ heart only contained one or two cell layers of myocardium (Fig. 4). Cell-cell adhesion among cardiocytes appeared to Wild type (A) embryo at E12.5 reveals yolk sac with well developed blood islands, while the PRIP Ϫ/Ϫ littermate (B) is smaller in size with fewer blood islands in its yolk sac (ys). C, external appearance of eye, bloodenriched liver, and distal part of hind limb with separated fingers (white arrow) in wild type embryo. D, PRIP Ϫ/Ϫ embryo shows dilated leaking small vessels (white arrowhead) and few large blood vessels. Pigmented tissue is thinner in the dorsal portion of the eye (ey). The liver (li) mass is small and pale in the PRIP Ϫ/Ϫ embryo. Finger separation does not occur in the distal part of hind limb (white arrow). Each pair of photographs was taken at the same magnification. be defective, which possibly contributes to leakage of blood cells into epicardial spaces. Cell proliferation in PRIP Ϫ/Ϫ myocardium was markedly reduced as compared with PRIP ϩ/ϩ myocardium (Fig. 4, C and D). The failure of ventricular myocardium of PRIP Ϫ/Ϫ embryos to stratify into the multilayer compact structure required to sustain adequate cardiac function appears similar to that noted in PBP null and PPAR␥ null mutants (22,24,30).
Defective Hepatopoiesis and Hepatic Hematopoiesis in PRIP Ϫ/Ϫ Mutants-Liver of PRIP null mutants appeared considerably smaller in size when compared with their littermates (Fig. 5, A and B). The function of the liver at E12.5 days is to become the major site of hematopoiesis so as to gradually replace yolk sac based hematopoiesis. Histological examination of PRIP Ϫ/Ϫ liver revealed reduction in hepatocyte population and an increase in hepatocyte apoptosis (Fig. 5D). A marked decrease in the number of erythroid progenitors was also evident, and these cells had large nuclei with scant minimally hemoglobinized cytoplasm. Liver exhibited large numbers of megakaryocytes.
Differential Reduction of Transactivation by Nuclear Receptors in PRIP Ϫ/Ϫ Primary Fibroblasts-To assess the impact of loss of PRIP on transcriptional activities of nuclear receptor, we isolated MEFs from PRIP Ϫ/Ϫ and PRIP ϩ/ϩ embryos. They were used for assaying the transcriptional activities of PPAR␥, RXR, and RAR. In wild type MEFs transfected with a RXR expressing vector and RXR-responsive element-linked reporter, the addition of RXR-ligand 9-cis-retinoic acid induced marked increase (ϳ76-fold) in the transcription (Fig. 6A). In PRIP Ϫ/Ϫ MEFs, the induction of ligand-mediated RXR transcription was markedly reduced (ϳ3-fold). Transcription assays with PPAR␥ (Fig. 6B) and RAR␣ (data not shown) showed that the influence of PRIP was only modest. These results demonstrated that nuclear receptors require PRIP to achieve their full transcriptional potential, although the contribution of PRIP to the activities of the three nuclear receptors examined here differed somewhat in that PRIP seemed to influence RXR maximally. DISCUSSION The nuclear hormone receptors comprise a superfamily of transcription factors that regulates coordinated expression of gene networks involved in developmental, physiological, and metabolic processes (1). Notable among this nuclear receptor superfamily is PPAR subfamily comprising of three isoforms, PPAR␣, PPAR␥, and PPAR␤/␦, since these receptors have emerged in recent years as a critical player in regulating energy metabolism. In an effort to understand the factors controlling cell and gene specific transcriptional events initiated by nuclear receptors, the ligand-binding domain of nuclear receptors was used in the yeast two-hybrid screen to identify receptor interacting proteins (2)(3)(4)(5)(6)(7)31). During the past 7 years, more than 25 nuclear receptor coactivators have been cloned raising the issue of redundancy, since these coactivators generally appear promiscuous in their coactivation potential.
To fully appreciate the in vivo biological functions of these coactivators, molecular genetic approaches are being increasingly employed. Previous studies have demonstrated that mice lacking SRC-1 or p/CIP/SRC-3 are viable and manifest either partial or full redundancy for certain nuclear receptor actions (32)(33)(34)(35)(36). In contrast, deletion of more general coactivators such as CBP/p300 and PBP in mice leads to embryonic lethality implying that these are essential coactivators (20 -24). Thus, there appear to be at least two broad classes of coactivators: essential and redundant. Our observations reported here now add coactivator PRIP to the class of essential coactivators because of the embryonic growth retardation, defects in placental, cardiac and hepatic development, and embryonic lethality. Embryonic lethality was noted between E11.5 and E12.5 days with no viable embryos at E13.5. Defects were noted in heart (reduced amount of myocardium and noncompaction), liver (small liver with reduction in hepatocyte population, and hepatocyte apoptosis), and defects in erythropoiesis (reduced hemoglobinization) and placenta (maturation block of trophoblast with vascularization defect). The placental defects, although not as pronounced as those encountered in PBP Ϫ/Ϫ placenta (22, 24), nevertheless compromise fetal-maternal exchange of nutrients, growth factors, and oxygen, which may contribute to embryonic growth retardation. The death of embryos at about mid-gestation has been reported in a number of null mutants induced by gene targeting, including PPAR␥ (30), RXR␣ (37), PBP (22)(23)(24), and N-myc (38).
It should be noted that we isolated both PRIP and PBP using PPAR␥ as bait in the yeast two-hybrid screen and identified them as nuclear receptor coactivators (9,10). PBP has since emerged as a central piece in large TRAP-DRIP-ARC-PRIC multiprotein cofactor complex (17)(18)(19)27). Recent studies have established that PBP is indispensable for embryonic development because PBP null mutation leads to embryonic death around E11.5 of mouse development (22)(23)(24). PBP null mutation also causes defects in the development of placental vasculature similar to those encountered in PPAR␥ mutants, supporting the requirement of PBP for PPAR␥ function in vivo (22,24,30). PBP null mutants also exhibited cardiac failure because of noncompaction of the ventricular myocardium and resultant ventricular dilatation (22)(23)(24). There was also paucity of retinal pigment, excessive systemic angiogenesis, a deficiency in the number of megakaryocytes, and an arrest in erythrocyte differentiation (24). We showed that PBP interacts with GATA family of transcription factors and thus influences the development of vital organ systems (24). Consistent with this view is that the gene encoding PBP is amplified and overexpressed in breast cancer (39). Like PBP, PRIP is also highly amplified/overexpressed in human breast and colon tumors (11), suggesting that both PBP and PRIP by virtue of their coactivating function may augment cell proliferation and neoplastic progression. Finally, the PRIP null MEFs exhibited marked repression of RXR-mediated transcriptional activity as compared with PPAR and RAR. These observations strongly suggest that PRIP has better preference for RXR than other nuclear receptors, and some of the abnormalities noted in PRIP null mutants may be due to inhibition of RXR function. Further studies are needed to examine the role of PRIP in various tissues by generating PRIP conditional null mice.