Removing the vertebrate-specific TBP N terminus disrupts placental beta2m-dependent interactions with the maternal immune system

Abstract

Mammalian TBP consists of a 180 amino acid core that is common to all eukaryotes, fused to a vertebrate-specific N-terminal domain. We generated mice having a modified tbp allele, tbp(DeltaN), that produces a version of TBP lacking 111 of the 135 vertebrate-specific amino acids. Most tbp(DeltaN/DeltaN) fetuses (>90%) died in midgestation from an apparent defect in the placenta. tbp(DeltaN/DeltaN) fetuses could be rescued by supplying them with a wild-type tetraploid placenta. Mutants also could be rescued by rearing them in immunocompromised mothers. In immune-competent mothers, survival of tbp(DeltaN/DeltaN) fetuses increased when fetal/placental beta2m expression was genetically disrupted. These results suggest that the TBP N terminus functions in transcriptional regulation of a placental beta2m-dependent process that favors maternal immunotolerance of pregnancy.

Figure 1

Targeted Mutagenesis of N-Terminal Protein-Coding Sequences of the Mouse tbp Gene (A) Targeting strategy and targeting vector design. The 5′ end of the wild-type mouse tbp gene (tbp+) is diagramed (fine horizontal line on top) indicating 5′ exons (thickened regions of line, labeled 1C, 1D, and 1E for alternate promoter/first exons [Ohbayashi et al., 1996; Schmidt et al., 1997], 2, 3, and 4), selected restriction sites (b, Bam HI; e, Eco RI; s, Sac I; x, Xho I), diagnostic PCR primers (arrows), and PCR product sizes (region between arrows, lengths indicated). Below is indicated the predominant splicing pattern (gray broken lines [Schmidt et al., 1997]) that yields the predominant TBP mRNA (yellow) and cognate TBP protein product (blue box). Translation initiates in the second exon and terminates in the eighth exon (Sumita et al., 1993). The N and C termini are indicated. Below is shown the targeting vector design, including the replacement of most of exon 3 with two tandem copies of the FLAG epitope tag (green box). Below this is indicated the targeted tbp allele still containing the loxP-flanked MC1p-neo gene, with the sizes of diagnostic PCR fragments from the primers shown above on the tbp⁺ allele indicated. At the bottom is shown the targeted allele after removal of the loxP-flanked MC1p-neo gene by Cre recombinase, with the resultant expressed somatic cell TBP mRNA and TBP protein indicated below. (B) Genotyping animals using the primer set that spans the ΔN mutation. (C) Expression of TBP and TBP-ΔN mRNA in mouse cells and tissues. RNase protection assays were performed on 10 μg of total RNA from the indicated tissues harvested from adult male mice (8- to 12-weeks-old) of the indicated genotypes, supplemented with yeast RNA to 50 μg (see Supplemental Data at http://www.cell.com/cgi/content/full/110/1/43/DC1). Positions of undigested probe, TBP mRNA, and TBP-ΔN mRNA are indicated at right. Abbreviations: P, 1:100 dilution of undigested probe; C, control lane containing probe hybridized to 50 μg yeast RNA; T, testis; K, kidney; S, spleen; L, liver; and B, brain. (D) Wild-type and mutant (ΔN) TBP protein expression in adult mouse spleen nuclei (right).

Figure 2

Histopathology of tbp^ΔN/ΔN Fetuses and Placentas (A) Wild-type (+/+) and homozygous mutant (ΔN/ΔN) 10.5 d.p.f. whole fetuses from the same litter. (B) Histology of 10.5 d.p.f. ΔN/ΔN fetus. (C) Evidence of hemorrhage in placenta, showing a blood sinus with a large clot of maternal blood associated with peripheral polymorphonuclear leukocytes, as well as mixing of maternal and embryonic blood. (D) Trophoblast giant cells form normally and have normal, large, polyploid nuclei; however, they are engorged with heme-filled vesicles, which suggests that the giant cells in mutant placentas are particularly active in hemophagocytosis. Abbreviations: ba, brachial arch; cl, clot; da, dorsal aorta; eRBC, embryonic red blood cells; fb, forebrain; g, gut; gcn, giant cell nucleus; h, heart; hv, hemophagic vesicles; li, liver; mRBC, maternal red blood cell; nt, neural tube; s, somite; and uc, umbilical cord.

Figure 3

Models of the Function of the N Terminus of TBP (A) Evolution’s embellishments on the basal transcription machinery. Model depicts the basal transcription machinery interacting with transcriptional regulators on promoters in archaea and in mammals. The diverse embellishments on the mammalian transcription machinery are posited to provide novel interaction surfaces that coevolved with the novel transcriptional regulators for advanced situation-specific gene regulation. (B) Three models of TBP-ΔN-mediated placental gene expression in the maternal/placental interaction. In models 1 and 2, signaling through the TBP N terminus represses expression of β2m-dependent antigen presentation, either by directly repressing expression of β2m (yellow, model 1) or expression of a MHC-I or MHC-I-like heavy chain (in pink, model 2). The β2m-dependent antigen presentation is then detected by an as yet unidentified maternal receptor (light green). In these two models, the regulator (labeled “Reg,” in purple) would be a repressor. The molecular interaction disrupted by the TBP-ΔN mutation is indicated by a large red “X.” Conversely, in model 3, the regulator may be an activator, and signaling through the TBP N terminus may induce expression of another target gene (labeled “TG,” in blue), which attenuates, modifies, or blocks placental β2m-dependent antigen presentation. This could occur intracellularly, as depicted, or extracellularly, by masking the β2m-dependent antigen from the maternal receptor. Points in the process that are affected by the rescuing mutations are indicated as “β2m^-/-,” “scid/scid,” and “rag1^-/-.”