Liver-specific γ-glutamyl carboxylase-deficient mice display bleeding diathesis and short life span

Liver-Specific γ-Glutamyl Carboxylase-Deficient Mice Display Bleeding Diathesis and Short Life Span. Azuma K, Tsukui T, Ikeda K, Shiba S, Nakagawa K, et al. PLOS ONE. 2014. 9(2) doi:10.1371/journal.pone.008864

Transcriptional Coactivator PC4 Stimulates Promoter Escape and Facilitates Transcriptional Synergy by GAL4-VP16

Positive cofactor 4 (PC4) is a coactivator that strongly augments transcription by various activators, presumably by facilitating the assembly of the preinitiation complex (PIC). However, our previous observation of stimulation of promoter escape in GAL4-VP16-dependent transcription in the presence of PC4 suggested a possible role for PC4 in this step. Here, we performed quantitative analyses of the stimulatory effects of PC4 on initiation, promoter escape, and elongation in GAL4-VP16-dependent transcription and found that PC4 possesses the ability to stimulate promoter escape in response to GAL4-VP16 in addition to its previously demonstrated effect on PIC assembly. This stimulatory effect of PC4 on promoter escape required TFIIA and the TATA box binding protein-associated factor subunits of TFIID. Furthermore, PC4 displayed physical interactions with both TFIIH and GAL4-VP16 through its coactivator domain, and these interactions were regulated distinctly by PC4 phosphorylation. Finally, GAL4-VP16 and PC4 stimulated both initiation and promoter escape to similar extents on the promoters with three and five GAL4 sites; however, they stimulated promoter escape preferentially on the promoter with a single GAL4 site. These results provide insight into the mechanism by which PC4 permits multiply bound GAL4-VP16 to attain synergy to achieve robust transcriptional activation

Bleeding diathesis in <i>Ggcx^{Δliver/Δliver}</i> mice.

<p>A, Decreased activity of coagulation factor in <i>Ggcx^{Δliver/Δliver}</i> mice. Activities of factors II and IX were significantly decreased in <i>Ggcx^{Δliver/Δliver}</i> mice compared with wild type (<i>Ggcx^+/+</i>) mice. Bars represent the mean value ± SEM (n = 8). Differences between the mean values were analyzed using the unpaired Student's <i>t</i>-test. ***<i>P</i><0.001. B, Prolonged bleeding time in <i>Ggcx^{Δliver/Δliver}</i> mice. Tail bleeding time was measured by a filter paper method. <i>Ggcx^{Δliver/Δliver}</i> mice continued bleeding for more than 30 min. Gray triangle: 0 min; Black triangle: 10 min. A representative figure is shown. C. Platelet counts of <i>Ggcx^{Δliver/Δliver}</i> mice. Hematological examination of male <i>Ggcx^{Δliver/Δliver}</i> mice (n = 6), female <i>Ggcx^{Δliver/Δliver}</i> mice (n = 12), male wild type mice (n = 10), and female wild type mice (n = 10) was performed. The platelet count was not significantly different between wild type mice and <i>Ggcx^{Δliver/Δliver}</i> mice. Bars represent the mean value ± SEM. NS: not significant.</p

Shorter life span of <i>Ggcx^{Δliver/Δliver}</i> mice.

<p>Cumulative life spans of male <i>Ggcx^{Δliver/Δliver}</i> mice (n = 10), female <i>Ggcx^{Δliver/Δliver}</i> mice (n = 11), male heterozygous littermates (<i>Ggcx^+/Δliver</i> mice) (n = 12), and female heterozygous littermates (<i>Ggcx^+/Δliver</i> mice) (n = 12) were calculated by the Kaplan-Meier method and compared using the log-rank test. P-values were adjusted by the Bonferroni method. Life span of male <i>Ggcx^{Δliver/Δliver}</i> mice was significantly shorter (<i>P</i><0.001) compared with that of heterozygous mice. The life spans of female <i>Ggcx^{Δliver/Δliver}</i> mice were significantly longer than those of male <i>Ggcx^{Δliver/Δliver}</i> mice (<i>P</i> = 0.044).</p

Liver-specific ablation of <i>Ggcx</i> gene.

<p>A, The albumin promoter is active only in hepatocytes. Alb-Cre or wild type (WT) mice were mated with ROSA26-LacZ mice. Livers were obtained postnatally from mice and stained with X-gal. β-galactosidase activity was detected in the hepatocytes of the mouse born from mating of Alb-Cre and ROSA26-LacZ mice (right panel). Liver of the mouse born from wild type was shown as a negative control (left panel). Representative figures are shown for each group. B, Genotyping using genomic tail DNA. Expected bands from Cre recombinase, <i>loxP</i> sequence (floxed allele), and wild type allele are shown. In mouse #2, #4 and #5, both alleles were replaced with <i>loxP</i> containing recombinant alleles, with at least one copy of Cre recombinase. A representative result is shown. C, Liver-specific ablation of <i>Ggcx</i> gene was confirmed with PCR analysis. DNA samples derived from liver, spleen, kidney and heart of 6-month week old <i>Ggcx^{Δliver/Δliver}</i> mice (4 male mice and 4 female mice) and control <i>Ggcx^+/+</i> mice (4 male mice and 4 female mice) were used as templates. D, Activity of Ggcx in the liver-specific Ggcx-deficient mice. Microsome was prepared from the livers of 6-week old <i>Ggcx^{Δliver/Δliver}</i> mice and control <i>Ggcx^+/+</i> mice of both sexes. The activity of Ggcx was measured by ¹⁴CO₂ incorporated into exogenous substrate in the presence of reduced vitamin K (222 µM). Bars represent the mean value ± SEM (n = 4). Differences between the mean values were analyzed using the unpaired Student's <i>t</i>-test. **<i>P</i><0.01.</p

Cre-recombinase-mediated tissue-specific excision of <i>Ggcx</i>.

<p>A, Strategy for homologous recombination. Targeting vector was designed to flank exon 6 of the <i>Ggcx</i> gene, and frame shift was generated by excision with Cre recombinase. Two <i>loxP</i> sequences (triangles) were inserted into introns 5 and 6. Neomycin cassette and EcoRI site were inserted into intron 6. B, Southern blot analysis of tail DNA. Homologous recombinant allele generated a 7.4-kb fragment by EcoRI digestion. A representative figure is shown.</p