Urethane Induced Tumors in Early <i>Cebpa</i> Deletion Study.

<p>(A): The tumor numbers, tumor size and tumor burden were all significantly higher in the lungs of <i>Cebpα^Δ/Δ</i> mice at 20 and 28 wk after urethane injection (10 wk: n = 10, 20 wk: n = 11, 28 wk: n = 17/group, *p<0.01). The bar indicates the average. (B): Appearance of the lung at 28 weeks after urethane injection. <i>Arrow</i>: surface tumors. Urethane injection induced lung tumors in both control and <i>Cebpα^Δ/Δ</i> mice. Small tumors were observed on the surface of the lung in control mice. In contrast, <i>Cebpα^Δ/Δ</i> mice developed multiple large tumors. Representative microphotographs of H&E stained sections showed the histology of the adenoma in control mice and adenocarcinomas with airway invasion in <i>Cebpα^Δ/Δ</i> mice. Immunohistochemistry showed strong C/EBPα expression in tumor of control mice, but no staining in <i>Cebpα^Δ/Δ</i> mice. Tu: Tumor. (C): <i>Cebpa</i> mRNA expression was evaluated by qRT-PCR. At 28 wk after urethane injection, <i>Cebpa</i> mRNA expression in control mice was significantly higher than saline injected mice (n = 4/group, p<0.05). The expression in <i>Cebpα^Δ/Δ</i> mice was not affected by urethane injection. In control mice, <i>Cebpa</i> mRNA expression was induced in tumor tissues and was significantly higher than that in non-tumor lung tissues (n = 4/group, p<0.05). (D): <i>Kaplan-Meier</i> survival curves of control and <i>Cebpα^Δ/Δ</i> mice after urethane injection (n = 20/group). <i>Cebpα^Δ/Δ</i> mice showed significantly shorter survival than did control mice (log rank test, p<0.00001, the average survival after urethane injection: control mice (63.5±2.7 wk), <i>Cebpα^Δ/Δ</i> mice (28.5±0.9 wk).</p

Cell Death and Proliferation in Urethane-Induced Tumors.

<p>(A): Immunohistochemistry for Ki-67 and pH 3 at 20 wk after urethane injection. (B): Mitotic index in the tumors of control and <i>Cebpα^Δ/Δ</i> mice (n = 7/group). <i>Cebpα^Δ/Δ</i> mice showed significantly higher numbers of Ki-67 positive cells in tumors (*p<0.01). The tumors in <i>Cebpα^Δ/Δ</i> mice were more proliferative than those in control mice. (C): Arrows indicate TUNEL stain and cleaved-caspase 3 positive cells in tumors. Apoptotic cells were not obvious in this model.</p

CCAAT/Enhancer-Binding Protein-α Suppresses Lung Tumor Development in Mice through the p38α MAP Kinase Pathway

<div><p>The transcription factor CCAAT/enhancer-binding protein α (C/EBPα) is a basic leucine zipper transcription factor and is expressed in alveolar type II cells, alveolar macrophages and Clara cells in the lung. Although decrease or absence of C/EBPα expression in human non-small cell lung cancer suggests a possible role of C/EBPα as a lung tumor suppressor, there is no direct proof for this hypothesis. In this study, we investigated, for the first time, the role of C/EBPα in lung tumors in vivo using transgenic mice with lung epithelial specific conditional deletion of <i>Cebpa</i> (<i>Cebpα^Δ/Δ</i> mice) and a urethane-induced lung tumor model. C/EBPα expression in the lung was dispensable, and its deletion was not oncogenic under unstressed conditions. However, at 28 wk after urethane injection, the number and size of tumors and the tumor burden were significantly higher in <i>Cebpα^Δ/Δ</i> mice than in littermate control mice. Urethane-injected <i>Cebpα^Δ/Δ</i> mice showed highly proliferative adenomas and adenocarcinomas in the lung, and survival time after urethane-injection was significantly shorter than that in control mice. In control mice, C/EBPα was strongly induced in the tumor tissues at 28 weeks after urethane-injection, but became weakened or absent as tumors progressed after long-term observation for over 1 year. Using intraperitoneal injection of p38 inhibitor (SB203580), we demonstrated that the induction of C/EBPα is strongly regulated by the p38 MAP kinase in murine alveolar epithelial cells. A high correlation was demonstrated between the expression of C/EBPα and p38α MAP kinase in tumor cells, suggesting that C/EBPα silencing in tumor cells is caused by down-regulation of p38α MAP kinase. In conclusion, the role of C/EBPα as a lung tumor suppressor was demonstrated for the first time in the present study, and the extinguished C/EBPα expression through p38α inactivation leads tumor promotion and progression.</p> </div

Natural C/EBPα Silencing and Promoter DNA Methylation in Urethane-Induced Tumors.

<p>(A): H&E staining and C/EBPα immunohistochemistry in urethane induced tumors in control mice evaluated 60 wk after urethane injection. <i>Left column</i>: C/EBPα-entirely positive tumor, <i>Middle column</i>: C/EBPα-partially positive tumor and <i>Right column</i>: C/EBPα-entirely negative tumor. The samples in each row were taken from serial sections. At least one partially positive tumor was observed in all mice (26/26). Entirely negative tumors were observed in 6 out of 26 mice. (B): Double immunofluorescence of C/EBPα and 5-mC in entirely-positive and entirely-negative tumor in long-observed model. C/EBPα-positive cells were weak or negative for 5-mC (upper row), while C/EBPα-negative cells stained positive for 5-mC (lower row), suggesting that C/EBPα-negative cells revealed DNA hypermethylation. (C): Bisulfite sequencing analysis for 3 entirely C/EBPα-negative tumors (M1, M2 and M3) and 2 C/EBPα entirely positive tumors (C1 and C2, 28 wk after urethane injection). <i>Arrow</i>, transcription start site; the diagrams of the core-promoter sites (−337 to +49) are drawn to scale. Promoter DNA methylation was weak in entirely C/EBPα-negative tumors. Each row represents an individual clone. <i>White circles, unmethylated CG dinucleotides; black circles, methylated CG dinucleotides</i>.</p

Regulation of C/EBPα through p38 MAP kinase.

<p>(A): Double immunofluorescence of C/EBPα and proSP-C. Alveolar type II cells and alveolar macrophages in vehicle-injected mice were positive for C/EBPα expression, while p38 MAP kinase inhibitor, SB203580, suppressed C/EBPα expression only in alveolar type II cells. Arrow head: alveolar macrophage. Arrow: alveolar type II cells. (B): qRT-PCR analysis of <i>Cebpa</i> mRNA expression. SB203580 significantly suppressed <i>Cebpa</i> mRNA expression in whole lung samples (n = 3, *p<0.05). C/EBPα expression is regulated by p38 MAP kinase <i>in vivo</i>. ((C): qRT-PCR for tumor tissue gene expression. The data were normalized to the expression in whole lung tissues from 6 wk old mice (n = 4, solid circles). Open circles represent tumor samples. <i>Cebpa</i> and <i>MAPK14</i> mRNA expressions in tumors were significantly correlated (n = 13, r² = 0.832, p<0.0001, <i>Spearman's</i> correlation). (D): Double immunofluorescence of C/EBPα and p38 in C/EBPα partially positive tumors. In white rectangle in upper row, a high magnification clearly shows double-positive or –negative for C/EBPα and p38 expressions in tumor cells.</p

<i>Xist</i> Exon 7 Contributes to the Stable Localization of Xist RNA on the Inactive X-Chromosome

<div><p>To equalize X-linked gene dosage between the sexes in mammalian females, Xist RNA inactivates one of the two X-chromosomes. Here, we report the crucial function of <i>Xist</i> exon 7 in X-inactivation. <i>Xist</i> exon 7 is the second-largest exon with a well-conserved repeat E in eutherian mammals, but its role is often overlooked in X-inactivation. Although female ES cells with a targeted truncation of the <i>Xist</i> exon 7 showed no significant differences in their <i>Xist</i> expression levels and RNA stability from control cells expressing wild-type <i>Xist</i>, compromised localization of Xist RNA and incomplete silencing of X-linked genes on the inactive X-chromosome (Xi) were observed in the exon 7-truncated mutant cells. Furthermore, the interaction between the mutant Xist RNA and hnRNP U required for localization of Xist RNA to the Xi was impaired in the <i>Xist</i> exon 7 truncation mutant cells. Our results suggest that exon 7 of Xist RNA plays an important role for stable Xist RNA localization and silencing of the X-linked genes on the Xi, possibly acting through an interaction with hnRNP U.</p></div

The exon 7 truncation in Xist RNA impairs the interaction between hnRNP U and Xist RNA.

<p>(A) Overview showing the generation of the FLAG-HA knock-in hnRNP U allele. The protein coding or 5´ UTR regions are shown as a gray or white box, respectively. The FLAG-HA tag is shown as a red box. The first ATG sequence of the translation start is labeled in blue. The protein coding or 5´ UTR regions are capitalized or lowercased, respectively. The sgRNA sequence is labeled in green. The protospacer-adjacent motif (PAM) sequence is labeled in red. (B) PCR genotyping analysis of the FLAG-HA tag knock-in hnRNP U allele. Genomic PCR using primers hnRNPU-utrF and hnRNPU-codeR amplified 212 or 284 bp PCR products from the wild-type or the FLAG-HA knock-in alleles, respectively. All FLAG-HA targeted knock-in ES cells shown in this work are homozygous knock-in clones. (C) Quantitative RT-PCR analysis of FLAGHA-hnRNP U using hnRNPU-F and hnRNPU-R in the parental and targeted ES cell lines at day 12 upon differentiation. The mean ± SEM from three independent experiments is shown. (D) A representative western blotting analysis using anti-FLAG, hnRNP U and tubulin antibodies with the parental and FLAG-HA hnRNP U knock-in ES clones at day 12 upon differentiation. (E) Quantification analysis of the hnRNP U western blotting, including Fig 5D. Each was normalized to the parental TST6 cells (set to 1). The mean ± SD values from three independent experiments are shown. (F) UV-crosslinking RIP analysis using Tsix^TST6 and Xist^delEx7Tsix^TST6 mutant ES cell lines expressing FLAG-HA-tagged hnRNP U upon differentiation at day 12. The mean ± SD bar from three independent experiments is shown with an unpaired t test <i>P</i> values (*p<0.05, **p<0.01).</p

The creation of the <i>Xist</i>/<i>Tsix</i> double truncation Xist^delE7Tsix^TST6 and <i>Tsix</i> truncation Tsix^TST6 mutant female ES cells.

<p>(A) A map of the <i>Xist</i>/<i>Tsix</i> locus. The positions of the primer pairs used for RT-PCR are indicated with asterisks. SA, splice acceptor; IRES, internal ribosome entry site; Hyg, hygromycin resistance gene; bpA, beta-actin polyadenylation signal; tpA, tandem polyadenylation signal. (B) 129 and Cast allele-specific RT-qPCR analysis for Tsix RNA at positions T1 and T2, as shown Fig 1A, in Tsix^TST6 and two Xist^delE7Tsix^TST6 undifferentiated ES cells (#1 and #2). Each value was normalized to that of the wild-type cells (WT) (set to 1), and Gapdh was used as an internal control. Values are given as the mean ± standard deviation (SD) of three independent experiments. (C and D) 129 allele-specific RT-qPCR analysis of the <i>Xist</i> expression at exons 7 and exons 1–3, respectively, was conducted. The expression values were normalized to those of WT at day 0 (set to 1) and Gapdh. The mean ± SD from three independent experiments is shown. (E) Representative allele-specific RT-PCRs for <i>Xist</i>. (F) Quantitative analysis of allele-specific RT-PCRs from three independent experiments including Fig 1E (mean ± SD). (G) Half-life assay for Xist RNA in undifferentiated ES cells and differentiated EBs on day8 upon differentiation. The mean ± SD values from two independent experiments are shown.</p

hnRNP U interacts directly with the first and last exons of mouse and human Xist RNA.

<p>(A) UV-crosslinking RIP analysis for Xist RNA and hnRNP U in mouse Neuro2A cells. A relative amount of each immunoprecipitated Xist RNA to input was quantified by RT-qPCR. The positions of the primer pairs are shown as arrowheads. The <i>Xist</i>-specific repeats (A-F) defined by Brockdorff et al. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005430#pgen.1005430.ref051" target="_blank">51</a>] are shown. (B) A relative amount of each immunoprecipitated Xist RNA to input was quantified by RT-qPCR. The positions of primer pairs are shown as arrowheads. The mean ± SD from four independent experiments is shown.</p

Truncation of exon 7 of Xist RNA affects the X-linked gene silencing on the Xi during EB differentiation.

<p>(A) 129 mutant allele-specific RT-qPCR analysis of X-linked <i>Mecp2</i> and <i>Pgk1</i> genes upon differentiation; each was normalized to undifferentiated cells (set to 1) and Gapdh. The mean ± SD values from three independent experiments are shown with the unpaired t test <i>P</i> values (*p<0.05, **p<0.01). (B) Representative allele-specific RT-PCR analysis for <i>Mecp2</i> and <i>Pgk1</i>. (C) Quantification of relative expression levels of <i>Mecp2</i> and <i>Pgk1</i> from the 129 allele. The mean ± SD values from the three independent experiments included in Fig 3B are shown. <i>P</i>-values were derived from an unpaired t-test (*p<0.05, **p<0.01).</p