The Tgif2 gene contains a retained intron within the coding sequence

BACKGROUND: TGIF and TGIF2 are homeodomain proteins, which act as TGFβ specific Smad transcriptional corepressors. TGIF recruits general repressors including mSin3 and CtBP. The related TGIF2 protein functions in a similar manner, but does not bind CtBP. In addition to repressing TGFβ activated gene expression, TGIF and TGIF2 repress gene expression by binding directly to DNA. TGIF and TGIF2 share two major blocks of similarity, encompassing the homeodomain, and a conserved carboxyl terminal repression domain. Here we characterize two splice variants of the Tgif2 gene from mouse and demonstrate that the Tgif2 gene contains a retained intron. RESULTS: By PCR from mouse cDNA, we identified two alternate splice forms of the Tgif2 gene. One splice variant encodes the full length 237 amino acid Tgif2, whereas the shorter form results in the removal of 39 codons from the centre of the coding region. The generation of this alternate splice form occurs with the mouse RNA, but not the human, and both splice forms are present in all mouse tissues analyzed. Human and mouse Tgif2 coding sequences contain a retained intron, which in mouse Tgif2 is removed by splicing from around 25–50% of RNAs, as assessed by RT-PCR. This splicing event is dependent on sequences within the mouse Tgif2 coding sequence. Both splice forms of mouse Tgif2 encode proteins which are active transcriptional repressors, and can repress both TGFβ dependent and independent transcription. In addition, we show that human and mouse Tgif2 interact with the transcriptional corepressor mSin3. CONCLUSION: These data demonstrate that the Tgif2 gene contains a retained intron, within the second coding exon. This retained intron is not removed from the human mRNA at a detectable level, but is spliced out in a significant proportion of mouse RNAs. This alternate splicing is dependent entirely on sequences within the mouse Tgif2 coding sequence, suggesting the presence of an exonic splicing enhancer. Both splice forms of mouse Tgif2 produce proteins which are functional transcriptional repressors

A Role for Non-Covalent SUMO Interaction Motifs in Pc2/CBX4 E3 Activity

Background: Modification of proteins by the small ubiquitin like modifier (SUMO) is an essential process in mammalian cells. SUMO is covalently attached to lysines in target proteins via an enzymatic cascade which consists of E1 and E2, SUMO activating and conjugating enzymes. There is also a variable requirement for non-enzymatic E3 adapter like proteins, which can increase the efficiency and specificity of the sumoylation process. In addition to covalent attachment of SUMO to target proteins, specific non-covalent SUMO interaction motifs (SIMs) that are generally short hydrophobic peptide motifs have been identified. Methodology/Principal Findings: Intriguingly, consensus SIMs are present in most SUMO E3s, including the polycomb protein, Pc2/Cbx4. However, a role for SIMs in SUMO E3 activity remains to be shown. We show that Pc2 contains two functional SIMs, both of which contribute to full E3 activity in mammalian cells, and are also required for sumoylation of Pc2 itself. Pc2 forms distinct sub-nuclear foci, termed polycomb bodies, and can recruit partner proteins, such as the corepressor CtBP. We demonstrate that mutation of the SIMs in Pc2 prevents Pc2-dependent CtBP sumoylation, and decreases enrichment of SUMO1 and SUMO2 at polycomb foci. Furthermore, mutational analysis of both SUMO1 and SUMO2 reveals that the SIM-interacting residues of both SUMO isoforms are required for Pc2-mediated sumoylation and localization to polycomb foci

Premature Senescence and Increased TGFβ Signaling in the Absence of Tgif1

Transforming growth factor β (TGFβ) signaling regulates cell cycle progression in several cell types, primarily by inducing a G1 cell cycle arrest. Tgif1 is a transcriptional corepressor that limits TGFβ responsive gene expression. Here we demonstrate that primary mouse embryo fibroblasts (MEFs) lacking Tgif1 proliferate slowly, accumulate increased levels of DNA damage, and senesce prematurely. We also provide evidence that the effects of loss of Tgif1 on proliferation and senescence are not limited to primary cells. The increased DNA damage in Tgif1 null MEFs can be partially reversed by culturing cells at physiological oxygen levels, and growth in normoxic conditions also partially rescues the proliferation defect, suggesting that in the absence of Tgif1 primary MEFs are less able to cope with elevated levels of oxidative stress. Additionally, we show that Tgif1 null MEFs are more sensitive to TGFβ-mediated growth inhibition, and that treatment with a TGFβ receptor kinase inhibitor increases proliferation of Tgif1 null MEFs. Conversely, persistent treatment of wild type cells with low levels of TGFβ slows proliferation and induces senescence, suggesting that TGFβ signaling also contributes to cellular senescence. We suggest that in the absence of Tgif1, a persistent increase in TGFβ responsive transcription and a reduced ability to deal with hyperoxic stress result in premature senescence in primary MEFs

Prostate Cancer Induced by Loss of Apc Is Restrained by TGFβ Signaling

Recent work with mouse models of prostate cancer (CaP) has shown that inactivation of TGFβ signaling in prostate epithelium can cooperate with deletion of the Pten tumor suppressor to drive locally aggressive cancer and metastatic disease. Here, we show that inactivating the TGFβ pathway by deleting the gene encoding the TGFβ type II receptor (Tgfbr2) in combination with a deletion of the Apc tumor suppressor gene specifically in mouse prostate epithelium, results in the rapid onset of invasive CaP. Micro-metastases were observed in the lymph nodes and lungs of a proportion of the double mutant mice, whereas no metastases were observed in Apc single mutant mice. Prostate-specific Apc;Tgfbr2 mutants had a lower frequency of metastasis and survived significantly longer than Pten;Tgfbr2 double mutants. However, all Apc;Tgfbr2 mutants developed invasive cancer by 30 weeks of age, whereas invasive cancer was rarely observed in Apc single mutant animals, even by one year of age. Further comparison of the Pten and Apc models of CaP revealed additional differences, including adenosquamous carcinoma in the Apc;Tgfbr2 mutants that was not seen in the Pten model, and a lack of robust induction of the TGFβ pathway in Apc null prostate. In addition to causing high-grade prostate intra-epithelial neoplasia (HGPIN), deletion of either Pten or Apc induced senescence in affected prostate ducts, and this restraint was overcome by loss of Tgfbr2. In summary, this work demonstrates that TGFβ signaling restrains the progression of CaP induced by different tumor suppressor mutations, suggesting that TGFβ signaling exerts a general tumor suppressive effect in prostate.This work was supported by a Program Project Grant from the National Cancer Institute (2P01CA104106 to B. Paschal and D. Wotton), and by a pilot grant from the UVA Cancer Center (funded from the CCSG P30 CA44579, the James and Rebecca CraigFoundation, and UVA Women's Oncology fund) to D. Wotton. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Sharon Birdsall for technical assistance, Anindya Dutta and Dan Gioeli for helpful discussions, and Chun-Song Yang for advice and reagent

The Polycomb Protein Pc2 Is a SUMO E3

AbstractPolycomb group (PcG) proteins form large multimeric complexes (PcG bodies) which are involved in the stable repression of gene expression. The human PcG protein, Pc2, has been shown to recruit the transcriptional corepressor, CtBP, to PcG bodies. We show that CtBP is sumoylated at a single lysine. In vitro, CtBP sumoylation minimally requires the SUMO E1 and E2 (Ubc9) and SUMO-1. However, Pc2 dramatically enhances CtBP sumoylation. In vivo, this is likely due to the ability of Pc2 to recruit both CtBP and Ubc9 to PcG bodies, thereby bringing together substrate and E2, and stimulating the transfer of SUMO to CtBP. These results demonstrate that Pc2 is a SUMO E3, and suggest that PcG bodies may be sumoylation centers

Tgif1 and Tgif2 Regulate Axial Patterning in Mouse

<div><p>Tgif1 and Tgif2 are transcriptional repressors that inhibit the transcriptional response to transforming growth factor β signaling, and can repress gene expression by direct binding to DNA. Loss of function mutations in <i>TGIF1</i> are associated with holoprosencephaly (HPE) in humans. In mice, embryos lacking both Tgif1 and Tgif2 fail to complete gastrulation, and conditional double null embryos that survive past gastrulation have HPE and do not survive past mid-gestation. Here we show that in mice of a relatively pure C57BL/6 strain background, loss of Tgif1 alone results in defective axial patterning and altered expression of <i>Hoxc6</i>. The primary defects in <i>Tgif1</i> null embryos are the presence of extra ribs on the C7 vertebra, consistent with a posterior transformation phenotype. In addition we observed defective cervical vertebrae, primarily C1-C5, in both adult mice and embryos that lacked Tgif1. The combination of <i>Tgif1</i> and <i>Tgif2</i> mutations increases the severity and penetrance of the posterior transformation phenotype, without altering the type of defects seen. Similarly, exposure of <i>Tgif1</i> mutant embryos to retinoic acid at E8.5 increased the severity and penetrance of the <i>Tgif1</i> phenotype. This suggests that Tgif1 and Tgif2 regulate axial patterning and that reduced TGIF function sensitizes embryos to the effects of retinoic acid.</p></div

Combination of <i>Tgif1</i> and <i>Tgif2</i> mutations.

<p>A) The axial formulas and the presence of cervical vertebra defects is shown for E18.5 embryos from <i>Tgif1;Tgif2</i> double heterozygous intercrosses. # = the embryo number. For example a total of 12 double heterozygous embryos were analyzed, with embryos 1 to 4 being normal. The phenotypes for embryos 5 to 12 are listed individually. B) The percentage of embryos of the indicated genotypes with either normal or posterior transformation is shown, based on the embryos detailed in 3B and 8A. C) Data for E18.5 embryos from <i>Tgif1</i> heterozygous intercrosses, in which the embryos were exposed to 10mg/kg retinoic acid (ATRA) at E8.5. D) the percentage of embryos with different axial formulas are shown for embryos from <i>Tgif1</i> intercrosses (from 3B) compared to those treated with ATRA.</p

Rib defects in <i>Tgif1</i> and <i>Tgif2</i> mutants.

<p>A) The number (and percentage) of embryos of each genotype with defects the cervical vertebrae are summarized. Defects observed were fusion of two adjacent vertebrae (fused, in figure), or loss of one vertebra on one side of the embryo (partial, in figure). B) Axial patterning defects are summarized for each genotype. Embryos with C6, T14, L6 are separated out depending on whether this posterior transformation was seen on both sides or on left (L) or right (R) only. The percentage with any defect is shown. C) The normal (C7, T13, L6) and defective (C6, T14, L6) patterns are shown schematically. D) Co-occurrence of the posterior transformation phenotype and defects in the cervical vertebrae is summarized for <i>Tgif1</i> null embryos.</p

Analysis of <i>Hoxc5</i> expression.

<p>Wild type and <i>Tgif1</i> null embryos at E10.5 were analyzed by whole-mount <i>in situ</i> hybridization, for <i>Hoxc5</i>. Images of representative embryos are shown, with a higher resolution image of part of the embryos below. Two of four wild type embryos had this expression pattern, with the other two having slightly higher staining in somite 9 on one side of the embryo. Of the five <i>Tgif1</i> null embryos, three had the pattern shown and the remaining two had slightly weaker expression in somite 9 on one side of the embryo. Somites 8 and 12 are indicated by stars, and the arrowhead shows the expression in somite 9.</p

Analysis of <i>Hoxc6</i> expression.

<p>Wild type and <i>Tgif1</i> null embryos at E9.5 were analyzed by whole-mount <i>in situ</i> hybridization, for <i>Hoxc6</i>. A higher resolution image of part of the embryos is shown to the right. The bracket indicates the extent of strong <i>Hoxc6</i> staining in the somites, and the arrows indicate somite boundaries.</p