Transcription Intermediary Factor 1γ Research Articles

Abstract P4-11-24: TIF1[gamma] interacts with the TGFβ1/SMAD4 signaling leading to poorer outcome in operable breast cancer

Abstract Background The Transforming growth factorβ (TGFβ) signaling has a paradoxical role in cancer development and outcome. It protects against tumorigenesis by inhibiting cell growth and promoting apoptosis, but at advanced stages, it promotes tumor progression. Besides, the prognostic significance of the TGFβ1, SMAD4 in breast cancer patients is also an area of many contradictions. Transcriptional intermediary factor 1γ (TIF1γ) is thought to interact with the TGFβ/SMAD signaling through different mechanisms. Our study aimed at defining the prognostic significance of TGFβ1, SMAD4 and TIF1γ expression in breast cancer patients in addition to detection of possible interactions among those markers that might affect the outcome and explain the contradictory results. Methods Immunohistochemistry was performed on TMA blocks prepared from samples of 248 operable breast cancer patients who presented at CLB between 1998 and 2001 using. The intensity and the percentage of stained tumor cells were integrated into a single score (0-6) and a cutoff was defined for high or low expression for each marker. Correlation was done between the TGFβ1, SMAD4 and TIF1γ expression with the clinico-pathologic parameters using Pearson’s chi-square test. Kaplan-Meier method was used to estimate distant metastasis free survival (DMFS), disease free survival (DFS) and overall survival (OS) and the difference between the groups was evaluated with log-rank test. Results 223 cases were assessable for TIF1γ, 204 for TGFβ1 and 173 for SMAD4. Median age at diagnosis was 55.8 years (range: 27 to 89 years). Tumors were larger than 20 mm in 49.2% and 45.2% had axillary lymph node (LN) metastasis (N1a to N3). 19.4% of the patients had SBR grade I tumors, 46.8% grade II tumors and 33.9% grade III tumors. ER was positive in 85.4%, PR in 75.5% and Her2-neu was over-expressed in 10% of the cases. Nuclear TIF1γ, cytoplasmic TGFβ1, nuclear and cytoplasmic SMAD4 staining was high in 35.9%, 30.4%, 27.7% and 52.6% respectively. TIF1γ expression was associated with younger age (p=0.006), higher SBR grade (p<0.0001), more ER negativity (p=0.035), and tumors larger than 2 cm (p=0.081), while TGFβ1 was not associated with any of the traditional prognostic factors. TGFβ1 expression in tumor cells was a marker of poor prognosis regarding DMFS (HR=2.28; 95%CI: 1.4 to 3.8; p=0.002), DFS (HR=2.00; 95% CI: 1.25 to 3.5; p=0.005) and OS (HR=1.89; 95%CI: 1.04 to 3.43; p=0.037). TIF1γ expression carried a tendency towards poorer DMFS (p=0.091), DFS (p=0.143) and OS (p=0.091). In the multivariate analysis TGFβ1 remained an independent predictor of shorter DMFS, DFS and OS after adjustment for age, tumor size, SBR grade and LN invasion. Moreover, the prognostic significance of TGFβ1 was more obvious in the TIF1γ high patient subgroup than in the patients with TIF1γ low expression. The subgroup expressing both markers had the worst DMFS (HR=3.2; 95%CI: 1.7 to 5.9; p<0.0001), DFS (HR=3.02; 95%CI: 1.6 to 5.6; p<0.0001) and OS (HR=2.7; 95%CI: 1.4 to 5.4; p=0.005). Conclusion There is a crosstalk between the TIF1γ and the TGFβ1/SMAD4 signaling that deteriorate the outcome of operable breast cancer patients and when combined together they can serve as an effective prognostic tool for those patients. Citation Format: Loay Kassem, Laurent Fattet, Jonathan Lopez, Goulvent Thibault, Emilie Lavergne, Sylvie Chabaud, Nicolas Carrabin, Nicolas Chopin, Thomas Bachelot, Germain Gillet, Isabelle Treilleux, Ruth Rimohk. TIF1[gamma] interacts with the TGFβ1/SMAD4 signaling leading to poorer outcome in operable breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-11-24.

Expression of the dermatomyositis autoantigen transcription intermediary factor 1γ in regenerating muscle.

Autoantibodies against transcription intermediary factor 1γ (TIF1γ) are found in many patients with dermatomyositis (DM). Although TIF1γ is known to play a role in the differentiation of other tissues, its functional role in muscle regeneration has not been elucidated. This study was undertaken to explore the regulation and functional role of this protein during muscle differentiation and regeneration. TIF1γ expression was analyzed in human muscle biopsy specimens using immunofluorescence microscopy. Immunofluorescence microscopy and immunoblotting analyses were used to study TIF1γ expression in a mouse model of muscle injury and repair. The effect of premature TIF1γ silencing on muscle differentiation was studied in cultured mouse myoblasts. In muscle biopsy specimens from DM patients, TIF1γ was expressed at low levels in the nuclei of histologically normal muscle cells but at high levels in the centralized nuclei of atrophic, perifascicular myofibers expressing markers of regeneration. TIF1γ levels were also increased in regenerating myonuclei following muscle injury in mice. Premature silencing of TIF1γ in vitro using small interfering RNA did not accelerate the expression of myogenin, a transcription factor that plays a central role in regulating relatively early stages of muscle differentiation. However, premature silencing of TIF1γ did accelerate myotube fusion and the expression of myosin heavy chain (MyHC), a later marker of muscle differentiation. The DM autoantigen TIF1γ is markedly up-regulated during muscle regeneration in human and mouse muscle cells. Premature silencing of this protein in cultured myoblasts accelerates MyHC expression and myoblast fusion. However, TIF1γ may function independently of, or downstream from, myogenin.

Tif1γ controls the TGF-β receptor on hematopoietic stem cells: implication in physiological aging

Hematopoietic stem cell (HSC) aging has been directly linked to the development of several hematological disorders including myeloproliferative diseases. We recently described that in elderly mice (20-month-old), physiological aging of the hematopoietic system is associated with a decreased expression of the Transcription Intermediary Factor 1γ (Tif1γ) gene in HSCs. In young mice (4-month-old), deleted for the Tif1γ gene in HSCs (Tif1γ -/- ), the hematopoiesis aging phenotype is intensified. We discovered that Tif1γ controls the TGF-b receptor 1 (Tgfbr1) and finely regulates the number of myeloid-restricted HSCs in bone marrow. Altogether, we established that young Tif1γ -/- mice develop a phenotype of premature hematopoietic aging, which may explain their tendency to myeloproliferative disease. We identified two populations of HSCs specifically discriminated by Tgfbr1 expression and afforded evidence of the capture of myeloid-restricted (Tgfbr1 hi ) and myeloid-lymphoid-balanced (Tgfbr1 lo ) HSC. In conclusion, our study proves that Tif1γ can regulate the balance between lymphoid and myeloid HSCs, through a modulation of the TGF-b signaling.

Tif1γ regulates the TGF-β1 receptor and promotes physiological aging of hematopoietic stem cells.

The hematopoietic system declines with age. Myeloid-biased differentiation and increased incidence of myeloid malignancies feature aging of hematopoietic stem cells (HSCs), but the mechanisms involved remain uncertain. Here, we report that 4-mo-old mice deleted for transcription intermediary factor 1γ (Tif1γ) in HSCs developed an accelerated aging phenotype. To reinforce this result, we also show that Tif1γ is down-regulated in HSCs during aging in 20-mo-old wild-type mice. We established that Tif1γ controls TGF-β1 receptor (Tgfbr1) turnover. Compared with young HSCs, Tif1γ(-/-) and old HSCs are more sensitive to TGF-β signaling. Importantly, we identified two populations of HSCs specifically discriminated by Tgfbr1 expression level and provided evidence of the capture of myeloid-biased (Tgfbr1(hi)) and myeloid-lymphoid-balanced (Tgfbr1(lo)) HSCs. In conclusion, our data provide a new paradigm for Tif1γ in regulating the balance between lymphoid- and myeloid-derived HSCs through TGF-β signaling, leading to HSC aging.

Most Patients With Cancer‐Associated Dermatomyositis Have Antibodies to Nuclear Matrix Protein NXP‐2 or Transcription Intermediary Factor 1γ

Since dermatomyositis (DM) is associated with an increased risk of malignancy, accurate identification of patients likely to harbor cancers is important. Using immunoprecipitations from radiolabeled cell lysates, several groups recently showed that anti-transcription intermediary factor 1γ (anti-TIF-1γ) antibodies are associated with malignancy in DM. We undertook this study to develop sensitive, specific assays to detect antibodies against TIF-1γ and nuclear matrix protein NXP-2 and to evaluate their association with malignancy in DM. To detect anti-TIF-1γ antibodies, immunoprecipitations were performed using lysates made from HeLa cells overexpressing TIF-1γ, with detection by immunoblotting. Anti-NXP-2 antibodies were assayed by immunoprecipitation using (35) S-methionine-labeled NXP-2 generated by in vitro transcription/translation. We analyzed patient sera from DM cohorts seen at the Stanford University Dermatology Clinic (n = 111) and the Johns Hopkins Myositis Center (n = 102). A total of 17% and 38% of patients had antibodies against NXP-2 and TIF-1γ, respectively. Reactivity against either NXP-2 or TIF-1γ identified 83% of patients with cancer-associated DM. In addition to older age and male sex, cancer was associated with antibodies to NXP-2 or TIF-1γ on multivariate analysis (odds ratio 3.78 [95% confidence interval 1.33-10.8]). Stratification by sex revealed that anti-NXP-2 was specifically associated with cancer in males (odds ratio 5.78 [95% confidence interval 1.35-24.7]). These studies demonstrate that anti-NXP-2 and anti-TIF-1γ antibodies are frequent DM specificities (found in 55% of patients) and are present in most patients with cancer-associated DM.

Transcriptional intermediary factor 1γ binds to the anaphase-promoting complex/cyclosome and promotes mitosis

The Anaphase-Promoting Complex/Cyclosome (APC/C) is an ubiquitin ligase that functions during mitosis. Here we identify the transcriptional regulator, Transcriptional Intermediary Factor 1γ, TIF1γ as an APC/C-interacting protein that regulates APC/C function. TIF1γ is not a substrate for APC/C-dependent ubiquitylation but instead, associates specifically with the APC/C holoenzyme and Cdc20 to affect APC/C activity and progression through mitosis. RNA interference studies indicate that TIF1γ knockdown results in a specific reduction in APC/C ubiquitin ligase activity, the stabilization of APC/C substrates, and an increase in the time taken for cells to progress through mitosis from nuclear envelope breakdown (NEBD) to anaphase. TIF1γ knockdown cells are also characterized by the inappropriate presence of cyclin A at metaphase, and an increase in the number of cells that fail to undergo metaphase-to-anaphase transition. Expression of a siRNA-resistant TIF1γ species relieves the mitotic phenotype imposed by TIF1γ knockdown and allows for mitotic progression. Binding studies indicate that TIF1γ is also a component of the APC/C-Mitotic Checkpoint Complex (MCC), but is not required for MCC dissociation from the APC/C once the Spindle Assembly Checkpoint (SAC) is satisfied. TIF1γ inactivation also results in chromosome misalignment at metaphase, and SAC activation; inactivation of the SAC relieves the mitotic block imposed by TIF1γ knockdown. Together these data define novel functions for TIF1γ during mitosis and suggest that a reduction in APC/C ubiquitin ligase activity promotes SAC activation.

Tif1γ Suppresses Murine Pancreatic Tumoral Transformation by a Smad4-Independent Pathway

Transcriptional intermediary factor 1γ (TIF1γ; alias, TRIM33/RFG7/PTC7/ectodermin) belongs to an evolutionarily conserved family of nuclear factors that have been implicated in stem cell pluripotency, embryonic development, and tumor suppression. TIF1γ expression is markedly down-regulated in human pancreatic tumors, and Pdx1-driven Tif1γ inactivation cooperates with the Kras(G12D) oncogene in the mouse pancreas to induce intraductal papillary mucinous neoplasms. In this study, we report that aged Pdx1-Cre; LSL-Kras(G12D); Tif1γ(lox/lox) mice develop pancreatic ductal adenocarcinomas (PDACs), an aggressive and always fatal neoplasm, demonstrating a Tif1γ tumor-suppressive function in the development of pancreatic carcinogenesis. Deletion of SMAD4/DPC4 (deleted in pancreatic carcinoma locus 4) occurs in approximately 50% of human cases of PDAC. We, therefore, assessed the genetic relationship between Tif1γ and Smad4 signaling in pancreatic tumors and found that Pdx1-Cre; LSL-Kras(G12D); Smad4(lox/lox); Tif1γ(lox/lox) (alias, KSSTT) mutant mice exhibit accelerated tumor progression. Consequently, Tif1γ tumor-suppressor effects during progression from a premalignant to a malignant state in our mouse model of pancreatic cancer are independent of Smad4. These findings establish, for the first time to our knowledge, that Tif1γ and Smad4 both regulate an intraductal papillary mucinous neoplasm-to-PDAC sequence through distinct tumor-suppressor programs.

Adenovirus E4orf3 Targets Transcriptional Intermediary Factor 1γ for Proteasome-Dependent Degradation during Infection

The ability of adenovirus early region proteins, E1B-55K and E4orf6, to usurp control of cellular ubiquitin ligases and target proteins for proteasome-dependent degradation during infection is well established. Here we show that the E4 gene product, E4orf3 can, independently of E1B-55K and E4orf6, target the transcriptional corepressor transcriptional intermediary factor 1γ (TIF1γ) for proteasome-mediated degradation during infection. Initial mass spectrometric studies identified TIF1 family members-TIF1α, TIF1β, and TIF1γ-as E1B-55K-binding proteins in both transformed and infected cells, but analyses revealed that, akin to TIF1α, TIF1γ is reorganized in an E4orf3-dependent manner to promyelocytic leukemia protein-containing nuclear tracks during infection. The use of a number of different adenovirus early region mutants identified the specific and sole requirement for E4orf3 in mediating TIF1γ degradation. Further analyses revealed that TIF1γ is targeted for degradation by a number of divergent human adenoviruses, suggesting that the ability of E4orf3 to regulate TIF1γ expression is evolutionarily conserved. We also determined that E4orf3 does not utilize the Cullin-based ubiquitin ligases, CRL2 and CRL5, or the TIF1α ubiquitin ligase in order to promote TIF1γ degradation. Further studies suggested that TIF1γ possesses antiviral activity and limits adenovirus early and late gene product expression during infection. Indeed, TIF1γ knockdown accelerates the adenovirus-mediated degradation of MRE11, while TIF1γ overexpression delays the adenovirus-mediated degradation of MRE11. Taken together, these studies have identified novel adenovirus targets and have established a new role for the E4orf3 protein during infection.

Antagonistic regulation of EMT by TIF1γ and Smad4 in mammary epithelial cells

TGF-β is a potent inducer of epithelial-to-mesenchymal transition (EMT), a process involved in tumour invasion. TIF1γ participates in TGF-β signalling. To understand the role of TIF1γ in TGF-β signalling and its requirement for EMT, we analysed the TGF-β1 response of human mammary epithelial cell lines. A strong EMT increase was observed in TIF1γ-silenced cells after TGF-β1 treatment, whereas Smad4 inactivation completely blocked this process. Accordingly, the functions of several TIF1γ target genes can be linked to EMT, as shown by microarray analysis. As a negative regulator of Smad4, TIF1γ could be crucial for the regulation of TGF-β signalling. Furthermore, TIF1γ binds to and represses the plasminogen activator inhibitor 1 promoter, demonstrating a direct role of TIF1γ in TGF-β-dependent gene expression. This study shows the molecular relationship between TIF1γ and Smad4 in TGF-β signalling and EMT.

Transcription intermediary factor 1γ is a tumor suppressor in mouse and human chronic myelomonocytic leukemia.

Transcription intermediary factor 1γ (TIF1γ) was suggested to play a role in erythropoiesis. However, how TIF1γ regulates the development of different blood cell lineages and whether TIF1γ is involved in human hematological malignancies remain to be determined. Here we have shown that TIF1γ was a tumor suppressor in mouse and human chronic myelomonocytic leukemia (CMML). Loss of Tif1g in mouse HSCs favored the expansion of the granulo-monocytic progenitor compartment. Furthermore, Tif1g deletion induced the age-dependent appearance of a cell-autonomous myeloproliferative disorder in mice that recapitulated essential characteristics of human CMML. TIF1γ was almost undetectable in leukemic cells of 35% of CMML patients. This downregulation was related to the hypermethylation of CpG sequences and specific histone modifications in the gene promoter. A demethylating agent restored the normal epigenetic status of the TIF1G promoter in human cells, which correlated with a reestablishment of TIF1γ expression. Together, these results demonstrate that TIF1G is an epigenetically regulated tumor suppressor gene in hematopoietic cells and suggest that changes in TIF1γ expression may be a biomarker of response to demethylating agents in CMML.

Adult Hematopoiesis is Regulated by TIF1γ, a Repressor of TAL1 and PU.1 Transcriptional Activity

Crosstalk between transcription factors and cytokines precisely regulates tissue homeostasis. Transcriptional intermediary factor 1γ (TIF1γ) regulates vertebrate hematopoietic development, can control transcription elongation, and is a component of the TGF-β signaling pathway. Here we show that deletion of TIF1γ in adult hematopoiesis is compatible with life and long-term maintenance of essential blood cell lineages. However, loss of TIF1γ results in deficient long-term hematopoietic stem cell (LT-HSC) transplantation activity, deficient short-term HSC (ST-HSC) bone marrow retention, and priming ST-HSCs to myelomonocytic lineage. These defects are hematopoietic cell-autonomous, and priming of TIF1γ-deficient ST-HSCs can be partially rescued by wild-type hematopoietic cells. TIF1γ can form complexes with TAL1 or PU.1-two essential DNA-binding proteins in hematopoiesis-occupy specific subsets of their DNA binding sites invivo, and repress their transcriptional activity. These results suggest a regulation of adult hematopoiesis through TIF1γ-mediated transcriptional repression of TAL1 and PU.1 target genes.

Transcription intermediary factor 1γ decreases protein expression of the transcriptional cofactor, LIM-domain-binding 1

LIM-domain-binding 1 (LDB1) is a cofactor that participates in formation of regulatory complexes involving transcription factors containing LIM domains as well as other factors. We have examined the ability of transcriptional intermediary factor 1γ (TIF1γ) to decrease LDB1 expression. An expression vector for TIF1γ was found to decrease expression of LDB1. A mutation which disrupts the ubiquitin ligase activity of TIF1γ was found to block the ability of TIF1γ to decrease LDB1 expression. Proteasome inhibitors were also able to block TIF1γ effects on LDB1. Immunoprecipitation studies provided evidence that LDB1 interacts with TIF1γ in intact cells. Knockdown of TIF1γ in zebrafish embryos led to increased expression of LDB1 providing evidence for a physiological role of TIF1γ in regulating LDB1 expression. Reporter gene assays demonstrated that TIF1γ can alter the activity of LIM-homeodomain transcription factor-responsive promoters. These studies are consistent with a model in which TIF1γ acts to ubiquitinate LDB1 leading to degradation of LDB1 and changes in transcription of LDB1-dependent promoters.

New Branch to TGF-β Pathway

Cytokines of the transforming growth factor-β (TGF-β) family have important roles in development, cancer, and other cellular processes. Abundant evidence shows that stimulated TGF-β receptors phosphorylate Smad2 and Smad3 proteins, which act in complexes with Smad4 to regulate transcription in the nucleus. New results from He et al. point to a distinct mechanism by which TGF-β can control gene regulation. The authors searched for binding partners for Smads 2 and 3 and identified the protein transcriptional intermediary factor 1γ (TIF1γ, also called TRIM33, RFG7, PTC7, or ectodermin), a protein that functions as a cofactor with nuclear receptors and other transcription factors and as a component of the histone deacetylase N-CoR1/HDAC3 complex. Exposure of human hematopoietic progenitor cells to TGF increased the usual association of Smads 2 and 3 with Smad4 but also increased formation of complexes of Smad2/3 with TIF1γ in an apparently competitive manner. In human hematopoietic stem cells, TGF-β promotes differentiation, and this effect required TIF1γ. Reduced abundance of TIF1γ after infection of cells with retroviruses encoding shRNA inhibited differentiation of the cells in culture. Depletion of Smad4 had no effect on differentiation. In other culture conditions in which stem cell proliferation is enhanced, TGF-β inhibits proliferation. This effect required the presence of Smads 2 and 3 but was not affected by depletion of TIF1γ. The authors therefore conclude that activated Smads 2 and 3 can trigger two separate transcriptional responses with distinct biological outcomes, depending on whether they act in association with Smad4 or TIF1γ. W. He, D. C. Dorn, H. Erdjument-Bromage, P. Tempst, M. A. S. Moore, J. Massagué, Hematopoiesis controlled by distinct TIF1γ and Smad4 branches of the TGFβ pathway. Cell 125 , 929-941 (2006). [PubMed]