T:G mismatch-specific thymine-DNA glycosylase (TDG) as a coregulator of transcription interacts with SRC1 family members through a novel tyrosine repeat motif

Gene activation involves protein complexes with diverse enzymatic activities, some of which are involved in chromatin modification. We have shown previously that the base excision repair enzyme thymine DNA glycosylase (TDG) acts as a potent coactivator for estrogen receptor-α. To further understand how TDG acts in this context, we studied its interaction with known coactivators of nuclear receptors. We find that TDG interacts in vitro and in vivo with the p160 coactivator SRC1, with the interaction being mediated by a previously undescribed motif encoding four equally spaced tyrosine residues in TDG, each tyrosine being separated by three amino acids. This is found to interact with two motifs in SRC1 also containing tyrosine residues separated by three amino acids. Site-directed mutagenesis shows that the tyrosines encoded in these motifs are critical for the interaction. The related p160 protein TIF2 does not interact with TDG and has the altered sequence, F-X-X-X-Y, at the equivalent positions relative to SRC1. Substitution of the phenylalanines to tyrosines is sufficient to bring about interaction of TIF2 with TDG. These findings highlight a new protein-protein interaction motif based on Y-X-X-X-Y and provide new insight into the interaction of diverse proteins in coactivator complexe

iNKT cell development is orchestrated by different branches of TGF-β signaling

Invariant natural killer T (iNKT) cells constitute a distinct subset of T lymphocytes exhibiting important immune-regulatory functions. Although various steps of their differentiation have been well characterized, the factors controlling their development remain poorly documented. Here, we show that TGF-β controls the differentiation program of iNKT cells. We demonstrate that TGF-β signaling carefully and specifically orchestrates several steps of iNKT cell development. In vivo, this multifaceted role of TGF-β involves the concerted action of different pathways of TGF-β signaling. Whereas the Tif-1γ branch controls lineage expansion, the Smad4 branch maintains the maturation stage that is initially repressed by a Tif-1γ/Smad4-independent branch. Thus, these three different branches of TGF-β signaling function in concert as complementary effectors, allowing TGF-β to fine tune the iNKT cell differentiation program

Epigenetic tethering of AID to the donor switch region during immunoglobulin class switch recombination

A complex of KAP1 and HP1 is needed to tether AID to the H3K9me3-marked donor switch region during CSR

Inactivation of TIF1γ Cooperates with KrasG12D to Induce Cystic Tumors of the Pancreas

Inactivation of the Transforming Growth Factor Beta (TGFβ) tumor suppressor pathway contributes to the progression of Pancreatic Ductal AdenoCarcinoma (PDAC) since it is inactivated in virtually all cases of this malignancy. Genetic lesions inactivating this pathway contribute to pancreatic tumor progression in mouse models. Transcriptional Intermediary Factor 1 gamma (TIF1γ) has recently been proposed to be involved in TGFβ signaling, functioning as either a positive or negative regulator of the pathway. Here, we addressed the role of TIF1γ in pancreatic carcinogenesis. Using conditional Tif1γ knockout mice (Tif1γlox/lox), we selectively abrogated Tif1γ expression in the pancreas of Pdx1-Cre;Tif1γlox/lox mice. We also generated Pdx1-Cre;LSL-KrasG12D;Tif1γlox/lox mice to address the effect of Tif1γ loss-of-function in precancerous lesions induced by oncogenic KrasG12D. Finally, we analyzed TIF1γ expression in human pancreatic tumors. In our mouse model, we showed that Tif1γ was dispensable for normal pancreatic development but cooperated with Kras activation to induce pancreatic tumors reminiscent of human Intraductal Papillary Mucinous Neoplasms (IPMNs). Interestingly, these cystic lesions resemble those observed in Pdx1-Cre;LSL-KrasG12D;Smad4lox/lox mice described by others. However, distinctive characteristics, such as the systematic presence of endocrine pseudo-islets within the papillary projections, suggest that SMAD4 and TIF1γ don't have strictly redundant functions. Finally, we report that TIF1γ expression is markedly down-regulated in human pancreatic tumors by quantitative RT–PCR and immunohistochemistry supporting the relevance of these findings to human malignancy. This study suggests that TIF1γ is critical for tumor suppression in the pancreas, brings new insight into the genetics of pancreatic cancer, and constitutes a promising model to decipher the respective roles of SMAD4 and TIF1γ in the multifaceted functions of TGFβ in carcinogenesis and development

Caractérisation fonctionnelle des facteurs intermédiaires de transcription TIF1alpha et TIF1delta chez la souris

STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Rôle de la protéine de régulation TIFa dans la signalisation par les récepteurs nucléaires (Analyse transcriptomique et implications physiopathologiques)

TIF1a est un corépresseur ligand-dépendant des récepteurs nucléaires in vitro. Nous avons montré que dans des fibroblastes traités à l acide rétinoïque (RA), TIF1a se fixe au promoteur de gènes cibles du RA et atténue leur induction. Les souris KO pour TIF1a ont établi que son contrôle sur l activité de RARa (RA Receptor a) est essentiel pour la suppression de l hépatocarcinogenèse. Une analyse transcriptomique a montré que les foies KO surexpriment les voies RA, IFNg, Wnt et TGFb dès 5semaines. Ces dérégulations et la formation de tumeurs sont supprimées chez les souris TIF1a-/-RARa+/-. À 14semaines, les foies KO résistent à ces dérégulations en surexprimant suppresseurs de tumeurs et inhibiteurs de ces voies. Néanmoins, des tumeurs surexprimant ces voies apparaissent dès 11mois.Les souris KO développent aussi des calcifications artérielles,qui corrèlent avec la surexpression de cibles de VDR (Vitamin D Receptor), confirmant le rôle d atténuateur de TIF1a sur les récepteurs nucléairesTIF1a act as a ligand-dependant corepressor in vitro. We showed that in retinoic acid (RA) treated fibroblasts, TIF1a is bound to the promoter of RA target genes and attenuates their induction. TIF1a knock out mice established that its control on RARa (RA receptor a) activity is essential for liver tumorigenesis suppression. Transcriptomic analyses showed that KO livers overexpress RA, IFNg, TGFb, and Wnt signaling at the age of 5 weeks. These deregulations as well as tumor formation are suppressed in the TIF1a-/-RARa+/- mice. At 14 weeks, KO livers resist to these deregulations by overexpressing tumor suppressors and inhibitors of these pathways. Nevertheless, tumors overexpressing these pathways appear at 11 months.The KO mice develop arterial calcifications as well. This artheriopathy correlates with an increased Vitamin D signaling, confirming the attenuating role of TIF1a on nuclear receptors activity.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Caractérisation et étude fonctionnelle du gène codant le cofacteur transcriptionnel TIF1g

STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Identification et caractérisation des gènes cibles du corépresseur TIF1b (Vers une meilleure compréhension de son rôle en tant que régulateur épigénétique de l'activité transcriptionnelle du génome murin)

Mon travail de thèse a porté sur l identification et la caractérisation des gènes directement régulés par TIF1b dans les cellules de carcinome embryonnaire F9. Par analyse transcriptomique et ChIP, nous avons identifié MEST comme un gène cible primaire de TIF1b. Nous avons ensuite démontré que TIF1b est essentiel à l établissement et à la maintenance d une structure de type hétérochromatine dans la région promotrice de ce gène, caractérisée par la triméthylation de H3K9 et de H4K20, l hyperméthylation de l ADN et un enrichissement en protéines HP1. Enfin, cette structure semble entrainer une localisation préférentielle du gène MEST à proximité de l hétérochromatine. Nous avons démontré que cette organisation nécessite l interaction entre TIF1b et les HP1, puisqu elle est complètement perturbée dans des cellules F9 qui expriment une protéine TIF1b n interagissant plus avec les protéines HP1. Dans ces cellules, le promoteur MEST est hypométhylé et caractérisé par une triméthylation de H3K27, associée à la réactivation de l expression de ce gène et à son éloignement de l hétérochromatine.Par des expériences de ChIP-seq réalisées dans des cellules F9 au cours de la différenciation cellulaire, nous avons mis en évidence un grand nombre de régions du génome enrichies en TIF1b correspondant principalement à des régions promotrices proximales et distales. Cette étude nous a permis d établir que TIF1b régule majoritairement des gènes impliqués dans l expression des gènes, la mort cellulaire, la croissance et la prolifération cellulaire ainsi que dans le cancer; cette analyse suggère aussi que TIF1b pourrait jouer un rôle au niveau des séquences répétées de type LTR et SINE.During my thesis, we were interested in the identification and characterization of genes directly regulated by TIF1b in F9 embryonal carcinoma cells. By transcriptomic and TIF1b chromatin-immunoprecipitation (ChIP) analysis, we first identified MEST as a TIF1b target gene. We then demonstrated that TIF1b, through its interaction with HP1 proteins, is essential in establishing and maintaining a local heterochromatin-like structure on the promoter region of this gene, characterized by H3K9 and H4K20 trimethylation, DNA hypermethylation, and enrichment in HP1 proteins that correlates with preferential association to foci of pericentromeric heterochromatin and transcriptional repression. We demonstrate that upon disruption of the interaction between TIF1b and HP1 proteins, TIF1b is released from the promoter region, leading to a switch from DNA hypermethylation and histone H3K9 trimethylation to DNA hypomethylation and histone H3K27 trimethylation associated with rapid reactivation of MEST expression. To identifie new TIF1b target genes, we have perfomed a large scale TIF1b ChIP (ChIP-seq) experiments in differentiating F9 cells. We were able to identified a large number of genomic regions enriched in TIF1b corresponding predominantly to promoter and promoter proximal regions (86%). We demonstrated that TIF1b is a major regulator of cell physiology by regulating genes implicated in gene expression, cell death, cell growth and proliferation, and cancer. This analysis also strongly suggests that TIF1b plays a role in the transcriptionnal regulation on LTR and SINE repeated sequences.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Association of the transcriptional corepressor TIF1β with heterochromatin protein 1 (HP1): an essential role for progression through differentiation

The transcriptional intermediary factor 1β (TIF1β) is a corepressor for KRAB-domain-containing zinc finger proteins and is believed to play essential roles in cell physiology by regulating chromatin organization at specific loci through association with chromatin remodeling and histone-modifying activities and recruitment of heterochromatin protein 1 (HP1) proteins. In this study, we have engineered a modified embryonal carcinoma F9 cell line (TIF1β(HP1box/-)) expressing a mutated TIF1β protein (TIF1β(HP1box)) unable to interact with HP1 proteins. Phenotypic analysis of TIF1β(HP1box/-) and TIF1β(+/-) cells shows that TIF1β–HP1 interaction is not required for differentiation of F9 cells into primitive endoderm-like (PrE) cells on retinoic acid (RA) treatment but is essential for further differentiation into parietal endoderm-like (PE) cells on addition of cAMP and for differentiation into visceral endoderm-like cells on treatment of vesicles with RA. Complementation experiments reveal that TIF1β–HP1 interaction is essential only during a short window of time within early differentiating PrE cells to establish a selective transmittable competence to terminally differentiate on further cAMP inducing signal. Moreover, the expression of three endoderm-specific genes, GATA6, HNF4, and Dab2, is down-regulated in TIF1β(HP1box/-) cells compared with wild-type cells during PrE differentiation. Collectively, these data demonstrate that the interaction between TIF1β and HP1 proteins is essential for progression through differentiation by regulating the expression of endoderm differentiation master players

Rigor mortis encodes a novel nuclear receptor interacting protein required for ecdysone signaling during Drosophila larval development

Pulses of the steroid hormone ecdysone trigger the major developmental transitions in Drosophila, including molting and puparium formation. The ecdysone signal is transduced by the EcR/USP nuclear receptor heterodimer that binds to specific response elements in the genome and directly regulates target gene transcription. We describe a novel nuclear receptor interacting protein encoded by rigor mortis (rig) that is required for ecdysone responses during larval development. rig mutants display defects in molting, delayed larval development, larval lethality, duplicated mouth parts, and defects in puparium formation--phenotypes that resemble those seen in EcR, usp, E75A and betaFTZ-F1 mutants. Although the expression of these nuclear receptor genes is essentially normal in rig mutant larvae, the ecdysone-triggered switch in E74 isoform expression is defective. rig encodes a protein with multiple WD-40 repeats and an LXXLL motif, sequences that act as specific protein-protein interaction domains. Consistent with the presence of these elements and the lethal phenotypes of rig mutants, Rig protein interacts with several Drosophila nuclear receptors in GST pull-down experiments, including EcR, USP, DHR3, SVP and betaFTZ-F1. The ligand binding domain of betaFTZ-F1 is sufficient for this interaction, which can occur in an AF-2-independent manner. Antibody stains reveal that Rig protein is present in the brain and imaginal discs of second and third instar larvae, where it is restricted to the cytoplasm. In larval salivary gland and midgut cells, however, Rig shuttles between the cytoplasm and nucleus in a spatially and temporally regulated manner, at times that correlate with the major lethal phase of rig mutants and major switches in ecdysone-regulated gene expression. Taken together, these data indicate that rig exerts essential functions during larval development through gene-specific effects on ecdysone-regulated transcription, most likely as a cofactor for one or more nuclear receptors. Furthermore, the dynamic intracellular redistribution of Rig protein suggests that it may act to refine spatial and temporal responses to ecdysone during development.J.A.O. was supported by a grant from the MCyT of Spain (`Ramón y Cajal'Program). J.G. was a Predoctoral Fellow and C.S.T. is an Investigator with the Howard Hughes Medical Institute.Peer reviewe