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

Caractérisation fonctionnelle de la protéine de l hétérochromatine HP1g chez la souris

Les protéines de la famille HP1 constituent l un des éléments principaux de l hétérochromatine. Les fonctions moléculaires de ces protéines ont été bien caractérisées mais les rôles physiologiques d HP1g sont encore mal connus ; afin d élucider les fonctions in vivo d HP1g, j ai étudié les conséquences de l inactivation du gène Cbx3, codant pour cette protéine, dans un modèle murin. Bien que certaines observations restent à confirmer, les résultats de mon travail suggèrent qu HP1g exerce d importantes fonctions dans plusieurs processus physiologiques. Nous avons ainsi montré l implication de cette protéine dans plusieurs mécanismes de la défense immunitaire : en effet, HP1g est essentielle pour la migration des lymphocytes Th au niveau de la rate et pour la maturation des lymphocytes B lors des stades finaux de leur développement. De plus, HP1g joue un rôle au niveau de la recombinaison de classe en favorisant la production de cellules exprimant l isotype d anticorps IgG1. Par ailleurs, HP1g est nécessaire à l initiation de la spermatogenèse ainsi qu à la libération des spermatozoïdes matures dans la lumière des tubes séminifères, et cette protéine semble également impliquée dans l organisation sub-nucléaire des cellules de Sertoli. Mes résultats suggèrent qu HP1g, mais non HP1a, permet l association au niveau de l hétérochromatine des protéines HP1b et TIF1b, et que ce recrutement pourrait permettre d établir et/ou de maintenir une organisation sub-nucléaire spécifique ayant des implications fonctionnelles. L ensemble de mes résultats apportent de nouvelles évidences que les protéines de la famille HP1 ont des fonctions non redondantes dans la physiologique murine.HP1 proteins are one of the main heterochromatin components. The molecular functions of these proteins have been well characterized but physiological roles of HP1g are still unknown. In order to elucidate the in vivo functions of HP1g, I have worked on the consequences of the inactivation of Cbx3 gene, coding for HP1g, in a murine model. Although some observations remain to be fully established, the results of my work suggest that HP1g exerts important functions in several physiological processes. We have shown the implication of this protein in several mechanisms of immune response : namely, HP1g is essential for Th cells migration to spleen and for B cells development during final maturation stages. Furthermore, HP1g plays a role in class switch recombination by promoting IgG1-expressing cells production. HP1g is also required for spermatogenesis initiation and for mature spermatozoa release in seminiferous tubes lumen, and this protein seems to be involved in sub-nuclear organization of Sertoli cells. My results suggest that HP1g, but not HP1a, allows the association of HP1b and TIF1b to heterochromatin, and that this recruitment allows to establish and/or maintain a specific sub-nuclear organization with functional implications. All in all, my results bring new evidences that HP1 family proteins have non-redundant functions in murine physiology.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

TIF1b, un nouveau régulateur épigénétique de la pluripotence des cellules ES

Initialement identifié en tant que corépresseur des protéines à domaine KRAB , le cofacteur TIF1 a été démontré interagir avec diverses machineries de modifications et de remodelage de la chromatine, ainsi qu avec les protéines de l hétérochromatine HP1. J ai participé à la caractérisation de MEST, un gène cible primaire de TIF1 dans les cellules du carcinome embryonnaire F9. Nous avons ainsi démontré que l interaction entre TIF1 et HP1 est essentiel à l établissement et au maintien d une structure de type hétérochromatine au niveau du promoteur du gène MEST. J ai ensuite caractérisé les fonctions de TIF1b dans les cellules souches embryonnaires ES. Ainsi, nous avons établit par recombinaison homologue une lignée de cellules ES portant les deux allèles de TIF1 floxés et exprimant une recombinase Cre modifiée inductible par l hydroxytamoxifène (OHT) (TIF1 L3/L3-CreERT2). Ces cellules présentent une mort cellulaire massive et une différenciation morphologique spontanée en présence d OHT. L analyse de l expression de nombreux gènes normalement exprimés spécifiquement dans les cellules différenciées et de gènes de pluripotence au cours d une cinétique de traitement à l OHT des cellules ES TIF1 L3/L3-CreERT2 a montré que le phénotype de différenciation spontanée observée dans les cellules mutantes est précédé par une augmentation rapide de l expression des gènes spécifiques des cellules différenciées, suivie d une diminution progressive de l expression de nombreux gènes connus pour jouer un rôle dans la pluripotence. Ces résultats montrent que TIF1 joue un rôle essentiel dans le mécanisme de maintenance de la pluripotence des cellules ES.The cofactor TIF1ß, initially identified as a corepressor for KRAB domain protein, has been shown to interact with several modifications machinery, chromatin-remodelling factors and with the heterochromatin proteins HP1. During my thesis, I participated in the characterization of Mest, a primary target gene of TIF1b in the embryonic carcinoma cells F9. Our results showed that the interaction between TIF1ß and HP1 is essential in the heterochromatin structure establishment and maintenance at Mest promoter. Later on, I characterized functions of TIF1b in the embryonic stem cells ES. By homologue recombination we established ES cell line carrying two TIF1b floxed alleles and expressing an inducible Cre-recombinase by the hydroxytamoxifène (OHT) (TIF1ßL3/L3-CreERT2). Cells treated with OHT show a massive cellular death and spontaneous morphological differentiation. The expression analysis of several specific genes normally expressed in differentiated cells and genes of pluripotency, during a kinetics of treatment with OHT of ES cells TIF1ßL3/L3-CreERT2, revealed that the phenotype of spontaneous differentiation observed in the mutant cells, is preceded by a quick increasing of expression of specific genes in differentiated cells, this increasing is followed by a progressive reduction of many genes expression known to play a role in pluripotency. These results show that TIF1b plays an essential role in the mechanism of pluripotency of ES cells.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

Correlation of the exon/intron organization to the conserved domains of the mouse transcriptional corepressor TIF1beta

TIF1beta, a member of the transcriptional intermediary factor 1 family, has been reported to function as a corepressor for the large class of KRAB domain-containing zinc finger proteins of the Krüppel type. In this study, we report the genomic organization and nucleotide sequence of the mouse TIF1beta gene. This gene comprises 17 coding exons located within 7 kb of genomic DNA. Exon sizes vary from 37 bp (exon 10) to 901 bp (exon 1), and intron sizes range from 71 bp to 1843 bp. All introns have the conserved GT and AG dinucleotides present at the donor and acceptor sites, respectively. The functional/homology regions of the TIF1beta protein are encoded by distinct exons. The amino-terminal RING finger is encoded by two exons interrupted by a small intron. The B boxes lie within individual exons. Similarly to the RING finger, the PHD finger is encoded by two exons. Three exons constitute the carboxy-terminal bromodomain, and their position correlates well with the secondary structure elements of the domain as predicted by computer modeling. Taken together, these results will facilitate the genetic manipulation of TIF1beta for future in vivo structure-function studies

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

HP1s modulate the S-Adenosyl Methionine synthesis pathway in liver cancer cells

International audienc

The histone subcode: poly(ADP-ribose) polymerase-1 (Parp-1) and Parp-2 control cell differentiation by regulating the transcriptional intermediary factor TIF1beta and the heterochromatin protein HP1alpha.

International audienceRecent advances reveal emerging unique functions of poly(ADP-ribose) polymerase-1 (Parp-1) and Parp-2 in heterochromatin integrity and cell differentiation. However, the chromatin-mediated molecular and cellular events involved remain elusive. Here we describe specific physical and functional interactions of Parp-1 and Parp-2 with the transcriptional intermediary factor (TIF1beta) and the heterochromatin proteins (HP1) that affect endodermal differentiation. We show that Parp-2 binds to TIF1beta with high affinity both directly and through HP1alpha. Both partners colocalize at pericentric heterochromatin in primitive endoderm-like cells. Parp-2 also binds to HP1beta but not to HP1gamma. In contrast Parp-1 binds weakly to TIF1beta and HP1beta only. Both Parps selectively poly(ADP-ribosyl)ate HP1alpha. Using shRNA approaches, we provide evidence for distinct participation of both Parps in endodermal differentiation. Whereas Parp-2 and its activity are required for the relocation of TIF1beta to heterochromatic foci during primitive endodermal differentiation, Parp-1 and its activity modulate TIF1beta-HP1alpha association with consequences on parietal endodermal differentiation. Both Parps control TIF1beta transcriptional activity. In addition, this work identifies both Parps as new modulators of the HP1-mediated subcode histone.-Qu?t, D., Gasser, V., Fouillen, L., Cammas, F., Sanglier-Cianferani, S., Losson, R., Dantzer, F. The histone subcode: poly(ADP-ribose) polymerase-1 (Parp-1) and Parp-2 control cell differentiation by regulating the transcriptional intermediary factor TIF1beta and the heterochromatin protein HP1alpha

Cell differentiation induces TIF1beta association with centromeric heterochromatin via an HP1 interaction

International audienceThe transcriptional intermediary factor 1 (TIF1) family protein TIF1beta is a corepressor for Krüppel-associated box (KRAB)-domain-containing zinc finger proteins and plays a critical role in early embryogenesis. Here, we examined TIF1beta distribution in the nucleus of mouse embryonic carcinoma F9 cells during retinoic-acid-induced primitive endodermal differentiation. Using confocal immunofluorescence microscopy, we show that, although TIF1beta is diffusely distributed throughout the nucleoplasm of undifferentiated cells, it relocates and concentrates into distinct foci of centromeric heterochromatin in differentiated cells characterized by a low proliferation rate and a well developed cytokeratin network. This relocation was not observed in isoleucine-deprived cells, which are growth arrested, or in compound RXR alpha(-/-)/RAR gamma(-/-) null mutant cells, which are resistant to RA-induced differentiation. Amino-acid substitutions in the PxVxL motif of TIF1beta, which abolish interaction with members of the heterochromatin protein 1 (HP1) family, prevent its centromeric localization in differentiated cells. Collectively, these data provide compelling evidence for a dynamic nuclear compartmentalization of TIF1beta that is regulated during cell differentiation through a mechanism that requires HP1 interaction

Endogenous Retroviral Sequences Behave as Putative Enhancers Controlling Gene Expression through HP1-Regulated Long-Range Chromatin Interactions

International audienceAbout half of the mammalian genome is constituted of repeated elements, among which endogenous retroviruses (ERVs) are known to influence gene expression and cancer development. The HP1 (Heterochromatin Protein 1) proteins are known to be essential for heterochromatin establishment and function and its loss in hepatocytes leads to the reactivation of specific ERVs and to liver tumorigenesis. Here, by studying two ERVs located upstream of genes upregulated upon loss of HP1, Mbd1 and Trim24, we show that these HP1-dependent ERVs behave as either alternative promoters or as putative enhancers forming a loop with promoters of endogenous genes depending on the genomic context and HP1 expression level. These ERVs are characterised by a specific HP1-independent enrichment in heterochromatin-associated marks H3K9me3 and H4K20me3 as well as in the enhancer-specific mark H3K4me1, a combination that might represent a bookmark of putative ERV-derived enhancers. These ERVs are further enriched in a HP1-dependent manner in H3K27me3, suggesting a critical role of this mark together with HP1 in the silencing of the ERVs, as well as for the repression of the associated genes. Altogether, these results lead to the identification of a new regulatory hub involving the HP1-dependent formation of a physical loop between specific ERVs and endogenous genes