![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
RESEARCH PAPER
with heterochromatin protein 1 (HP1): an essential role for progression through differentiation
Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France, BP10142, 67404 Illkirch-Cedex, France
 |
Abstract |
|---|
 Top Abstract ResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
|---|
(TIF1) 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 (TIF1HP1box/-) expressing a mutated TIF1 protein (TIF1HP1box) unable to interact with HP1 proteins. Phenotypic analysis of TIF1HP1box/- and TIF1+/- cells shows that TIF1–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 TIF1–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 TIF1HP1box/- cells compared with wild-type cells during PrE differentiation. Collectively, these data demonstrate that the interaction between TIF1 and HP1 proteins is essential for progression through differentiation by regulating the expression of endoderm differentiation master players.
[Keywords: Transcriptional silencing; KRAB zinc finger proteins; F9 EC cells; retinoic acid; cAMP; nuclear compartmentalization]
Received March 19, 2004; revised version accepted July 15, 2004.
). The isolation and biochemical characterization of several complexes involved in chromatin remodeling, including SWI/SNF, NuRD, and ISWI, led to major breakthroughs into the mechanisms of chromatin structure regulation (for review, see Narlikar et al. 2002). The identification of chromatin-modifying enzymes (e.g., histone acetylases, deacetylases, and methyltransferases) and determination of their substrate specificities suggested the existence of a histone code (Jenuwein and Allis 2001). However, how these activities are coordinated to respond properly to any environmental stimulus in vivo remains largely unknown.
TIF1 (Le Douarin et al. 1996), also known as KAP-1 (Friedman et al. 1996) or KRIP-1 (Kim et al. 1996), is a member of an emerging family of transcriptional regulators designated as TIF1 (transcriptional intermediary factor 1). The TIF1 family includes TIF1
(Le Douarin et al. 1995), TIF1
(Venturini et al. 1999; Yan et al. 2004) in mammals, and Bonus in Drosophila (Beckstead et al. 2001). TIF1 proteins are defined by the presence of two conserved amino acid regions: an N-terminal RBCC (RING finger, B boxes, coiled-coil) motif that is involved in homo- and heterodimerization (Peng et al. 2002), and a C-terminal bromodomain preceded by a PHD (plant homeodomain) finger. These latter two motifs are often associated and present in a number of transcriptional cofactors acting at the chromatin level (Aasland et al. 1995; Jeanmougin et al. 1997; Zeng and Zhou 2002).
Recent genetic studies in mice have provided evidence that TIF1 is a developmental regulatory protein that exerts cellular function(s) essential for early embryonic development (Cammas et al. 2000) and for spermatogenesis (Weber et al. 2002). Furthermore, TIF1 displays several biochemical properties suggesting that it could be a coordinator in epigenetic regulation of transcription: (1) TIF1 is a universal corepressor for a large family of transcription factors, the Krüppel associated box (KRAB)-domain containing zinc finger proteins (Friedman et al. 1996; Kim et al. 1996; Moosmann et al. 1996; Abrink et al. 2001); (2) TIF1 is an intrinsic component of two chromatin remodeling and histone deacetylase complexes, N-CoR1 and NuRD (Underhill et al. 2000; Schultz et al. 2001); (3) TIF1 directly interacts with the histone methyltransferase SETDB1, which specifically methylates Lys 9 of histone H3 preferentially within euchromatin (Schultz et al. 2002); and (4) TIF1 interacts with members of the heterochromatin protein 1 (HP1) family (Le Douarin et al. 1996; Nielsen et al. 1999; Ryan et al. 1999).
HP1 is a structurally and functionally highly conserved protein with family members found in a variety of eukaryotic organisms ranging from Schizosaccharomyces pombe to humans (Eissenberg et al. 1990; Wang et al. 2000). These proteins participate in chromatin packaging and have a well-established function in heterochromatin-mediated silencing (for review, see Eissenberg and Elgin 2000). Mice and humans each have three different HP1 proteins (HP1, , and ) that are associated, although not exclusively, with pericentromeric heterochromatin (Nielsen et al. 1999). The structure of the HP1-like proteins comprises a conserved N-terminal region called chromo-domain (CD) and a conserved C-terminal chromo shadow domain (CSD) separated by a less conserved hinge region (Eissenberg 2001). The HP1 CD binds methylated Lys 9 of histone H3 (Lachner et al. 2001; Nakayama et al. 2001), as well as the histone fold motif of histone H3 (Nielsen et al. 2001a). HP1s interact with a large number of proteins and in particular with several proteins known to function at the transcriptional level through a specific pentapeptide PxVxL called HP1 box (Le Douarin et al. 1996; Thiru et al. 2004). These proteins include the chromatin remodeling factor BRG1 (Nielsen et al. 2002), the TBP-associated factor TAF130 (Vassallo and Tanese 2002), the retinoblastoma protein Rb (Nielsen et al. 2001b), and the transcriptional intermediary factors TIF1 and TIF1 (Le Douarin et al. 1996; Nielsen et al. 1999). It is currently speculated that HP1 serves as a bridging protein, connecting histones through interaction with the CD to nonhistone chromosomal proteins through interaction with the CSD (Li et al. 2002). We and others have previously demonstrated that the interaction between TIF1 and HP1 is required for the TIF1 transcriptional repression activity, which also requires histone deacetylase activity (Nielsen et al. 1999; Ryan et al. 1999). In mouse embryonal carcinoma (EC) F9 cells that resemble the pluripotent inner cell mass cells of the early embryo and can be induced to differentiate into primitive endoderm-like (PrE) cells or into parietal endoderm-like (PE) cells on treatment with retinoic acid (RA) alone or RA plus cAMP, respectively (Strickland and Mahdavi 1978; Strickland et al. 1980), we have shown that the interaction between TIF1 and HP1 proteins was essential for the relocation of TIF1 from eu- to heterochromatin that accompanies PrE differentiation (Cammas et al. 2002). Thus TIF1–HP1 interaction may play an essential role during cell differentiation.
In this paper, we report the first demonstration of the functional relevance of the interaction between TIF1 and HP1 proteins. We have introduced by homologous recombination in F9 EC cells a mutation in the TIF1 HP1 box motif that had previously been shown to disrupt the interaction between TIF1 and HP1 proteins (Cammas et al. 2002). We demonstrate that TIF1–HP1 interaction is essential for F9 cell differentiation into PE cells and into visceral endoderm-like (VE) cells obtained by treatment of vesicles with RA.
|
Results |
|---|
|
TopAbstractResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
|---|
HP1 box
To disrupt the interaction between TIF1 and HP1 proteins in F9 EC cells, we specifically mutated the HP1 box coding sequence of one TIF1 allele and inactivated the other TIF1 allele. Two targeting vectors, pTIF1LHL:HP1box and pTIF1LNL:L (Fig. 1A), were constructed. pTIF1LNL:L was obtained by introduction of a PGK-Neo selection cassette flanked by two LoxP sites in intron 3 and a LoxP site in intron 14 (Fig. 1A; see Materials and Methods; Cammas et al. 2000). pTIF1LHL:HP1box was obtained by introduction of a PGK-Hygro selection cassette flanked by two LoxP sites in intron 3 and mutation of the TIF1 HP1 box-coding sequence within exon 12 (substitution of two amino acids, V488L490/AA), which was previously shown to disrupt the interaction between TIF1 and HP1, , and (Fig. 1A; Materials and Methods; Cammas et al. 2002). This mutation generated an Eco47III restriction site in the targeted TIF1 allele (E47, Fig. 1A).
|
LNL:L. One G-418-resistant clone, TIF1L3/+, with homologous recombination of a single copy of the targeting vector as assessed by Southern blot with three different probes (3', 5', and Neo probes [Fig. 1A,B]; data not shown) was isolated and was transfected with the pTIF1LHL:HP1box targeting construct. A clone resistant to both G-418 and hygromycin and having integrated a single copy of pTIF1LHL:HP1box into the second TIF1 allele (5' and Hygro probes [Fig. 1B]; PCR analysis with primers flanking the HP1box, followed by digestion with Eco47III [Fig. 1C,D]; data not shown) was isolated and called TIF1L3/L2HP1box. To excise the LoxP-flanked sequences, we transiently transfected TIF1L3/L2HP1box cells with the Cre-recombinase encoding expression vector pPGK-Cre, yielding a clone, TIF1LHP1box/L-, with complete excision in one allele, whereas the other allele had the HP1box motif point mutations (Southern blot analysis with three different probes: 5', Neo, and Hygro [Fig. 1B]; data not shown; PCR analysis followed by Eco47III digestion [Fig. 1C,D]). The heterozygous clone TIF1+/L- was obtained by transfection of TIF1+/L3 cells with pPGK-Cre (Fig. 1A,B).
The level of TIF1 gene expression in both TIF1+/L- and TIF1LHP1box/L- F9 cell lines was compared with the level of TIF1 expression in wild-type F9 cells. A TIF1 monoclonal antibody (mAb) revealed a decrease by 
50% in TIF1+/L- and TIF1LHP1box/L- F9 cells in comparison with wild-type F9 cells. Thus, the HP1 box mutation had no effect on the level of expression of the mutated TIF1 allele (Fig. 1E). TIF1+/L- and TIF1LHP1box/L- F9 cell lines, thereafter designated as TIF1+/- and TIF1HP1box/- cells, respectively, expressed the wild-type (TIF1+) and mutant (TIF1HP1box) proteins, respectively.
The HP1 box mutation has previously been shown to disrupt the interaction between TIF1 and HP1, HP1, and HP1 (Weber et al. 2002). To investigate whether the TIF1HP1box mutant protein was associated with HP1 proteins in TIF1HP1box/- cells, we performed immunoprecipitation (IP). Whole-cell extracts (WCE) from wild-type and TIF1HP1box/- F9 cells were immunoprecipitated with TIF1 mAb. As indicated by Western blot analysis, HP1, HP1, and HP1 were found in wild-type immunoprecipitates, but not in TIF1HP1box/- immunoprecipitates (Fig. 1F). Thus, the TIF1HP1box mutant protein is not associated with HP1 proteins within TIF1HP1box/- cells.
TIF1 interaction with HP1 proteins is not required for PrE cell differentiation
Treatment of wild-type, TIF1+/-, and TIF1HP1box/- cells with 1 µM RA led to an enlargement and flattening of the cells, which are morphological features characteristic of F9 cell differentiation into PrE cells (Fig. 2, panels a–f). To confirm the PrE identity of these cells, we analyzed expression of Troma-1, a marker for F9 cell differentiation (Kemler et al. 1981; Verheijen et al. 1999). Wild-type, TIF1+/-, and TIF1HP1box/- cells were grown for 4 d in the absence or presence of 1µM RA and analyzed by immunocytochemistry. Troma-1 was weakly expressed and localized exclusively in nuclei of nontreated wild-type, TIF1+/-, and TIF1HP1box/- cells (Fig. 3A, panels a,b,e,f,i,j), whereas it was highly expressed and distributed in the cytoplasm of RA-treated cells (Fig. 3A, panels c,d,g,h,k,l), indicating that these cells have features of PrE cells.
|
|
from eu- to heterochromatin (Cammas et al. 2002). The localization of TIF1HP1box was analyzed during PrE differentiation. wild-type, TIF1+/-, and TIF1HP1box/- cells were grown for 4 d in the absence or presence of 1 µM RA and analyzed by immunocytochemistry. In nontreated cells, TIF1 labeling was rather uniformly distributed within the nucleoplasm and largely excluded from the nucleoli of wild-type, TIF1+/-, and TIF1HP1box/- cells (Fig. 3B, panels a,b,d,e; data not shown). After 4 d of RA treatment, wild-type and TIF1+/- cells displayed an enrichment of TIF1 within heterochromatin characterized by bright spots of Hoechst staining, whereas TIF1HP1box remained diffusely distributed throughout TIF1HP1box/- nuclei (Fig. 3B, panels g,h,j,k). The PrE identity of these cells exhibiting diffused distribution of TIF1HP1box was confirmed by expression of Troma-1 (Fig. 3B, panels i,l). Taken together, these results indicate that the interaction between TIF1 and HP1 proteins is not required for PrE differentiation.
TIF1 interaction with HP1 proteins is required for terminal differentiation into PE cells and into VE cells
Wild-type, TIF1+/-, and TIF1HP1box/- cells were treated for 4 d with RA, and then with 250 µM dbcAMP and 1 µM RA for 2 d. wild-type and TIF1+/- cells differentiated into cells exhibiting a very stringent core body and neuronal-like outgrowths that are morphological changes characteristic of PE differentiation (Fig. 2, panels g,h). In contrast, TIF1HP1box/- cells differentiated into PrE cells but did not further differentiate into PE cells (Fig. 2, panels d–f,i). Expression of Troma-1 in these cells confirmed their differentiated status (Fig. 4A, panel f). To investigate the RA + cAMP responsiveness of TIF1HP1box/- cells at the molecular level, we analyzed the expression of thrombomodulin (TM), a molecular marker unique to PE cells (Weiler-Guettler et al. 1992). RNA from wild-type, TIF1+/-, and TIF1HP1box/- cells grown for 6 d with either vehicle or 1 µM RA or for 4 d with 1 µM RA followed by addition of 250 µM dbcAMP for 2 d were analyzed by semiquantitative RT–PCR. No TM expression was detected in either nontreated or RA-treated cell lines (Fig. 4B; lanes 1–6). In contrast, TM expression was induced after treatment with RA-db-cAMP in wild-type and TIF1+/- cells, whereas it could not be detected in TIF1HP1box/- cells (Fig. 4B; lanes 7–9). Thus, in contrast to wild-type and TIF1+/- cells, TIF1HP1box/- cells do not undergo PE differentiation after treatment with RA-dbcAMP. Note that TM expression was lower in TIF1+/- cells when compared with wild-type cells, in agreement with the observation that 20%–30% of the TIF1+/- cell population failed to morphologically differentiate into PE cells, indicating that the correct gene dosage of TIF1 is required for full differentiation into PE cells (data not shown). It is noteworthy that TIF1HP1box/- cells treated for 6 d with RA and dbcAMP added simultaneously were also unable to undergo PE differentiation (data not shown). Altogether, these results demonstrate that differentiation of TIF1HP1box/- cells on RA-dbcAMP treatment is blocked at the PrE stage, and therefore that the interaction between TIF1 and HP1 proteins is essential for differentiation of PrE cells into PE cells.
|
–HP1 interaction for VE differentiation, we grew wild-type, TIF1+/-, and TIF1HP1box/- cells in bacterial Petri dishes to allow formation of vesicles and treated them for 6 d with 50 nM RA as described previously (Casanova and Grabel 1988). Wild-type and TIF1+/- cells treated with RA formed vesicles with an outer layer of large stringent cells typical of VE differentiation (Fig. 2B, panels d,e). In contrast, TIF1HP1box/- cells rapidly degenerated from 2 d (data not shown) to 6 d of RA treatment at a stage at which there were only few residual vesicles left, as illustrated in Figure 2B, panel f. This effect is specific of VE differentiation and not of the growth conditions because TIF1HP1box/- cells grown on bacterial Petri dishes in the absence of RA treatment formed vesicles resembling wild-type and TIF1+/- vesicles (Fig. 2B, panels a–c). These results indicate that the interaction between TIF1 and HP1 is essential for VE differentiation.
We have previously shown and confirmed in the present study that association of TIF1 to heterochromatin was not observed within differentiated PE nuclei (Fig. 4A, panel b; Cammas et al. 2002). However, PE differentiation is a two-step differentiation process including an RA-dependent phase followed by a cAMP-dependent phase. It is therefore conceivable that TIF1 could concentrate within heterochromatin at specific stages during the process of PE differentiation. To address this question, we treated F9 cells for 6 d with either 1 µM RA alone or with 1 µM RA plus 250 µM dbcAMP. Cells were analyzed each day of the differentiation processes by immunofluorescence with a TIF1 mAb. As previously observed (Cammas et al. 2002), RA treatment led to relocation of TIF1 from eu- to heterochromatin in a progressively increasing number of nuclei to reach a maximum of 50% after 4 d of treatment (Fig. 4C). This percentage remained unchanged at all subsequent points (Fig. 4C; data not shown). In the presence of RA plus dbcAMP, TIF1 also relocates from eu- to heterochromatin until 3 d of treatment to reach a maximum of 20%–25%. Thereafter, the percentage of cells with heterochromatic TIF1 decreased to reach 0%–5% after 6 d of PE differentiation (Fig. 4C). The kinetics of replacement of the cell population with heterochromatic TIF1 by a cell population with diffused nuclear TIF1 correlated with the morphological changes associated with the differentiation of PrE cells into PE cells (data not shown). It is noteworthy that the homogenous localization of TIF1 within PE nuclei is also observed when F9 cells are sequentially treated 4 d with 1 µM RA followed by 2 d with 1 µM RA plus 250 µM dbcAMP (data not shown). Altogether, these results demonstrated that TIF1 concentration within heterochromatin is a transient status during PE differentiation, whereas it is permanently established during PrE differentiation. Interestingly, TIF1 relocation from eu- to heterochromatin also occurs during VE differentiation of F9 cells (data not shown). These results suggest that the relocation of TIF1 from eu- to heterochromatin during the RA-treated phase of endodermal differentiation might play a role in the regulation of TIF1 concentration within differentiated cell nuclei. The level of TIF1 and TIF1HP1box was assessed in wild-type and TIF1HP1box/- cells, respectively, during PE differentiation. Western blot analysis with a TIF1 mAb revealed that TIF1 concentration is constant for up to 2 d of RA treatment and then decreases of 10-fold after 4 d of treatment (Fig. 4D, cf. lanes 5 and 1). The level of TIF1 further decreased by 1.7-fold after treatment with dbcAMP (Fig. 4D, lanes 6,7). In TIF1HP1box/- cells, the level of TIF1HP1box was also decreased after 4 d of RA treatment (fourfold) but did not further decrease after RA-dbcAMP treatment (Fig. 4D). This expression of TIF1 within F9 PE cells is in contrast with our previous observation that TIF1 transcript was not detected, by in situ hybridization, within PE and VE layers of embryonic day 6.5 (E6.5) and E7.5 embryos (Cammas et al. 2000). We therefore analyzed TIF1 expression within embryos by immunohistochemistry using the TIF1 pAb, PF64. This study revealed that, as in F9 cells, TIF1 is expressed in both PE and VE of E6.5 and PE of E7.5 embryos (data not shown).
Ectopic expression of TIF1 into TIF1HP1box/- cells restores their ability to differentiate into PE cells
To unequivocally demonstrate that the failure of TIF1HP1box/- cells to differentiate into PE cells reflects the requirement of an interaction between TIF1 and HP1 proteins, we investigated whether ectopically expressed TIF1 or TIF1HP1box mutant proteins into TIF1HP1box/- cells could restore PE differentiation. TIF1HP1box/- cells were transfected with three constructs to perform doxycycline (Dox)-inducible expression of TIF1: (1) an expression cassette encoding the reverse tetracycline-controlled transactivator (rtTA; Gossen et al. 1995); (2) a construct containing the mouse Flag-tagged TIF1 or TIF1HP1box cDNAs (f.TIF1 and f.TIF1HP1box, respectively) under the control of tetracycline operators; and (3) a PGK-Hygro selection cassette-expressing vector (Fig. 5A). Clones resistant to hygromycin were amplified, grown in the presence or absence of 1 µg/mL Dox for 3 d, and analyzed by Western blotting with an anti-Flag mAb. Two cell lines expressing either f.TIF1 or f.TIF1HP1box proteins selectively in the presence of Dox were isolated and designated TIF1HP1box/-/rtTA-f.TIF1 and TIF1HP1box/-/rtTA-f.TIF1HP1box, respectively (Fig. 5B). Western blotting using a TIF1-specific mAb showed that in the presence of Dox, TIF1HP1box/-/rtTA-f.TIF1 cells express a total amount of TIF1 protein similar to wild-type cells, whereas TIF1HP1box/-/rtTA-f.TIF1HP1box cells express a slightly lower level (Fig. 5B, lane TIF1). The ability of these two cell lines to undergo PE differentiation on RA-dbcAMP treatment was analyzed in the absence or presence of 1 µg/mL Dox. TIF1HP1box/-/rTA-f.TIF1 cells treated with RA-dbcAMP in the presence of Dox differentiated into PE cells as assessed by morphological analysis, whereas the same cells differentiated only into PrE cells in the absence of Dox (Fig. 5C, panels f,b). In contrast, TIF1HP1box/-/rTA-f.TIF1HP1box cells treated with RA-dbcAMP in the presence or absence of Dox only differentiated into PrE-like cells (Fig. 5C, panels d,h). These results were confirmed by the expression of the PE marker TM. As expected, TM was expressed in RA-dbcAMP-treated wild-type, but not TIF1HP1box/- cells, independently of the presence of Dox (Fig. 5D, lanes 1–8). In TIF1HP1box/-/rTA-f.TIF1 cells, but not in TIF1HP1box/-/rTA-f.TIF1HP1box cells, TM was induced specifically in the presence of RA-dbcAMP and Dox, thus confirming that ectopic expression of TIF1 enabled TIF1HP1box/- cells to differentiate into PE cells, whereas TIF1HP1box did not (Fig. 5D, lanes 9–16). Note that the level of TM induction in TIF1HP1box/-/rTA-F.TIF1 was reduced in comparison with wild-type cells, confirming that PE differentiation is sensitive to TIF1 dosage.
|
interaction with HP1 proteins is indispensable to enable F9 cells to differentiate into PE cells, whereas it is not required for PrE differentiation.
Interaction between TIF1 and HP1 is essential during RA-induced PrE differentiation to enable further PE differentiation
To establish whether the TIF1–HP1 interaction was required throughout PE differentiation, we took advantage of the temporally controlled inducibility of expression of f.TIF1 cDNA in TIF1HP1box/-/rtTA-f.TIF1 cells. These cells were grown in PE differentiation-inducing conditions: (1) 3 d in the absence of treatment (Fig. 6A, d-3 to d0), followed by (2) 4 d in the presence of 1 µM RA (Fig. 6A, d0 to d4), and finally (3) 2 d in the presence of 1 µM RA plus 250 µM dbcAMP (Fig. 6A, d4 to d6). f.TIF1 expression was induced in these cells by addition of 1 µg/mL Dox at various times of the differentiation process (Fig. 6A). Western blot analysis using an anti-Flag antibody revealed that f.TIF1 was expressed during different phases of the differentiation process (Fig. 6B). Actin was used as an equal loading control (data not shown).
|
expression, we performed morphological analysis in the previously described growing conditions (Fig. 6A), with cell dilutions allowing us to count individually each clone arising from single cells. The percentage of clones containing PE cells in each growing condition is plotted in Figure 6C. Note that in the same experimental conditions, 100% of wild-type cells underwent PE differentiation (data not shown). This morphological analysis indicated that no PE cells were detected in TIF1HP1box/- cells expressing TIF1 only from d-2 to d1 (Fig. 6B,C, lane 3), whereas PE differentiation was induced in cells expressing TIF1 from d0 (Fig. 6B,C, lane 8). Therefore, expression of f.TIF1 before RA treatment is neither necessary (Fig. 6B,C, lanes 7,8) nor sufficient (Fig. 6B,C, lane 3) to enable TIF1HP1box/-/rtTA-f.TIF1 cells to undergo PE differentiation. In contrast, TIF1 must be expressed between at least the first and second day of RA treatment in order for PE differentiation to occur (Fig. 6B,C, lanes 5–9). Strikingly, cells that expressed TIF1 only after 2–3 d of RA treatment were mostly blocked at the PrE stage of differentiation (Fig. 6B,C, lanes 10,11), demonstrating that the TIF1–HP1 interaction was essential for PE differentiation well before the addition of dbcAMP. This morphological analysis was confirmed at the molecular level by the expression of TM, which was only induced in cells expressing f.TIF1 between at least the first and second day of RA treatment (Fig. 6D, lanes 5–9).
Altogether, these results indicated that the essential role of the interaction between TIF1 and HP1 proteins for PE differentiation is restricted to an early time window during the initial RA-dependent phase of the differentiation process.
Interaction between TIF1 and HP1 is essential to regulate expression of endoderm-specific genes
To further investigate the RA responsiveness of TIF1HP1box/- cells, we analyzed the expression of several genes, including (1) genes known to play essential roles in mouse endoderm differentiation: the transcription factor genes GATA6 (Morrisey et al. 1998), HNF4 (Chen et al. 1994), and Oct3/4 (Niwa et al. 2000) and the signal transduction adapter protein Disabled-2 (Dab2; Yang et al. 2002); and (2) genes whose expression is known to be induced by RA treatment of F9 cells: the transcription factor genes RAR2 and Hoxb1, and genes encoding the components of the extracellular matrix procolIagen type IV (ColIV) and laminin B1 (LamB1; Harris and Childs 2002). Wild-type, TIF1+/-, and TIF1HP1box/- cells were either not treated or treated for 24 h or 48 h with 1 µM RA. These time points corresponded to the window of time during which TIF1–HP1 interaction is indispensable for PE differentiation. Expression was analyzed by semiquantitative RT–PCR analysis. We found that, in the absence of RA treatment, only two genes, HNF4 and Oct3/4, had a detectable level of expression and were both down-regulated in TIF1HP1box/- cells compared with wild-type cells (seven- and threefold, respectively; Fig. 7, lanes 1–3). For Oct3/4, the difference between wild-type and TIF1HP1box/- cells was maintained constant in the presence of RA treatment, even though, as expected, Oct3/4 expression was repressed by RA treatment in wild-type and TIF1+/- cells (2.5-fold after 24 h of RA treatment; Fig. 7, cf. lanes 4,5 and 1,2). For all of the other genes tested, RA induced their expression in wild-type, TIF1+/-, and TIF1HP1box/- cells (Fig. 7, cf. lanes 4–9 and 1–3). However, the extent of induction was highly dependent on cell genotypes. After 24 h of RA treatment, GATA6 and HNF4 expression was, respectively, four and three times lower in TIF1HP1box/- cells in comparison with wild-type cells, whereas the expression of all of the other genes was not significantly different between the two cell lines. After 48 h of RA treatment, GATA6, Dab2, HNF4, ColIV, and lamB1 displayed reduced expression in TIF1HP1box/- cells in comparison with wild-type cells (Fig. 7; eight-, seven-, 40-, 1.5-, and twofold, respectively). In sharp contrast, expression of RAR2 was equivalent in wild-type and TIF1HP1box/- cells, and Hoxb1 expression was higher in TIF1HP1box/- cells in comparison with wild-type cells (Fig. 7, threefold).
|
and HP1 proteins is essential to regulate the expression of specific genes in both nondifferentiated and differentiated F9 cells, and in particular is critical for the induction of endoderm master players in response to RA treatment. |
Discussion |
|---|
|
TopAbstractResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
|---|
relocates from eu- to heterochromatin during RA-induced PrE differentiation of F9 cells via its interaction with HP1 proteins (Cammas et al. 2002). In this study, we demonstrate, by specifically mutating in EC F9 cells the HP1-interacting PxVxL motif of TIF1, that the association between TIF1 and HP1 proteins is essential for F9 cells to differentiate into PE and VE cells.
The TIF1–HP1 association is required for progression through differentiation
Differentiation is a highly regulated process that comprises different phases including self-renewal, commitment, initiation, and progression through terminal differentiation. These different phases correlate with specific combinations of gene induction and repression orchestrated by a limited number of master players (for review, see Fisher 2002). When grown as a monolayer, EC F9 cells treated either sequentially or in combination with RA plus cAMP differentiate first into PrE cells, and then into terminally differentiated PE cells (Strickland et al. 1980). This progression through differentiation is marked by characteristic morphological changes as well as by a bimodal expression pattern of hundreds of genes (Harris and Childs 2002). We have shown here that the disruption of the interaction between TIF1 and HP1 does not affect the ability of EC F9 cells (TIF1HP1box/- cells) to morphologically differentiate into PrE cells, but completely abrogates their capacity to differentiate into PE cells on further treatment with cAMP, indicating that the TIF1–HP1 interaction is not required to initiate differentiation, but is essential at later stages to allow terminal differentiation. Furthermore, using a temporally controlled system of TIF1 expression to re-establish the interaction between TIF1 and HP1 proteins within TIF1HP1box/- cells, we have established that (1) TIF1 expression during the noninduced phase up to the first day of RA treatment, or during a period extending from the second day after RA treatment through the end of the differentiation process, is neither necessary nor sufficient to allow TIF1HP1box/- cells to differentiate into PE cells; and (2) TIF1 expression during the noninduced phase up to the second day of RA treatment or during a period of time extending from the first day of RA treatment through the end of the differentiation process enables TIF1HP1box/- cells to differentiate into PE cells. Altogether, these results indicate that (1) the interaction between TIF1 and HP1 proteins is essential only during a short window of time early within differentiating PrE cells, whereas it is not required during the cAMP-dependent phase of PE differentiation; and (2) the deleterious effect of the TIF1 HP1box mutation on PE cell differentiation, once established in PrE early differentiating cells, cannot be corrected by re-establishing the interaction between TIF1 and HP1 proteins. This strongly suggests that the TIF1–HP1 association is required within early differentiating PrE cells to establish a selective, transmittable competence to respond to the further cAMP differentiation signal.
TIF1–HP1 interaction is involved in control of expression of endoderm-specific genes
Gene expression analysis in differentiating PrE cells has revealed that expression of GATA6, HNF4, Dab2, and Oct3/4 is severely down-regulated in the absence of interaction between TIF1 and HP1 proteins, whereas the expression of RAR2 and Hoxb1 was either not modified or moderately increased, respectively. In keeping with the competence of TIF1HP1box/- cells to morphologically differentiate into PrE cells, these data demonstrate that the TIF1–HP1 association does not play a general role in F9 cell RA responsiveness, but rather is critical for the regulation of a specific set of genes. Of particular interest, GATA6, HNF4, Oct3/4, and Dab2, but not RAR2 or Hoxb1, have been shown to play essential roles in endoderm differentiation during early mouse embryogenesis (Chen et al. 1994; Gavalas et al. 1998; Morrisey et al. 1998, Dupe et al. 1999; Koutsoukaris et al. 1999; Niwa et al. 2000; Yang et al. 2002). Mice having either GATA6, HNF4, or Dab2 genes inactivated can develop normally until the blastocyst stage displaying normal PrE differentiation, but fail to gastrulate with specific defects into extraembryonic visceral endoderm cells, demonstrating that none of these factors is essential for primitive endoderm differentiation, but rather they are master players for the progression of extraembryonic endoderm differentiation (Chen et al. 1994; Morrisey et al. 1998; Koutsoukaris et al. 1999; Yang et al. 2002). We therefore propose that the inability of TIF1HP1box/- cells to differentiate into PE cells is a consequence of the down-regulation of one of these genes. Furthermore, differential expression of HNF4 between wild-type and TIF1HP1box/- cells is detectable prior differential expression of GATA6, although it has previously been shown that GATA6 is epistatic to HNF4 during endoderm differentiation (Morrisey et al. 2000), strongly suggesting that GATA6 and HNF4 genes are independently down-regulated in TIF1HP1box/- cells. From the available literature and our present findings, we propose that the interaction between TIF1 and HP1 proteins plays an essential role during PrE differentiation for proper induction of GATA6, HNF4, and/or Dab2 that are essential for PE and VE differentiation. We have previously shown that TIF1 is not critical for the formation of either PE- or VE-like layers within early embryos (Cammas et al. 2000). Whether TIF1 is required for appropriate differentiation of embryonic PE cells remains to be determined, but we have clearly shown that TIF1 is critical for the complete differentiation of the VE layer into squamous cells (Cammas et al. 2000), strongly suggesting that the inappropriate endoderm differentiation ability of TIF1HP1box/- F9 cells has some functional relevance.
The TIF1–HP1 association represses a factor that negatively controls the expression of endoderm-specific genes
How are TIF1–HP1 interactions involved in the control of expression of endoderm-specific genes? From studies showing that TIF1 interacts with the transcriptional repressors KRAB-ZFPs and recruits histone deacetylase and methyltransferase complexes together with HP1 proteins to specific chromatin sites, it is currently assumed that TIF1 acts as a corepressor of KRAB-ZFPs (Ayyanathan et al. 2003; and see introduction). It is therefore unlikely that the TIF1–HP1 association directly regulates the expression of the genes that are down-regulated in TIF1HP1box/- cells. Rather, we propose that the TIF1–HP1 association indirectly controls the expression of these endoderm-specific genes by repressing the expression of a factor(s) that normally down-regulates their expression. This factor(s) could belong to different classes of regulators: (1) It could be a transcriptional repressor, the expression of which would maintain the expression of endoderm-specific genes either low (HNF4) or fully repressed (GATA6) in nondifferentiated cells, whereas its repression by TIF1–HP1 after RA treatment would allow the induction of endoderm-specific gene expression. Similar mechanisms of regulation of differentiation progression by transcriptional repressors have been described for different differentiation pathways. For example, the repressor REST (Ballas et al. 2001) and the nuclear receptor corepressor N-CoR (Hermanson et al. 2002) play essential roles in neural stem cell self-renewal and their decreased expression led to neuronal and astroglial cell differentiation, respectively. The transcription factor Pax-5 has been shown to play essential roles in B-lineage commitment by repressing lineage-promiscuous transcription (Nutt et al. 1999; Mikkola et al. 2002); (2) It could correspond to factor(s) that inactivate, either by posttranslational modification or by degradation, an activator(s) of endoderm-specific gene expression. In this respect, we note that RXR phosphorylation has been shown to be crucial for the expression of several RA-responsive genes in F9 cells (Bastien et al. 2002) and also that the ligand-dependent degradation of RARs and RXRs by the ubiquitin proteasome pathway could be important for the magnitude and duration of the effect of retinoid signals in F9 cells (Kopf et al. 2000). (3) It could be a chromatin silencing factor(s) such as histone methyltransferase, deacetylase, and/or DNA methyltransferase, which have all been shown to be involved in control of embryonic development and cellular differentiation (for reviews, see Ansel et al. 2003; Ehrlich 2003). The identification of the genes that are misexpressed in TIF1HP1box/- cells during the early phase of PrE differentiation will allow discrimination between these various possibilities.
It is noteworthy that the window of time during which the TIF1–HP1 interaction is essential for progression through differentiation strikingly correlates with the normal onset of TIF1 relocation from eu- to heterochromatin domains (Cammas et al. 2002). We therefore propose that, in the presence of RA, TIF1–HP1 triggers the translocation of the direct target genes from eu- to heterochromatin for their silencing. In this respect, we note that Matsuda et al. (2001) have shown the colocalization of two KRAB-ZFP proteins, KRAZ1 and KRAZ2, with TIF1 and HP1 proteins within pericentromeric heterochromatin of a fibroblast population. We note also that the translocation of specific euchromatic genes near or within heterochromatin has been proposed for the transcription factor Ikaros that regulates the expression of genes involved in T-cell activation (Brown et al. 1997, 1999).
In conclusion, this report is the first demonstration that the interaction between TIF1 and HP1 proteins plays a critical role in vivo, that is, for the progression of F9 cells through terminal differentiation. Although EC F9 cells represent a relatively simple model of differentiation, it is likely that the regulatory mechanism uncovered in this study will apply to other differentiation pathways. It is indeed noteworthy that we have also observed TIF1 relocation from eu- to heterochromatin during differentiation of ES cells into cardiomyocytes and neuronal cell types (Cammas et al. 2002), which suggests that the TIF1–HP1 association is also playing a pivotal role in these differentiation pathways.
|
Materials and methods |
|---|
|
TopAbstractResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
|---|
Construction of the targeting vector
The targeting vector pTIF1LNL:L for inactivation of the first TIF1 allele was described previously (Cammas et al. 2000), with the addition of a positive selection cassette containing the coding sequence for the diphterin toxin A (DTA) under the control of the TK promoter (DTA cassette; McCarrick et al. 1993; gift from J.W. McCarrick, Wistar Institute, Philadelphia, PA) at the 3' end of the construct. The pTIF1(LNL:L) ClaI fragment was ligated in the ClaI site of pSL1190-DTA to give pTIFLNL:L. To mutate the HP1 box motif on the second allele of TIF1, we exchanged the PGK-Neo cassette of pTIF1(LNL:L) with a PGK-Hygro (gift from D. Metzger, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France) and introduced the previously described HP1 box mutation (Cammas et al. 2002) in the TIF1 gene. The HindIII PGK-HygroMod fragment was ligated in purified HindIII TIF1B11 fragment to give TIF1B40 (see Cammas et al. 2000). The double mutation in the HP1 box of TIF1 (leading to the V488A/L490A substitutions) was generated by site-directed PCR mutagenesis with the following internal primers (the codon change is shown in boldface): ABP88 (5'-GAAGGTGCCACGCGCGAGCGCTGAAC GCCTGG-3') and ABP89 (5'-CCAGGCGTTCAGCGCTCGC GCGTGGCACCTTC-3'). The mutation in the HP1 box was introduced by PCR in TIF1B4 (see Cammas et al. 2000). Two PCR reactions were first performed with oligonucleotides ACC242 (5'-CACCAGGAACACATTTTGGCG-3') and ABP89 and with oligonucleotides ACC243 (5'-GGGACGGCATG GTTCATGGC-3') and ABP88 using TIF1B4 as template. These two PCR fragments were then mixed and used as the template for a third PCR with oligonucleotides ACC242 and ACC243. This PCR fragment was then digested with EcoRI and NdeI and ligated in NdeI/EcoRI-digested TIF1B4 plasmid, to give TIF1B41. The 4.5-kb XhoI/XhoI fragment from TIF1B41 was then cloned in the XhoI site of TIF1B40 to give TIF1B23. The DTA cassette was added to this construct as described earlier to give pTIF1LHL:L.
For the temporally controlled expression of the Flag-tagged TIF1 and TIF1HP1box cDNAs, the reverse tetracycline system was used. The expression vector pDG1-rtTA, in which the expression of the rtTA (Gossen et al. 1995) is under the control of the PGK promoter, was a gift from Dr. Metzger. The Flag-tagged TIF1 and TIF1HP1box cDNA EcoRI fragments from pCX-Flag-TIF1 and pCX-Flag-TIF1HP1box (Cammas et al. 2002
