Oncogene (2005) 24, 6993–7001 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc ORIGINAL PAPERS acts as Pax 8 transcriptional coactivator

Stefania Miccadei1,4, Claudia Provenzano2,4, Martin Mojzisek1,3, Pier Giorgio Natali1 and Donato Civitareale*,1,2

1Molecular Pathology Laboratory, Regina Elena Institute, Via delle Messi d’Oro 156, 00158 Rome, Italy; 2Institute of Neurobiology and Molecular Medicine, National Council Research, Via del Fosso del Cavaliere 100, 00133 Rome, Italy; 3Department of Medical Biology and Genetics, Charles University, Faculty of Medicine in Hradec Kra´love´, Simkova 870, 500 01 Hradec Kralove, Czech Republic

Control of cell proliferation and differentiation by the binding partners. It interacts with the virally encoded retinoblastoma protein (pRb) depends on its interactions transforming proteins Large T antigen (Tag), E1A and with key cellular substrates. Available data indicate that E7 of the DNA tumor viruses SV40, adenovirus and pRb and the factor Pax 8 play a crucial role human papilloma virus (Chellappan et al., 1992). in the differentiation of thyroid follicular cells. In this in the pRb-binding domain prevent cellular study, we show that pRb takes part in the complex transformation by these transforming viral proteins. assembled on the thyroperoxidase acting as These results led to the preliminary conclusion that pRb a transcriptional coactivator of Pax 8. Accordingly, pRb binds E2F transcription factors and negatively regulates interacts with and potentiates Pax 8 transcriptional their activity in cell cycle progression (Nevins, 2001). activity. In addition, we show that the downregulation of Like Large Tag, E1A and E7, E2F binds only the pRb , in thyrocytes, through RNA hypophosphorylated forms of pRb present during the interference results in a reduction of the thyroperoxidase G1 phase of the cell cycle. Thus, phosphorylation of gene mediated by the Pax 8-binding site. pRb, mainly mediated by cyclinD/cdk4 and cyclin In agreement with these results and with the ability of the E/cdk2, increases the activity of promoters containing adenoviral protein E1A to bind pRb, we show that E1A the E2F-binding sites and thereby induces the transcrip- downregulates Pax 8 activity and that such inhibition tion of several important involved in cell cycle requires the E1A–Rb interaction. Furthermore, we show regulation (Adams, 2001). Thus, most of the growth that the Pax 8/pRb synergy plays a role on the sodium/ suppressive properties of pRb and of its related ‘pocket iodide symporter gene expression as well. proteins’ p107 and p130 are mediated through their Oncogene (2005) 24, 6993–7001. doi:10.1038/sj.onc.1208861; modulation of the activity of E2F factors (Harbour and published online 27 June 2005 Dean, 2000). Furthermore, a regulatory loop has been proposed as one of the mechanisms by which pRb Keywords: Pax 8; retinoblastoma protein; thyroid; couples permanent exit from mitosis with tissue-specific transcription; coactivator gene expression. pRb also induces the expression of p21 (Decesse et al., 2001). This cell cycle-dependent kinase inhibitor leads to inactivation of multiple cyclin/cdk complexes and allows pRb to remain hypophosphoryl- ated and active. Introduction Studies with cells have shown that the ability of MyoD and other members of the basic helix– Terminal differentiation invariably involves two closely loop–helix family of myogenic transcription factors to linked phenomena: permanent withdrawal from the cell regulate differentiation depends on the presence of pRb cycle and biochemical differentiation characterized by (Puri and Sartorelli, 2000). Similarly, pRb positively the tissue-specific gene expression. The retinoblastoma regulates terminal adipocyte differentiation through gene product, pRb has been implicated in mediating direct interaction with C/EBPs (Chen et al., 1996a). In both the permanent cell cycle arrest and upregulation of addition, in hematopoietic cells, it regulates NF-IL6 tissue-specific genes in a wide variety of tissues (Wein- activity (Chen et al., 1996b). Furthermore, it has been berg, 1995; Lipinski and Jacks, 1999). In deciphering the demonstrated that, in osteogenic differentiation, the function of pRb, it has been crucial to identify its central role exerted by the CBFA1 requires pRb and that loss of pRb blocks late osteoblast *Correspondence: D Civitareale, Institute of Neurobiology and differentiation. Coexpression of CBFA1 and pRb results Molecular Medicine, Italian National Council Research, Via del Fosso in the association of both proteins with an osteoblast- del Cavaliere 100, 00133 Rome, Italy; specific promoter in vivo and consequent transcriptional E-mail: [email protected] 4These two authors contributed equally to this work activation. Thus, in osteoclast cells, pRb works as a Received 1 July 2003; revised 16 May 2005; accepted 18 May 2005; positive transcriptional coactivator (Thomas et al., published online 27 June 2005 2001). Role of pRb in thyroid-specific gene expression S Miccadei et al 6994 Iuliano et al. have reported that pRB plays a pivotal amino acids 379–928) fusion protein was expressed in role in thyroid cell differentiation and transformation. Escherichia coli and bound to the glutathione-Sepharose They have shown that the interaction of E1A with pRb resin (Amersham-Pharmacia). On this affinity column, is necessary to block differentiation, while an E1A we have loaded the protein cell extract from HeLa cell mutant unable to bind pRb does not affect thyroid cell transfected with Pax 8-expressing vector. We collected differentiation. Furthermore, they have demonstrated three fractions from the column: the flow-through, that pRb is required for the expression of the thyroid the wash and the elution step at 1 M NaCl. To identify differentiation markers thyroglobulin (Tg) and thyro- the fraction containing Pax 8, they were subsequently peroxidase (TPO) (Iuliano et al., 2000). analysed in a band-shift assay. As shown in Figure 1A, The activity of Pax 8 is crucial in these pathways being Pax 8 was identified in the elution step at high salt a paired-domain-containing transcription factor with a concentration. In a control experiment, where the key role in thyrocyte differentiation (Pasca di Magliano affinity column was prepared with the GST protein et al., 2000). Pax 8 gene knockout mice have smaller only, hence in absence of pRbLP, Pax 8 was identified in thyroids with normal calcitonin-producing parafollicular the flow-through fraction (data not shown). In addition, C cells but no follicular cells, thus they suffer from severe to demonstrate that pRb and Pax 8 directly interact, hypothyroidism (Mansouri et al., 1998). Furthermore, in Figure 1B we show the pull-down experiments in humans, mutations in the Pax 8 gene are associated performed using the chimeric protein GST-pRbLP and with dysgenesis of the thyroid gland (Macchia et al., the in vitro translated Pax 8, Figure 1B, lane WT. Using 1998). It has been shown that Pax 8 activates the gene the same assay and deletion mutants of Pax 8, we have expression of the thyrocyte differentiation marker been able to identify in the C-terminal region of Pax 8, genes: Tg, TPO and sodium/iodide symporter (NIS) the domain involved in the interaction, Figure 1B, lane gene (Fabbro et al., 1998; Zannini et al., 1992; Esposito Dad. Therefore, differently from the pRb/Pax 5 interac- et al., 1998; Ohno et al., 1999). The Pax 8 role in TPO tion where pRb binds the residual homeodomain of Pax gene transcription is very well characterized. Both TPO 5 (Eberhard and Busslinger, 1999), our experiment gene promoter and TPO element are bound suggests that pRb interacts with the activation domain and activated by Pax 8 (Zannini et al., 1992; Esposito of Pax 8, Figure 1B. As shown in Figure 1B, lane DHD, et al., 1998). Recently, it has been described that Pax 8 Pax 8 deleted of the residual homeodomain is still able and a second tissue-specific transcription factor termed to interact with pRbLP, although the reduced intensity thyroid transcription factor 1 (TTF-1) act synergistically of the Pax 8 band (lane 3) compared to the input (lane 1) in the activation of both TPO and Tg gene transcription would suggest that the DHD domain could partially (Miccadei et al., 2002; Di Palma et al., 2003). contribute to the interaction. Moreover, to verify the Furthermore, we have previously reported that Pax 8 interaction in vivo, we expressed the two proteins in activation of the TPO promoter requires, as a transcrip- transiently transfected HeLa cells. The Pax 8–pRb tional coactivator, p300 (De Leo et al., 2000). complexes were immunoprecipitated with an anti-Pax 8 Based on the above-mentioned findings, pointing to a antibody from the HeLa cells extracts and detected by possible role of pRb as transcriptional coactivator and Western blot analysis with an anti-Rb antibody, regulator of TPO gene expression, we have envisaged Figure 1C, lane b. In Figure 1C, lane a, is shown the that Pax 8 and pRb could act cooperatively. This same experiment using unspecific IgG in the immuno- hypothesis is also supported by studies that show a precipitation. Furthermore, in order to demonstrate that direct interaction between pRb and factors containing the pRb/Pax 8 interaction is not the result of the high paired-like homeodomains (Wiggan et al., 1998). Inter- protein concentration achieved in transfected Hela cells, estingly, Eberhard and Busslinger (1999) have demon- the antibody against pRb conjugated with agarose beads strated the direct interaction between Pax 5 and pRb (Santa Cruz Biothecnology) was used to precipitate the both in vitro and in vivo and it has been reported that endogenous pRb from nuclear proteins extracted from pRb interacts with Pax 2 (Yuan et al., 2002). It is worth FRTL-5 cells. This is a stable cell line, derived from rat to point out that structure–function analysis revealed thyrocytes, able to maintain in culture the differentiated that Pax 2, Pax 5 and Pax 8 proteins are structurally and characteristic of the follicular thyroid cells (Ambesi- functionally very similar (Dorfler and Busslinger, 1996). Impiombato et al., 1980). The FRTL-5 nuclear extract We show here that Pax 8 and pRb not only do interact and the immunoprecipitated proteins were analysed by but that pRb acts as a Pax 8 transcriptional coactivator Western blot with the Pax 8-specific antibody, on the TPO gene promoter. Figure 1C, lane c and lane d, respectively. The pRb/ Pax 8 complex is demonstrated by the presence of the Pax 8 in the immunoprecipitated proteins. Hence, the Pax8/pRb complex is present in vivo in the thyrocytes Results nucleous. Pax 8/pRb interaction Pax8/pRb synergism In order to provide experimental evidence for the Pax 8/ pRb protein–protein interaction, we have followed a In order to assign a functional role to the Pax 8/pRb biochemical approach. A GST-RbLP (glutathione S- interaction, we took advantage of the strong activity transferase fused to the large pocked of human pRB, shown by Pax 8 on the TPO promoter in non-thyroid

Oncogene Role of pRb in thyroid-specific gene expression S Miccadei et al 6995 HeLa cells (Zannini et al., 1992). As shown in Figure 2a, show that Rb does not cooperate, in the tranfection cotransfection of a plasmid containing the luciferase assays, with Pax 8 in the activation of the Tg gene reporter gene under the control of the TPO gene promoter, whereas it enhances Pax 8 activity on NIS promoter (TPO-Luc, De Leo et al., 2000) with the Pax gene transcription. Hence, the Pax 8/pRb synegy is 8 encoding plasmid results in strong activation of the promoter specific. TPO promoter. Cotransfection of pRb and TPO-Luc Since the adenoviral protein E1A has been shown to results in very weak reporter gene expression, whereas block thyrocyte differentiation (Berlingieri et al., 1993), the combined activity of pRb and Pax 8 results in to downregulate TPO gene expression (Iuliano et al., cooperative activation of TPO gene promoter activity. 2000) and to bind the pRb pocket domain (Whyte et al., This synergism requires Pax 8 bound to the promoter 1988), we wondered whether E1A and Pax 8 could since a mutated TPO promoter, TPO-Pm, which does compete for the binding of pRb. To test this hypothesis, not bind Pax 8 (Zannini et al., 1992) cannot be activated we overexpressed, in transient transfection experiments by Pax 8 and the coexpression of pRb and Pax 8 does in HeLa cells, E1A and Pax 8. As shown in Figure 2c, not affect TPO-Pm promoter activity (data not shown). E1A inhibits Pax 8 trans-activation whereas the E1A As shown in Figure 2a, pRb synergy with Pax 8 is dose mutant (E1AM), unable to interact with pRb, does not dependent and is not limited to HeLa cells but can also affect Pax 8 activity. To further support our hypothesis, be demonstrated in SAOS-2 cells, Figure 2a. we show that the E1A inhibition of the Pax 8 activity is Since Pax 8 activates the Tg and NIS gene expression reduced by the forced expression of pRb in a dose- (Fabbro et al., 1998; Ohno et al., 1999), we addressed dependent way. Thus, E1A inhibition of Pax 8 activity is the question whether pRb is required on these gene mediated by pRb. promoters as well as on TPO promoter. In Figure 2b we In Table 1, we list the values obtained from transfection experiments in HeLa cells using the phosphorylation mutant of pRb, pRbDcdk (Lukas et al., 1999) and the other pocket proteins (Grana et al., 1998). Although to different extents, both p130 and p107 can cooperate with Pax 8. In addition, the results with the pRbDcdk mutant suggest that the pRb nonphosphorylated form can cooperate with Pax 8. This result is in agreement with the pull-down experiments shown in Figure 1 where the bacterially expressed pRb binds Pax 8.

Figure 1 In vitro and in vivo interaction of Pax 8 and pRb. (A) Band-shift assay of the input protein nuclear extract and fractions of the GST affinity column. Lane 1 shows the band-shift assay with the nuclear extract from HeLa cells overexpressing Pax 8. Lane 2 shows the same assay with nuclear extract from HeLa cells transfected with an empty vector. Lanes 3, 4 and 5 show the band- shift assays performed with the flow-through, wash and high salt elution step, respectively, of the GST affinity column loaded with the nuclear extract from HeLa cells overexpressing Pax 8. (B) Pull- down experiments with the full-length Pax 8 (lane WT); with Pax8Dad (lane Dad) and with Pax8DHD (lane DHD). Row 1 shows 10% of the input protein. Row 2, pull-down experiment with the GST control protein. Row 3, pull-down experiment with the GST- pRbLP protein. Schematic diagram of the Pax 8 full-length and mutants are shown on the right. The paired domain (pd), octapeptide (o), residual homeodomain (hd) and activation domain (ad) are indicated as well. In the Supplementary Information, we provide further controls to the pull-down experiment. (C) In vivo interaction of Pax 8 and pRb. Cell lysates from HeLa over- expressing both Pax 8 and pRb were precipitated with unspecific IgG, lane a, or with Pax 8 specific antibody, lane b. The immunoprecipitated proteins were analysed by Western blot and the filter was probed with the pRb-specific antibody C15. The arrowheads indicate the migration of the molecular weight markers of 85 and 118 kDa as indicated. In lane d, we show the co- immunoprecipitation from 10 mg of FRTL-5 nuclear extracts using the beads-conjugated anti-Rb antibody. The immunoprecipitated proteins were analysed by Western blot and the filter was probed with the Pax 8-specific antibody. As control, we show in lane c the Western blot of the FRTL-5 nuclear proteins probed with the same Pax 8 antibody. The arrowheads indicate the migration of the molecular weight markers of 47 and 85 kDa, as indicated

Oncogene Role of pRb in thyroid-specific gene expression S Miccadei et al 6996

Figure 2 pRb cooperates with Pax 8 in TPO gene promoter activation and it is required for E1A inhibition of Pax 8 activity. HeLa cells were transiently transfected with the TPO-Luc plasmid and were cotransfected with the vector encoding the indicated proteins. The relative luciferase activity of the cells transfected with TPO-Luc only was normalized to 1, and the other activities are expressed relative to this. Values of fold of activation and standard deviations are reported. The amount of the transfected plasmids is reported in the Meterials and methods section unless specifically indicated in the figure. (a) We show the synergy between Pax 8 and pRb in the activation of TPO gene promoter. Furthermore, we show the Pax 8/pRb synergy in SAOS-2 cells as well. Data obtained with HeLa cells are shown with black bar-graphs and the experiments with SAOS-2 cells are shown in gray. The results from three independent experiments are reported. (b) We have tested, in transfection experiments in HeLa cells, the role of pRb on Pax 8 activity on the thyroid-specific gene promoters, Tg and NIS. The plasmid containing the NIS transcriptional regulatory elements in front of the luciferase gene, termed pNISLUC9, was kindly provided by R Di Lauro, Naples (Ohno et al., 1999). The standard deviations were in the same range as those reported in the experiments shown in Figure 2a; they have been omitted because they are negligible. (c) Pax 8 activity is downregulated by E1A and such inhibition requires the E1A capability to bind pRb. E1AM is the point mutant of E1A gene at nucleotide 928 inhibiting the pRb binding (Moran, 1993). The pRb-encoding vector was tranfected at 10 ng per well and the experiment with 50 ng is indicated. The results from three independent experiments are reported

pRb binds on and is required for the activity of the we have performed chromatin immunoprecipitation thyroid-specific TPO promoter (ChIP) experiments using subconfluent FRTL5 cells. In Figure 3a we show that an anti-pRb antibody In order to further substantiate the role of pRb as Pax 8 immunoprecipitates, from FRTL5 chromatin, the TPO coactivator, we show by that pRb is involved in the gene promoter whereas the same antibody does not complex assembled on the TPO gene promoter. Hence, precipitate neither a GAPDH gene sequence nor the

Oncogene Role of pRb in thyroid-specific gene expression S Miccadei et al 6997 Table 1 Role of pRbDcdk, p130 and p107 on Pax 8 activity Reporter vector FA +Pax 8 FA +Pocket protein FA +Pax8+pocket protein FA

TPO-Luc 1 Pax 8 27.5 pRb 1.6 Pax 8+pRb 59.7 TPO-Luc 1 Pax 8 28.6 pRbDcdk 1.4 Pax 8+pRbDcdk 55.0 TPO-Luc 1 Pax 8 27.9 p130 1.8 Pax 8+p130 46.1 TPO-Luc 1 Pax 8 26.9 p107 1.4 Pax 8+p107 38.2

We have performed, in HeLa cells, the same experiments shown in Figure 2a expressing the other pocket proteins p130 or p107 or pRbDcdk. The results with pRb are reported as well. FA stands for fold of activation. Similarly to the experiments shown in Figure 2, the FA of the reporter vector alone was arbitrarily set to 1. The standard deviations were in the same range as those reported in the experiments shown in Figure 2; they have been omitted because they are negligible

Table 2 pRb downregulation, in FRTL5 cells, decreases TPO promoter activity via the Pax 8-binding site Transfected vectors Luc gene expression

pGL3 Promoter+pSUPER 100 pGL3 Promoter+pSUPER-iRbA 9878 pTPO-Luc+pSUPER 100 pTPO-Luc+pSUPER-iRbA 3976 pTPO-Pm-Luc+pSUPER 100 pTPO-Pm-Luc+pSUPER-iRbA 105710

The FRTL5 cells were transiently transfected with the indicated luciferase-encoding plasmids. Each plasmid was cotransfected with pSuper-iRbA or with pSuper as control. The luciferase activity measured in the control experiments was set as 100%,, and the luciferase activity in the experiments with pSuper-iRbA refers to the respective control. The means of the row luciferase/b-galactosidase data for pGL3 Promoter, pTPO-Luc and pTPO.Pm-Luc in the control experiments were 850 000/260, 20 000/260 and 3000/280, respectively. We show, as Supplementary Information, some experiments that we have performed to control the pSuper-iRbA activity, Figures 4 and 5 Figure 3 pRb is involved in the complex assembled on the TPO gene promoter. Chromatin from crosslinked subconfluent FRTL5 cells was immunoprecipitated with anti-pRb antibody. (a) GAPDH, lanes 1 and 4, or TPO promoter, lanes 2 and 5, or TSHr promoter sequences were detected, by PCR analysis, using show that pSUPER-iRbA does not affect luciferase gene input DNA, lanes 1, 2 and 3, or immunoprecipitated DNA, lanes 4, expression controlled by SV40 promoter in pGL3 5 and 6. (b) As control of the PCR conditions utilized in ChIP Promoter vector but downregulates the same gene experiments, we show that there is linearity between the PCR expression when transcription occurs under the TPO products and the amount of DNA template (input DNA). Lane 1, gene promoter control. The same interfering plasmid 2 and 3 are with 0.5, 1.0 and 2.0 ml of input DNA, respectively does not inhibit the activity of the TPO gene promoter mutated in the Pax 8-binding site, TPO.Pm (Zannini et al., 1992). This suggests that in thyrocytes, pRb stimulates TPO gene transcription through Pax 8 promoter of the thyroid-specific TSH receptor gene. activity. We used these negative controls because the GAPDH gene expression is not activated by pRb and the TSHr gene promoter is not activated by Pax 8 (Civitareale et al., 1993; Decary et al., 2002). Figure 3b shows the Discussion linear dependence of the amount of PCR amplicons to the amount of DNA template. In this study, we have provided experimental evidence In order to obtain experimental evidence for the that pRb interacts and functionally cooperates with the role of pRb on TPO gene transcription in the context of thyrocyte-specific transcription factor Pax 8. Hence, the thyroid follicular cells, we took advantage of the pRb acts as a positive transcriptional coactivator in possibility to inhibit gene expression by short interfering thyroid follicular cells. This finding identifies a mole- RNA (Sharp, 1999). We utilized the pSUPER plasmid cular mechanism underlying the relevant role of pRb in (Brummelkamp et al., 2002) to construct the pSUPER- thyrocytes differentiation (Iuliano et al., 2000). pRb has iRbA vector which directs the synthesis of small been shown to regulate differentiation in several other interfering RNAs containing the rat pRb gene target cell types (Lipinski and Jacks, 1999). In osteoclasts, pRb sequence. This plasmid was used in transient transfec- acts as a transcriptional coactivator of CBFA1 and, in tion experiments with FRTL-5 cells. The data in Table 2 muscle cells, it cooperates with the myogenic bHLH

Oncogene Role of pRb in thyroid-specific gene expression S Miccadei et al 6998 transcription factors (Puri and Sartorelli, 2000). Pax 8 activity. Hence, our results would indicate that Furthermore, direct or indirect interaction of pRb Pax 8, like E1A, simultaneously interacts with both p300 with several transcription factors, including C/EBPs, and pRb. Several transcription factors, including NF-IL6, Ap1, ATF2, Sp1 and Sp3 was demonstrated CBFA1 (Sierra et al., 2003), MyoD, MEF2, AP2, C/ to enhance their transcriptional activity (Lipinski EBP, require both p300 and pRb as coactivators (Grana and Jacks, 1999). The synergy of pRb with these trans- et al., 1998; Goodman and Smolik, 2000). These cription factors suggests a potential mechanism for observations are compatible with the hypothesis that integrating the process of cellular proliferation and pRb and p300 function as molecular matchmakers, differentiation. It is conceivable that a molecule such as assembling different protein complexes in various cells pRb capable of inhibiting cell cycle progression is also and as crucial regulators of the differentiation process required for the activity of the differentiation ‘master (Wang, 1997). Furthermore, it has been shown that E1A genes’. Thus, the synergy between Pax 8 and pRb could is able to simultaneously recruit both p300 and pRb into indicate a molecular mechanism resulting in the a multimeric-protein complex, which results in the permanent withdrawal from the cell cycle and the stimulation of pRb by the acetylase activity expression of the differentiation markers in thyrocytes. of p300 (Chan et al., 2001). This post-translational pRb directly interacts with several Pax genes, it binds modification appears to positively regulate the growth- all the members of the Pax subfamily, Pax 2/5/8 suppressing and the differentiation-inducing capabilities (Eberhard and Busslinger, 1999; Yuan et al., 2002; and of pRb, given that acetylated pRb is a poorer substrate this study). Since it has been reported that pRb increases for phosphorylation by cyclin dependent kinases. Thus, the DNA-binding activity of several transcription it is tempting to speculate that in thyrocytes, the Pax 8– factors (Lipinski and Jacks, 1999), we have tested this p300–pRb complex could allow, on TPO gene promoter, hypothesis but we did not identified such a role of pRb both the formation of the active transcription complex on Pax 8 DNA-binding activity (data not shown). and the preservation of the pRb active form, resulting in Therefore, how pRb can enhance Pax 8 transcriptional the maintenance of the differentiated state and the exit activity remains an open question. In this regards, it is from the cell cycle. worth mentioning that Pax 8 activity is inhibited by Id proteins (Roberts et al., 2001), and that pRb binds Id2 (Lasorella et al., 1996). Although we have not addressed this hypothesis experimentally, it is possible to imagine Materials and methods that pRb could displace Id2 from Pax 8 and thereby Plasmid construction and cell transfection rescue the Pax 8 transcriptional activity. In analogy, it has been shown that pRb binds the inhibitory factor To construct pcDNA3k-Pax8, the Pax 8 gene was amplified EID-1 rescuing the transcriptional activity of MyoD from pCMV5-Pax 8 (Zannini et al., 1992) with the oligo- nucleotides bP8F (50-cgcggatccgccaccatgcctcacaactcgatcag-30) (MacLellan et al., 2000; Miyake et al., 2000). 0 0 We have shown that Pax 8/pRb synergy is promoter- and xP8R (5 -ggtttctagactacagatggtcaaaggctg-3 ), the ampli- con, digested with BamH1 and Xba1, was cloned in pcDNA3 specific. It can be detected on both TPO and NIS gene digested with the same enzymes. The plasmid pcDNA3k- transcription but not in the Tg gene promoter activity. Pax8Dad was similarly constructed using the oligonucleotide These findings are in agreement with other published dP8ad (50-cttatctagattatgagaggagggcctggc-30) instead of xP8R. results (Perrone et al., 2000), indicating that Pax 8 The plasmid encoding Pax 8 lacking the residual home- activates its target genes using different molecular odomain (pcDNA3k-Pax 8DHD) was constructed, according requirements. to Ho et al. (1989), using pcDNA3-mPax8 as template in the A remarkable aspect of the Pax 8/pRb synergy polymerase chain reaction (PCR) with the oligonucleotides emerged from the experiments with E1A (Figure 2b). bP8F and P8-HDR (50-ccctttggtgtggctgggggaggcggtgtccgtac gaaggtgctttcg-30) and with the oligonuclotides P8-HDF We have shown that the downregulation of thyrocyte 0 0 differentiation, mediated by E1A, correlates with its (5 -cgaaagcaccttcgtacggacaccgcctcccccagccacaccaaaggg-3 ) and xP8R. The two amplicons were mixed and used as template in ability to inhibit Pax 8 activity and that this inhibition a PCR with the oligonucleotides bP8F and xP8R. The final relies on pRb binding. We suggest that repression by PCR product, digested with BamH1 and Xba1, was cloned in E1A results from the sequestration of pRb that is pcDNA3 digested with the same enzymes. Thus, the resulting required for Pax 8-mediated activation. Such a role of plasmid encodes a mutated mouse Pax 8 lacking the amino pRb in thyroid-specific gene expression would explain acids 229– 250. pSuper-iRbA was constructed annealing the how E1A can abrogate thyroid follicular cell differentia- oligonucleotides Ri-RbA-Fw (50-gatcccccacatcatctggactctgttt- tion with concomitant downregulation of the thyroid- caagagaacagagtccagatgatgtgtttttggaaa-30) and Ri-RbA-Rv specific gene expression (Berlingieri et al., 1993; Iuliano (50-agcttttccaaaaacacatcatctggactctgttctcttgaaacagagtccagatga 0 et al., 2000). The adenoviral protein can sequester the tgtgggg-3 ) and cloning the resulting double-stranded oligonu- coactivator, thus hampering the activity of Pax 8. cleotide in pSuper (Brummelkamp et al., 2002) digested with Bgl2 and Hind3. The pTg-Luc plasmid expressing the We have shown that E1A inhibits Pax 8 activity, luciferase under the rat Tg gene promoter was constructed whereas the E1A mutant, lacking the pRb interaction cloning in pGL3 Basic, digested with Bgl2 and Hind3, the domain, does not. In line with these results, we have BamH1–Hind3 fragment obtained from 50–41 plasmid, kindly previously reported that Pax 8 and p300 cooperate in the provided by R Di Lauro, Naples (Musti et al., 1987). The new induction of TPO gene expression and that the E1A constructs were confirmed by restriction enzyme digestions or mutant deficient in p300 binding was not able to inhibit by direct sequencing analysis.

Oncogene Role of pRb in thyroid-specific gene expression S Miccadei et al 6999 Hela, Saos-2 and FRTL-5 cells were transfected by transfected HeLa cells, 24 h after transfection, the cells were Lipofectamine 2000 Reagent (Invitrogen) according to the resuspended in hypotonic buffer (10 mM HEPES pH 7.9, protocol suggested by the manufacturer. The following 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, amount of the indicated plasmids were transfected: 0.4 mg, 0.5 mM PMSF, 0.5% NP40), the nuclei were pelletted by 30 s luciferase reporter plasmids; 10 ng of the Pax 8, wild type or centrifugation at maximum speed in the microcentrifuge. mutant, encoding plasmids; 10 ng of the E1A, wild type or Crude nuclear extracts were prepared in 20 mM HEPES pH mutant, expressing vectors; 10 ng of pocket protein-expressing 7.9, 350 mM NaCl, 30 mM MgCl2, 0.5% NP-40, 5 mM b- plasmids (pCMV-Rb, pCMV-p107 and pCMV-p130). We glycerophosphate, 0.1 mM Na3VO4,10mM NaF, 1 mM DTT), have tested in control experiments that the proteins, encoded supplemented with Roche protease inhibitors. After 30 min by the transfected plasmids, were expressed in comparable centrifugation at maximum speed in the microcentrifuge, the amounts by EMSA and/or Western blot experiments, these supernatant was diluted in the same buffer deprived of NaCl experiments are presented as Figure 6 in Supplementary and NP40 to lower the salt concentration to 150 mM and the Information. In HeLa and Saos-2 cells, the efficiency of detergent to 0.2%. Crude HeLa nuclear extract were transfection was assayed with 20 ng of the pCMV-b-galacto- immunoprecititated with 2 mg of normal rabbit IgG (Santa sidase plasmid, whereas in FRTL-5 cells it was tested Cruz Biotechnology) or with 2 mg affinity-purified polyclonal transfecting 100 ng of pRSV-CAT (chloramphenicol acetyl- rabbit anti-Pax 8 antibody (kindly provided by R Di Lauro, transferase) plasmid. At 48 h after transfection, cell extracts Naples, Italy). The FRTL5 nuclear proteins were extracted were prepared using the Reporter Lysis Buffer (Promega). according to Shapiro et al. (1998). The co-immunoprecipita- Luciferase, b-galactosidase and CAT assays were performed as tion from FRTL5 nuclear proteins were performed according previously reported (Civitareale et al., 1993; De Leo et al., to Klenova et al., 2002. For Western blot analysis, the extracts 2000). Transfection experiments were carried out in duplicate or the immunoprecipitated products were separated by SDS– or in triplicate and repeated at least three times. For each PAGE followed by liquid transfer to Hybond P membranes experiment, we report the mean of three independent experi- (Amersham-Pharmacia Biotech). Membranes were blocked ments and the standard deviations are shown. The statistical with 5% nonfat dry milk in Tris-buffered-saline (TBS), 0.1% analysis performed utilizing the row data of all transfection Tween 20 and incubated with primary antibody, anti-pRb C15 experiments resulted in Po0.05. For each experiment, the (Santa Cruz Biotechnology). Membranes were washed and t-test (the probability associated with the t-test) was calculated incubated with horseradish peroxidase-conjugated secondary versus the respective control group. antibody (Santa Cruz Biotechnology). Filters were then processed for enhanced chemiluminescence detection (ECL kit from Amersham-Pharmacia Biotech). Affinity column and band-shift assay GST and GST-pRbLP were expressed in the E.coli strain Chromatin immunoprecipitation BL21. Bacteria growth, expression induction and purification of the recombinant protein was performed according to Chromatin immunoprecipitations were performed using the Eberhard and Busslinger, 1999. Bacterially expressed GST- anti-pRb C-15 (Santa Cruz Biotechnology) according to pRbLP fusion protein was bound to Glutathione-Sepharose Ferreira et al. (2001). Subconfluent FRTL-5 cells were treated 4B resin (Amersham Biosciences). The column was equili- with formaldehyde at a final concentration of 1% for 7 min at brated in buffer A (30 mM HEPES, 0.1 mM EGTA, 0.5 mM room temperature. Chemical crosslinking was terminated by DTT, 0.5 mM PMSF) þ 80 mM NaCl and loaded with the addition of glycine to a final concentration of 0.125 M, nuclear extract prepared from HeLa cells overexpressing Pax followed by additional incubation for 5 min. After a wash 8. The nuclear extract, prepared according to Suzuki et al. with cold phosphate-buffered saline, cells were suspended in (1998), was diluted with buffer A to decrease the salt lysis buffer (5 mM piperazine N,N0-bis(2-ethanesulfonic acid) concentration to 80 mM. The column was washed with buffer (pH 8.0), 85 mM KCl, 0.5% NP-40) and disrupted using a A þ 80 mM NaCl and eluted with buffer A þ 1 M NaCl. The Dounce homogenizer. Nuclei were then pelleted and sus- flow-through, wash and high salt step elution fractions were pended in nuclear lysis buffer (50 mM Tris-HCl (pH 8.1), concentrated on Centricon 30 and assayed in band-shift 10 mM EDTA, 1% SDS. Chromatin was sonicated with 16 experiments using the double-strand oligonucleotide with the 10-s pulses (50 W; amplitude, 80%). After centrifugation, the Pax 8-binding site, oligo C (Civitareale et al., 1989). The band- supernatant was diluted 10-fold with TNE buffer (16.7 mM shift assays were performed as previously reported (Esposito Tris-HCl (pH 8.1), 167 mM NaCl, 0.01% SDS, 1.1% Triton et al., 1998). X-100, 1.2 mM EDTA). Diluted chromatin was precleared with protein A/G agarose beads (Santa Cruz) saturated with bovine serum albumin and salmon sperm DNA and then incubated Protein–protein interaction assay overnight at 41C using anti-Rb C-15 (Santa Cruz) and Glutathione-Sepharose beads coated with 3 mg of GST or immunoprecipitated with protein A/G agarose beads. The GST-pRbLP recombinant proteins were incubated for 2 h at beads were extensively washed, and then chromatin was eluted 41C with 5 mlof14C-proteins that were synthesized by a from beads by incubation during vortexing in elution buffer coupled in vitro transcription– system (TNT, (50 mM NaHCO3, 1% SDS). Crosslinks were then reversed by Promega) in the presence of [14C]Leucine. Binding assay were overnight incubation at 651C in elution buffer containing in performed in buffer BC100 (20 mM Tris-Cl pH 8.0, 100 mM addition 300 mM NaCl and 30 mg of RNase A/ml. An KCl, 0.1 mM EDTA 5 mM MgCl2, 20% glycerol, 1 mM DTT, equivalent amount of diluted chromatin was similarly pro- 0.5 mM PMSF) containing 0.2% NP40 and 0.5 mg/ml BSA. cessed without immunoprecipitation and noted as ‘input’ After washing with 400 volumes of binding buffer, bound afterwards. DNA samples were then purified by phenol– proteins were eluted from the beads by boiling in 2 Â SDS chloroform extraction, ethanol precipitated, and further (sodium dodecyl sulfate) sample buffer and were applied to analysed by PCR. In each experiment, linearity of the signal SDS–PAGE (polyacrylamide gel electrophoresis). Image was verified by amplifying increasing amount of the DNA analysis was performed by using a Molecular Dynamics template. For each experiment, PCRs were performed with Model Storm (860) PhosphorImager. For coprecipitation of different numbers of cycles or with dilution series of input

Oncogene Role of pRb in thyroid-specific gene expression S Miccadei et al 7000 DNA to determine the linear range of the amplification; all Acknowledgements results shown fall within this range. The primer sequences to We thank Thomas Wagner and Armando Felsani for critical amplify the TPO gene promoter are: TPO2Fw (50-gctaaca reading of the manuscript. Various colleagues kindly provided cacctagcaggaaggg-30) and TPO.Pr4 (50-gtgaatctcgagtactttctg many of the plasmids used in this study and we thank them for gagacttggttacccaccatataaatggactccatgc-30). To amplify the their kindness. The E1A- and E1A-Rb-encoding plasmids were minimal TSHr gene promoter, we used the previously a gift of B Moran (Moran, 1993). pCMV-Rb and pGST- described oligonucleotides (Civitareale et al., 1993). The PCR RbLP, pCMV-107 and pCMV-130 were provided by A Felsani to amplify the GAPDH gene fragment was performed with the e M Caruso. pRbDcdk was kindly provided by WJ Harbour. oligonucleotides GAPDH 1F (50-cctggccaaggtcatccatgacaa-30) We thank MR Nicotra for immunohistochemistry. MM was and GAPDH 1R (50-gcctgcttcaccaccttcttgatg-30). PCR condi- supported by a fellowship of Marie Curie Training site tions were: 951C for 5 s followed by 35 cycles at 941C for 40 s, program number HPMT-CT-2000-0010. This study was in 561C for 30 s and 721C for 30 s. The images from the agarose part supported by MURST-FIRB n.RBAU19HHA-001 and gels were acquired by using the Chemi-Doc (Bio Rad). by grant no. 210 Life Sciences 2002 from ASI to DC.

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