TIF1�, a Novel HP1-interacting Member of the TranscriptionalIntermediary Factor 1 (TIF1) Family Expressed byElongating Spermatids*

Received for publication, April 29, 2004, and in revised form, August 16, 2004Published, JBC Papers in Press, August 20, 2004, DOI 10.1074/jbc.M404779200

Konstantin Khetchoumian‡§, Marius Teletin¶, Manuel Mark‡¶, Thierry Lerouge‡,Margarita Cervino‡, Mustapha Oulad-Abdelghani‡, Pierre Chambon‡¶, and Regine Losson‡�

From the ‡Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM/ULP/College de France and¶Institut Clinique de la Souris, BP 10142, 67 404 Illkirch-Cedex, France

TIF1 (transcriptional intermediary factor 1) proteinsare encoded by an expanding family of developmentaland physiological control genes that are conserved fromflies to man. These proteins are characterized by anN-terminal RING-B box-coiled-coil (RBCC) motif and aC-terminal PHD finger/bromodomain unit, and havebeen implicated in epigenetic mechanisms of transcrip-tional repression involving histone modifiers and het-erochromatin-binding proteins. We describe here theisolation and functional characterization of a fourthmurine TIF1 gene, TIF1�. The predicted TIF1� proteindisplays all the structural hallmarks of a bona fide TIF1family member and resembles the other TIF1s in that itcan exert a deacetylase-dependent silencing effect whentethered to a promoter region. Moreover, like TIF1� andTIF1�, TIF1� can homodimerize and contains a PXVXLmotif necessary and sufficient for HP1 (heterochroma-tin protein 1) binding. Although TIF1� and TIF1� alsobind nuclear receptors and Kruppel-associated boxesspecifically and respectively, TIF1� appears to lack nu-clear receptor- and Kruppel-associated box binding ac-tivity. Furthermore, TIF1� is unique among the TIF1family proteins in that its expression is largely re-stricted to the testis and confined to haploid elongatingspermatids, where it associates preferentially with HP1isotype � (HP1�) and forms discrete foci dispersedwithin the centromeric chromocenter and the surround-ing nucleoplasm. Collectively, these data are consistentwith specific, nonredundant functions for the TIF1 fam-ily members in vivo and suggest a role for TIF1� inheterochromatin-mediated gene silencing during post-meiotic phases of spermatogenesis.

Transcriptional regulation of gene expression in eukaryotesin response to developmental or environmental signals is acomplex multistep process that requires the concerted action ofmany cellular factors. Central players in this elaborate processare sequence-specific transcription factors that positivelyand/or negatively control transcription through interactions

with transcriptional intermediary factors (TIFs1; also desig-nated as coactivators and corepressors), whose ultimate func-tion is to remodel chromatin structure (1, 2), to stimulate orinhibit (pre)initiation complex formation (3), or to associatetarget genes with specialized nuclear compartments (4, 5).

TIF1s are members of a growing family of chromatin-associ-ated/related TIFs, a subset that has emerged as key regulatorsof developmental and physiological processes (6). Included inthis family are three members (TIF1�, -�, and -�) (7–9) inmammals and one (Bonus) (10) in Drosophila, and all consist oftwo conserved amino acid regions: an N-terminal RING-B boxcoiled-coil (RBCC) domain with potential self-assembly proper-ties (11, 12); and a C-terminal region containing a PHD fingerand a bromodomain, two well conserved signature motifswidely distributed among nuclear proteins known to functionat the chromatin level (13–15).

TIF1�, the founding member of the family, was initiallyidentified in a yeast genetic screen for proteins modulating thetransactivation potential of retinoid X receptor (RXR) (7) andwas subsequently found to interact via a single LXXLL motifwith the AF-2 transcriptional activation domain of severalnuclear receptors, including retinoic acid (RAR), thyroid (TR),vitamin D3 (VDR), and estrogen (ER) receptors (8, 16). TIF1� isa euchromatin-enriched chromosomal protein expressed ubiq-uitously and early in development (17, 18) as well as in manyadult tissues (7). In mouse NIH 3T3 cells, TIF1� was reportedto play a role in the growth-suppressive activity of RXR/RARand to exhibit a transforming activity when fused to a trun-cated B-Raf (19). Supporting the notion that the biologicalfunctions of TIF1� are achieved through modulation of chro-matin states, TIF1� has been demonstrated to possess an in-trinsic transcriptional silencing activity that requires histonedeacetylation (8, 20). Moreover, TIF1� has the ability to inter-act directly with members of the heterochromatin protein 1(HP1) family (8), a class of nonhistone chromosomal proteinsthat serve as dose-dependent regulators of higher order chro-matin structures to promote silencing on euchromatic genes(reviewed in Refs. 21 and 22). Mapping of the HP1-interacting

* This work was supported in part by the CNRS, the INSERM, lesHopitaux Universitaires de Strasbourg, the Association pour la Recher-che sur le Cancer, the College de France, and the Fondation pour laRecherche Medicale. The costs of publication of this article were de-frayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

§ Supported by the Ligue Nationale Contre le Cancer.� To whom correspondence should be addressed. Tel.: 33-3-88-65-34-

71; Fax: 33-3-88-65-32-01; E-mail: losson@igbmc.u-strasbg.fr.

1 The abbreviations used are: TIF, transcriptional intermediaryfactor; RBCC, RING-B box-coiled-coil; NR, nuclear receptors; KRAB,Kruppel-associated boxes; RXR, retinoid X receptor; RAR, retinoicacid receptor; TR, thyroid receptor; VDR, vitamin D3 receptor; ER,estrogen receptor; mAb, monoclonal antibody; ORF, open readingframe; DAPI, 4,6-diamidino-2-phenylindole; PBS, phosphate-bufferedsaline; RT, reverse transcription; NGS, normal growth serum; GST,glutathione S-transferase; RACE, rapid amplification of cDNA ends;AAD, acidic activation domain; OMPdecase, orotidine 5�-monophos-phate decarboxylase; TSA, trichostatin A; PtdInsPs, phosphoinosi-tides; ERE, estrogen-response element; CAT, chloramphenicol acetyl-transferase; DBD, DNA binding domain.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 46, Issue of November 12, pp. 48329–48341, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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domain in TIF1� led to the identification of a conserved PXVXLmotif located in its central region that binds directly to theC-terminal chromoshadow domain of HP1 proteins (8) andexists in other potential transcriptional regulatory targets (23).

The identification of TIF1� established the TIF1 family oftranscriptional cofactors (8). TIF1� (also known as KAP-1 orKRIP-1) was isolated by virtue of its ability to interact withmouse HP1� (8) and with the KRAB domain of the humanKruppel-like proteins KOX1 (24, 25) and Kid-1 (26). The KRABtranscriptional repression domain is a widely distributed motiffrequently found at the N termini of zinc finger proteins ofKruppel Cys2-His2-type (27). This domain contains a conservedKRAB A box and is usually followed by a KRAB B box (or adiverged b box; Ref. 28). All variants of the KRAB domainstudied so far function through the recruitment of TIF1� (29).Consistent with a role in chromatin organization, TIF1� si-lences transcription through a mechanism that involves his-tone deacetylation (20, 30), histone H3 Lys-9 methylation (31),and recruitment of HP1 proteins by a PXVXL motif (20, 32).Most importantly, this motif is also required to trigger therelocalization of TIF1� into regions of centromeric heterochro-matin during cell differentiation (33). In mouse, TIF1� is ex-pressed ubiquitously throughout development (34) and inmany adult tissues (26). We have recently shown that disrup-tion of TIF1� in mice leads to an embryonic lethal phenotypedue to a developmental arrest at the egg cylinder stage, prior tothe onset of gastrulation (34), thus demonstrating that TIF1�

exerts essential and nonredundant functions during early post-implantation development. Subsequently, the use of a condi-tional germ line-specific disruption of TIF1� in the adult testisunveiled a later and essential nonredundant function of TIF1�

in the homeostasis of the seminiferous epithelium (35).The third mammalian member of the TIF1 family, TIF1�,

was discovered via low stringency hybridization screens usingTIF1� as a probe (9). Amino acid comparison revealed that,among the three mammalian members of the family, TIF1� iscloser to TIF1� than to TIF1� (50% overall identity betweenTIF1� and TIF1� and �30% identity among the other TIF1s)(9, 36). In vitro, TIF1� and TIF1� hetero-oligomerize as effi-ciently as they homo-oligomerize, whereas TIF1� does homo-oligomerize but does not hetero-oligomerize with TIF1� orTIF1� (12, 37). Moreover, it has been shown that an overex-pression of TIF1� in transiently transfected cells can interferewith the transrepression activity of TIF1� (12). Further evi-dence supporting a cross-talk between TIF1� and TIF1� is therecent identification of two novel types of RET rearrangementsin childhood papillary thyroid carcinomas, PTC6 and PTC7,having in common the RET receptor tyrosine kinase domainfused to the RBCC domains of TIF1� (PTC6) and TIF1�

(PTC7), respectively (38). In both human and mouse, TIF1�

transcripts are widely expressed at varying levels in adult andfetal tissues (9, 36). Similar to the other TIF1 family members,TIF1� contains an intrinsic transcriptional silencing function(9); however, the downstream targets that mediate gene silenc-ing have yet to be identified.

Recently, we isolated and characterized the only ortholog ofmammalian TIF1s in the Drosophila genome, the so-calledBonus (10). Bonus is a chromatin-associated protein that isexpressed throughout development. Mutational analysis re-vealed that Bonus is required for male viability, molting, andnumerous events in metamorphosis that are associated withgenes implicated in the ecdysone pathway, a nuclear hormonereceptor pathway required throughout Drosophila develop-ment (10). Bonus shares with TIF1� the ability to interact withthe nuclear receptor AF-2 activation domain (10). Direct evi-dence for the biological relevance of this evolutionarily con-

served interaction was provided by genetic studies showingthat reduction in the level of Bonus suppresses the phenotypeassociated with a partial loss of function mutation of the nu-clear receptor �FTZ-F1 (10), thus defining Bonus (and by anal-ogy TIF1�) as negative regulators of nuclear receptor-depend-ent transcription.

Here we report the isolation and functional analysis of a fourthmurine TIF1 gene, TIF1�. Over the entire length of the 1344-amino acid protein, TIF1� shares �30% identity with the otherTIF1s and also displays a potent trichostatin A (TSA, a specificinhibitor of histone deacetylases)-sensitive repression function.However, TIF1� differs remarkably from the other TIF1 familymembers in that its expression is largely restricted to testis,where it was seen by immunohistochemistry only in male germcells that have completed meiosis, at the early elongating sper-matid stages. TIF1� was localized by confocal laser-scanningmicroscopy in discrete nuclear foci distributed throughout thenucleus including the chromocenter, a condensed structureformed by the association of centromeric heterochromatin. Fur-thermore, a preferential association between TIF1� and HP1isotype � (HP1�) has been identified through coimmunoprecipi-tation. Taken together, these data suggest a role for TIF1� as anegative regulator of postmeiotic genes acting through HP1�complex formation and centromere association.

EXPERIMENTAL PROCEDURES

Clone Isolation and Sequencing—Four exon-specific primers de-signed according to the nucleotide sequences of predicted human TIF1�exons in the KIAA0298 genomic sequence (GenBankTM accession num-ber AJ400879) were used to amplify two DNA probes by RT-PCR.Primers used are as follows: (a) forward primer, 5�-AACCCAAGATG-GCCAGGAAC-3� (AAU20) and reverse primer, 5�-GCCCTGGACATTT-GTCCACC-3� (AAU21), and (b) forward primer, 5�-CAGAGCCT-TCAATAGTGAGC-3� (AAU22) and reverse primer 5�-TGCC-TTGGCTGGGCAAACCG-3� (AAU23). RT-PCR amplification was per-formed on 0.5 �g of HeLa total RNA using the Clostridium thermocel-lum polymerase One-step RT-PCR system (Roche Applied Science). Theamplified DNA fragments were used as probes to screen a �ZAPIImouse brain-derived cDNA library (Stratagene) according to the man-ufacturer’s instructions. These screens resulted in the isolation of twomouse cDNA clones, TIF1�-a (nucleotides �212 to �1322) and TIF1�-b(nucleotides �2011 to �4111) (see Fig. 1B). Complete sequence infor-mation of the coding region was achieved by RT-PCR using 1.5 �g ofmouse testis total RNA as template and the following primer pairs: (a)TIF1� exon 9 forward primer, 5�-TTCCAGTCTCCAGCACTGTG-3�(ACD251), and TIF1� exon 10 reverse primer, 5�-AGAGCTGGCTTT-GGGCCCTC-3� (ACD252), designed from the TIF1�-a and TIF1�-b se-quences, respectively; and (b) TIF1� exon 1 forward primer, 5�-ATA-ACTTGGACCTCGGAGAC-3� (ADA27), and TIF1� exon 3 reverseprimer, 5�-ATGTGTGCTGCCCGCTTCTC-3� (ACM266), designed onthe basis of the nucleotide sequences of putative TIF1� exon 1 (Gen-BankTM accession number AJ307670) and TIF1�-a cDNA clone, respec-tively. The resulting RT-PCR products (TIF1�-c (nucleotides �1240 to�2045), TIF1�-d1 (nucleotides �183 to �353), and TIF1�-d2 (nucleo-tides �183 to �353 containing a deletion of 63 nucleotides), see Fig. 1,B and D) were gel-purified and subcloned into pBluescript II SK(�)(Stratagene). Three independent clones of amplified cDNAs from eachRT-PCR were subjected to DNA sequence analysis. To extend the TIF1�cDNA sequence toward its 3� end, a 3�-RACE experiment was per-formed using the SMART RACE kit (Clontech), with mouse testis totalRNA and the gene-specific forward primer, TIF1� exon 19 primer,5�-TACATGCAGGAAGGCATCCA-3� (AFA51). The fragment amplifiedby 3�-RACE (TIF1�-e (nucleotides �3931 to �4254)) was purified,cloned, and sequenced.

Northern Blot Analysis—Multiple mouse tissue Northern blotscontaining poly(A)� RNA from various tissues (Clontech) were hy-bridized with a cDNA fragment encoding amino acids 671–976 ofTIF1� (nucleotides �2011 to �2930) or a �-actin cDNA fragment(control) labeled with [�-32P]dCTP by random priming according tothe manufacturer’s instructions.

Antibodies—Specific antibodies used include the following: (a) mouseanti-TIF1� monoclonal antibody (mAb), PG124, raised against TIF1�-(132–151) for immunohistochemistry, and rabbit anti-TIF1� polyclonalantibody, PG78, raised against TIF1�-(695–721) for Western blot anal-

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ysis. The specificity of these antibodies was established with an extractof COS-1 cells transfected with a eukaryotic expression plasmid encod-ing FLAG-tagged TIF1� (Fig. 5 and data not shown). (b) mouse anti-HP1� mAbs, 2HP1H5 for immunoprecipitation and immunohistochem-istry and 2HP2G9 for Western blot analysis (18); (c) mouse anti-HP1�mAb, 1Mod1A9 (20); (d) mouse anti-HP1� mAb, 2Mod1G6 (20). For thedouble immunostaining experiments, anti-HP1� and -HP1� antibodieswere purified by the caprylic acid/ammonium sulfate method and thencoupled to biotin (Sulfo-NHS-LC-Biotin kit, ES-Link, Pierce) or to Cy3(Cy3 mAb labeling kit, Amersham Biosciences); mouse anti-FLAG mAb,2Fl1B11 (gift of M. Oulad-Abdelghani, IGBMC, France); mouse anti-VP16 mAb, 2GV4 (39); mouse anti-ER� mAb, F3, raised against the Fregion of human ER� (39); and mouse anti-�-actin mAb, AC-15 (Sigma).

Immunohistochemistry—Testes from 3-month-old mice were fixedin 4% paraformaldehyde in PBS (12 h at 4 °C) and then embedded inParaplast. Five-�m-thick sections were collected on Superfrost slides(Kindler, Freiburg, Germany), dried for 16 h at 37 °C, and kept at4 °C until use. Sections were dried again for 15 min at 57 °C, de-waxed, hydrated, then placed into 0.01 M sodium citrate buffer, pH6.0, and exposed to microwave treatment (power output 900 watts; 10min). After cooling down to room temperature, sections were rinsed inPBS, treated with 5% NGS in PBS, 0.05% Tween 20 (PBS/NGS/Tween) for 30 min at room temperature to block nonspecific antibodybinding to the tissue, and incubated for 16 h at 4 °C with the anti-TIF1� antibody PG124 (ascites dilution: 1/1000 in PBS/NGS/Tween).Sections were then washed (PBS; three times for 5 min), incubatedwith the secondary antibody (Cy3-coupled goat anti-mouse, diluted1/500, The Jackson Laboratories) for 1 h at room temperature,washed in PBS, and mounted in Vectashield (Vector Laboratories)containing DAPI (Roche Applied Science) at 10 �g/�l. As negativecontrols of the immunostaining procedure, histological sections wereincubated with a mixture of the primary antibody (diluted 1/1000)and immunizing peptide (5 �g/ml).

Double immunostaining experiments were performed as follows.Following incubation with the primary anti-TIF1� antibody PG124 asdescribed above, testis sections were then washed (PBS; three timesfor 5 min), incubated with the secondary antibody (Alexa488-coupledgoat anti-mouse, diluted 1/250, The Jackson Laboratories) for 45 minat room temperature, washed in PBS, and incubated with eitherBiotin-coupled anti-HP1� antibody 1Mod1A9 (dilution 1/150 in PBS)or Cy3-coupled anti-HP1� antibody 2Mod1G6 (dilution 1/50 in PBS)for 12 h at 4 °C. TIF1�/HP1� double-labeled sections were thenwashed (PBS, three times for 5 min), incubated with Cy3-coupledstreptavidin (dilution 1/250 in PBS, The Jackson Laboratories) for 45min at room temperature, washed in PBS, fixed in 4% formalin (10min, room temperature), then washed again in PBS and mounted inVectashield (Vector Laboratories) containing DAPI at 10 �g/�l.Washing, fixation, and mounting were also done for TIF1�/HP1�double-labeled sections. Confocal images were obtained using a Leicaconfocal scanning microscope (SP1) and were superposed using home-made software.

Immunoprecipitation and Western Blot Analysis—Testes from adultmice were homogenized with a Dounce homogenizer in ice-cold lysisbuffer (EBC: 50 mM Tris-HCl, pH 8.0, 170 mM NaCl, 0.5% Nonidet P-40,50 mM NaF) containing 1 mM phenylmethylsulfonyl fluoride and aprotease inhibitor mixture of aprotinin, leupeptin, chymostatin, andpepstatin at 2.5 �g/ml each. Intact cells and debris were removed bycentrifugation at 13,000 rpm for 15 min at 4 °C, and supernatants werecollected. Protein concentration was determined with Bio-Rad proteinassay reagent (Bio-Rad). To immunoprecipitate the HP1 proteins, tes-ticular lysates (15 mg) were first incubated with 50 �l of protein G-Sepharose beads for 1 h at 4 °C. The beads were removed by centrifu-gation at 2000 rpm for 2 min, and 5 �l of specific antibody was thenadded. Samples were incubated for 3 h at 4 °C, followed by incubationovernight with 50 �l of Protein G-Sepharose beads at 4 °C. Immunecomplexes were collected by centrifugation at 2000 rpm for 2 min andwashed three times with 3 ml of EBC buffer. The final pellets were thenresuspended in 50 �l of 2� Laemmli SDS buffer (100 mM Tris-HCl, pH6.8, 4% SDS, 20% glycerol, 2% �-mercaptoethanol, 0.002% bromphenolblue) and analyzed by SDS-PAGE and immunoblotting as describedpreviously (40).

Transient Transfection—Transient transfection in COS-1 cells as wellas CAT and �-galactosidase assays were performed as described (8).

Yeast Two-hybrid Interaction Assays—DBD and acidic activationdomain (AAD) fusion proteins were expressed from the yeast multicopyplasmids pBL1 and pASV3, respectively (39). These plasmids expressinserts under the control of the phosphoglycerate kinase promoter.PBL1 contains the HIS3 marker and directs the synthesis of epitope

(region F of ER�)-tagged ER� DBD fusion proteins. pASV3 contains theLEU2 marker and a cassette expressing a nuclear localized VP16 AAD.The reporter strain PL3 (MAT� leu2-�1 ura3-�1 his3-�200trp1::(ERE)3-URA3) was as described elsewhere (39). Yeast cells grownin yeast extract/peptone/dextrose or selective medium were trans-formed by the lithium acetate procedure. Transformants were grownexponentially for about five generations in selective medium supple-mented with uracil. Yeast extracts were prepared and assayed fororotidine 5�-monophosphate decarboxylase (OMPdecase) activity as de-scribed previously (39).

In Vitro Binding Assays—GST pull-down assays were performed asdescribed (20). GST and GST-HP1 fusion proteins were expressed inEscherichia coli and purified on glutathione-Sepharose beads (Amer-sham Biosciences), as described by the manufacturer. For expression of35S-labeled TIF1�, the coding sequence of TIF1� was inserted into thepSG5 vector, and coupled transcription/translation was performed us-ing the T7 RNA polymerase with the TNT lysate system (Promega).

RESULTS

Isolation of a Fourth Murine TIF1 Gene, TIF1�—To identifyadditional members of the TIF1 family, we performedBLASTTM searches against GenBankTM and dbEST data basesusing the mouse TIF1� protein (7) (GenBankTM accession num-ber Q64127) as a query sequence. These searches revealed highsimilarity to a putative gene residing at corresponding loca-tions on human chromosome 11p15.4 and mouse chromosome 7(GenBankTM accession numbers AJ400879 and AJ307670, re-spectively). By using oligonucleotide primers designed accord-ing to the predicted open reading frame (ORF) of this gene, aset of overlapping mouse cDNA clones was isolated by a com-bination of cDNA library screening, RT-PCR, and 3�-RACE (see“Experimental Procedures”; Fig. 1B). After compilation of thesecDNAs, a contig of 4437 bp (hereafter called TIF1� cDNA) wasobtained (Fig. 1A), including a potential start codon in framewith an ORF of 4035 bp flanked by a 183-bp 5�- and a 219-bp3�-untranslated region (Fig. 1A; GenBankTM accession numberAY572454). Comparison of the nucleotide sequence of thiscDNA to the corresponding genomic sequence identified 20exons located within 54.4 kb of genomic DNA with 19 intronsranging in size from 142 to 21,602 bp (Fig. 1C and datanot shown).

A TIF1� cDNA variant lacking a stretch of 63 nucleotidesbetween nucleotides �149 and �211 was also isolated byRT-PCR (Fig. 1, A and B; GenBankTM accession numberAY572455). The presence or absence of this 63-nucleotidesegment in the TIF1� cDNA reflects the use of two alterna-tive 5�-splicing donor sites located 63 bp apart at the 3� end ofexon 1 (Fig. 1D). Excision of the segment maintains an in-phase ORF but causes a premature termination of transla-tion at codon 50 (Fig. 1D). Due to a possible downstreamreinitiation of translation at methionine codon 103, theshorter transcript is predicted to produce an N-terminallytruncated TIF1� protein lacking the first 102 amino acids(see legends to Fig. 1D). All subsequent studies were per-formed with the longest cDNA isoform that encodes a 1344-amino acid protein containing all the characteristic motifs ofTIF1 proteins (see Fig. 1A and Fig. 2B).

Over the entire length of the protein, TIF1� is 35 and 29%identical to mouse TIF1� and TIF1�, respectively (7, 8), 37%identical to human TIF1� (9), and 26% identical to DrosophilaBonus (10) (Fig. 2A). Like the other family members, TIF1�contains several evolutionary conserved domains (Figs. 1A and2B). At the N terminus, a C3HC4 zinc finger motif or RINGfinger is followed by two cysteine-rich zinc binding regions (Bboxes) and a predicted �-helical coiled-coil domain forming atripartite motif designated RBCC (7) (Figs. 1A and 2B). At theC terminus, a bromodomain (14, 15) is preceded by a C4HC3zinc finger motif or PHD finger (13) (Figs. 1A and 2B). Inaddition to these conserved domains found in a number of

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FIG. 1. Mouse TIF1� cDNA sequence, protein domain structure, and genomic structure. A, nucleotide and deduced amino acid sequenceof mouse cDNA encoding TIF1�. The 4437 nucleotides of TIF1� cDNA, the putative translation start site (in boldface and underlined), the1344-amino acid ORF, and the 5�-flanking termination codons (underlined) are shown. The alternatively spliced sequence (nucleotides �149 to�211) is shown in brackets. The putative translational start site for the shortest isoform of TIF1� (see D) is also shown in boldface and underlined.Cysteine and histidine residues belonging to the Cys/His-rich clusters (RING finger, B boxes, and PHD finger) are highlighted by circles. Thehydrophobic amino acids defining the heptad repeats of the putative coiled-coil structure are highlighted by gray circles. Highly conserved aminoacid residues in the HP1 box (underlined) and the bromodomain (boxed) are in boldface. Residues of the bromodomain that are essential for theacetyl-lysine binding are underlined. An LXXLL motif (amino acids 617–621) and a bipartite nuclear targeting sequence (amino acids 1318–1336)are underlined with a dashed line. B, schematic representation of the TIF1� cDNA sequence. ORF is depicted as a box and untranslated regionsas horizontal lines. The sequence was reconstituted from the indicated cDNA clones. TIF1�-a and TIF1�-b were obtained by screening a mouseadult brain cDNA library, whereas the others were isolated by RT-PCR (TIF1�-c, TIF1�-d1, and TIF1�-d2) or 3�-RACE (TIF1�-e) using mouse testisRNA as template. TIF1�-d2 differs from TIF1�-d1 by a 63-nucleotide deletion between positions �149 and �211. C, genomic organization of themouse TIF1� gene. Exons are represented as numbered boxes and introns as connecting lines. The scale of the genomic map is indicated below. Exon1 contains the ATG (from nucleotides �183 to �211) and exon 20 contains the stop codon TGA. D, alternative splicing of the mouse TIF1� genebetween exons 1 and 2. The 5�-splice donor gt, and 3�-splice acceptor ag are in italics. Translation start and stop codons defining open readingframes are in boldface.

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transcriptional cofactors (see “Discussion”), we identified a 39-amino acid segment in the central region of TIF1� (amino acids982–1020; underlined in Fig. 1A) which is highly similar to theHP1 box of TIF1� and TIF1�, in which the integrity of theconserved PXVXL motif (where X is any amino acid) has beenshown to be required for HP1 binding (8, 20) (Fig. 2C). Similarto TIF1� and Bonus, the central region of TIF1� also containsan LXXLL motif (amino acids 617–621; underlined in Fig. 1A),which, however, did not allow TIF1� to directly interact withnuclear receptors (see below; Fig. 10A). Note that a cluster ofglutamine and proline residues is also present in the centralregion of TIF1� from position 554 to 572 (Fig. 1A), whereas theC-terminal part of the protein contains a bipartite nuclearlocalization signal between residues 1318 and 1336 (Fig. 1A).

TIF1� Expression Is Predominant in Testis and Occurs Spe-cifically in Elongating Spermatids—TIF1� (7), TIF1� (26), andTIF1� (36) were shown previously to be expressed in a widevariety of mouse adult tissues. Using a TIF1�-specific probe(see Experimental Procedures), we analyzed the tissue distri-bution of TIF1� mRNA in the adult mouse by Northern blothybridization and found two transcripts of �5.2 and 6.0 kb onlyin the poly(A)� RNA from testis (Fig. 3). Prolonged exposure ofthe autoradiogram failed to reveal hybridizing species of the

same size in any tissue examined, although in both brain andtestis a faint hybridization to high molecular weight tran-scripts (�10 kb) was seen (indicated by * in Fig. 3; and data notshown). Thus, TIF1� differs remarkably from the other TIF1family members in that its expression in the adult mice islargely restricted to the testis.

Testicular cells expressing TIF1� were identified by indirectimmunofluorescence using a specific monoclonal antibodyraised against a 20-amino acid peptide of TIF1� (amino acids132–151; see “Experimental Procedures”). An intense fluores-cent signal was detected in the nuclei of elongating, steps 9–11,spermatids (St9 and St10 in Fig. 4, D–I, and data not shown).Immunostaining became fainter in early condensing, step 12,spermatids (St12 in Fig. 4, J–L) and was undetectable in al-most mature, i.e. elongated and condensed, spermatids popu-lating tubules at stages I to VIII of the seminiferous epitheliumcycle (II–VI and VII–VIII in Fig. 4, A–C, and data not shown).No immunostaining was observed in nuclei of (i) round sper-matids (data not shown), (ii) proliferative and meiotic germcells (i.e. spermatogonia and spermatocytes; G, LP, P, and Z inFig. 4, D–L, and data not shown), and (iii) somatic cell types(i.e. Sertoli, myoepithelial, and Leydig cells; S, M, and L in Fig.4, A–L). Negative controls incubated with the mixture of theprimary antibody and immunizing peptide yielded faint cyto-plasmic signals in both the seminiferous epithelium and Leydigcells but no nuclear signals (data not shown). These resultsindicate that TIF1� expression within the testis is confined topostmeiotic, haploid, and elongating spermatids, in contrast tothat of other TIF1s (i.e. � and �) that are produced by prolifer-ative, meiotic, and postmeiotic germ cells as well as Sertoli cells(35, 36).2

Western blot analysis was also employed to follow the testic-ular expression of TIF1� during puberty. No significant expres-sion was found in 2- and 3-week-old mouse testes (Fig. 5A, lane3 and 4, respectively), whereas the TIF1� protein was clearlydetectable from the age of 4 weeks onward (Fig. 5A, lane 5), a

2 K. Khetchoumian, M. Teletin, M. Mark, T. Lerouge, M. Cervino, M.Oulad-Abdelghani, P. Chambon, and R. Losson, unpublished results.

FIG. 2. TIF1� is structurally related to the other previouslycloned TIF1 family members. A, amino acid identities/similarities(in %) among the TIF1 proteins. The proteins shown are mouse TIF1�(GenBankTM accession number Q64127) and TIF1� (GenBankTM acces-sion number Q62318), human TIF1� (GenBankTM accession numberQ9UPN9), and Drosophila Bonus (GenBankTM accession numberAAF19646). Sequence comparisons were performed with the Bestfitprogram of the Wisconsin GCG package. B, schematic representation ofthe TIF1 protein family. Amino acid numbers indicate the size of eachTIF1, and amino acid identities/similarities are given between TIF1�and each other protein. C, sequence alignment of the HP1-interactingdomain of TIF1�, TIF1�, and CAF-1 with TIF1� amino acids 982–1020.Residues that are conserved between TIF1� and the other proteins areshaded. Asterisks indicate residues that are identical in all proteins.The pentameric consensus defining the conserved PXVXL motif re-quired for HP1 binding is shown.

FIG. 3. Tissue specificity of TIF1� expression. RNA blots contain-ing poly(A)� RNA from the indicated mouse tissues were hybridizedwith a TIF1�-specific cDNA fragment (nucleotides �2010 to �2930).Two mRNAs of 5.2- and 6.0-kb were seen, as indicated by arrowheads.Traces of a high molecular weight transcript (�10-kb), indicated by anasterisk, were also detected in both brain and testis. Hybridization to a�-actin probe is shown as a control for mRNA integrity and loading. Thepositions of RNA size markers (kb) are shown on the left.

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time when elongating spermatids become abundant (41). Thisobservation is consistent with the above immunohistochemicaldata, indicating that expression of TIF1� only occurs at latestages of spermatogenesis (summarized in Fig. 5B).

TIF1� Can Function as a Transcriptional Repressor—TIF1� (8), TIF1� (20), and TIF1� (9) have been shown pre-viously to repress transcription when directly tethered to apromoter region in mammalian cells. To investigate whetherTIF1� also possesses intrinsic repressor activity, we fused thecoding sequence of TIF1� to the GAL4 DNA binding domain(amino acids 1–147), and the resulting fusion protein wastested for its ability to repress transcription activated byER(C)-VP16, a chimeric activator containing the estrogenreceptor DNA binding domain fused to VP16 (8). GAL4-TIF1�and ER(C)-VP16 were transiently transfected into COS-1cells, together with a GAL4 reporter containing two GAL4-binding sites (17 M2) and an estrogen-response element(ERE) in front of a �-globin (G) promoter-CAT fusion (17M2-ERE-G-CAT; see Ref. 8 and also see Fig. 6A). As shown in Fig.6B, GAL4-TIF1� efficiently repressed transcription in a dose-dependent manner. In contrast, no significant reduction inCAT activity was detected by coexpressed TIF1� unfused tothe GAL4 DBD, indicating that repression by TIF1� is en-tirely dependent on DNA binding. Thus, like the other TIF1family proteins, TIF1� can function as a repressor of tran-scription when fused to a heterologous DBD.

Because in the case of TIF1�- and TIF1�-mediated repres-sion, recruitment of histone deacetylases has been involved (20,30), we analyzed the effects of the potent histone deacetylaseinhibitor trichostatin A (TSA) (42) on the repression activity ofTIF1�. Treatment of the transfected cells with TSA signifi-cantly reduced GAL4-TIF1� repression (Fig. 6C), indicatingthat the repressive effects of TIF1� on transcription are medi-ated, at least in part, by recruitment of deacetylase activity.

TIF1� Interacts with the Nonhistone Chromosomal ProteinsHP1�, -�, and -�—TIF1� (8) and TIF1� (20, 32) both interact

with the HP1 family proteins through a 25-amino acid segmentcontaining a conserved pentapeptide motif (PXVXL), alsotermed the HP1 box (8, 23). Because a similar pentapeptidesequence is present between residues 982 and 1020 of TIF1�(see Figs. 1A and 2C), the ability of TIF1� to interact with theHP1 proteins was tested using the yeast two-hybrid system.Full-length TIF1� was fused to the estrogen receptor DNAbinding domain, and the resulting hybrid protein (DBD-TIF1�;Fig. 7A) was coexpressed with either “unfused” AAD (as acontrol) or AAD chimeric proteins consisting of the AAD ofVP16 (amino acids 411–490) fused to any one of the HP1 familymembers (AAD-HP1�, AAD-HP1�, and AAD-HP1�; Fig. 7A) inthe yeast reporter strain PL3 containing an ERE-URA3 re-porter gene (see Fig. 7A) (39). Activation of the reporter wasdetermined by measuring the orotidine 5�-monophosphate de-carboxylase (OMPdecase) activity of the URA3 gene product.When coexpressed with the AAD or DBD controls, none of thehybrid proteins transactivated the URA3 reporter (Fig. 7B). Incontrast, coexpression of DBD-TIF1� with either AAD-HP1� orAAD-HP1� or AAD-HP1� resulted in �20–30-fold increases inthe reporter gene activity, indicating that TIF1� can interactwith HP1�, -�, and -� in yeast. Most interestingly, an interac-tion was also observed with a DBD fusion protein bearingTIF1� residues 982–1020 (Fig. 7B; DBD-TIF1�-(982–1020)),thus providing strong evidence for the presence of an HP1 boxwithin the central region of TIF1�. To demonstrate that TIF1�actually binds HP1 proteins through this HP1 box, two pointmutations were introduced into the PXVXL motif, replacing theconserved hydrophobic residues Val-991 and Leu-993 by ala-nine residues (Fig. 7A). In contrast to wild type TIF1�, themutant protein (DBD-TIF1�V991A/L993A; Fig. 7B) was dras-tically impaired in its ability to interact with HP1�, HP1�, andHP1�. Taken together, these data indicate that TIF1�, likeTIF1� and TIF1�, contains a conserved HP1 box that is neces-sary and sufficient for specific interaction with the HP1proteins.

FIG. 4. Stage-specific expression ofTIF1� in male germ cells. Histologicalsections from 3-month-old testes wereincubated with the anti-TIF1� mAbPG124, whose binding to cell structureswas then revealed with a Cy3-conju-gated secondary antibody (red signal),and nuclei were counterstained withDAPI (blue signal). Roman numerals re-fer to stages of the seminiferous epithe-lium cycle. Abbreviations: G, spermato-gonia; L, Leydig cells; LP, leptotenespermatocyte; M, peritubular myoepi-thelial cells; P, pachytene spermato-cytes; S, Sertoli cells; Sc2, type 2 sper-matocytes; St9, St10, and St12, step 9,10, and 12 spermatids; Z, zygotene sper-matocyte. Bar: 80 �m (A–C) and 40 �m(D–L).

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To identify the HP1 sequences to which TIF1� binds, a de-letion analysis of HP1� was performed using the yeast two-hybrid system. Various HP1� deletion mutants expressed asDBD fusion proteins (8) were tested for interaction with TIF1�fused to the VP16 AAD in the yeast strain PL3 (Fig. 7C). Nointeraction was detected with a DBD fusion protein bearingHP1� residues 1–66 or 67–113, which include the chromodo-main and the less conserved central region, respectively (Fig.7C). In contrast, an interaction was observed in the presence ofan N-terminally truncated fusion protein lacking the chromo-domain (DBD-HP1�-(67–191); Fig. 7C) or in the presence of afusion protein bearing residues 114–180 of the chromoshadowdomain (DBD-HP1�-(114–180); Fig. 7C). Similarly, HP1� andHP1� derivatives containing the chromoshadow domain only(see DBD-HP1�-(110–177) and DBD-HP1�-(104–170) in Fig.7D) interacted with TIF1� (Fig. 7D). Thus, the chromoshadowdomain of either HP1�, HP1�, or HP1� is sufficient for bindingto TIF1�.

Binding assays between TIF1� and the HP1 proteins werealso performed in vitro. GST-HP1 fusion proteins, GST-HP1�,

GST-HP1�, and GST-HP1�, were expressed in E. coli, immo-bilized on glutathione-Sepharose beads, and subsequently in-cubated with in vitro synthesized 35S-labeled TIF1�. After ex-tensive washing, the matrix-associated TIF1� protein waseluted and visualized by SDS-PAGE and autoradiography. Asshown in Fig. 7E, TIF1� was retained on beads coupled toeither GST-HP1� (lane 3), GST-HP1� (lane 4), or GST-HP1�(lane 5) but not on “control” GST beads (lane 2). Thus, TIF1�can interact with the HP1 proteins both in yeast and in vitro.

FIG. 5. A, developmental expression of TIF1� in testis from pre-pubertal and adult mice. Whole cell extracts (100 �g) from mouse testesof different postnatal ages (lane 3, 2-week-old; lane 4, 3-week-old; lane5, 4-week-old; lane 6, 3-month-old) were resolved along with control(lane 1) and TIF1� (lane 2)-transfected COS-1 cell extracts by 8%SDS-PAGE and were immunoblotted with the anti-TIF1� polyclonalantibody PG78. The sizes of the protein markers (kDa) are indicated onthe left side of the figure, and the arrowhead on the right side specifiesthe position of the TIF1� protein. �-Actin was used as a loading control.B, summary of TIF1� expression during spermatogenesis. The diagramis adapted from Russell et al. (62). The red and blue areas indicate areasof strong and weak immunoreactivity, respectively. Abbreviations: A,type A spermatogonia; B, type B spermatogonia; D, diplotene; In, in-termediate spermatogonia; L, leptotene; P, pachytene; PL, prelepto-tene; S, Sertoli cells; SC2, type 2 spermatocytes; Z, zygotene.

FIG. 6. TIF1� harbors an autonomous repression function. A,schematic representation of the 17M2-ERE-G-CAT reporter gene used.GAL4 UAS are represented by filled squares, the ERE by an open oval,and the transcription initiation site by an arrow. B, TIF1� repressesactivated transcription in a dose-dependent manner when tethered toDNA. The 17M2-ERE-G-CAT reporter (1 �g) and 1 �g of pCH110(expressing �-galactosidase) were cotransfected into COS-1 cells withthe indicated pSG5-based vectors expressing the activator ER(C)-VP16(100 ng) and the unfused GAL4 DBD (GAL4: 200 ng) or TIF1� fused tothe GAL4 DBD (GAL4-TIF1�: 2, 20, or 200 ng) or TIF1� tagged with theFLAG epitope (TIF1�: 200 ng). CAT activities are expressed relative tothe activity measured in the presence of ER(C)-VP16 and the unfusedGAL4 (taken as 100%). Values (�10%) represent the averages of threeindependent and duplicated transfection experiments after normaliza-tion to �-galactosidase activities. C, TSA treatment partially relievesTIF1� repression. COS-1 cells were transfected as described in B. Co-transfected cells were treated with 300 nM TSA 18 h prior to harvesting.CAT activities are expressed as in B.

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TIF1� Associates Preferentially with HP1� and Forms Mul-tiple Foci throughout the Nucleus of Elongating Spermatids—The ability of TIF1� to interact with HP1�, -�, and -� in vitroprompted us to investigate whether these proteins could bephysically associated in vivo. Whole cell extracts from adultmouse testis were immunoprecipitated with either anti-HP1�,-�, or -� antibodies, and the immunoprecipitates were probedfor the presence of TIF1�. Coimmunoprecipitation of endoge-nous TIF1� was clearly detected in each HP1 immunoprecipi-tate (Fig. 8A, lanes 4–6) but not in control immunoprecipitatesusing an irrelevant antibody (lane 3). Quantification by densi-tometry of the TIF1� levels in the load material (input) versuspellets (HP1 IP) from three separate experiments indicated

that less than 1% of total TIF1� could be immunoprecipitatedwith HP1� (0.5 � 0.1%; Fig. 8A and data not shown), whereasHP1� and HP1� antibodies were equally potent in immunopre-cipitating �5–10% of TIF1� from the whole testis extract (7.6 �3.3% and 8.5 � 3.9%, respectively; Fig. 8A and data not shown).However, these immunoprecipitations were done under condi-tions where the percent recovery of immunoprecipitated HP1�was quite low (8.4 � 4.1%; see Fig. 8A, lanes 13 and 14,and datanot shown) as compared with HP1� (19.7 � 3.3%; lanes 7–9)and HP1� (29.8 � 8.2%; lanes 10–12). Thus, after normaliza-tion to the respective levels of HP1 recovery, it was estimatedthat endogenous TIF1� was mostly associated with HP1� and,to a lesser extent (�25%), with HP1�, whereas a small propor-

FIG. 7. TIF1� interacts with HP1�, -�, and -�. A, schematic representation of the yeast two-hybrid system used in this study. The DBD of thehuman ER� (amino acids 176–282) and the AAD of VP16 (amino acids 411–490) unfused or fused to the proteins tested for interaction are shown.The HP1 chromodomain (CD) and chromoshadow domain (CSD) are represented. In the TIF1� protein, boxes indicate the conserved domains (seeFig. 2B). The pentameric consensus defining the conserved PXVXL motif, referred to as the HP1 box, is shown. The URA3 reporter gene, whichis regulated by three estrogen response elements (ERE3X) in the yeast reporter strain PL3, is represented below. B, TIF1� interacts with all threeHP1s through a 39-amino acid segment containing the conserved PXVXL motif. Plasmids expressing the indicated DBD fusion proteins,DBD-TIF1�, DBD-TIF1�-(982–1020), and DBD-TIF1�V991A/L993A, were introduced into PL3 together with either the unfused VP16 AAD or AADfusions containing HP1�, HP1�, or HP1�. Transformants were grown in liquid medium containing uracil. OMPdecase activities determined oneach cell-free extract are expressed in nanomoles of substrate/min/mg protein. The values (� 20%) are the average of at least three independenttransformants. C and D, the C-terminal chromoshadow domains of HP1�, HP1�, and HP1� are sufficient for TIF1� interaction. A schematicdiagram of the chimeras is shown on the left. The indicated DBD-HP1 fusions were assayed for two-hybrid interaction with either the VP16 AADor an AAD fusion containing TIF1� (AAD-TIF1�). Two-hybrid interaction assays were performed as in B. Note that in B–D, expression of all DBDand AAD fusion proteins was confirmed by Western blotting using the antibodies F3 against the F region of ER� and 2GV4 against VP16,respectively (data not shown). E, in vitro binding of TIF1� to HP1s. In vitro 35S-labeled TIF1� was incubated in a batch assay with “control” GST(lane 2), GST-HP1� (lane 3), GST-HP1� (lane 4), or GST-HP1� (lane 5). Bound TIF1� was resolved on SDS-PAGE and visualized by autoradiog-raphy. Lane 1 represents 1/10 the amount of input labeled TIF1�.

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tion (less than 5%) may also be present as stable complexwith HP1�.

Confocal laser-scanning microscopy was then employed tofurther investigate the distribution and subnuclear localizationof TIF1� in elongating spermatids with respect to the threeHP1 proteins as well as the chromocenter, a condensed struc-ture formed by the association of centromeric heterochromatin(43). The anti-TIF1� monoclonal antibody revealed a micro-granular signal comprising numerous small nuclear foci (seeFig. 8, B, I and C, I; step 9 elongating spermatids and data notshown). These foci were distributed throughout the nucleus,including the characteristic DAPI-dense block of centromericheterochromatin that constitutes the chromocenter (Fig. 8, B,III, and C, III). As reported previously (44), no specific stainingfor HP1� was detectable in elongating spermatids (data notshown), suggesting that these cells express very low levels, ifany, of HP1�. In contrast, using anti-HP1� or anti-HP1� anti-bodies, we were able to detect significant levels of expression ofHP1� and HP1� in elongating spermatids (see Fig. 8, B, IV,and C, IV, respectively). Within the nuclei of these cells, HP1�and HP1� both exhibited a predominant localization to thechromocenter with a mainly diffuse distribution; however, asobserved previously in various cell lines (20, 40, 45), HP1� andeven more so HP1� were detected in many additional sitesdispersed within the nucleoplasm that surrounds the chromo-center (Fig. 8, B, IV, and C, IV). Double immunostaining with

TIF1� and HP1� revealed an association of some TIF1�-en-riched foci with the HP1� signal within the chromocenter butnot outside of it (Fig. 8B, V). This result is consistent with thecoimmunoprecipitation data showing that not all but only afraction of the pool of TIF1� is physically associated with HP1�(Fig. 8A). Double staining with TIF1� and HP1� revealed ahigh number of TIF1� foci that showed significant overlap withthe HP1� signals within both the chromocenter and the sur-rounding nucleoplasmic compartment (Fig. 8C, V). Althoughthe significance of these coimmunolocalizations is difficult tointerpret because of the rather homogeneous staining patternof HP1� within the nucleus, the results are in complete agree-ment with the immunoprecipitation data indicating that al-most all of the TIF1� protein in elongating spermatids is stablyassociated with HP1� isotype (Fig. 8A).

Evidence for TIF1� Self-association—Because of the previousdemonstration that TIF1s (�, -�, and -�) can form homodimers(12, 37) as well heterodimers (TIF1�-TIF1�) (12), we examinedthe dimerization capacities of TIF1� using the yeast two-hybridsystem. In this assay, DBD-TIF1� was coexpressed with AADfusion proteins between the AAD of VP16 and any one of thefamily members (AAD-TIF1�, AAD-TIF1�, AAD-TIF1�, andAAD-TIF1�; Fig. 9A) in the yeast reporter strain PL3. Nosignificant increase in the reporter gene activity above the AADcontrol was detected with either AAD-TIF1�, AAD-TIF1�, orAAD-TIF1�, whereas under the same conditions, a reporter

FIG. 8. TIF1� preferentially associates with HP1� in elongating spermatids and localizes to discrete nuclear foci within both thechromocenter and the surrounding nucleoplasm. A, detection of endogenous TIF1� in HP1 immunoprecipitates. Whole cell extracts frommouse testis were analyzed by Western blotting either directly (input) or following immunoprecipitation (IP) with mAbs against HP1� (HP1� IP),-� (HP1� IP) or -� (HP1� IP) or with an irrelevant antibody (anti-FLAG antibody; control IP). Western blots (WB) of the HP1 immunoprecipitateswere probed with either TIF1� (lanes 1–6), HP1� (lanes 7–9), HP1� (lanes 10–12), or HP1� (lanes 13 and 14) antibodies. Arrowheads indicate theposition of the protein recognized by each antibody. B and C, distribution and colocalization of TIF1� with HP1� (B) and HP1� (C) in step 9 (St9)elongating spermatids (ES). Confocal images of single optical sections through the nucleus of representative individual spermatids are shown.Panels I and IV correspond to immunodetection with specific mAbs against TIF1� or HP1 isotype � or �, as indicated. Panels II show the DAPI DNAstaining, which highlights A/T-rich repeat sequences found within the centromeric heterochromatin of the chromocenter (C) (bright blue patches).

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activation was observed in the presence of AAD-TIF1� (Fig.9A). These results indicate that TIF1� can homodimerize but isunable to heterodimerize with TIF1�, TIF1�, or TIF1�.

A similar specificity of dimerization was observed using aC-terminally truncated DBD-TIF1� fusion protein bearing theN-terminal residues 1–350 and spanning the RBCC motif(DBD-TIF1�(RBCC); Fig. 9B). In the presence of AAD-TIF1� orAAD-TIF1�(RBCC), DBD-TIF1�(RBCC) significantly inducedthe reporter gene above the level of unfused AAD, whereas noactivation was detected with AAD-TIF1�, AAD-TIF1�, or AAD-TIF1�. Thus, the RBCC motif of TIF1� contains a uniquedimerization interface that is capable of mediating the forma-tion of homodimers but not heterodimers.

TIF1� Shows Neither Nuclear Receptor- nor KRAB-bindingProperties—TIF1� and TIF1� have been shown previously tointeract with various nuclear receptor (NR) family membersand several different Kruppel-associated box (KRAB) domains,respectively (7, 8, 16, 24, 26, 29). The yeast two-hybrid systemwas therefore used to investigate whether TIF1� could exhibitsome NR and/or KRAB binding activity. As shown in Fig. 10A,no significant increase in the reporter gene activity above theAAD control was detected when DBD-TIF1� was coexpressedwith AAD-NR fusion proteins (AAD-RAR�, AAD-RXR�, AAD-TR�(DE), AAD-VDR(DE), and AAD-ER�(DEF)) (16) in eitherthe presence or absence of the appropriate NR ligand, whereasunder the same conditions, coexpression of DBD-TIF1� withany of the AAD-NR fusions tested resulted in a ligand-depend-ent stimulation of the reporter gene (Fig. 10A). Similarly, nosignificant activation above the AAD control was detectedwhen individual members of the three KRAB domain subfam-ilies, KOX1(AB), MTZ1(Ab), and MZF13(A), fused to the DBDof ER� were coexpressed with AAD-TIF1�, whereas the pres-ence of AAD-TIF1� did stimulate expression of the reportergene (Fig. 10B; see also Ref. 2). Thus, like TIF1� and TIF1�,TIF1� does not display the NR binding activity found in TIF1�(7, 8, 16). Furthermore, like TIF1� and TIF1�, TIF1� does not

interact with the KRAB repression domains to which TIF1�binds (24, 26, 29).

DISCUSSION

TIF1�, a Fourth Mammalian Member of the TIF1 Gene Fam-ily—In the present study we report the isolation and charac-terization of a novel mouse gene, TIF1�, encoding a protein of1344 amino acids with all of the structural hallmarks of a bonafide TIF1 family member. Like Drosophila Bonus (10) andmammalian TIF1� (7), TIF1� (8, 24–26), and TIF1� (9, 36),TIF1� contains an N-terminal RBCC motif and a C-terminalbromodomain preceded by a PHD finger.

The RBCC motif (also known as the TRIpartite Motif orTRIM) (46) is a widely distributed motif that is composed ofthree subdomains, a C3HC4 zinc finger motif (RING), a B box

FIG. 9. Yeast two-hybrid analysis of the homo- and het-erodimerization properties of TIF1�. A, TIF1� homodimerizes butdoes not heterodimerize with TIF1�, -�, or -�. A plasmid expressingDBD-TIF1� was introduced into the yeast reporter strain PL3 togetherwith either the VP16 AAD (as a control) or the VP16 AAD fused toTIF1�, TIF1�, TIF1�, or TIF1�. OMPdecase activities determined oneach cell-free extracts are expressed in nanomoles of substrate/min/mgof protein. The values (�20%) are the average of at least three inde-pendent transformants. B, the RBCC motif of TIF1� is sufficient forself-association. A DBD fusion containing amino acids 1–350 of TIF1�(DBD-TIF1�(RBCC)) was coexpressed into PL3 with the unfused AADor the indicated AAD fusion proteins. Two-hybrid interaction assayswere performed as in A. Note that in A and B, expression of all DBD andAAD fusion proteins was confirmed by Western blotting using theantibodies F3 against the F region tag of the ER� DBD and 2GV4against VP16, respectively (data not shown).

FIG. 10. Yeast two-hybrid analysis of the interaction betweenTIF1� and numerous nuclear receptors and KRAB domains. A,TIF1� does not interact with any of the nuclear receptor ligand bindingdomains (region DE/F) known to interact with TIF1�. Plasmids ex-pressing TIF1� or TIF1� fused to the ER� DBD were introduced intothe yeast reporter strain PL3 together with VP16 AAD (as a control) orAAD fusion receptors, as indicated to the left. Transformants weregrown in the presence (�) or absence (�) of the cognate ligand (1 �M

all-trans-retinoic acid for RAR�, 1 �M 9-cis-retinoic acid for RXR�, 5 �M

T3 for TR�, 5 �M vitamin D3 for VDR, and 1 �M E2 for ER�). OMPde-case activities determined on each cell-free extract are given on theright; values are expressed as in Fig. 9A. B, TIF1� does not interact withany of the KRAB domains known to interact with TIF1�. The indicatedDBD-KRAB(AB), KRAB(Ab), and KRAB(A) domain fusions were as-sayed for two-hybrid interaction with AAD-TIF1� and AAD-TIF1� as inFig. 8C. In both panels, the values (�20%) represent the averages of atleast three independent transformants.

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type 1 (B1), and B box type 2 (B2) zinc-binding motifs of theCHC3H2 form and a predicted �-helical coiled-coil domain. Thespatial conservation of these subdomains among family mem-bers of diverse functions and their presence in large multipro-tein cellular complexes has led to the suggestion of a generalfunction of RBCC motifs in the organization of functional mac-romolecular scaffolds (11). In support of this, the RBCC motif ofTIF1� has been demonstrated recently to function as a singleintegrated unit with each subdomain contributing to ho-modimerization/homomultimerization, a prerequisite for asso-ciation of TIF1� to the KRAB domain (12, 47). In this study, wehave shown that TIF1� can interact with itself in the context oftwo-hybrid assays. This interaction is mediated by the RBCCmotif and was not detected with either TIF1�, TIF1�, or TIF1�,whereas under similar experimental conditions TIF1� andTIF1� were found to heterodimerize (data not shown and seeRef. 12). These results suggest that, in contrast to TIF1� andTIF1�, TIF1� may participate in the formation of specific cel-lular complex(es) in vivo.

Whereas RING fingers have been found in many proteins ofdiverse functions, the bromodomain is a characteristic signa-ture motif of proteins involved in chromatin-dependent regula-tion of transcription. For instance, the P/CAF histone acetyl-transferase and the chromatin-remodeling factor SWI2/SNF2are bromodomain-containing proteins (14). Recent structuraland biochemical studies of three independent bromodomainsfrom P/CAF, TAFII250, and GCN5p have clearly established arole for this domain in the molecular recognition of amino acidsequences in which lysines are acetylated, as shown for histonetails (48) as well as for MyoD (49) and Tat (50) transactivators.These studies have shown that the bromodomain has a con-served structural fold and mode of ligand binding (51). Thedomain forms an atypical left-handed four-helix bundle (helices�Z, �A, �B, and �C), and the acetyllysine side chain binds withina hydrophobic pocket formed by the ZA and BC loops of thedomain. Most interestingly, comparison of the primary se-quence of the TIF1� bromodomain with P/CAF, TAFII250, andGCN5p revealed conservation of the amino acids in the ZA andBC loops that are essential for the acetyl-lysine binding (seelegends to Fig. 1A), which suggests an acetylation-dependentmechanism of recognition for potentially interacting partnersof the TIF1� bromodomain.

In TIF1� and all members of the TIF1 gene family, thebromodomain is preceded by a PHD finger, an arrangementalso found in SP140 (52), ACF1 (53), and TIP5 (54). The PHDfinger is a conserved C4HC3 zinc-binding motif that binds twozinc atoms in a cross-brace fashion, reminiscent of the zinccoordination found in the RING finger (55, 56). Diverse humandisorders including cancer have been described to result frommutations within PHD fingers (57–59), thus emphasizing thebiological importance of this domain. Recently, PHD fingersderived from different chromatin-associated proteins (ING2,ACF1, and WSTF) have been demonstrated to bind phosphoi-nositides (PtdInsPs) (60). However, under similar binding con-ditions, the finding that the PHD finger from the Mi-2� subunitof the nucleosome remodeling and histone deacetylation NuRDcomplex does not bind to PtdInsPs (60) raises the possibilitythat PtdInsP binding is specific to a subclass of PHDs ratherthan a general property of PHDs. Another related observationis that the stretches of basic residues critical for PtdInsP in-teraction in the ING2 PHD finger (60) are not conserved amongall PHDs; in particular, absent from the list of those havingthese key residues are the PHD fingers of Mi2-�, TIF1�, -�, -�,and -� (50),2 suggesting that these PHDs may not function asPtdInsP-binding modules.

TIF1�, a Potential Epigenetic Regulator of Postmeiotic GeneExpression—Spermatogenesis is a complex multistep processof cell proliferation and differentiation in which proliferativespermatogonia generate differentiating spermatogonia thatare irreversibly committed toward the production of spermato-zoa (61, 62). This process begins by mitotic divisions of germcell spermatogonia to give rise to diploid spermatocytes, whichthemselves replicate their DNA content, before undergoing thetwo successive meiotic divisions, which results in the produc-tion of haploid round spermatids. Subsequently, these cellsenter spermiogenesis and undergo an elongation phase inwhich they are sculptured into the shape of mature spermato-zoa. During this phase, the nucleus adopts its elongated shape,the rate of transcription declines, the histones are almost com-pletely removed, and the chromatin appears as smooth fibersand then becomes highly condensed. This reorganization of thechromosomal material results from a gradual replacement ofpart of the somatic histone variants by transition proteins,which are subsequently replaced by highly basic nuclear pro-teins, the protamines (63–65). Each of these steps requires aparticular combination of expression of genes, some of whichare testis- and cell type-specific (66, 67).

We have shown here that, in contrast to the nearly ubiq-uitous expression of TIF1�, TIF1�, and TIF1� in many mouseadult tissues (7, 26, 36), expression of TIF1� is largely re-stricted to testis. Moreover, immunohistochemistry has re-vealed that, in contrast to TIF1� and TIF1� expression inproliferative, meiotic, and postmeiotic germ cells as well as inSertoli cells (35, 36), the expression of TIF1� within the testisis confined to postmeiotic, haploid-elongating spermatids.The protein first appears in elongating spermatids of step 9at the stage IX of the seminiferous epithelium cycle, reachesa maximum at step 10, and disappears during step 12 (seeFig. 5B). This timing of protein expression immediately pre-cedes the histone replacement by transition proteins (63, 64,68) and coincides precisely with the earliest evidence for bothtranscriptional quiescence and chromatin remodeling (69–71), raising the possibility that TIF1� is involved in theseprocesses.

Several lines of evidence presented here support the notionthat TIF1� could be involved in the organization of higher ordertranscriptionally repressive chromatin structures. First, tran-sient transfection and reporter assays have shown that TIF1�

can confer dose-dependent repression on a heterologous DNAbinding domain by a deacetylation inhibitor-sensitive mecha-nism. Second, by means of confocal laser-scanning microscopy,we have localized TIF1� in the nuclei of elongating spermatidsas discrete foci, some of which are associated to the blocks ofcentromeric heterochromatin that constitute the chromocenter.Most importantly, immunoprecipitation has revealed that al-most all of the endogenous TIF1� protein forms a complex withHP1�, a member of a highly conserved family of heterochroma-tin-associated proteins that have been implicated in gene si-lencing at both centromeric and noncentromeric positions (re-viewed in Refs. 21 and 22).

The isotype-preferential association of TIF1� with HP1� inelongating spermatids probably results from differences be-tween the three HP1 isotypes in their expression and sub-nuclear distribution pattern. Indeed, in agreement with pre-viously reported results (44), we failed to detect expression ofHP1� in elongating spermatids. Moreover, although thesecells are capable of expressing significant levels of expressionof HP1� and HP1�, we have shown here that, in addition toa chromocenter localization, these two isotypes also localizein the surrounding nucleoplasm, but HP1� does so abun-dantly. In accordance with this, we found by double immu-

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nostaining that most of the TIF1� foci present in the nucleo-plasm show significant colocalization with HP1� but notHP1�, whereas an association of some TIF1� foci with bothHP1� and HP1� was seen within the chromocenter. Finally,using yeast two-hybrid as well as GST pull-down experi-ments, we have demonstrated that TIF1� can bind all threerecombinant HP1 proteins, suggesting that the preferentialin vivo association of TIF1� with the HP1� isotype does notsimply reflect differences between the three HP1 proteins intheir binding affinity to TIF1�. Consistent with this, interac-tion was shown to involve a pentapeptide sequence (PYVRL)resembling the consensus HP1 box PXVXL (20, 23), which isknown to be necessary and sufficient for binding the chro-moshadow domains of the three HP1 family members.

Recently, a multisubunit chromatin-modifying complex hasbeen purified from somatic cell lines that contains HP1� butnot HP1� or HP1� (72). Most interestingly, this complex, whichcontains other chromatin modifiers such as Lys-9-H3 histonemethyltransferases and polycomb group proteins, was shownby chromatin immunoprecipitation to occupy silent promotersof several euchromatic genes in quiescent cells (72), indicatingthat it may contribute to the silencing of these genes. As his-tone H3 methylated at Lys-9 represents a high affinity bindingsite for the HP1 proteins (73, 74), a model of epigenetic genesilencing has been proposed in which binding of HP1s to meth-ylated Lys-9-H3 ultimately induces the local packaging of thetarget gene into a condensed, transcriptionally inactive hetero-chromatin-like structure and/or its relocation to heterochro-matic nuclear compartments (72, 75). This mechanism of genesilencing is thought to be widespread in the genome (75–77).Supporting a role in regulating postmeiotic gene expression,methylated Lys-9-H3 has been described in postmeiotic cells(78), and an essential testis-specific Lys-9-H3 histone methyl-transferase (Suv39h2) has been reported to be expressed inthese cells (79, 80). In light of these observations and ourpresent data, we suggest that the nuclear foci enriched inTIF1� protein in postmeiotic, steps 9 and 10, elongating sper-matids represent heterochromatin or heterochromatin-like re-pressive structures at multiple genomic sites. To gain a betterunderstanding of the biological significance of these foci, addi-tional studies are in progress involving targeted disruption ofTIF1�. These experiments are likely to provide new insightsinto the mechanisms of heterochromatin-associated gene si-lencing controlling the postmeiotic stages of male germcell development.

Acknowledgments—We thank S. Vicaire for DNA sequencing,F. Ruffenach for oligonucleotide synthesis, and the cell culture staff andour colleagues of the Department of Physiological Genetics and NuclearSignaling for helpful discussions. We also thank F. Cammas,J.-V. Fougerolle, M. Herzog, and J. Tisserand for constant support.

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Cerviño, Mustapha Oulad-Abdelghani, Pierre Chambon and Régine LossonKonstantin Khetchoumian, Marius Teletin, Manuel Mark, Thierry Lerouge, Margarita

1 (TIF1) Family Expressed by Elongating Spermatids, a Novel HP1-interacting Member of the Transcriptional Intermediary FactorδTIF1

doi: 10.1074/jbc.M404779200 originally published online August 20, 20042004, 279:48329-48341.J. Biol. Chem. 

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