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Originally published In Press as doi:10.1074/jbc.M404779200 on August 20, 2004

J. Biol. Chem., Vol. 279, Issue 46, 48329-48341, November 12, 2004
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TIF1{delta}, a Novel HP1-interacting Member of the Transcriptional Intermediary Factor 1 (TIF1) Family Expressed by Elongating Spermatids*

Konstantin Khetchoumian{ddagger}§, Marius Teletin¶, Manuel Mark{ddagger}, Thierry Lerouge{ddagger}, Margarita Cerviño{ddagger}, Mustapha Oulad-Abdelghani{ddagger}, Pierre Chambon{ddagger}, and Régine Losson{ddagger}||

From the {ddagger}Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France and Institut Clinique de la Souris, BP 10142, 67 404 Illkirch-Cedex, France

Received for publication, April 29, 2004 , and in revised form, August 16, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
TIF1 (transcriptional intermediary factor 1) proteins are encoded by an expanding family of developmental and physiological control genes that are conserved from flies to man. These proteins are characterized by an N-terminal RING-B box-coiled-coil (RBCC) motif and a C-terminal PHD finger/bromodomain unit, and have been implicated in epigenetic mechanisms of transcriptional repression involving histone modifiers and heterochromatin-binding proteins. We describe here the isolation and functional characterization of a fourth murine TIF1 gene, TIF1{delta}. The predicted TIF1{delta} protein displays all the structural hallmarks of a bona fide TIF1 family member and resembles the other TIF1s in that it can exert a deacetylase-dependent silencing effect when tethered to a promoter region. Moreover, like TIF1{alpha} and TIF1{beta}, TIF1{delta} can homodimerize and contains a PXVXL motif necessary and sufficient for HP1 (heterochromatin protein 1) binding. Although TIF1{alpha} and TIF1{beta} also bind nuclear receptors and Krüppel-associated boxes specifically and respectively, TIF1{delta} appears to lack nuclear receptor- and Krüppel-associated box binding activity. Furthermore, TIF1{delta} is unique among the TIF1 family proteins in that its expression is largely restricted to the testis and confined to haploid elongating spermatids, where it associates preferentially with HP1 isotype {gamma} (HP1{gamma}) and forms discrete foci dispersed within the centromeric chromocenter and the surrounding nucleoplasm. Collectively, these data are consistent with specific, nonredundant functions for the TIF1 family members in vivo and suggest a role for TIF1{delta} in heterochromatin-mediated gene silencing during postmeiotic phases of spermatogenesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Transcriptional regulation of gene expression in eukaryotes in response to developmental or environmental signals is a complex multistep process that requires the concerted action of many cellular factors. Central players in this elaborate process are sequence-specific transcription factors that positively and/or negatively control transcription through interactions with transcriptional intermediary factors (TIFs1; also designated as coactivators and corepressors), whose ultimate function is to remodel chromatin structure (1, 2), to stimulate or inhibit (pre)initiation complex formation (3), or to associate target genes with specialized nuclear compartments (4, 5).

TIF1s are members of a growing family of chromatin-associated/related TIFs, a subset that has emerged as key regulators of developmental and physiological processes (6). Included in this family are three members (TIF1{alpha}, -{beta}, and -{gamma}) (7-9) in mammals and one (Bonus) (10) in Drosophila, and all consist of two conserved amino acid regions: an N-terminal RING-B box coiled-coil (RBCC) domain with potential self-assembly properties (11, 12); and a C-terminal region containing a PHD finger and a bromodomain, two well conserved signature motifs widely distributed among nuclear proteins known to function at the chromatin level (13-15).

TIF1{alpha}, the founding member of the family, was initially identified in a yeast genetic screen for proteins modulating the transactivation potential of retinoid X receptor (RXR) (7) and was subsequently found to interact via a single LXXLL motif with the AF-2 transcriptional activation domain of several nuclear receptors, including retinoic acid (RAR), thyroid (TR), vitamin D3 (VDR), and estrogen (ER) receptors (8, 16). TIF1{alpha} is a euchromatin-enriched chromosomal protein expressed ubiquitously and early in development (17, 18) as well as in many adult tissues (7). In mouse NIH 3T3 cells, TIF1{alpha} was reported to play a role in the growth-suppressive activity of RXR/RAR and to exhibit a transforming activity when fused to a truncated B-Raf (19). Supporting the notion that the biological functions of TIF1{alpha} are achieved through modulation of chromatin states, TIF1{alpha} has been demonstrated to possess an intrinsic transcriptional silencing activity that requires histone deacetylation (8, 20). Moreover, TIF1{alpha} has the ability to interact directly with members of the heterochromatin protein 1 (HP1) family (8), a class of nonhistone chromosomal proteins that serve as dose-dependent regulators of higher order chromatin structures to promote silencing on euchromatic genes (reviewed in Refs. 21 and 22). Mapping of the HP1-interacting domain in TIF1{alpha} led to the identification of a conserved PXVXL motif located in its central region that binds directly to the C-terminal chromoshadow domain of HP1 proteins (8) and exists in other potential transcriptional regulatory targets (23).

The identification of TIF1{beta} established the TIF1 family of transcriptional cofactors (8). TIF1{beta} (also known as KAP-1 or KRIP-1) was isolated by virtue of its ability to interact with mouse HP1{alpha} (8) and with the KRAB domain of the human Krüppel-like proteins KOX1 (24, 25) and Kid-1 (26). The KRAB transcriptional repression domain is a widely distributed motif frequently found at the N termini of zinc finger proteins of Krüppel Cys2-His2-type (27). This domain contains a conserved KRAB A box and is usually followed by a KRAB B box (or a diverged b box; Ref. 28). All variants of the KRAB domain studied so far function through the recruitment of TIF1{beta} (29). Consistent with a role in chromatin organization, TIF1{beta} silences transcription through a mechanism that involves histone 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 the relocalization of TIF1{beta} into regions of centromeric heterochromatin during cell differentiation (33). In mouse, TIF1{beta} is expressed ubiquitously throughout development (34) and in many adult tissues (26). We have recently shown that disruption of TIF1{beta} in mice leads to an embryonic lethal phenotype due to a developmental arrest at the egg cylinder stage, prior to the onset of gastrulation (34), thus demonstrating that TIF1{beta} exerts essential and nonredundant functions during early post-implantation development. Subsequently, the use of a conditional germ line-specific disruption of TIF1{beta} in the adult testis unveiled a later and essential nonredundant function of TIF1{beta} in the homeostasis of the seminiferous epithelium (35).

The third mammalian member of the TIF1 family, TIF1{gamma}, was discovered via low stringency hybridization screens using TIF1{alpha} as a probe (9). Amino acid comparison revealed that, among the three mammalian members of the family, TIF1{gamma} is closer to TIF1{alpha} than to TIF1{beta} (50% overall identity between TIF1{alpha} and TIF1{gamma} and ~30% identity among the other TIF1s) (9, 36). In vitro, TIF1{alpha} and TIF1{gamma} hetero-oligomerize as efficiently as they homo-oligomerize, whereas TIF1{beta} does homooligomerize but does not hetero-oligomerize with TIF1{alpha} or TIF1{gamma} (12, 37). Moreover, it has been shown that an overexpression of TIF1{gamma} in transiently transfected cells can interfere with the transrepression activity of TIF1{alpha} (12). Further evidence supporting a cross-talk between TIF1{alpha} and TIF1{gamma} is the recent identification of two novel types of RET rearrangements in childhood papillary thyroid carcinomas, PTC6 and PTC7, having in common the RET receptor tyrosine kinase domain fused to the RBCC domains of TIF1{alpha} (PTC6) and TIF1{gamma} (PTC7), respectively (38). In both human and mouse, TIF1{gamma} transcripts are widely expressed at varying levels in adult and fetal tissues (9, 36). Similar to the other TIF1 family members, TIF1{gamma} contains an intrinsic transcriptional silencing function (9); however, the downstream targets that mediate gene silencing have yet to be identified.

Recently, we isolated and characterized the only ortholog of mammalian TIF1s in the Drosophila genome, the so-called Bonus (10). Bonus is a chromatin-associated protein that is expressed throughout development. Mutational analysis revealed that Bonus is required for male viability, molting, and numerous events in metamorphosis that are associated with genes implicated in the ecdysone pathway, a nuclear hormone receptor pathway required throughout Drosophila development (10). Bonus shares with TIF1{alpha} the ability to interact with the nuclear receptor AF-2 activation domain (10). Direct evidence for the biological relevance of this evolutionarily conserved interaction was provided by genetic studies showing that reduction in the level of Bonus suppresses the phenotype associated with a partial loss of function mutation of the nuclear receptor {beta}FTZ-F1 (10), thus defining Bonus (and by analogy TIF1{alpha}) as negative regulators of nuclear receptor-dependent transcription.

Here we report the isolation and functional analysis of a fourth murine TIF1 gene, TIF1{delta}. Over the entire length of the 1344-amino acid protein, TIF1{delta} shares ~30% identity with the other TIF1s and also displays a potent trichostatin A (TSA, a specific inhibitor of histone deacetylases)-sensitive repression function. However, TIF1{delta} differs remarkably from the other TIF1 family members in that its expression is largely restricted to testis, where it was seen by immunohistochemistry only in male germ cells that have completed meiosis, at the early elongating spermatid stages. TIF1{delta} was localized by confocal laser-scanning microscopy in discrete nuclear foci distributed throughout the nucleus including the chromocenter, a condensed structure formed by the association of centromeric heterochromatin. Furthermore, a preferential association between TIF1{delta} and HP1 isotype {gamma} (HP1{gamma}) has been identified through coimmunoprecipitation. Taken together, these data suggest a role for TIF1{delta} as a negative regulator of postmeiotic genes acting through HP1{gamma} complex formation and centromere association.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Clone Isolation and Sequencing—Four exon-specific primers designed according to the nucleotide sequences of predicted human TIF1{delta} exons in the KIAA0298 genomic sequence (GenBankTM accession number AJ400879 [GenBank] ) were used to amplify two DNA probes by RT-PCR. Primers used are as follows: (a) forward primer, 5'-AACCCAAGATGGCCAGGAAC-3' (AAU20) and reverse primer, 5'-GCCCTGGACATTTGTCCACC-3' (AAU21), and (b) forward primer, 5'-CAGAGCCTTCAATAGTGAGC-3' (AAU22) and reverse primer 5'-TGCCTTGGCTGGGCAAACCG-3' (AAU23). RT-PCR amplification was performed on 0.5 µg of HeLa total RNA using the Clostridium thermocellum polymerase One-step RT-PCR system (Roche Applied Science). The amplified DNA fragments were used as probes to screen a {lambda}ZAPII mouse brain-derived cDNA library (Stratagene) according to the manufacturer's instructions. These screens resulted in the isolation of two mouse cDNA clones, TIF1{delta}-a (nucleotides +212 to +1322) and TIF1{delta}-b (nucleotides +2011 to +4111) (see Fig. 1B). Complete sequence information of the coding region was achieved by RT-PCR using 1.5 µg of mouse testis total RNA as template and the following primer pairs: (a) TIF1{delta} exon 9 forward primer, 5'-TTCCAGTCTCCAGCACTGTG-3' (ACD251), and TIF1{delta} exon 10 reverse primer, 5'-AGAGCTGGCTTTGGGCCCTC-3' (ACD252), designed from the TIF1{delta}-a and TIF1{delta}-b sequences, respectively; and (b) TIF1{delta} exon 1 forward primer, 5'-ATAACTTGGACCTCGGAGAC-3' (ADA27), and TIF1{delta} exon 3 reverse primer, 5'-ATGTGTGCTGCCCGCTTCTC-3' (ACM266), designed on the basis of the nucleotide sequences of putative TIF1{delta} exon 1 (GenBankTM accession number AJ307670 [GenBank] ) and TIF1{delta}-a cDNA clone, respectively. The resulting RT-PCR products (TIF1{delta}-c (nucleotides +1240 to +2045), TIF1{delta}-d1 (nucleotides -183 to +353), and TIF1{delta}-d2 (nucleotides -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 each RT-PCR were subjected to DNA sequence analysis. To extend the TIF1{delta} cDNA sequence toward its 3' end, a 3'-RACE experiment was performed using the SMART RACE kit (Clontech), with mouse testis total RNA and the gene-specific forward primer, TIF1{delta} exon 19 primer, 5'-TACATGCAGGAAGGCATCCA-3' (AFA51). The fragment amplified by 3'-RACE (TIF1{delta}-e (nucleotides +3931 to +4254)) was purified, cloned, and sequenced.



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  		FIG. 1.
Mouse TIF1{delta} cDNA sequence, protein domain structure, and genomic structure. A, nucleotide and deduced amino acid sequence of mouse cDNA encoding TIF1{delta}. The 4437 nucleotides of TIF1{delta} cDNA, the putative translation start site (in boldface and underlined), the 1344-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{delta} (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. The hydrophobic amino acids defining the heptad repeats of the putative coiled-coil structure are highlighted by gray circles. Highly conserved amino acid residues in the HP1 box (underlined) and the bromodomain (boxed) are in boldface. Residues of the bromodomain that are essential for the acetyl-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{delta} cDNA sequence. ORF is depicted as a box and untranslated regions as horizontal lines. The sequence was reconstituted from the indicated cDNA clones. TIF1{delta}-a and TIF1{delta}-b were obtained by screening a mouse adult brain cDNA library, whereas the others were isolated by RT-PCR (TIF1{delta}-c, TIF1{delta}-d1, and TIF1{delta}-d2) or 3'-RACE (TIF1{delta}-e) using mouse testis RNA as template. TIF1{delta}-d2 differs from TIF1{delta}-d1 by a 63-nucleotide deletion between positions +149 and +211. C, genomic organization of the mouse TIF1{delta} gene. Exons are represented as numbered boxes and introns as connecting lines. The scale of the genomic map is indicated below. Exon 1 contains the ATG (from nucleotides -183 to +211) and exon 20 contains the stop codon TGA. D, alternative splicing of the mouse TIF1{delta} gene between exons 1 and 2. The 5'-splice donor gt, and 3'-splice acceptor ag are in italics. Translation start and stop codons defining open reading frames are in boldface.

 
Northern Blot Analysis—Multiple mouse tissue Northern blots containing poly(A)+ RNA from various tissues (Clontech) were hybridized with a cDNA fragment encoding amino acids 671-976 of TIF1{delta} (nucleotides +2011 to +2930) or a {beta}-actin cDNA fragment (control) labeled with [{alpha}-32P]dCTP by random priming according to the manufacturer's instructions.

Antibodies—Specific antibodies used include the following: (a) mouse anti-TIF1{delta} monoclonal antibody (mAb), PG124, raised against TIF1{delta}-(132-151) for immunohistochemistry, and rabbit anti-TIF1{delta} polyclonal antibody, PG78, raised against TIF1{delta}-(695-721) for Western blot analysis. The specificity of these antibodies was established with an extract of COS-1 cells transfected with a eukaryotic expression plasmid encoding FLAG-tagged TIF1{delta} (Fig. 5 and data not shown). (b) mouse anti-HP1{alpha} mAbs, 2HP1H5 for immunoprecipitation and immunohistochemistry and 2HP2G9 for Western blot analysis (18); (c) mouse anti-HP1{beta} mAb, 1Mod1A9 (20); (d) mouse anti-HP1{gamma} mAb, 2Mod1G6 (20). For the double immunostaining experiments, anti-HP1{beta} and -HP1{gamma} antibodies were purified by the caprylic acid/ammonium sulfate method and then coupled 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{alpha} mAb, F3, raised against the F region of human ER{alpha} (39); and mouse anti-{beta}-actin mAb, AC-15 (Sigma).



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  		FIG. 5.
A, developmental expression of TIF1{delta} in testis from prepubertal and adult mice. Whole cell extracts (100 µg) from mouse testes of different postnatal ages (lane 3, 2-week-old; lane 4, 3-week-old; lane 5, 4-week-old; lane 6, 3-month-old) were resolved along with control (lane 1) and TIF1{delta} (lane 2)-transfected COS-1 cell extracts by 8% SDS-PAGE and were immunoblotted with the anti-TIF1{delta} polyclonal antibody PG78. The sizes of the protein markers (kDa) are indicated on the left side of the figure, and the arrowhead on the right side specifies the position of the TIF1{delta} protein. {beta}-Actin was used as a loading control. B, summary of TIF1{delta} expression during spermatogenesis. The diagram is adapted from Russell et al. (62). The red and blue areas indicate areas of strong and weak immunoreactivity, respectively. Abbreviations: A, type A spermatogonia; B, type B spermatogonia; D, diplotene; In, intermediate spermatogonia; L, leptotene; P, pachytene; PL, preleptotene; S, Sertoli cells; SC2, type 2 spermatocytes; Z, zygotene.

 
Immunohistochemistry—Testes from 3-month-old mice were fixed in 4% paraformaldehyde in PBS (12 h at 4 °C) and then embedded in Paraplast. Five-µm-thick sections were collected on Superfrost slides (Kindler, Freiburg, Germany), dried for 16 h at 37 °C, and kept at 4 °C until use. Sections were dried again for 15 min at 57 °C, dewaxed, hydrated, then placed into 0.01 M sodium citrate buffer, pH 6.0, and exposed to microwave treatment (power output 900 watts; 10 min). After cooling down to room temperature, sections were rinsed in PBS, treated with 5% NGS in PBS, 0.05% Tween 20 (PBS/NGS/Tween) for 30 min at room temperature to block nonspecific antibody binding to the tissue, and incubated for 16 h at 4 °C with the anti-TIF1{delta} antibody PG124 (ascites dilution: 1/1000 in PBS/NGS/Tween). Sections were then washed (PBS; three times for 5 min), incubated with the secondary antibody (Cy3-coupled goat anti-mouse, diluted 1/500, 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 µg/µl. As negative controls of the immunostaining procedure, histological sections were incubated with a mixture of the primary antibody (diluted 1/1000) and immunizing peptide (5 µg/ml).

Double immunostaining experiments were performed as follows. Following incubation with the primary anti-TIF1{delta} antibody PG124 as described above, testis sections were then washed (PBS; three times for 5 min), incubated with the secondary antibody (Alexa488-coupled goat anti-mouse, diluted 1/250, The Jackson Laboratories) for 45 min at room temperature, washed in PBS, and incubated with either Biotin-coupled anti-HP1{beta} antibody 1Mod1A9 (dilution 1/150 in PBS) or Cy3-coupled anti-HP1{gamma} antibody 2Mod1G6 (dilution 1/50 in PBS) for 12 h at 4 °C. TIF1{delta}/HP1{beta} double-labeled sections were then washed (PBS, three times for 5 min), incubated with Cy3-coupled streptavidin (dilution 1/250 in PBS, The Jackson Laboratories) for 45 min at room temperature, washed in PBS, fixed in 4% formalin (10 min, room temperature), then washed again in PBS and mounted in Vectashield (Vector Laboratories) containing DAPI at 10 µg/µl. Washing, fixation, and mounting were also done for TIF1{delta}/HP1{gamma} double-labeled sections. Confocal images were obtained using a Leica confocal scanning microscope (SP1) and were superposed using home-made software.

Immunoprecipitation and Western Blot Analysis—Testes from adult mice were homogenized with a Dounce homogenizer in ice-cold lysis buffer (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 a protease inhibitor mixture of aprotinin, leupeptin, chymostatin, and pepstatin at 2.5 µg/ml each. Intact cells and debris were removed by centrifugation at 13,000 rpm for 15 min at 4 °C, and supernatants were collected. Protein concentration was determined with Bio-Rad protein assay reagent (Bio-Rad). To immunoprecipitate the HP1 proteins, testicular lysates (15 mg) were first incubated with 50 µl of protein G-Sepharose beads for 1 h at 4 °C. The beads were removed by centrifugation at 2000 rpm for 2 min, and 5 µl of specific antibody was then added. Samples were incubated for 3 h at 4 °C, followed by incubation overnight with 50 µl of Protein G-Sepharose beads at 4 °C. Immune complexes were collected by centrifugation at 2000 rpm for 2 min and washed three times with 3 ml of EBC buffer. The final pellets were then resuspended in 50 µl of 2x Laemmli SDS buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 2% {beta}-mercaptoethanol, 0.002% bromphenol blue) and analyzed by SDS-PAGE and immunoblotting as described previously (40).

Transient Transfection—Transient transfection in COS-1 cells as well as CAT and {beta}-galactosidase assays were performed as described (8).

Yeast Two-hybrid Interaction Assays—DBD and acidic activation domain (AAD) fusion proteins were expressed from the yeast multicopy plasmids pBL1 and pASV3, respectively (39). These plasmids express inserts under the control of the phosphoglycerate kinase promoter. PBL1 contains the HIS3 marker and directs the synthesis of epitope (region F of ER{alpha})-tagged ER{alpha} DBD fusion proteins. pASV3 contains the LEU2 marker and a cassette expressing a nuclear localized VP16 AAD. The reporter strain PL3 (MAT{alpha} leu2-{Delta}1 ura3-{Delta}1 his3-{Delta}200 trp1::(ERE)3-URA3) was as described elsewhere (39). Yeast cells grown in yeast extract/peptone/dextrose or selective medium were transformed by the lithium acetate procedure. Transformants were grown exponentially for about five generations in selective medium supplemented with uracil. Yeast extracts were prepared and assayed for orotidine 5'-monophosphate decarboxylase (OMPdecase) activity as described previously (39).

In Vitro Binding Assays—GST pull-down assays were performed as described (20). GST and GST-HP1 fusion proteins were expressed in Escherichia coli and purified on glutathione-Sepharose beads (Amersham Biosciences), as described by the manufacturer. For expression of 35S-labeled TIF1{delta}, the coding sequence of TIF1{delta} was inserted into the pSG5 vector, and coupled transcription/translation was performed using the T7 RNA polymerase with the TNT lysate system (Promega).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation of a Fourth Murine TIF1 Gene, TIF1{delta}—To identify additional members of the TIF1 family, we performed BLASTTM searches against GenBankTM and dbEST data bases using the mouse TIF1{alpha} protein (7) (GenBankTM accession number Q64127) as a query sequence. These searches revealed high similarity to a putative gene residing at corresponding locations on human chromosome 11p15.4 and mouse chromosome 7 (GenBankTM accession numbers AJ400879 [GenBank] and AJ307670 [GenBank] , respectively). By using oligonucleotide primers designed according to the predicted open reading frame (ORF) of this gene, a set of overlapping mouse cDNA clones was isolated by a combination of cDNA library screening, RT-PCR, and 3'-RACE (see "Experimental Procedures"; Fig. 1B). After compilation of these cDNAs, a contig of 4437 bp (hereafter called TIF1{delta} cDNA) was obtained (Fig. 1A), including a potential start codon in frame with an ORF of 4035 bp flanked by a 183-bp 5'- and a 219-bp 3'-untranslated region (Fig. 1A; GenBankTM accession number AY572454 [GenBank] ). Comparison of the nucleotide sequence of this cDNA to the corresponding genomic sequence identified 20 exons located within 54.4 kb of genomic DNA with 19 introns ranging in size from 142 to 21,602 bp (Fig. 1C and data not shown).

A TIF1{delta} cDNA variant lacking a stretch of 63 nucleotides between nucleotides +149 and +211 was also isolated by RT-PCR (Fig. 1, A and B; GenBankTM accession number AY572455 [GenBank] ). The presence or absence of this 63-nucleotide segment in the TIF1{delta} cDNA reflects the use of two alternative 5'-splicing donor sites located 63 bp apart at the 3' end of exon 1 (Fig. 1D). Excision of the segment maintains an inphase ORF but causes a premature termination of translation at codon 50 (Fig. 1D). Due to a possible downstream reinitiation of translation at methionine codon 103, the shorter transcript is predicted to produce an N-terminally truncated TIF1{delta} protein lacking the first 102 amino acids (see legends to Fig. 1D). All subsequent studies were performed with the longest cDNA isoform that encodes a 1344-amino acid protein containing all the characteristic motifs of TIF1 proteins (see Fig. 1A and Fig. 2B).



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  		FIG. 2.
TIF1{delta} is structurally related to the other previously cloned TIF1 family members. A, amino acid identities/similarities (in %) among the TIF1 proteins. The proteins shown are mouse TIF1{alpha} (GenBankTM accession number Q64127) and TIF1{beta} (GenBankTM accession number Q62318), human TIF1{gamma} (GenBankTM accession number Q9UPN9), and Drosophila Bonus (GenBankTM accession number AAF19646 [GenBank] . Sequence comparisons were performed with the Bestfit program of the Wisconsin GCG package. B, schematic representation of the TIF1 protein family. Amino acid numbers indicate the size of each TIF1, and amino acid identities/similarities are given between TIF1{delta} and each other protein. C, sequence alignment of the HP1-interacting domain of TIF1{alpha}, TIF1{beta}, and CAF-1 with TIF1{delta} amino acids 982-1020. Residues that are conserved between TIF1{delta} and the other proteins are shaded. Asterisks indicate residues that are identical in all proteins. The pentameric consensus defining the conserved PXVXL motif required for HP1 binding is shown.

 
Over the entire length of the protein, TIF1{delta} is 35 and 29% identical to mouse TIF1{alpha} and TIF1{beta}, respectively (7, 8), 37% identical to human TIF1{gamma} (9), and 26% identical to Drosophila Bonus (10) (Fig. 2A). Like the other family members, TIF1{delta} contains several evolutionary conserved domains (Figs. 1A and 2B). At the N terminus, a C3HC4 zinc finger motif or RING finger is followed by two cysteine-rich zinc binding regions (B boxes) and a predicted {alpha}-helical coiled-coil domain forming a tripartite motif designated RBCC (7) (Figs. 1A and 2B). At the C terminus, a bromodomain (14, 15) is preceded by a C4HC3 zinc finger motif or PHD finger (13) (Figs. 1A and 2B). In addition to these conserved domains found in a number of transcriptional cofactors (see "Discussion"), we identified a 39-amino acid segment in the central region of TIF1{delta} (amino acids 982-1020; underlined in Fig. 1A) which is highly similar to the HP1 box of TIF1{alpha} and TIF1{beta}, in which the integrity of the conserved PXVXL motif (where X is any amino acid) has been shown to be required for HP1 binding (8, 20) (Fig. 2C). Similar to TIF1{alpha} and Bonus, the central region of TIF1{delta} also contains an LXXLL motif (amino acids 617-621; underlined in Fig. 1A), which, however, did not allow TIF1{delta} to directly interact with nuclear receptors (see below; Fig. 10A). Note that a cluster of glutamine and proline residues is also present in the central region of TIF1{delta} from position 554 to 572 (Fig. 1A), whereas the C-terminal part of the protein contains a bipartite nuclear localization signal between residues 1318 and 1336 (Fig. 1A).



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  		FIG. 10.
Yeast two-hybrid analysis of the interaction between TIF1{delta} and numerous nuclear receptors and KRAB domains. A, TIF1{delta} does not interact with any of the nuclear receptor ligand binding domains (region DE/F) known to interact with TIF1{alpha}. Plasmids expressing TIF1{alpha} or TIF1{delta} fused to the ER{alpha} DBD were introduced into the yeast reporter strain PL3 together with VP16 AAD (as a control) or AAD fusion receptors, as indicated to the left. Transformants were grown in the presence (+) or absence (-) of the cognate ligand (1 µM all-trans-retinoic acid for RAR{alpha}, 1 µM 9-cis-retinoic acid for RXR{alpha}, 5 µM T3 for TR{alpha}, 5 µM vitamin D3 for VDR, and 1 µM E2 for ER{alpha}). OMPdecase activities determined on each cell-free extract are given on the right; values are expressed as in Fig. 9A. B, TIF1{delta} does not interact with any of the KRAB domains known to interact with TIF1{beta}. The indicated DBD-KRAB(AB), KRAB(Ab), and KRAB(A) domain fusions were assayed for two-hybrid interaction with AAD-TIF1{beta} and AAD-TIF1{delta} as in Fig. 8C. In both panels, the values (±20%) represent the averages of at least three independent transformants.

 
TIF1{delta} Expression Is Predominant in Testis and Occurs Specifically in Elongating Spermatids—TIF1{alpha} (7), TIF1{beta} (26), and TIF1{gamma} (36) were shown previously to be expressed in a wide variety of mouse adult tissues. Using a TIF1{delta}-specific probe (see Experimental Procedures), we analyzed the tissue distribution of TIF1{delta} mRNA in the adult mouse by Northern blot hybridization and found two transcripts of ~5.2 and 6.0 kb only in the poly(A)+ RNA from testis (Fig. 3). Prolonged exposure of the autoradiogram failed to reveal hybridizing species of the same size in any tissue examined, although in both brain and testis a faint hybridization to high molecular weight transcripts (~10 kb) was seen (indicated by * in Fig. 3; and data not shown). Thus, TIF1{delta} differs remarkably from the other TIF1 family members in that its expression in the adult mice is largely restricted to the testis.



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  		FIG. 3.
Tissue specificity of TIF1{delta} expression. RNA blots containing poly(A)+ RNA from the indicated mouse tissues were hybridized with a TIF1{delta}-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 an asterisk, were also detected in both brain and testis. Hybridization to a {beta}-actin probe is shown as a control for mRNA integrity and loading. The positions of RNA size markers (kb) are shown on the left.

 
Testicular cells expressing TIF1{delta} were identified by indirect immunofluorescence using a specific monoclonal antibody raised against a 20-amino acid peptide of TIF1{delta} (amino acids 132-151; see "Experimental Procedures"). An intense fluorescent 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 almost mature, i.e. elongated and condensed, spermatids populating tubules at stages I to VIII of the seminiferous epithelium cycle (II-VI and VII-VIII in Fig. 4, A-C, and data not shown). No immunostaining was observed in nuclei of (i) round spermatids (data not shown), (ii) proliferative and meiotic germ cells (i.e. spermatogonia and spermatocytes; G, LP, P, and Z in Fig. 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 the primary antibody and immunizing peptide yielded faint cytoplasmic signals in both the seminiferous epithelium and Leydig cells but no nuclear signals (data not shown). These results indicate that TIF1{delta} expression within the testis is confined to postmeiotic, haploid, and elongating spermatids, in contrast to that of other TIF1s (i.e. {beta} and {gamma}) that are produced by proliferative, meiotic, and postmeiotic germ cells as well as Sertoli cells (35, 36).2



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  		FIG. 4.
Stage-specific expression of TIF1{delta} in male germ cells. Histological sections from 3-month-old testes were incubated with the anti-TIF1{delta} mAb PG124, whose binding to cell structures was then revealed with a Cy3-conjugated secondary antibody (red signal), and nuclei were counterstained with DAPI (blue signal). Roman numerals refer to stages of the seminiferous epithelium cycle. Abbreviations: G, spermatogonia; L, Leydig cells; LP, leptotene spermatocyte; M, peritubular myoepithelial cells; P, pachytene spermatocytes; S, Sertoli cells; Sc2, type 2 spermatocytes; St9, St10, and St12, step 9, 10, and 12 spermatids; Z, zygotene spermatocyte. Bar: 80 µm (A-C) and 40 µm (D-L).

 

Western blot analysis was also employed to follow the testicular expression of TIF1{delta} during puberty. No significant expression was found in 2- and 3-week-old mouse testes (Fig. 5A, lane 3 and 4, respectively), whereas the TIF1{delta} protein was clearly detectable from the age of 4 weeks onward (Fig. 5A, lane 5), a time when elongating spermatids become abundant (41). This observation is consistent with the above immunohistochemical data, indicating that expression of TIF1{delta} only occurs at late stages of spermatogenesis (summarized in Fig. 5B).

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



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  		FIG. 6.
TIF1{delta} 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{delta} represses activated transcription in a dose-dependent manner when tethered to DNA. The 17M2-ERE-G-CAT reporter (1 µg) and 1 µg of pCH110 (expressing {beta}-galactosidase) were cotransfected into COS-1 cells with the indicated pSG5-based vectors expressing the activator ER(C)-VP16 (100 ng) and the unfused GAL4 DBD (GAL4: 200 ng) or TIF1{delta} fused to the GAL4 DBD (GAL4-TIF1{delta}: 2, 20, or 200 ng) or TIF1{delta} tagged with the FLAG epitope (TIF1{delta}: 200 ng). CAT activities are expressed relative to the activity measured in the presence of ER(C)-VP16 and the unfused GAL4 (taken as 100%). Values (±10%) represent the averages of three independent and duplicated transfection experiments after normalization to {beta}-galactosidase activities. C, TSA treatment partially relieves TIF1{delta} 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.

 
Because in the case of TIF1{alpha}- and TIF1{beta}-mediated repression, recruitment of histone deacetylases has been involved (20, 30), we analyzed the effects of the potent histone deacetylase inhibitor trichostatin A (TSA) (42) on the repression activity of TIF1{delta}. Treatment of the transfected cells with TSA significantly reduced GAL4-TIF1{delta} repression (Fig. 6C), indicating that the repressive effects of TIF1{delta} on transcription are mediated, at least in part, by recruitment of deacetylase activity.

TIF1{delta} Interacts with the Nonhistone Chromosomal Proteins HP1{alpha}, -{beta}, and -{gamma}—TIF1{alpha} (8) and TIF1{beta} (20, 32) both interact with the HP1 family proteins through a 25-amino acid segment containing a conserved pentapeptide motif (PXVXL), also termed the HP1 box (8, 23). Because a similar pentapeptide sequence is present between residues 982 and 1020 of TIF1{delta} (see Figs. 1A and 2C), the ability of TIF1{delta} to interact with the HP1 proteins was tested using the yeast two-hybrid system. Full-length TIF1{delta} was fused to the estrogen receptor DNA binding domain, and the resulting hybrid protein (DBD-TIF1{delta}; Fig. 7A) was coexpressed with either "unfused" AAD (as a control) or AAD chimeric proteins consisting of the AAD of VP16 (amino acids 411-490) fused to any one of the HP1 family members (AAD-HP1{alpha}, AAD-HP1{beta}, and AAD-HP1{gamma}; Fig. 7A) in the yeast reporter strain PL3 containing an ERE-URA3 reporter gene (see Fig. 7A) (39). Activation of the reporter was determined by measuring the orotidine 5'-monophosphate decarboxylase (OMPdecase) activity of the URA3 gene product. When coexpressed with the AAD or DBD controls, none of the hybrid proteins transactivated the URA3 reporter (Fig. 7B). In contrast, coexpression of DBD-TIF1{delta} with either AAD-HP1{alpha} or AAD-HP1{beta} or AAD-HP1{gamma} resulted in ~20-30-fold increases in the reporter gene activity, indicating that TIF1{delta} can interact with HP1{alpha}, -{beta}, and -{gamma} in yeast. Most interestingly, an interaction was also observed with a DBD fusion protein bearing TIF1{delta} residues 982-1020 (Fig. 7B; DBD-TIF1{delta}-(982-1020)), thus providing strong evidence for the presence of an HP1 box within the central region of TIF1{delta}. To demonstrate that TIF1{delta} actually binds HP1 proteins through this HP1 box, two point mutations were introduced into the PXVXL motif, replacing the conserved hydrophobic residues Val-991 and Leu-993 by alanine residues (Fig. 7A). In contrast to wild type TIF1{delta}, the mutant protein (DBD-TIF1{delta}V991A/L993A; Fig. 7B) was drastically impaired in its ability to interact with HP1{alpha}, HP1{beta}, and HP1{gamma}. Taken together, these data indicate that TIF1{delta}, like TIF1{alpha} and TIF1{beta}, contains a conserved HP1 box that is necessary and sufficient for specific interaction with the HP1 proteins.



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  		FIG. 7.
TIF1{delta} interacts with HP1{alpha},-{beta}, and -{gamma}. A, schematic representation of the yeast two-hybrid system used in this study. The DBD of the human ER{alpha} (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{delta} protein, boxes indicate the conserved domains (see Fig. 2B). The pentameric consensus defining the conserved PXVXL motif, referred to as the HP1 box, is shown. The URA3 reporter gene, which is regulated by three estrogen response elements (ERE3X) in the yeast reporter strain PL3, is represented below. B, TIF1{delta} interacts with all three HP1s through a 39-amino acid segment containing the conserved PXVXL motif. Plasmids expressing the indicated DBD fusion proteins, DBD-TIF1{delta}, DBD-TIF1{delta}-(982-1020), and DBD-TIF1{delta}V991A/L993A, were introduced into PL3 together with either the unfused VP16 AAD or AAD fusions containing HP1{alpha}, HP1{beta}, or HP1{gamma}. Transformants were grown in liquid medium containing uracil. OMPdecase activities determined on each cell-free extract are expressed in nanomoles of substrate/min/mg protein. The values (± 20%) are the average of at least three independent transformants. C and D, the C-terminal chromoshadow domains of HP1{alpha}, HP1{beta}, and HP1{gamma} are sufficient for TIF1{delta} interaction. A schematic diagram of the chimeras is shown on the left. The indicated DBD-HP1 fusions were assayed for two-hybrid interaction with either the VP16 AAD or an AAD fusion containing TIF1{delta} (AAD-TIF1{delta}). Two-hybrid interaction assays were performed as in B. Note that in B-D, expression of all DBD and AAD fusion proteins was confirmed by Western blotting using the antibodies F3 against the F region of ER{alpha} and 2GV4 against VP16, respectively (data not shown). E, in vitro binding of TIF1{delta} to HP1s. In vitro 35S-labeled TIF1{delta} was incubated in a batch assay with "control" GST (lane 2), GST-HP1{alpha} (lane 3), GST-HP1{beta} (lane 4), or GST-HP1{gamma} (lane 5). Bound TIF1{delta} was resolved on SDS-PAGE and visualized by autoradiography. Lane 1 represents 1/10 the amount of input labeled TIF1{delta}.

 
To identify the HP1 sequences to which TIF1{delta} binds, a deletion analysis of HP1{alpha} was performed using the yeast two-hybrid system. Various HP1{alpha} deletion mutants expressed as DBD fusion proteins (8) were tested for interaction with TIF1{delta} fused to the VP16 AAD in the yeast strain PL3 (Fig. 7C). No interaction was detected with a DBD fusion protein bearing HP1{alpha} residues 1-66 or 67-113, which include the chromodomain and the less conserved central region, respectively (Fig. 7C). In contrast, an interaction was observed in the presence of an N-terminally truncated fusion protein lacking the chromodomain (DBD-HP1{alpha}-(67-191); Fig. 7C) or in the presence of a fusion protein bearing residues 114-180 of the chromoshadow domain (DBD-HP1{alpha}-(114-180); Fig. 7C). Similarly, HP1{beta} and HP1{gamma} derivatives containing the chromoshadow domain only (see DBD-HP1{beta}-(110-177) and DBD-HP1{gamma}-(104-170) in Fig. 7D) interacted with TIF1{delta} (Fig. 7D). Thus, the chromoshadow domain of either HP1{alpha}, HP1{beta}, or HP1{gamma} is sufficient for binding to TIF1{delta}.

Binding assays between TIF1{delta} and the HP1 proteins were also performed in vitro. GST-HP1 fusion proteins, GST-HP1{alpha}, GST-HP1{beta}, and GST-HP1{gamma}, were expressed in E. coli, immobilized on glutathione-Sepharose beads, and subsequently incubated with in vitro synthesized 35S-labeled TIF1{delta}. After extensive washing, the matrix-associated TIF1{delta} protein was eluted and visualized by SDS-PAGE and autoradiography. As shown in Fig. 7E, TIF1{delta} was retained on beads coupled to either GST-HP1{alpha} (lane 3), GST-HP1{beta} (lane 4), or GST-HP1{gamma} (lane 5) but not on "control" GST beads (lane 2). Thus, TIF1{delta} can interact with the HP1 proteins both in yeast and in vitro.

TIF1{delta} Associates Preferentially with HP1{gamma} and Forms Multiple Foci throughout the Nucleus of Elongating Spermatids— The ability of TIF1{delta} to interact with HP1{alpha}, -{beta}, and -{gamma} in vitro prompted us to investigate whether these proteins could be physically associated in vivo. Whole cell extracts from adult mouse testis were immunoprecipitated with either anti-HP1{alpha}, -{beta}, or -{gamma} antibodies, and the immunoprecipitates were probed for the presence of TIF1{delta}. Coimmunoprecipitation of endogenous TIF1{delta} was clearly detected in each HP1 immunoprecipitate (Fig. 8A, lanes 4-6) but not in control immunoprecipitates using an irrelevant antibody (lane 3). Quantification by densitometry of the TIF1{delta} levels in the load material (input) versus pellets (HP1 IP) from three separate experiments indicated that less than 1% of total TIF1{delta} could be immunoprecipitated with HP1{alpha} (0.5 ± 0.1%; Fig. 8A and data not shown), whereas HP1{beta} and HP1{gamma} antibodies were equally potent in immunoprecipitating ~5-10% of TIF1{delta} 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 conditions where the percent recovery of immunoprecipitated HP1{gamma} was quite low (8.4 ± 4.1%; see Fig. 8A, lanes 13 and 14,and data not shown) as compared with HP1{alpha} (19.7 ± 3.3%; lanes 7-9) and HP1{beta} (29.8 ± 8.2%; lanes 10-12). Thus, after normalization to the respective levels of HP1 recovery, it was estimated that endogenous TIF1{delta} was mostly associated with HP1{gamma} and, to a lesser extent (~25%), with HP1{beta}, whereas a small proportion (less than 5%) may also be present as stable complex with HP1{alpha}.



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  		FIG. 8.
TIF1{delta} preferentially associates with HP1{gamma} in elongating spermatids and localizes to discrete nuclear foci within both the chromocenter and the surrounding nucleoplasm. A, detection of endogenous TIF1{delta} in HP1 immunoprecipitates. Whole cell extracts from mouse testis were analyzed by Western blotting either directly (input) or following immunoprecipitation (IP) with mAbs against HP1{alpha} (HP1{alpha} IP), -{beta} (HP1{beta} IP) or -{gamma} (HP1{gamma} IP) or with an irrelevant antibody (anti-FLAG antibody; control IP). Western blots (WB) of the HP1 immunoprecipitates were probed with either TIF1{delta} (lanes 1-6), HP1{alpha} (lanes 7-9), HP1{beta} (lanes 10-12), or HP1{gamma} (lanes 13 and 14) antibodies. Arrowheads indicate the position of the protein recognized by each antibody. B and C, distribution and colocalization of TIF1{delta} with HP1{beta} (B) and HP1{gamma} (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{delta} or HP1 isotype {beta} or {gamma}, as indicated. Panels II show the DAPI DNA staining, which highlights A/T-rich repeat sequences found within the centromeric heterochromatin of the chromocenter (C) (bright blue patches).

 
Confocal laser-scanning microscopy was then employed to further investigate the distribution and subnuclear localization of TIF1{delta} in elongating spermatids with respect to the three HP1 proteins as well as the chromocenter, a condensed structure formed by the association of centromeric heterochromatin (43). The anti-TIF1{delta} monoclonal antibody revealed a microgranular signal comprising numerous small nuclear foci (see Fig. 8, B, I and C, I; step 9 elongating spermatids and data not shown). These foci were distributed throughout the nucleus, including the characteristic DAPI-dense block of centromeric heterochromatin that constitutes the chromocenter (Fig. 8, B, III, and C, III). As reported previously (44), no specific staining for HP1{alpha} was detectable in elongating spermatids (data not shown), suggesting that these cells express very low levels, if any, of HP1{alpha}. In contrast, using anti-HP1{beta} or anti-HP1{gamma} antibodies, we were able to detect significant levels of expression of HP1{beta} and HP1{gamma} in elongating spermatids (see Fig. 8, B, IV, and C, IV, respectively). Within the nuclei of these cells, HP1{beta} and HP1{gamma} both exhibited a predominant localization to the chromocenter with a mainly diffuse distribution; however, as observed previously in various cell lines (20, 40, 45), HP1{beta} and even more so HP1{gamma} were detected in many additional sites dispersed within the nucleoplasm that surrounds the chromocenter (Fig. 8, B, IV, and C, IV). Double immunostaining with TIF1{delta} and HP1{beta} revealed an association of some TIF1{delta}-enriched foci with the HP1{beta} signal within the chromocenter but not outside of it (Fig. 8B, V). This result is consistent with the coimmunoprecipitation data showing that not all but only a fraction of the pool of TIF1{delta} is physically associated with HP1{beta} (Fig. 8A). Double staining with TIF1{delta} and HP1{gamma} revealed a high number of TIF1{delta} foci that showed significant overlap with the HP1{gamma} signals within both the chromocenter and the surrounding nucleoplasmic compartment (Fig. 8C, V). Although the significance of these coimmunolocalizations is difficult to interpret because of the rather homogeneous staining pattern of HP1{gamma} within the nucleus, the results are in complete agreement with the immunoprecipitation data indicating that almost all of the TIF1{delta} protein in elongating spermatids is stably associated with HP1{gamma} isotype (Fig. 8A).

Evidence for TIF1{delta} Self-association—Because of the previous demonstration that TIF1s ({alpha}, -{beta}, and -{gamma}) can form homodimers (12, 37) as well heterodimers (TIF1{alpha}-TIF1{gamma}) (12), we examined the dimerization capacities of TIF1{delta} using the yeast two-hybrid system. In this assay, DBD-TIF1{delta} was coexpressed with AAD fusion proteins between the AAD of VP16 and any one of the family members (AAD-TIF1{alpha}, AAD-TIF1{beta}, AAD-TIF1{gamma}, and AAD-TIF1{delta}; Fig. 9A) in the yeast reporter strain PL3. No significant increase in the reporter gene activity above the AAD control was detected with either AAD-TIF1{alpha}, AAD-TIF1{beta}, or AAD-TIF1{gamma}, whereas under the same conditions, a reporter activation was observed in the presence of AAD-TIF1{delta} (Fig. 9A). These results indicate that TIF1{delta} can homodimerize but is unable to heterodimerize with TIF1{alpha}, TIF1{beta}, or TIF1{gamma}.



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  		FIG. 9.
Yeast two-hybrid analysis of the homo- and heterodimerization properties of TIF1{delta}. A, TIF1{delta} homodimerizes but does not heterodimerize with TIF1{alpha}, -{beta}, or -{gamma}. A plasmid expressing DBD-TIF1{delta} was introduced into the yeast reporter strain PL3 together with either the VP16 AAD (as a control) or the VP16 AAD fused to TIF1{alpha}, TIF1{beta}, TIF1{gamma}, or TIF1{delta}. OMPdecase activities determined on each cell-free extracts are expressed in nanomoles of substrate/min/mg of protein. The values (±20%) are the average of at least three independent transformants. B, the RBCC motif of TIF1{delta} is sufficient for self-association. A DBD fusion containing amino acids 1-350 of TIF1{delta} (DBD-TIF1{delta}(RBCC)) was coexpressed into PL3 with the unfused AAD or the indicated AAD fusion proteins. Two-hybrid interaction assays were performed as in A. Note that in A and B, expression of all DBD and AAD fusion proteins was confirmed by Western blotting using the antibodies F3 against the F region tag of the ER{alpha} DBD and 2GV4 against VP16, respectively (data not shown).

 
A similar specificity of dimerization was observed using a C-terminally truncated DBD-TIF1{delta} fusion protein bearing the N-terminal residues 1-350 and spanning the RBCC motif (DBD-TIF1{delta}(RBCC); Fig. 9B). In the presence of AAD-TIF1{delta} or AAD-TIF1{delta}(RBCC), DBD-TIF1{delta}(RBCC) significantly induced the reporter gene above the level of unfused AAD, whereas no activation was detected with AAD-TIF1{alpha}, AAD-TIF1{beta}, or AAD-TIF1{gamma}. Thus, the RBCC motif of TIF1{delta} contains a unique dimerization interface that is capable of mediating the formation of homodimers but not heterodimers.

TIF1{delta} Shows Neither Nuclear Receptor- nor KRAB-binding Properties—TIF1{alpha} and TIF1{beta} have been shown previously to interact with various nuclear receptor (NR) family members and several different Krüppel-associated box (KRAB) domains, respectively (7, 8, 16, 24, 26, 29). The yeast two-hybrid system was therefore used to investigate whether TIF1{delta} could exhibit some NR and/or KRAB binding activity. As shown in Fig. 10A, no significant increase in the reporter gene activity above the AAD control was detected when DBD-TIF1{delta} was coexpressed with AAD-NR fusion proteins (AAD-RAR{alpha}, AAD-RXR{alpha}, AAD-TR{alpha}(DE), AAD-VDR(DE), and AAD-ER{alpha}(DEF)) (16) in either the presence or absence of the appropriate NR ligand, whereas under the same conditions, coexpression of DBD-TIF1{alpha} with any of the AAD-NR fusions tested resulted in a ligand-dependent stimulation of the reporter gene (Fig. 10A). Similarly, no significant activation above the AAD control was detected when individual members of the three KRAB domain subfamilies, KOX1(AB), MTZ1(Ab), and MZF13(A), fused to the DBD of ER{alpha} were coexpressed with AAD-TIF1{delta}, whereas the presence of AAD-TIF1{beta} did stimulate expression of the reporter gene (Fig. 10B; see also Ref. 2). Thus, like TIF1{beta} and TIF1{gamma}, TIF1{delta} does not display the NR binding activity found in TIF1{alpha} (7, 8, 16). Furthermore, like TIF1{alpha} and TIF1{gamma}, TIF1{delta} does not interact with the KRAB repression domains to which TIF1{beta} binds (24, 26, 29).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
TIF1{delta}, a Fourth Mammalian Member of the TIF1 Gene Family—In the present study we report the isolation and characterization of a novel mouse gene, TIF1{delta}, encoding a protein of 1344 amino acids with all of the structural hallmarks of a bona fide TIF1 family member. Like Drosophila Bonus (10) and mammalian TIF1{alpha} (7), TIF1{beta} (8, 24-26), and TIF1{gamma} (9, 36), TIF1{delta} contains an N-terminal RBCC motif and a C-terminal bromodomain preceded by a PHD finger.

The RBCC motif (also known as the TRIpartite Motif or TRIM) (46) is a widely distributed motif that is composed of three subdomains, a C3HC4 zinc finger motif (RING), a B box type 1 (B1), and B box type 2 (B2) zinc-binding motifs of the CHC3H2 form and a predicted {alpha}-helical coiled-coil domain. The spatial conservation of these subdomains among family members of diverse functions and their presence in large multiprotein cellular complexes has led to the suggestion of a general function of RBCC motifs in the organization of functional macromolecular scaffolds (11). In support of this, the RBCC motif of TIF1{beta} has been demonstrated recently to function as a single integrated unit with each subdomain contributing to homodimerization/homomultimerization, a prerequisite for association of TIF1{beta} to the KRAB domain (12, 47). In this study, we have shown that TIF1{delta} can interact with itself in the context of two-hybrid assays. This interaction is mediated by the RBCC motif and was not detected with either TIF1{alpha}, TIF1{beta}, or TIF1{gamma}, whereas under similar experimental conditions TIF1{alpha} and TIF1{gamma} were found to heterodimerize (data not shown and see Ref. 12). These results suggest that, in contrast to TIF1{alpha} and TIF1{gamma}, TIF1{delta} may participate in the formation of specific cellular complex(es) in vivo.

Whereas RING fingers have been found in many proteins of diverse functions, the bromodomain is a characteristic signature motif of proteins involved in chromatin-dependent regulation of transcription. For instance, the P/CAF histone acetyl-transferase and the chromatin-remodeling factor SWI2/SNF2 are bromodomain-containing proteins (14). Recent structural and biochemical studies of three independent bromodomains from P/CAF, TAFII250, and GCN5p have clearly established a role for this domain in the molecular recognition of amino acid sequences in which lysines are acetylated, as shown for histone tails (48) as well as for MyoD (49) and Tat (50) transactivators. These studies have shown that the bromodomain has a conserved structural fold and mode of ligand binding (51). The domain forms an atypical left-handed four-helix bundle (helices {alpha}Z, {alpha}A, {alpha}B, and {alpha}C), and the acetyllysine side chain binds within a hydrophobic pocket formed by the ZA and BC loops of the domain. Most interestingly, comparison of the primary sequence of the TIF1{delta} bromodomain with P/CAF, TAFII250, and GCN5p revealed conservation of the amino acids in the ZA and BC loops that are essential for the acetyl-lysine binding (see legends to Fig. 1A), which suggests an acetylation-dependent mechanism of recognition for potentially interacting partners of the TIF1{delta} bromodomain.

In TIF1{delta} and all members of the TIF1 gene family, the bromodomain is preceded by a PHD finger, an arrangement also found in SP140 (52), ACF1 (53), and TIP5 (54). The PHD finger is a conserved C4HC3 zinc-binding motif that binds two zinc atoms in a cross-brace fashion, reminiscent of the zinc coordination found in the RING finger (55, 56). Diverse human disorders including cancer have been described to result from mutations within PHD fingers (57-59), thus emphasizing the biological importance of this domain. Recently, PHD fingers derived from different chromatin-associated proteins (ING2, ACF1, and WSTF) have been demonstrated to bind phosphoinositides (PtdInsPs) (60). However, under similar binding conditions, the finding that the PHD finger from the Mi-2{alpha} subunit of the nucleosome remodeling and histone deacetylation NuRD complex does not bind to PtdInsPs (60) raises the possibility that PtdInsP binding is specific to a subclass of PHDs rather than a general property of PHDs. Another related observation is that the stretches of basic residues critical for PtdInsP interaction in the ING2 PHD finger (60) are not conserved among all PHDs; in particular, absent from the list of those having these key residues are the PHD fingers of Mi2-{alpha}, TIF1{alpha}, -{beta}, -{gamma}, and -{delta} (50),2 suggesting that these PHDs may not function as PtdInsP-binding modules.

TIF1{delta}, a Potential Epigenetic Regulator of Postmeiotic Gene Expression—Spermatogenesis is a complex multistep process of cell proliferation and differentiation in which proliferative spermatogonia generate differentiating spermatogonia that are irreversibly committed toward the production of spermatozoa (61, 62). This process begins by mitotic divisions of germ cell spermatogonia to give rise to diploid spermatocytes, which themselves replicate their DNA content, before undergoing the two successive meiotic divisions, which results in the production of haploid round spermatids. Subsequently, these cells enter spermiogenesis and undergo an elongation phase in which they are sculptured into the shape of mature spermatozoa. During this phase, the nucleus adopts its elongated shape, the rate of transcription declines, the histones are almost completely removed, and the chromatin appears as smooth fibers and then becomes highly condensed. This reorganization of the chromosomal material results from a gradual replacement of part of the somatic histone variants by transition proteins, which are subsequently replaced by highly basic nuclear proteins, the protamines (63-65). Each of these steps requires a particular combination of expression of genes, some of which are testis- and cell type-specific (66, 67).

We have shown here that, in contrast to the nearly ubiquitous expression of TIF1{alpha}, TIF1{beta}, and TIF1{gamma} in many mouse adult tissues (7, 26, 36), expression of TIF1{delta} is largely restricted to testis. Moreover, immunohistochemistry has revealed that, in contrast to TIF1{beta} and TIF1{gamma} expression in proliferative, meiotic, and postmeiotic germ cells as well as in Sertoli cells (35, 36), the expression of TIF1{delta} within the testis is confined to postmeiotic, haploid-elongating spermatids. The protein first appears in elongating spermatids of step 9 at the stage IX of the seminiferous epithelium cycle, reaches a maximum at step 10, and disappears during step 12 (see Fig. 5B). This timing of protein expression immediately precedes the histone replacement by transition proteins (63, 64, 68) and coincides precisely with the earliest evidence for both transcriptional quiescence and chromatin remodeling (69-71), raising the possibility that TIF1{delta} is involved in these processes.

Several lines of evidence presented here support the notion that TIF1{delta} could be involved in the organization of higher order transcriptionally repressive chromatin structures. First, transient transfection and reporter assays have shown that TIF1{delta} can confer dose-dependent repression on a heterologous DNA binding domain by a deacetylation inhibitor-sensitive mechanism. Second, by means of confocal laser-scanning microscopy, we have localized TIF1{delta} in the nuclei of elongating spermatids as discrete foci, some of which are associated to the blocks of centromeric heterochromatin that constitute the chromocenter. Most importantly, immunoprecipitation has revealed that almost all of the endogenous TIF1{delta} protein forms a complex with HP1{gamma}, a member of a highly conserved family of heterochromatin-associated proteins that have been implicated in gene silencing at both centromeric and noncentromeric positions (reviewed in Refs. 21 and 22).

The isotype-preferential association of TIF1{delta} with HP1{gamma} in elongating spermatids probably results from differences between the three HP1 isotypes in their expression and subnuclear distribution pattern. Indeed, in agreement with previously reported results (44), we failed to detect expression of HP1{alpha} in elongating spermatids. Moreover, although these cells are capable of expressing significant levels of expression of HP1{beta} and HP1{gamma}, we have shown here that, in addition to a chromocenter localization, these two isotypes also localize in the surrounding nucleoplasm, but HP1{gamma} does so abundantly. In accordance with this, we found by double immunostaining that most of the TIF1{delta} foci present in the nucleoplasm show significant colocalization with HP1{gamma} but not HP1{beta}, whereas an association of some TIF1{delta} foci with both HP1{beta} and HP1{gamma} was seen within the chromocenter. Finally, using yeast two-hybrid as well as GST pull-down experiments, we have demonstrated that TIF1{delta} can bind all three recombinant HP1 proteins, suggesting that the preferential in vivo association of TIF1{delta} with the HP1{gamma} isotype does not simply reflect differences between the three HP1 proteins in their binding affinity to TIF1{delta}. Consistent with this, interaction was shown to involve a pentapeptide sequence (PYVRL) resembling the consensus HP1 box PXVXL (20, 23), which is known to be necessary and sufficient for binding the chromoshadow domains of the three HP1 family members.

Recently, a multisubunit chromatin-modifying complex has been purified from somatic cell lines that contains HP1{gamma} but not HP1{alpha} or HP1{beta} (72). Most interestingly, this complex, which contains other chromatin modifiers such as Lys-9-H3 histone methyltransferases and polycomb group proteins, was shown by chromatin immunoprecipitation to occupy silent promoters of several euchromatic genes in quiescent cells (72), indicating that it may contribute to the silencing of these genes. As histone H3 methylated at Lys-9 represents a high affinity binding site for the HP1 proteins (73, 74), a model of epigenetic gene silencing has been proposed in which binding of HP1s to methylated Lys-9-H3 ultimately induces the local packaging of the target gene into a condensed, transcriptionally inactive heterochromatin-like structure and/or its relocation to heterochromatic nuclear compartments (72, 75). This mechanism of gene silencing 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 methyltransferase (Suv39h2) has been reported to be expressed in these cells (79, 80). In light of these observations and our present data, we suggest that the nuclear foci enriched in TIF1{delta} protein in postmeiotic, steps 9 and 10, elongating spermatids represent heterochromatin or heterochromatin-like repressive structures at multiple genomic sites. To gain a better understanding of the biological significance of these foci, additional studies are in progress involving targeted disruption of TIF1{delta}. These experiments are likely to provide new insights into the mechanisms of heterochromatin-associated gene silencing controlling the postmeiotic stages of male germ cell development.


   FOOTNOTES
 
* This work was supported in part by the CNRS, the INSERM, les Hôpitaux Universitaires de Strasbourg, the Association pour la Recherche sur le Cancer, the Collège de France, and the Fondation pour la Recherche Médicale. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Supported by the Ligue Nationale Contre le Cancer. Back

|| To whom correspondence should be addressed. Tel.: 33-3-88-65-34-71; Fax: 33-3-88-65-32-01; E-mail: losson{at}igbmc.u-strasbg.fr.

1 The abbreviations used are: TIF, transcriptional intermediary factor; RBCC, RING-B box-coiled-coil; NR, nuclear receptors; KRAB, Krüppel-associated boxes; RXR, retinoid X receptor; RAR, retinoic acid receptor; TR, thyroid receptor; VDR, vitamin D3 receptor; ER, estrogen receptor; mAb, monoclonal antibody; ORF, open reading frame; DAPI, 4,6-diamidino-2-phenylindole; PBS, phosphate-buffered saline; RT, reverse transcription; NGS, normal growth serum; GST, glutathione S-transferase; RACE, rapid amplification of cDNA ends; AAD, acidic activation domain; OMPdecase, orotidine 5'-monophosphate decarboxylase; TSA, trichostatin A; PtdInsPs, phosphoinositides; ERE, estrogen-response element; CAT, chloramphenicol acetyl-transferase; DBD, DNA binding domain. Back

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


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



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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