Human Molecular Genetics

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Human Molecular Genetics, 2002, Vol. 11, No. 19 2341-2346
© 2002 Oxford University Press

Functional substitution for TAF_II250 by a retroposed homolog that is expressed in human spermatogenesis

P. Jeremy Wang and David C. Page^*

Howard Hughes Medical Institute, Whitehead Institute and Department of Biology, Massachusetts Institute of Technology, 9 Cambridge Center, Cambridge, MA 02142, USA

Received May 28, 2002; Accepted July 15, 2002

DDBJ/EMBL/GenBank accession nos

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
TAF_II250, the largest subunit of the general transcription factor TFIID, is expressed from the human X chromosome, at least in somatic cells. In male meiosis, however, the sex chromosomes are transcriptionally silenced, while the autosomes remain active. How then are protein-encoding genes transcribed during human male meiosis? Here we present a novel autosomal human gene, TAF1L, which is homologous to TAF_II250 and is expressed specifically in the testis, apparently in germ cells. We hypothesize that during male meiosis, transcription of protein-encoding genes relies upon TAF1L as a functional substitute for TAF_II250. Like TAF_II250, the human TAF1L protein can bind directly to TATA-binding protein, an essential component of TFIID. Most importantly, transfection with human TAF1L rescued the temperature-sensitive lethality of a hamster cell line mutant in TAF_II250. TAF1L lacks introns and evidently arose by retroposition of a processed TAF_II250 mRNA during primate evolution. The observation that TAF1L can functionally replace TAF_II250 provides experimental support for the hypothesis that during male meiosis, autosomes provide cellular functions usually supplied by the X chromosome in somatic cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
In mammalian spermatocytes (male meiotic cells), the activities of autosomal and sex-linked genes differ starkly. The autosomes are transcriptionally active. In contrast, the entireties of the X and Y chromosomes are condensed to form heterochromatin and are segregated into a special nuclear compartment, the ‘XYbody’, where Pol II (RNA polymerase II) is absent, as are critical splicing factors. There appears to be no transcription within this sex chromosome compartment (1–4). (This phenomenon is distinct from somatic ‘X inactivation’, which is the silencing of most but not all genes on one of the two X chromosomes in female cells.) In humans, sex chromosome silencing during male meiosis lasts roughly 15 days, during which time ongoing transcription of protein-encoding genes by Pol II is likely required (5). The largest of the essential factors engaged in Pol II transcription is TAF_II250, which, paradoxically, is encoded by the mammalian X chromosome (6–8). Since the TAF_II250 gene is transcriptionally inactive, perhaps another protein acts as a substitute in male meiosis. Specifically, we wondered whether the autosomes encode a TAF_II250 isoform that enables or modulates Pol II transcription during male meiosis.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
TAF1L is a retroposed homolog of TAF_II250
We examined the human genome, where the availability of extensive draft sequence facilitated an electronic search for TAF_II250 homologs (9,10). The TAF_II250 gene (also known as TAF1 or CCG1) spans 100 kb on the human X chromosome (6–8). Within the genome, we identified only one homologous locus, which we named TAF1L. TAF1L is autosomal, residing on chromosome 9. The TAF1L genomic locus exhibits >94% nucleotide identity to TAF_II250's 38 exons, but it completely lacks the latter's 37 introns (Fig. 1) and thus spans only 6.5 kb. This suggested that TAF1L is a retroposed derivative of TAF_II250.

View larger version (11K): [in this window] [in a new window]   Figure 1. Comparison of exon/intron structures of human TAFII250 and TAF1L genes. Exons 1 and 38 in TAFII250 are labeled; there appear to be no introns in TAF1L. Coding regions are shown in black. Introns are not drawn to scale. Percentage identities in coding and 3' untranslated regions (UTR) for both nucleotide and (where applicable) predicted amino acid sequences are indicated; na, not applicable. 5'-UTR display 91% nucleotide identity. In comparison with TAFII250, the TAF1L coding region displays two small deletions (3 bp each) and three insertions. The first two insertions add 29 residues to the predicted TAF1L protein but do not shift the reading frame. The third insertion (10 bp), near the 3' end, shifts the reading frame and thus the position of the termination codon, TGA, as shown. Note that we extended the published TAFII250 cDNA sequence (8) to include the 3'-UTR and AATAAA polyadenylation signal by electronically assembling overlapping expressed sequence tags (GenBank accession nos AU123960, AW959904, W05157 and W55984).

 
In the human genome, retroposed copies of genes are commonplace, but most are transcriptionally silent pseudogenes whose protein-coding regions have been disrupted by mutation (11). In contrast, we found that TAF1L is transcribed (Fig. 2) and that its protein-coding potential appeared intact. We confirmed this by sequencing TAF1L cDNA clones isolated from a human testis library. The TAF1L cDNA and genomic sequences proved to be co-linear, and the coding region is 96% identical to that found in long TAF_II250 transcripts (Fig. 1). [Alternative splice acceptor sites in exon 5 of TAF_II250 yield two distinct transcripts, one being 63 nucleotides longer than the other (6).] We concluded that TAF1L was created by reverse transcription and subsequent autosomal integration of a properly spliced TAF_II250 transcript. The result is a single-exon gene that encodes an 1826-residue protein with 95% amino acid identity to human TAF_II250 (Fig. 1). Interestingly, there is no apparent remnant of a poly(A) tail in the TAF1L gene, as is observed in many but not all retroposons.

View larger version (30K): [in this window] [in a new window]   Figure 2. Expression of TAFII250 and TAF1L in human tissues assayed by RT–PCR. (A) Normal human tissues. (B) Normal and germ-cell-deficient human testis biopsies. FTH1 (ferritin heavy chain; ubiquitously expressed) and RBMY (RNA-binding-motif protein; expressed only in male germ cells) served as controls (30). RT+ or RT- indicates presence or absence of reverse transcriptase in the reaction.

 
TAF1L displays testis-specific expression
If TAF1L assumes the role of TAF_II250 in human spermatocytes, TAF1L should be transcribed in testes. Indeed, this is the only human organ in which we have detected TAF1L expression (Fig. 2A). More specifically, our model predicts that TAF1L should be expressed in testicular germ cells. Our data suggest that TAF1L expression may be restricted to such cells: TAF1L is transcribed in normal testis but not in testis lacking germ cells (Fig. 2B). (Alternatively, it is formally possible that TAF1L is expressed in the somatic protein of the testis in a germ-cell-dependent manner.) By contrast, the X-linked TAF_II250 gene is expressed widely in human somatic tissues, including germ-cell-deficient testis (Fig. 2.).

Ideally, we would test our assumption that the X-linked TAF_II250 gene is transcriptionally silenced in male meiotic cells by RNA in situ hybridization on human testis sections. Unfortunately, this experiment is not feasible because of the high nucleotide sequence identity between TAF_II250 and TAF1L (96% in the coding region and 94% in the 3'-UTR; Fig. 1). RNA probes of sufficient length to detect these modest-abundance transcripts would likely lack the ability to discriminate between the two genes' transcripts.

TAF1L interacts with human TBP
We then tested whether the TAF1L and TAF_II250 proteins share functional characteristics. TAF_II250 has been shown to bind directly to TATA-binding protein (TBP) (6,7). To test whether TAF1L protein interacts with TBP, we performed a two-hybrid analysis in yeast cells. We fused the TAF1L protein to the DNA-binding domain of GAL4, and separately we fused human TBP to the activation domain of GAL4. Neither of the two fusion proteins alone activated a GAL4-responsive lacZ reporter gene. In combination, however, the two fusion proteins strongly activated the reporter gene (Fig. 3), indicating a direct interaction, in eukaryotic cells, of TAF1L and TBP. This supports the hypothesis that TAF1L functions as a TBP-associated factor (TAF).

View larger version (12K): [in this window] [in a new window]   Figure 3. Human TAF1L protein interacts with human TBP protein in a yeast two-hybrid assay. Quantitative assays for ß-galactosidase activity were performed with liquid cultures of yeast cells transformed with either TBP–GAL4-AD, or TAF1L–GAL4-BD or both. ONPG (o-nitrophenyl ß-D-galactopyranoside; Sigma) was used as substrate. GAL4-AD, GAL4 activation domain; GAL4-BD, GAL4 DNA-binding domain.

 
TAF1L rescues the temperature-sensitive lethality of TAF_II250 mutant cells
We then tested the ability of the human TAF1L protein to functionally replace TAF_II250 in mammalian cells. We took advantage of a hamster cell line, ts13, which is temperature-sensitive because of a single amino acid substitution (G690D) in TAF_II250 (12). When shifted to 39.5°C, ts13 cells arrest in the G₁ phase of the cell cycle and undergo apoptosis (13,14). The human TAF_II250 gene had been cloned by transfecting ts13 cells with human genomic DNA and selecting for growth at 39.5°C (13). The human TAF1L gene that we report was not identified in this transfection screen, suggesting that either (i) the TAF1L protein is not functionally equivalent to TAF_II250 or (ii) the TAF1L gene's testis-specific promoter is inactive in ts13 cells, which derive from hamster kidney (8).

To distinguish between these two possibilities, we transfected ts13 cells at the permissive temperature (33.5°C) with a DNA construct in which a ubiquitously expressed viral promoter (cytomegalovirus, CMV) drove expression of human TAF1L coding sequences. We assayed the rescue capability of this pCMN–TAF1L construct by selection at 39.5°C. As controls, we conducted parallel experiments with pCMV alone, and also with a construct bearing a single amino acid substitution (G714D) in TAF1L—a mutation analogous to that in TAF_II250 in ts13 cells. At 33.5°C, transfections of ts13 with experimental and control constructs generated neomycin-resistant colonies at similar efficiencies (Fig. 4B). At 39.5°C, neither the pCMV vector alone nor the mutant TAF1L construct yielded any viable cells, but wild-type TAF1L transfectants were viable and readily formed colonies (Fig. 4). By western blotting, we confirmed the presence of wild-type and mutant TAF1L proteins in their respective transfectants (Fig. 4C), indicating that the inability of the mutant TAF1L protein to complement the ts13 mutation is not due to protein degradation. Taken together, our results demonstrate that the human TAF1L protein is functionally interchangeable with TAF_II250 in mammalian cells.

View larger version (34K): [in this window] [in a new window]   Figure 4. Rescue of temperature sensitivity of TAFII250-mutant hamster cells (ts13) by human TAF1L. Following transfection with vector alone, or vector expressing wild-type human TAF1L, or vector expressing mutant human TAF1L (G714D), cells were split equally onto two plates: one incubated at the permissive temperature (33.5°C) with 400 µg/ml neomycin and one incubated at the non-permissive temperature (39.5°C) without neomycin. (A) Normarski images of cells 6 days after transfection. In control experiments with no DNA transfected, all cells died (not shown). (B) Numbers of colonies present 10 days after transfection. (C) Western blot of hemagglutinin (HA)-epitope-tagged TAF1L proteins expressed in transfected cells. Protein extracts from transfected cells grown at 33.5°C or 39.5°C were run on a 7.5% SDS–polyacrylamide gel. Mouse monoclonal antibody 12CA5 (Roche) was used to recognize the HA epitope. Lanes 1, 2 and 3: neomycin-resistant cells populations transfected with HA3-TAF1L (lane 1), mutant HA3-TAF1L (G714D) (lane 2) or pCMV–tag2 vector (Stratagene) (lane 3). Lane 4: HA3-TAF1L stable cell line isolated from a single colony at 39.5°C. The positions of TAF1L and molecular weight standards (in kDa) are indicated on the left.

 
TAF1L retroposition occurred during primate evolution
To determine when in human evolution the TAF1L retroposition event occurred, we searched for TAF1L orthologs in the genomes of other mammals. We employed PCR primers that would likely amplify TAF1L but not TAF_II250 genomic sequences. We found that TAF1L is present in apes and Old World monkeys but is absent in New World monkeys and rodents (Fig. 5A). We sequenced TAF1L orthologs in six apes and Old World monkeys (chimpanzee, gorilla, orangutan, gibbon, baboon and macaque), and found that the 5.5 kb open reading frame is conserved and intact in all six species, in each case displaying >97% nucleotide identity and >95% predicted protein sequence identity to human TAF1L. Analysis of rates of non-synonymous substitution (K_a) and synonymous substitution (K_s) in TAF1L orthologs revealed that the K_a/K_s ratio is consistently <1 (Fig. 5B), indicating that the TAF1L protein has been subject to functional constraint and purifying selection. Taken together, these results suggest two conclusions. First, the TAF1L retroposition probably occurred 25–40 million years ago, prior to the radiation of extant Old World monkey lineages, but after their divergence from New World monkeys (15). Second, the functional integrity of the TAF1L protein apparently has been conserved among diverse Old World monkeys and apes, including humans.

View larger version (24K): [in this window] [in a new window]   Figure 5. Orthologs of human TAF1L in apes and Old World monkeys. (A) TAF1L orthologs were sought by PCR using genomic DNA from 11 mammalian species, arrayed here phylogenetically (15). The 257 bp PCR products shown were obtained using primers selected from human TAF1L in regions of complete nucleotide sequence identity to human, mouse and hamster TAFII250 cDNAs. In the mammalian TAFII250 genomic loci, these conserved primers are interrupted by introns, and thus no amplification occurs. The results shown here were confirmed with two additional pairs of PCR primers (see Materials and Methods). NWM, New World monkeys; OWM, Old World monkeys; Mya, million years ago. (B) Ka/Ks ratios calculated from pairwise comparisons of TAF1L orthologs in seven species. TAF1L coding sequences were aligned using CLUSTAL W (31), and Ka and Ks values were calculated using Li's method as implemented in GCG software (32).

 

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
In this study, we identified an autosomal retrogene that encodes a functional substitute for the product of its X-linked progenitor. The autosomal gene, TAF1L, appears to be expressed only in spermatogenic cells, whereas the X-linked homolog, TAF_II250, is widely expressed. Several other examples of active, testis-specific autosomal retrogenes with X-linked progenitors have been identified in human and/or mouse (16–24). In each known case, the autosomal retrogene is specifically expressed in testis, while the X-linked source gene is widely expressed. However, apart from TAF_II250 and TAF1L, functional comparisons of X- and autosome-encoded isoforms have yet to be conducted.

Our study raises questions about evolutionary pressures that may have favored the functional substitution of essential somatic factors by gametogenesis-specific homologs. It has been hypothesized that autosomal, testis-specific retrogenes evolved to compensate for inactivation of their X-linked source genes during male meiosis (16). Our discovery of the functional interchangeability of X-encoded TAF_II250 and autosome-encoded TAF1L is an important step in validating this hypothesis.

Human TAF_II250 joins a small group of widely expressed TFIID components for which tissue-selective homologs have been functionally characterized. In mice, a homolog of TAF_II130 is essential for ovarian follicle development (25). Spermiogenesis in mice requires TRF2, a TBP homolog (26), while spermatogenesis in Drosophila requires a testis-specific homolog of dTAF_II80 (27). To date, all functionally characterized, tissue-selective homologs of TFIID components appear to be engaged in gametogenesis. Among these examples, the case of human TAF_II250 is unique in that we are cognizant of specific adaptive pressures (stemming from X inactivation during male meiosis) that may have driven the evolution of the tissue-specific homolog, TAF1L.

Autosomal retropositioning of X-linked genes appears to have been a recurrent, if infrequent, event throughout mammalian evolution. Among the few molecularly documented events, the most ancient involves PGK2, which arose via retroposition at least 130 million years ago, prior to the divergence of the marsupial and eutherian mammalian lineages (28). More recent events involving PHDA2, G6pd2 and Zfa have been reported (18–20,29). Our present findings indicate that TAF1L arose during primate evolution, after our species' lineage diverged from that of New World monkeys (Fig. 5A). The coming availability of complete DNA sequences of the human, mouse and other mammalian genomes will provide unprecedented opportunity to assess systematically the history and extent of retroposition of all X-linked genes during mammalian evolution.

	MATERIALS AND METHODS

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cDNA sequencing by PCR screening of library subpools
Lambda phage lysates were prepared from 24 subpools (80 000 clones each) of an amplified human testis cDNA library (Clontech). TAF1L-positive subpools were identified by PCR using primer pairs selected from the human TAF1L genomic locus (GenBank accession AL355811). Eleven TAF1L-positive subpools were identified. We then amplified 5' and 3' cDNA fragments separately from the positive subpools by two successive PCRs. In the first PCR (35 cycles), one TAF1L-specific primer and one vector primer were used. A 1 : 100 dilution of the first PCR product served as template in the second, nested PCR (25 cycles), where a second TAF1L-specific primer and a second vector primer were used to further amplify the cDNA fragment. The resulting PCR products were sequenced directly using the nested primers. Using these cDNA fragment sequences, a composite full-length cDNA sequence of TAF1L was assembled electronically.

Expression analysis by RT–PCR
Normal human tissue cDNA samples were purchased from Clontech. Human normal testis and germ-cell-deficient testis (Sertoli-cell-only-syndrome) biopsy samples were kindly provided by S. Silber (St Louis, MO). To construct bulk cDNA from testis biopsies, total RNA prepared using TRIzol reagent (Gibco BRL) was treated with DNase I to digest residual genomic DNA, and reverse-transcribed. Gene-specific PCR primers were designed to avoid cross-amplification between TAF_II250 and TAF1L. PCR conditions and primer sequences have been deposited at GenBank.

Yeast two-hybrid assay
Using the vector pAS2-1 (Clontech), the entire human TAF1L coding region was cloned in frame with the GAL4 DNA-binding domain, generating the construct TAF1L–GAL4-BD. The entire human TBP coding region was PCR-amplified from bulk human testis cDNA and fused with the GAL4 activation domain in the vector pACT2 (Clontech), generating the construct TBP–GAL4-AD. The human inserts of both resulting constructs were sequenced to confirm their integrity. Plasmids were transformed into yeast strain Y187 (GAL1_UAS –GAL1_TATA–lacZ). Quantitative liquid culture assays for ß-galactosidase activity were performed as described by the manufacturer (Clontech).

Transfection constructs
The entire human TAF1L coding region was cloned into the pCMV–Tag2A vector (Stratagene), resulting in pCMV–TAF1L.

A second construct, pCMV–TAF1L (G714D), was derived from pCMV–TAF1L by oligonucleotide-directed mutagenesis. The following four primers were used: primer 1, CATCT CTGAAGAAGAGTCGAA; primer 2, TCTTGGTTG CCATGTCAACCTGCATCATTA; primer 3, TAATGATG CAGGTTGACATGGCAACCAAGA; and primer 4, AGGTTGTTCTCAAGTGCCTG. Primers 1 and 4 are outer primers. Primers 2 and 3 are inner primers; these two primers are complementary to each other and contain the desired nucleotide change (underlined). Two DNA fragments (501 and 172 bp) were amplified by PCR with primers 1 and 2 or primers 3 and 4, respectively. The overlapping PCR products were mixed and subjected to PCR amplification with primers 1 and 4. The resulting fragment (643 bp) harbors the desired G-to-A substitution at nucleotide 2141 of the TAF1L coding region. Restriction digestion yielded an EcoRI–PstI fragment that was used to replace the corresponding portion of pCMV–TAF1L, resulting in pCMV–TAF1L (G714D).

Influenza hemagglutinin (HA) epitope-tagged versions of pCMV–TAF1L and pCMV–TAF1L (G714D) were then derived. DNA encoding three tandem copies of HA was PCR-amplified from pCU180 using primers GGAAGA TCTTTACCCATACGATGTTCCT and GGAAGATC TTGAGCAGCGTAATCTGGA. The amplified DNA was digested with BglII and ligated into BamHI-restricted pCMV–TAF1L and pCMV–TAF1L (G714D), generating the constructs pCMV–HA3–TAF1L and pCMV–HA3–TAF1L (G714D), respectively.

Transfection of ts13 cells
Prior to transfection, hamster ts13 cells were cultured at 33.5°C in 10% CO₂ in Dulbecco's modified Eagle's medium with 10% fetal calf serum and penicillin–streptomycin (8). All transfections were performed using LIPOFECTIN reagent (Life Technologies), and in each case 3 µg plasmid DNA was used as suggested by the manufacturer. Two days after transfections, cells were split 1 : 12 and subjected to selection with neomycin or at 39.5°C.

TAF1L orthologs
To identify TAF1L orthologs, we performed PCR on genomic DNAs from diverse mammals using three different pairs of primers. PCR conditions and primer sequences have been deposited at GenBank: accession nos G73370 (shown in Fig. 5A), G73371 (not shown) and G73372 (not shown).

GenBank accession numbers
Primer sequences and RT–PCR conditions for human genes: TAF_II250, G73373; TAF1L, G73374; FTH1, G65764; and RBMY, G73375. TAF1L sequences: human cDNA, AF390562; chimp genomic, AF390563; gorilla genomic, AF390564; orangutan genomic, AF390565; gibbon genomic, AF390566; baboon genomic, AF390567; and macaque genomic, AF390568.

	ACKNOWLEDGEMENTS

 
We thank S. Silber for testis biopsy samples, R. Tjian for ts13 cells, T. Huffaker for the HA3 plasmid, Yerkes Primate Center for chimpanzee, gorilla, orangutan and gibbon DNA samples, Joerg Gromoll for a macaque DNA sample, K. Rice for a baboon DNA sample, N. Watson for advice on microscopy, and A. Arango, A. Baltus, J. Bradley, L. Brown, K. Kleene, F. Lewitter, D. Menke, J. Potash, S. Rozen and H. Skaletsky for comments on the manuscript. P.J.W. is the recipient of a Medical Foundation postdoctoral fellowship.

	FOOTNOTES

 
^* To whom correspondence should be addressed. Tel: +1 6172585203; Fax: +1 6172585578; Email: page_admin{at}wi.mit.edu

AF390562–AF390568 and G73370–G73375

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