TAF7

Abstract: Transcription regulation in male germ cells can involve specialised mechanisms and testis-specific paralogues of the general transcription machinery. Here we describe TAF7L, a germ-cell-specific paralogue of the TFIID subunit TAF7. TAF7L is expressed through most of the male germ-cell differentiation programme, but its intracellular localisation is dynamically regulated from cytoplasmic in spermatogonia and early spermatocytes to nuclear in late pachytene spermatocytes and haploid round spermatids. Import of TAF7L into the nucleus coincides with decreased TAF7 expression and a strong increase in nuclear TBP expression, which suggests that TAF7L replaces TAF7 as a TFIID subunit in late pachytene spermatocytes and in haploid cells. In agreement with this, biochemical experiments indicate that a subpopulation of TAF7L is tightly associated with TBP in both pachytene and haploid cells and TAF7L interacts with the TFIID subunit TAF1. We further show that TAF3, TAF4 and TAF10 are all strongly expressed in early spermatocytes, but that in contrast to TBP and TAF7L, they are downregulated in haploid cells. Hence, different subunits of the TFIID complex are regulated in distinct ways during male germ-cell differentiation. These results show for the first time how the composition of a general transcription factor such as TFIID and other TAF-containing complexes are modulated during a differentiation programme highlighting the unique nature of the transcription regulatory machinery in spermatogenesis.

Abstract: TFIID is a general transcription factor required for transcription of most protein-coding genes by RNA polymerase II. TAF7L is an X-linked germ cell-specific paralogue of TAF7, which is a generally expressed component of TFIID. Here, we report the generation of Taf7l mutant mice by homologous recombination in embryonic stem cells by using the Cre-loxP strategy. While spermatogenesis was completed in Taf7l−/Y mice, the weight of Taf7l−/Y testis decreased and the amount of sperm in the epididymides was sharply reduced. Mutant epididymal sperm exhibited abnormal morphology, including folded tails. Sperm motility was significantly reduced, and Taf7l−/Y males were fertile with reduced litter size. Microarray profiling revealed that the abundance of six gene transcripts (including Fscn1) in Taf7l−/Y testes decreased more than twofold. In particular, FSCN1 is an F-action-bundling protein and thus may be critical for normal sperm morphology and sperm motility. Although deficiency of Taf7l may be compensated in part by Taf7, Taf7l has apparently evolved new specialized functions in the gene-selective transcription in male germ cell differentiation. Our mouse studies suggest that mutations in the human TAF7L gene might be implicated in X-linked oligozoospermia in men.

Abstract: The transcription factor TFIID, composed of the TATA box-binding protein (TBP) and 14 TBP-associated factors (TAFs), plays a key role in the regulation of gene expression by RNA polymerase II. The structure of yeast TFIID, as determined by electron microscopy and digital image analysis, is formed by three lobes, labelled A–C, connected by thin linking domains. Immunomapping revealed that TFIID contains two copies of the WD-40 repeat-containing TAF5 and that TAF5 contributes to the linkers since its C- and N-termini were found in different lobes. This property was confirmed by the finding that a recombinant complex containing TAF5 complexed with six histone fold containing TAFs was able to form a trilobed structure. Moreover, the N-terminal domain of TAF1 was mapped in lobe C, whereas the histone acetyltransferase domain resides in lobe A along with TAF7. TBP was found in the linker domain between lobes A and C in a way that the N-terminal 100 residues of TAF1 are spanned over it. The implications of these data with regard to TFIID function are discussed.

Abstract: The general transcription factor IID (TFIID) initiates RNA polymerase II-mediated eukaryotic transcription by nucleating pre-initiation complex formation at the core promoter of protein-encoding genes. TAF1, the largest integral subunit of TFIID, contains an evolutionarily conserved yet poorly characterized central core domain, whose specific mutation disrupts cell proliferation in the temperature-sensitive mutant hamster cell line ts13. Although the impaired TAF1 function in the ts13 mutant has been associated with defective transcriptional regulation of cell cycle genes, the mechanism by which TAF1 mediates transcription as part of TFIID remains unclear. Here, we present the crystal structure of the human TAF1 central core domain in complex with another conserved TFIID subunit, TAF7, which biochemically solubilizes TAF1. The TAF1-TAF7 complex displays an inter-digitated compact architecture, featuring an unexpected TAF1 winged helix (WH) domain mounted on top of a heterodimeric triple barrel. The single TAF1 residue altered in the ts13 mutant is buried at the junction of these two structural domains. We show that the TAF1 WH domain has intrinsic DNA-binding activity, which depends on characteristic residues that are commonly used by WH fold proteins for interacting with DNA. Importantly, mutations of these residues not only compromise DNA binding by TAF1, but also abrogate its ability to rescue the ts13 mutant phenotype. Together, our results resolve the structural organization of the TAF1-TAF7 module in TFIID and unveil a critical promoter-binding function of TAF1 in transcription regulation.