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First published online 11 March 2003
doi: 10.1242/jcs.00391

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The intracellular localisation of TAF7L, a paralogue of transcription factor TFIID subunit TAF7, is developmentally regulated during male germ-cell differentiation

¹ Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, 1 Rue Laurent Fries, 67404 Illkirch Cédex, France
² Department of Anatomy, University of Turku, 20520 Turku, Finland

View larger version (74K): [in a new window]   Fig. 1. Cloning of TAF7L and characterisation of anti-TAF7L antibodies. (A) Alignment of the sequences of TAF7L from human and mouse with those of TAF7 from mouse, Drosophila melanogaster and yeast (Saccharomyces cerevisiae). Highly conserved amino acids are shown in white on a black background. Positions conserved in at least three of the proteins are boxed in grey. Amino acids were classified as follows. Small residues: P, A, G, S, T; hydrophobic: L, I, V, A, F, M, C, Y, W; polar/acidic: D, E, Q, N; basic: R, K, H. The peptide used to generate the antibodies is underlined. (B) Cos cells were transfected with a TAF7L (lane 2) or empty (lane 1) expression vector and the recombinant TAF7L detected with monoclonal antibody 46TA. TAF7L is also visible in the testis nuclear extract (lane 3). (C) TAF7L, TBP, TAF12 and TAF13 were detected in total extracts from the tissues shown above each lane.

View larger version (89K): [in a new window]   Fig. 2. Developmental expression profiles of TAF7L, TBP and TAF7. (A) Double labelling immunodetection of TAF7L and TBP in developing male germ cells. TAF7L and TBP are detected in microdissected segments of seminiferous tubules from the stages shown in each panel. (B) Double labeling immunodetection of TAF7L and TAF7. Representative examples of cell types are indicated. ES, elongating spermatids; LS, leptotene spermatocytes; MDS, meiotic dividing spermatocytes; PS, pachytene spermatocytes; RS, haploid round spermatids; ZS, zygotene spermatocytes. Magnification, 40x.

View larger version (77K): [in a new window]   Fig. 4. Changes in intracellular localisation of TAF7L during differentiation. (A) Confocal images of staged squash sections. Double staining with TBP and TAF7L. The right hand panel shows the overlayed image of TAF7L labeling and Hoechst stained DNA. (B) Confocal image of meiotic dividing spermatocytes showing different cellular localisations of TAF7L and TBP. Magnification 100x. Representative examples of cell types are indicated. B-SG, type B spermatogonia; C, chromocenter; ERS, early haploid round spermatids; ES, elongating spermatids; MDS, meiotic dividing spermatocytes; RS, haploid round spermatids. Magnification, 40x.

View larger version (142K): [in a new window]   Fig. 3. Developmental expression profiles of TAF7L and TAF10. Double labeling immunodetection of TAF7L and TAF10 in developing male germ cells. Representative examples of cell types are indicated. ERS, early haploid round spermatids; MDS, meiotic dividing spermatocytes; PLS, preleptotene spermatocytes; PS, pachytene spermatocytes; RS, haploid round spermatids. Magnification, 40x.

View larger version (78K): [in a new window]   Fig. 5. (A-F) Summary of TBP and TAF expression during spermatogenesis. The spermatogenic differentiation programme is illustrated and the representative cell types are indicated below the bottom panels. Expression of TAFs is indicated in shades of red and that of TBP in panel B in shades of green.

View larger version (42K): [in a new window]   Fig. 6. Co-immunoprecipitation of TBP and TAF7L from testis extracts. (A) Immunoblots detecting TBP, TAF7 and TAF7L in cytoplasmic extract (CE) and nuclear extract (NE). (B) Immunoprecipitation of TBP from cytoplasmic extract. SN is the immunoprecipitate supernatant; IP the immunoprecipitated peptide-eluted fraction. (C) Immunoprecipitation of TBP from nuclear extract. (D) Detection of the presence of TBP and TAFs in total extracts from elutriation purified pachytene spermatocytes and haploid cells. The blot was first probed with antibodies against TBP, TAF7 and TAF7L and then reprobed with antibody against ACT and subsequently with TAF4. (E-F) The spermatocyte and haploid cell extracts (E) were immunoprecipitated with anti-TBP antibody. SN and IP are as described above. A small amount of IgG heavy chain which has leaked from the resin is visible in panel F, lane 3. The immunoblots were first probed with antibody against TBP, TAF7 and TAF7L (upper panel) and then reprobed with anti-TAF6 (lower panel). The presence of non-specific signal in lower panel F is indicated by asterisks (*).

View larger version (15K): [in a new window]   Fig. 7. Two hybrid interactions. (A) The TAF7L bait and the prey cDNA library are shown schematically. AD, activation domain; DBD, DNA binding domain. Graphical representation of quantitative two hybrid ß-galactosidase assays. The GAL4-DBD TAF7L chimera shown above the graph was tested with the GAL4-AD chimeras shown below each column. The results obtained with the two independent TAF1(1070-1226) clones are shown. — indicates negative controls with the GAL4-AD alone. The values represent the average of two independent experiments. (B) Schematic representation of TAF1 and the isolated clones. TAF1 is shown along with the histone acetyl-transferase (HAT) and RAPiD domains. A comparison of the TAF1 RAPiD domain and the region found in the two hybrid clones is also depicted. The numbers above represent the TAF1 amino acids at the N and C-termini of the domains.

View larger version (143K): [in a new window]   Fig. 8. Immunodetection of TAF4 in developing male germ cells. Immunofluorescence on sectioned mouse seminiferous tubules. TAF4 is shown in red. Representative examples of cell types are indicated. EES, early elongating spermatids; ES, elongating spermatids; PS, pachytene spermatocytes; RS, haploid round spermatids; ZS, zygotene spermatocytes. Magnification, 40x.