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First published online 29 August 2007
doi: 10.1242/dev.004481

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The first bromodomain of Brdt, a testis-specific member of the BET sub-family of double-bromodomain-containing proteins, is essential for male germ cell differentiation

¹ The Institute of Human Nutrition, Columbia University Medical Center, New York, NY 10032, USA.
² Department of Genetics and Development, Columbia University Medical Center, New York, NY 10032, USA.
³ The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.
⁴ Department of Obstetrics and Gynecology, Columbia University Medical Center, New York, NY 10032, USA.
⁵ The Center for Reproductive Sciences, Columbia University Medical Center, New York, NY 10032, USA.
⁶ The Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, NY 10032, USA.

View larger version (31K): [in this window] [in a new window]   Fig. 1. Targeting of the mouse Brdt locus. (A) Targeting construct used to generate Brdt mutant mice. The top line depicts the genomic region of the Brdt gene, along with the PCR primers and probes used for genotyping targeted embryonic stem (ES) cells and subsequent progeny; the middle line depicts the targeting construct with a Neo and TK cassette; the bottom line indicates the organization of the resulting recombined allele. (B) Northern hybridization revealed that the mutant allele is still transcribed but produces an mRNA that is smaller in size (3.4 kb). (C) Western blot showing that the shorter mRNA is translated in frame to produce a protein that is recognized by our -CT antibody. (D) Splicing of the mutant Brdt mRNA and the predicted corresponding protein product. Reverse transcription (RT)-PCR of the mutant testicular mRNA was used to define the exact nature of the mRNA and the putative truncated protein. The mRNA results from splicing from exon 1 to exon 5; there is an in-frame ATG at the beginning of exon 5. The original translation start codon (ATG) is located in exon 3, which has been deleted in the mutant allele. The allele that produces this altered mRNA is referred to as BrdtBD1.

View larger version (159K): [in this window] [in a new window]   Fig. 2. Immunostaining of testicular sections with anti-Brdt antibodies. DAB staining produces a brownish color at the site of antibody localization. The -CT antibody was used in the staining. Roman numerals within each tubule denote the stage of the tubule as defined previously (Russell et al., 1990). (A) A wild-type stage XI tubule showing the expression of Brdt protein in the nucleus of pachytene (P) but not zygotene (Z) spermatocytes. (B) A mutant stage IX tubule showing the expression of BrdtBD1 protein in the nucleus of pachytene (P) but not leptotene (L) spermatocytes. (C) A stage XII wild-type tubule showing that, during the meiotic divisions (M), Brdt protein was distributed throughout the cell, but after the meiotic divisions, Brdt protein was predominantly found in the nucleus. (D) A stage XII mutant tubule showing a similar expression pattern and sub-cellular distribution of the truncated Brdt protein in the BrdtBD1/BD1 testes. (E) Stage I, V-VI and VIII wild-type tubules showing the expression of Brdt protein in round spermatids (rs) but not in elongating spermatids (es). (F) Stage II-III, IV and IX mutant tubules showing the expression of the truncated Brdt protein in round spermatids (rs) but not in elongating spermatids (es). L, leptotene spermatocytes; Z, zygotene spermatocyte; P, pachytene spermatocyte; M, meiotic M phase; rs, round spermatid; es, elongating spermatid. Scale bar: 40 µm.

View larger version (52K): [in this window] [in a new window]   Fig. 3. Brdt mutant epididymal sperm are abnormal. (A) Morphology of epididymis from controls and homozygous mutants showing that very few sperm are present in the epididymis of the BrdtBD1/BD1 mutant mice. (B) Light microscopy showing epididymal sperm of wild-type and BrdtBD1/BD1 mutant mice. The photomicrographs show the variations of the abnormalities in the mutants, which occur throughout the sperm, including in the sperm head, mid-piece (M) and tail. (C) Electron microscopy of sperm reveals variation in the extent of nuclear condensation, as reflected by regions of reduced electron density and also by a failure of the acrosome to form normally. Residual cytoplasm, which should have been discarded as the residual body, is seen and it contains aberrant mitochondria. Magnifications in C: WT, x13,000; mutant, upper right, x8,300; lower left, x10,000; lower right, x8,300. A, acrosome; N, nucleus; M, midpiece. Scale bar: 5 µm.

View larger version (157K): [in this window] [in a new window]   Fig. 4. The abnormalities of spermatogenesis in BrdtBD1/BD1 mice were apparent at the elongating spermatid stage. (A,B) PAS staining of histological sections of testes from wild-type (A) and Brdt mutant (B) mice showing the abnormal shape of the elongating spermatids (B; arrow). (C,D) Immunostaining of histological sections of testes from wild-type (C) and Brdt mutant (D) mice with anti-Prm1 antibody, showing that the expression of Prm1 appeared on schedule, although the morphogenesis of the spermatids was abnormal in the mutants. Scale bar: 40 µm.

View larger version (82K): [in this window] [in a new window]   Fig. 5. Ultrastructural analysis of the elongating spermatids. Electron microscopy revealed that the normally occurring foci of heterochromatin (A, arrow) at the nuclear envelope are absent in Brdt mutant elongating spermatids (B). Magnifications: WT, x8,300; mutant, x13,000.

View larger version (69K): [in this window] [in a new window]   Fig. 6. Brdt protein is involved in the regulation of expression of histone H1t. (A) Histone H1t expression is upregulated in BrdtBD1/BD1 mutant testes. Real-time reverse transcription (RT)-PCR was used to quantify changes in the levels of expression of selected genes from control and BrdtBD1/BD1 mutant testes. Among the genes examined for expression was Brdt itself, using primers specifically designed such that they would not recognize the mutant transcripts. As predicted, no normal Brdt mRNA was detected (first bar). The expression levels are presented as relative levels of the wild-type expression and the wild-type expression levels are set as one. The results, corrected with GAPDH expression, are means±s.e.m. of at least three experiments in three pairs of animals. (B) Immunoblot of H1t protein in wild-type and BrdtBD1/BD1 mutant testes. H1t protein level was elevated in the mutant testis as compared with control. ß-tubulin was used as a loading control. (C) Immunostaining of testicular sections with anti-H1t antibody, showing that H1t was expressed in pachytene spermatocytes and spermatids and that H1t protein level was elevated in the mutant testis. (D) Chromatin immunoprecipitation (ChIP) assay showing that Brdt protein binds the histone H1t promoter. Two pairs of PCR primers corresponding to the histone H1t promoter proximal and distal regions were used. Total testicular cells were prepared from wild-type and BrdtBD1/BD1 mutant testis and used for ChIP experiments with anti-Brdt C-terminal antibody. The cartoon in the upper panel shows the positions of the primers in the H1t promoter. I, input chromatin; C, Brdt peptide blocked control Brdt antibody; E, Brdt unblocked antibody experimental.