Bromodomain-dependent stage-specific male genome programming by Brdt

Abstract

Male germ cell differentiation is a highly regulated multistep process initiated by the commitment of progenitor cells into meiosis and characterized by major chromatin reorganizations in haploid spermatids. We report here that a single member of the double bromodomain BET factors, Brdt, is a master regulator of both meiotic divisions and post-meiotic genome repackaging. Upon its activation at the onset of meiosis, Brdt drives and determines the developmental timing of a testis-specific gene expression program. In meiotic and post-meiotic cells, Brdt initiates a genuine histone acetylation-guided programming of the genome by activating essential genes and repressing a 'progenitor cells' gene expression program. At post-meiotic stages, a global chromatin hyperacetylation gives the signal for Brdt's first bromodomain to direct the genome-wide replacement of histones by transition proteins. Brdt is therefore a unique and essential regulator of male germ cell differentiation, which, by using various domains in a developmentally controlled manner, first drives a specific spermatogenic gene expression program, and later controls the tight packaging of the male genome.

Figure 1

Brdt is activated at the onset of meiosis. The expression of Brdt/Brdt was analysed either by RT–qPCR (A) or by western blots (B). The histograms represent the values of biological duplicates (normalized with respect to Actin as a control gene and to Brdt mean expression in 50-day-old wt testes). β-Galactosidase activity, driven by Brdt gene promoter, was assessed in seminiferous tubules sections (C, lower panels: ‘X-gal’). The corresponding sections were stained by Hoechst (C, upper panels: ‘Hoechst’). Scale bars, 10 μm. Figure source data can be found with the Supplementary data.

Figure 2

Brdt is required at the late stages of male meiotic prophase. (A) Testis histological sections, from 50 d.p.p. Brdt^+/− and Brdt^−/− animals, either stained by PAS/HE (Periodic Acid Schiff/Haematoxyline Eosine), or analysed by immunohistochemistry (IH) of the post-meiotic specific acrosome antigen, Sp56, without counter staining (left and middle panels, respectively). Scale bars, 50 μm. A higher magnification of adult testis sections at 50 d.p.p., stained with Hoechst is represented (right panels). A dotted line separates meiotic cells (lower left side of the panel) from post-meiotic cells (upper right side of the panel). Scale bars, 10 μm. (B) Testis histological sections, from 50 d.p.p. Brdt^+/− and Brdt^−/− animals stained by IH with an antibody against S10 phosphorylated histone H3. Scale bars, 50 μm. (C) Synaptonemal complex components, Sycp1 and Sycp3, and sex body marker γH2AX, were co-detected as indicated, in seminiferous tubule sections of Brdt^+/− and Brdt^−/− mice. Scale bars, 5 μm.

Figure 3

Brdt is a major testis-specific transcriptional regulator. (A) The transcriptome of the Brdt^−/− testes at 20 d.p.p. shows a total of 3017 genes affected by the absence of Brdt, of which 1153 genes were repressed by Brdt (upregulated in Brdt^−/− testes compared to heterozygous testes) and 1864 genes activated by Brdt (downregulated in Brdt^−/− testes compared to heterozygous testes). The genes differentially expressed in the testes of Brdt^−/− mice at 17 d.p.p. (n=337) were all included in those downregulated in the Brdt^−/− testes at 20 d.p.p. The genes affected by the absence of the first bromodomain BD1 (ΔBD1/ΔBD1) (n=1040) nearly all corresponded to a subset of downregulated genes in the Brdt^−/− testes. (B, C) RNA extracted from testes at 17 and 20 d.p.p. from Brdt^+/− (Ctrl, n=6) or Brdt^−/− (n=6), as well as from 20 d.p.p. testes from Brdt^+/+ (n=5) and Brdt^ΔBD1/ΔBD1 (n=5) was used for a transcriptomic analysis. The differentially expressed genes (fold change threshold of 1.5), between Brdt^+/− and Brdt^−/− mice at 17 d.p.p. and at 20 d.p.p., as well as between Brdt^ΔBD1/ΔBD1 and wild-type tests at 20 d.p.p., were respectively classified as ‘Brdt-activated genes’ (genes repressed in the absence of Brdt or in the presence of ΔBD1 Brdt), or as ‘Brdt-repressed genes’ (genes activated in the absence of Brdt). Their respective expression status was then retrieved from mouse tissue transcriptomes (B) and staged spermatogenic cell transcriptomes (C) and shown as heatmaps. The low versus high expression levels are represented on a green to red scale. ‘Soma’ represents the following mouse tissues from left to right: ovary, embryonic stem cells, placenta, foetus, brain, eye, pituitary, adipose tissue, adrenal gland, bone marrow, heart, kidney, liver, lung, muscle, salivary gland, seminal gland, small intestine, spleen and thymus.

Figure 4

Brdt binding and histone acetylation directed stage-specific gene expression. (A) Chromatin-bound Brdt was immunoprecipitated from fractionated spermatocyte populations and round spermatids and the associated DNA sequenced and position and the intensity of Brdt peaks determined. The SeqMiner software (Ye et al, 2010) was used to illustrate Brdt (this work) and histone acetylation peaks, which we recently reported from the same cell populations (Tan et al, 2011), at ±5 kb around gene TSSs. Left panels: heatmaps and quantification profiles of Brdt or histone acetylation peaks in TSS regions in meiotic and post-meiotic male germ cells corresponding to two categories of Brdt-bound TSSs: the upper panels show TSS associated with an increase of Brdt binding in post-meiotic cells as compared to meiotic cells, whereas the lower panels show TSS with a decrease or no Brdt binding in post-meiotic cells; The histograms (right) indicate the proportion of genes with meiotic and post-meiotic profile of expression in each of these two gene categories. The gene expression profiles were established following the strategy described for Figure 3C (but only genes with fold changes >1.2 between the meiotic and post-meiotic cells expression levels were taken into account). (B) Respective proportions of Brdt-activated and Brdt-repressed genes (see Figure 3) whose TSS is associated with a Brdt peak and enriched in acetylated histones: nearly half of the Brdt-activated genes show colocalized Brdt and histone acetylation peaks in TSS, whereas this is the case of only a minority of Brdt-repressed genes.

Figure 5

Brdt is a testis-specific ‘Brd4-like’ factor and a major driver of meiosis I. (A) Extracts from Brdt^+/+ testes were used for an immunoprecipitation using an anti-Cdk9, an anti-cyclin T1 antibody or an irrelevant antibody (pre-immune rabbit serum). Immunoprecipitated proteins were detected with the indicated antibodies. (B) RNA was extracted from testes at the indicated postnatal ages and the expression of three meiotic genes Ccna, H1t and Hspa2 (Dix et al, 1996) was monitored by RT–qPCR. The right panels show the expression of the same genes in testis from the three indicated genotypes, at 20 d.p.p. The histograms represent the values of biological duplicates, normalized with respect to Actin as a control gene, and to Brdt mean expression in 50-day-old wt testes. (C) The synaptonemal complex component, Sycp3 (green), and centromeric proteins CREST (red), were co-detected as indicated, in seminiferous tubule sections of Brdt^+/+ and Brdt^−/− mice. The panels show higher magnifications of two representative spermatocytes either wild type or Brdt^−/−. A general view of the spermatocytes is also shown. Scale bars, 5 μm. Figure source data can be found with the Supplementary data.

Figure 6

C-terminal-tagged Brdt severely interferes with spermatogenic cell differentiation. (A) The degree of the contribution of AT1 Brdt^+/tag ES cells (129) to embryogenesis was evaluated by coat colour (left panel) and more specifically to testis formation by genome type-specific PCR on testis DNA thanks to the strain-specific polymorphism of the D2Mit94 locus (middle lower panel). Chimeric mice testes were grouped into three classes according to the PCR-based estimation of their level of chimerism and their respective weights were plotted as a function of postnatal age (right panel). Representative testes from 49-day-old mice belonging to the three indicated classes are shown on the middle upper panel. (B) Testes from the three classes of 14-day-old chimeric mice were fixed and stained by Hoechst (scale bars, 50 μm). (C) Tunel assay was carried out on age-matched wild-type or chimeric mouse testes harvested at 14 d.p.p. Scale bars, 50 μm. (D) HeLa cells were transfected with the indicated GFP-Brdt constructs. ΔC corresponds to GFP-Brdt deleted of its C-terminal region (Pivot-Pajot et al, 2003). FL and FL TAG correspond to the full-length GFP-Brdt without or with the C-terminal tag, respectively (using the same tag as in the knock-in approach). After immunoprecipitation using an anti-cyclin T1 (upper panels) or an anti-Cdk9 antibody (lower panels), the presence of co-immunoprecipitated GFP-Brdt was monitored using an anti-GFP antibody. The positions of the GFP-Brdt fusion proteins in the input and immunoprecipitated materials are indicated by red arrows. (E) Cos7 cells were transfected with the same constructs as above but the extracts were used to test Brdt H4 histone tail binding. Extracts from cells expressing the indicated constructs were divided into three and incubated with streptavidin beads or streptavidin beads coupled with biotinylated H4 or tetra-acetylated H4 peptides (indicated). Bound proteins were then revealed by an anti-GFP antibody. ∅ represents extracts used in the pull down from non-transfected cells. Figure source data can be found with the Supplementary data.

Figure 7

Post-meiotic functions of Brdt’s first bromodomain. (A) An immunofluorescence detection of TP2 and of Prm1 and Prm2 (B) as well as a co-detection of the histone TH2B (green) and of Prm1 (red) were performed; Scale bars, 5 μm. (C) A comparative observation by electronic microscopy (EM) of two representative elongating spermatids at the same stage (as evaluated by the percentage of coverage of the spermatids’ heads by the acrosome) from wild-type (+/+) and Brdt^ΔBD1/ΔBD1 mice is shown. Scale bars, 1 μm.