Atad2 is highly expressed in post-meiotic male germ cells.

(A) The exon/intron organization of Atad2 gene and the two ATAD2-encoding transcriptional units (encoding for ATAD2-long and ATAD2-short) are indicated (Dyer et al. 2025). The red arrows indicate the position of insertion of the Lac-Z-Lox-Neo-Lox cassette. (B) Gene targeting strategy for the generation of the Atad2 KO allele is represented. Exon number and relative position, selection gene cassettes, LoxP sites and Frt sites are indicated. Crossing mice bearing this construct with CMV-Cre mice resulted in the generation of the Atad2 LacZΔNeo-Exon12 allele, which can be verified through PCR amplification of the genomic DNA (shown on the ethidium bromide-stained gel). The expected amplified bands for each genotype allowing the detection of both wild-type and KO alleles of Atad2 are indicated. E = Exon. (C) Extracts from wild-type and Atad2 KO mice testes were probed with anti-ATAD2 and anti-H3 antibodies as indicated. Four different mice were used for this experiment. (D) Seminiferous tubules sections from wild-type and Atad2 KO mice were stained with X-gal to visualize lacZ gene expression and β-galactosidase activity under the endogenous Atad2 gene promoter. Testes from three different wild-type and Atad2 KO mice were used to generate the represented sections. Scale bar: 200 μm

Atad2 gene inactivation leads to the accumulation of HIRA.

(A) Total testes’ extracts from individual wild-type or Atad2 KO mice (n =5) were used to detect HIRA and H3 by immunoblotting. The panel shows a representative immunoblotting after HIRA detection. The histogram shown in the lower left panel represents quantification of HIRA immunoblotting signal normalized to H3. Mean ± Standard Deviation is 0.86± 0.21 and 1.15±0.06 for wild type and Atad2 KO samples respectively and the p value is 0.02 for unpaired Student t-test. (B) Extracts from fractionated spermatogenic cells (pool of testes from 3 individuals per genotype) were used to detect HIRA and H3.

Enhanced H3.3 detection in Atad2 KO post-meiotic cells A specific antibody against H3.3 was used to detect H3.3 in round spermatid cells from WT and Atad2 KO mice.

Round spermatids are recognizable by their distinctive Hoechst - bright chromocenter (upper panels). The lower panels represent the magnification of selected cells shown at a lower magnification in the upper panels. The images show H3.3 labelling and Hoechst staining alone or the merged images of these staining as indicated.

ATAD2 controls active gene TSS accessibility.

A) Gene selection according to expression in round spermatids. The plot displays the mean expression levels on the y-axis and the corresponding standard deviations on the x-axis, calculated from three independent biological replicates of isolated round spermatids. The genes were divided into 4 groups (grp1 in blue, grp2 in cyan, grp3 in green and grp4 in orange) according to the quartile of their mean expression. B) Heatmap on ATAC-seq signal. The heatmap shows the ATAC-seq signal of each group (grp1 in blue, grp2 in cyan, grp3 in green and grp4 in orange) in rows, for 4 ATAD2 WT and 4 ATAD2 KO samples in column. C) Profiles according to selected genes. The panels show for each group the mean profile (± 2 s.e.m) (solid line) of four ATAD2 WT replicates (in blue) and 4 ATAD2 KO (in red) (transparency) over the four replicates.

ATAD2 enhances H3.3 gene regulatory functions.

(A) Expression profiles of post-meiotic H3.3-activated genes. The heatmap (left panel) displays the normalized expression levels of genes identified by Fontaine and colleagues as upregulated in the absence of histone H3.3, (Fontaine et al. 2022) for ATAD2 WT and ATAD2 KO samples at days 20, 22, 24, and 26 PP. The colour scale represents the z-score of log-transformed DESeq2-normalized counts. The box plots (middle panel) show the pooled normalized expression levels, aggregated across replicates and genes, for each condition (WT and KO) and time point (D20 to D26). Statistical significance between WT and KO conditions was determined using a two-sided t-test, with p-values indicated as follows: * for p-value<0.05, ** for p-value<0.01, *** for p-value<0.001. The GSEA results (right panel) highlight a significant enrichment of the “post-meiotic H3.3-activated genes” gene set in KO samples relative to WT samples at D26. The coloured vertical bars represent the core genes that account for the gene set’s enrichment signal. (B) As in (A) for the “post-meiotic H3.3-repressed genes” gene set. (C) As in (A) for the “ sex chromosome-linked genes “ gene set.

Atad2 is required for efficient histone-to-protamine replacement.

(A) Spermatogenic cell preparations from wild-type and Atad2 KO mice were stained with antibodies against TH2B (in green) and transition protein 1 (TP1) or protamine 1 (PRM1) (in red). A yellow signal indicates the co-detection of TH2B along with TP1 or PRM1. The indicated fields are shown at higher magnification in panel “B”. Scale bar: 10 μm. (B) Fields shown in panel “A” are ordered to illustrate the transition from the histone associated genome to its packaging by protamines, considering both wild-type and Atad2 KO spermatogenic cells.

Atad2 is required for proper pre-protamine 2 processing and protamine assembly

(A) A specific antibody raised against the processed part of pre-PRM2 (Rezaei-Gazik et al. 2022) was used to detect pre-PRM2 expression and localization in tubular elongating spermatid cells. The lower panels represent the magnification of selected cells. (B) The amount of pre-PRM2 was detected using soluble extracts from testis of wild-type and Atad2 KO mice using ELISA. Histograms show the normalized values (pre-PRM2/tubulin) of 4 measurements of sedimentation samples of Elongating/Condensing fractions. Mean ± Standard Deviation is 1.6 ± 0.2 and 2.1 ± 0.1 for wild type and Atad2 KO samples respectively and the p value equals 0.0158 when applying unpaired Student t-test.

ATAD2s’ function in sperm genome compaction and fertility parameters

(A) Epididymal sperm cells were isolated by the swim-up method from wild-type or Atad2 KO mice and underwent a decompaction test and the sizes of the sperm heads were measured and represented as box plots (n = 75 sperm heads / box plot). The value ranges (mean) were 10.4 ∼ 19.3 (16.0) for wild type sperms without decompaction; 11.4 ∼ 20.1 (16.4) for Atad2 KO sperms without decompaction; 33.4 ∼ 61.8 (48.5) for wild type sperms after decompaction; 45.0 ∼ 78.9 (62.6) Atad2 KO sperms after decompaction. Tukey’s HSD post two-way ANOVA test was used. ****p < 0.001. (B) Pools of oocytes from C57BL6 females were obtained and used for in vitro fertilization (IVF) with spermatozoa from WT or Atad2 KO males (the experiment was repeated 4 times independently). The fertilization success rate is indicated as a percentage of oocytes giving rise to stage 2 embryos. Mean ± Standard Deviation is 25.5 ± 14 and 12 ± 5.2 for wild type and Atad2 KO experiments respectively and the p = 0.002 for ratio paired Student t-test. (C) Wild-type (n = 5) and Atad2 KO (n = 5) male mice were crossed with C57BL6 female mice and the number of pups was counted and reported as box plots (n = 28 and 30, respectively). The average numbers of pups are respectively 8.0 ± 2.1 and 7.1 ± 2.5 and the p-value of Student t-test = 0.1.

Atad2 gene inactivation does not significantly disrupt testis histology, the occurrence of H4K5ac, or the cell-specific accumulation of H2A.L.2, TP2, PRM1 and PRM2.

Sections of paraffin-embedded testes from wild-type or Atad2 KO mice were used to visualize the accumulation H4K5ac or expression of H2A.L.2, TP2, PRM1 and PRM2.

Atad2 depletion affects testis weight and mature sperm counts.

(A) Testes from six 21- to 27-week-old wild-type and Atad2 KO mice were collected, weighted (mg) and their weights normalized to body weight and only same-age mice were compared pairwise. Histograms represent the mean of the measured weights and error bars the Standard Deviation. The p-value of pairwise Student t-test = 0.004 (upper panel). The lower panel shows representative testes from wild-type and Atad2 KO mice. (B) Spermatozoa counts (Cells /ml) were performed for pairs of wild type and Atad2 KO (n=6). The average numbers of spermatozoa counts are respectively 21.6 ± 5.8 and 13.7 ± 4,. and the p-value of paired Student t-test = 0.001.

Primary Antibodies

Reagents / Products used