Fig 1.
Pattern of BRDT protein expression during late stages of prophase I in male meiosis.
(A-F) Immunolocalization of BRDT (green) and SYCP3 (red) during prophase I and metaphase I in WT spermatocyte spreads. Right upper insets show enlargements of the X and Y sex chromosomes (XY body) outlined in B—E. (A) BRDT signal is absent in zygonema. (B) BRDT is faintly detected in early pachynema. (C,D) In mid and late pachytene, and (E) diplotene spermatocytes, BRDT appears as a strong signal throughout the chromatin of autosome chromosomes and weakly in the XY body (upper right inset in C-E). (F) In metaphase I, BRDT is faintly observed in the cytoplasm. (G) Quantification of the signal intensity of BRDT in the chromatin of autosomes and sex chromosomes during each stage of prophase I. *p<0.05, **p<0.01. Error bars indicate standard deviation. Spreads were prepared from three 3 month-old WT mice; n = 10 leptotene, 10 zygotene, 20 early pachytene, 30 mid pachynema, late pachynema, early diplonema and mid/late diplonema spermatocytes respectively per each mouse.
Fig 2.
Depletion of BRDT does not affect the global DSB repair and synapsis of autosomes, but results in asynapsis of the sex chromosomes during pachynema.
(A-D, F-I) Chromosome spreads of wild type (WT) and Brdt-/- early and mid pachytene spermatocytes. Upper insets show an enlargement of the sex chromosomes. A schematic representation of each inset is shown below the original, with the X chromosome represented in purple and the Y chromosome in red. (A-D) Immunolocalization of γH2AX (green) and SYCP3 (red). (A, C) In both WT and Brdt-/- early pachytene spermatocytes, γH2AX is present in the sex chromosomes and as foci in the chromatin adjacent to the SC. (B, D) At mid pachynema, γH2AX is observed only in the X and Y chromosomes, including those in complete asynapsis (D, inset). (E) Quantification of sex chromosomes with synapsis defects at the PAR in WT (black bar) and Brdt-/- (grey bar) spermatocytes. Samples were obtained from three WT and Brdt-/- 3 month-old mice. n = 62 and 99 early pachytene, and 125 and 132 mid/ late pachytene WT and Brdt-/- spermatocytes respectively. **p<0.01, ***p<0.001. (F-I) Immunolocalization of SYCP1 (green) and SYCP3 (red). In WT early (F) and mid pachynema (G), all chromosomes are fully synapsed, including the sex chromosomes. (H-I) Brdt-/- spermatocytes exhibit fully synapsed autosomal bivalents, but numerous sex chromosomes are completely unsynapsed (I, inset).
Fig 3.
The dynamics of localization and incorporation of histone modifications linked to chromatin condensation/transcriptional repression is altered in absence of BRDT.
(A-D, F-I) Chromosome spreads of wild type (WT) and Brdt-/- late pachytene and diplotene spermatocytes. Insets show an enlargement of the sex chromosomes. (A-D) Immunolocalization of H3K9me3 (green) and SYCP3 (red). (A, C) In late pachynema of both WT and Brdt-/- spermatocytes, H3K9me3 localizes only in the pericentric heterochromatin (PH) and as a very small signal in the PAR of the sex chromosomes. (B) In WT diplonema, H3K9me3 signal is intensely visualized in the PH and sex chromosomes. (D) In Brdt-/- diplotene spermatocytes, H3K9me3 staining persists in the PH but is weakly observed in the X and Y (insets). (E) Quantification of the signal intensity of H3K9me3 in the sex chromosomes in both WT (black square) and Brdt-/- (white circles) spermatocytes. Error bars indicate standard deviation. **p = 0.0072, ***p = 0.001. Samples were obtained from three 3 month-old WT and Brdt-/- mice. n = 10 and 17 early pachytene, 40 mid and 40 late pachytene, 30 early and 40 mid/late diplotene WT and Brdt-/- spermatocytes, respectively, per mouse. (F-I) Immunolocalization of H3K4me1 (green) and SYCP3 (red). (F) In WT late pachytene spermatocytes, H3K4me1 localizes throughout the chromatin of the autosomes and is slightly more intense in the sex chromosomes. (G) In diplonema, the signal intensity of H3K4me1 in the X and Y is notably increased (inset). (H) In Brdt-/- late pachytene spermatocytes, H3K4me1 signal starts to appear very faint throughout the chromatin from both autosomes and sex chromosomes (inset). (I) In diplonema, H3K4me1 remains weak in the X and Y (inset). (J) Quantification of the signal intensity of H3K4me1 in the sex chromosomes in both WT (black square) and Brdt-/- (white circles) spermatocytes. Error bars indicate standard deviation. *** p = 0.001. Samples were obtained from three 3 month-old WT and Brdt-/- mice. n = 13 early pachytene, 30 mid pachytene, 40 and 50 late pachytene, 30 early and 30 mid/late diplotene WT and Brdt-/- spermatocytes, respectively. (K-N) The same chromosome spreads of WT and Brdt-/- late pachytene and diplotene spermatocytes shown in F-I, but only H3K4me1 is displayed. (K’-N”) Enlargement of the chromosomes demarcated by the white rectangle in (K-N), with the chromosomes identified by SYCP3 (grey) in (K’-N’). (K-N) Yellow arrow indicates the sex body. (J-K”) In WT spermatocytes H3K4me1 is homogeneously distributed throughout the chromatin and is more concentrated in the sex chromosomes (yellow arrow). (M-N”) In the absence of BRDT, H3K4me1 is mainly located in the chromatin adjacent to the SC (white arrows, M-N”‘).
Fig 4.
Depletion of BRDT disrupts the timing of removal of transcriptional activation markers in the X and Y chromosomes.
(A-D, F-I) Chromosome spreads of wild type (WT) and Brdt-/- late pachytene and diplotene spermatocytes. Insets show an enlargement of the sex chromosomes. (A-D) Immunolocalization of H3K9ac (green) and SYCP3 (red). (A-B) In WT late pachytene and diplotene spermatocytes, H3K9ac localizes in the chromatin of autosomal chromosomes but is absent in the sex chromosomes (XY; inset). (C-D) In Brdt-/- late pachytene and diplotene spermatocytes, H3K9ac signal is intense and localizes in the chromatin of both autosome and sex chromosomes (XY; inset). (E) Quantification of the signal intensity of H3K9ac in the sex body in both WT (black squares) and Brdt-/- (white circles) spermatocytes. *p = 0.05, ***p = 0.001. Samples were obtained from three 3 month-old WT and Brdt-/- mice. n = 10 and 12 early pachytene, 24 and 25 mid pachytene, 30 late pachytene, 20 early and 40 mid/late diplotene WT and Brdt-/- spermatocytes, respectively, per mouse. Error bars indicate standard deviation. (F-I) Immunolocalization of RNA pol II (green) and SYCP3 (red). (F-G) In WT late pachytene and diplotene spermatocytes, RNA pol II localizes throughout the chromatin of autosomes but is barely detected in the sex chromosomes (XY, inset). (H, I) In Brdt-/- spermatocytes, RNA pol II is present in the chromatin of both autosomes and sex chromosomes. (J) Quantification of the signal intensity of RNA pol II in the sex chromosomes in both WT (black squares) and Brdt-/- (white circles) spermatocytes. ***p = 0.001. Samples were obtained from three 3 month-old WT and Brdt-/- mice. n = 15 early pachytene, 25 mid pachytene, 40 late pachynema, 25 early and 35 mid/late diplotene WT and Brdt-/- spermatocytes, respectively per mouse. Error bars indicate standard deviation.
Fig 5.
BRDT contributes to the transcriptional silencing of the X chromosome in spermatocytes.
(A, B) Volcano plot representation of differentially expressed X-linked genes in Brdt-/- 17dpp (A) and 20dpp testes (B). The X axis represents the log fold-change (FC) in WT vs Brdt-/- spermatogenic cells. The Y axis represents the adjusted p-value. Only genes with an adjusted p-value of ≤0.05 were considered as differentially expressed genes (DEGs). (B, D) Number of X-linked genes with no change in expression, under-regulation or up-regulation in Brdt-/- 17dpp (B) and 20dpp (D) testes as compared to WT testes.
Fig 6.
Absence of BRDT leads to a global chromatin condensation and a decrease in the length of the SC of autosomal chromosomes.
Chromatin from seminiferous tubules from 17 day old WT and Brdt-/- testes was digested with 1U/ul MNase. In WT chromatin, MNase digestion produced low molecular weight bands corresponding mainly to mono- (band 1), di- (band 2) and tri- (band 3) nucleosomes. MNase digestion of Brdt-/- chromatin leads to digestion products with the bulk of chromatin ranging between 1500 and 4000 kb in size (bands 4 and 5) and a concomitant reduction in the mono-, di- and tri nucleosome fractions. Four WT and Brdt-/- mice were used per experiment; 3 experiments total. (B) Quantification of the intensity of the bands obtained after digestion with MNase of WT (black bars) and Brdt-/- (grey bars) chromatin. Error bars indicate standard deviation. *** p<0.001. (C) The length of each SC from autosome chromosomes in WT (black circles) and Brdt-/- (white circles) spermatocytes was measured and quantified in micrometers. **p<0.0028, ***p<0.0001. Error bars indicate standard deviation. Samples were obtained from three WT and Brdt-/- mice. n = 22 and 39 early pachytene; 92 and 45 mid pachytene, 147 and 105 late pachytene; 38 and 53 early diplotene, and 158 and 65 mid/late diplotene WT and Brdt-/- spermatocytes, respectively.
Fig 7.
BRDT is required for the formation and distribution of CO during pachynema and chiasmata in metaphase.
(A, B) Immunolocalization of MLH1 (green) and SYCP3 (red) in WT (A) and Brdt-/- (B) spermatocytes. Insets enlarge the PAR. White arrow indicates the presence or absence of the MLH1 focus in the PAR. Yellow arrow in (B) indicates autosomes with no MLH1 foci. (C) Quantification of the number of MLH1 foci per nucleus in WT (black squares) and Brdt-/- (white circles) pachytene spermatocytes. ***p<0.0001. Error bars represent standard deviation. Samples were obtained from three mice per genotype. n = 45 and 39 WT and Brdt-/- spermatocytes, respectively. (D) Quantification of sex chromosomes with an MLH1 focus in the PAR in WT (black bar) and Brdt-/- (grey bar) pachytene spermatocytes. ***p<0.0001. Error bars represent standard deviation. Samples were obtained from three each WT and Brdt-/- mice. n = 45 cells per genotype. (E) Scheme representing how each SC (in red) was divided into 10 equal length intervals (SC rank) from the centromere to the distal telomere (centromere is shown in green). (F) Relative distance of localization of each CO focus along autosomes in WT (black squares and line) and Brdt-/- (red circles and line) pachytene spermatocytes. Each point represents the percentage of MLH1 foci in each SC segment. (Samples were obtained from two mice per genotype. n = 15 WT and 15 Brdt-/- spermatocytes). Error bars represent standard deviation. (G,H) Chromosome spreads of diakinesis/metaphase I spermatocytes from WT and Brdt-/- mice stained with DAPI. White arrows show chromosomes with chiasmata. Yellow arrows in (H) show achiasmatic chromosomes. (I) Quantification of the number of chiasmata per nucleus in WT (black circles) and Brdt-/- (white circles) diakinesis/metaphase I spermatocytes (n = 6 and 4 respectively). ***p<0.0001. Error bars represent standard deviation. Samples were obtained from two mice per genotype. n = 4 and 6 WT and Brdt-/- spermatocytes, respectively.
Fig 8.
Chromatin organization at the CO hotspot of the PAR changes in the absence of BRDT.
(A) Concentration-course of chromatin digestion from testicular cells with a 6 point MNase titration (0.125U/ul, 0.25U/ul, 0.5U/ul, 1U/ul, 2U/ul, 3U/ul). Samples were obtained from four WT mice per experiment. Three experiments total. (B) DNA enrichment density map of the region 166,425 to 166,446 kb of the X chromosome, obtained by tiled qPCR after MNase digestion of pachytene spermatocytes. Triplicates per each reaction were used in each genotype (four WT and Brdt-/- mice per experiment). (C) Graphic representation of the DNA enrichment position and density levels based on the graph displayed in (B) The color code represents the level of DNA enrichment based on the R value shown in (B). Blue arrows indicate protein occupancy areas present in Brdt-/- chromatin that are not observed in WT chromatin. Green arrows indicate lower protein occupancy areas in Brdt-/- chromatin.