GGNBP2 regulates histone ubiquitination and methylation in spermatogenesis

Figures & data

Figure 1. Ggnbp2 controls both chromatin compaction and nucleosome stability.

(a) Nuclei isolated from the WT and Ggnbp2KO GC-2 cells treated with increasing MNase concentrations (0.2, 0.4 and 0.6 KU/µL). N1, N2 and N3 indicate that the DNA length wrapped around 1, 2 or 3 nucleosomes, respectively. (b) Nuclei isolated from the WT and Ggnbp2KO GC-2 cells treated with MNase (0.6 KU/µL) with increasing incubation time. (c) Soluble H3 obtained by resuspension of nuclei in solutions containing low NaCl concentration was analysed. The amount of soluble H3 obtained following low salt wash was normalized to that obtained from 2.0 M NaCl wash (Input). (d) Quantitative analysis of H3 normalized by input H3. All the values are represented as mean ± SEM

Figure 2. Ggnbp2 deletion leads to the up-regulation of H2A_K119ubi and the down-regulation of H2B_K120ubi.

(a, b, c, d) Western-blotting analysis of H2A_K119ubi and H2B_K120ubi in WT and Ggnbp2KO GC-2 cells, as well as spermatocyte-enriched postnatal day 18 testis lysate, respectively. (e, f) Quantitative analysis of H2A_K119ubi and H2A_K120ubi normalized by H2A and H2B in GC-2 cells and 18D testis. (g) Immunofluorescence staining on spread chromosomes from 4-week-old male primary spermatocytes. The cells were imaged using an Olympus fluorescence microscope. Chromosome spreads were stained with antibodies against H2A_K119ubi. Scale bar = 5 μm. All the values are represented as mean ± SEM; *p < 0.05, **p < 0.01

Abbreviations: WT, wild type. 18D,18-day testis

Figure 3. GGNBP2 interacts and colocalizes with ASXL1. The absence of GGNBP2 affects ASXL1 and BAP1 interaction as well as BAP1 translocation.

(a,b) Reciprocal IP reveals that GGNBP2 interacts with ASXL1 in the mouse testis. (c,d) BAP1 bound with (d) ASXL1, (c) but not GGNBP2. Loss of GGNBP2 disrupts BAP1 and ASXL1 interaction. (e) Germ cells were prepared from wild-type testes and processed for immunofluorescence staining as indicated. GGNBP2 colocalized with ASXL1 in spermatocytes. (f) Western blotting analysis of ASXL1 and BAP1 using postnatal day 60 testis lysate. (g) Quantitative analysis of ASXL1 and BAP1 normalized by β-Tubulin. (h, i) Chromosome spreads of pachytene spermatocytes are stained with SYCP3 antibody (red), ASXL1 and BAP1 (green) as indicated, which shows GGNBP2 loss changes the localization of (i) BAP1, (h) but not ASXL1. (j) Western blotting analysis of BAP1 on the cytoplasmic and nuclear extract from postnatal day 18 testis. (k) Quantitative analysis of BAP1 in cytoplasmic and nuclear extract Representative images of three WT and Ggnbp2KO mice are shown. All the values are represented as mean ± SEM; *p < 0.05, **p < 0.01 Scale bar = 5 μm. Abbreviations: IP, immunoprecipitation; IB: immunoblotting.

Figure 4. GGNBP2 interacts and colocalizes with UBE2B, and GGNBP2 loss affects UBE2B and RNF40 interaction.

(a) IP revealing that GGNBP2 interacts with UBE2B in the mouse testis. (b, c) RNF40 bound with (b) UBE2B, (c) but not GGNBP2. Loss of GGNBP2 disrupted UBE2B and RNF40 interaction. (d) Western blotting analysis of RNF40 and UBE2B using postnatal day 60 testis lysate. (e) Quantitative analysis of RNF40 and UBE2B normalized by β-Tublin. (f) Germ cells were prepared from wild-type testis and processed for immunofluorescence staining as indicated. GGNBP2 colocalized with UBE2B in spermatocytes. (g, h) Chromosome spreads of pachytene spermatocytes are stained with SYCP3 antibody (red), UBE2B and RNF40 (green) as indicated, which shows that GGNBP2 loss does not change the localization of (g) UBE2B and (h) RNF40. Representative images of three WT and Ggnbp2KO mice are shown. All the values are represented as mean ± SEM; **p < 0.01.

Scale bar = 5 μm. Abbreviations: IP, immunoprecipitation.

Figure 5. GGNBP2 regulates histone H2A and H2B ubiquitination levels in vitro.

(a) GGNBP2 knockout increased H2A and decreased H2B ubiquitination levels in GC-2 cells. Ggnbp2 overexpression in Ggnbp2KO cells could restore H2A and H2B ubiquitination levels. (b) Quantitative data are from three experimental replicates. (c) Western blotting assay of WT and Ggnbp2KO cells transfected with control scrambled siRNA and siRNA against ASXL1. (d) Western blotting assay of Ggnbp2KO cells transfected with control scrambled siRNA, siRNA against ASXL1, or Ggnbp2overexpression plasmid. (e) Western blotting assay of WT and Ggnbp2KO cells transfected with control scrambled siRNA and siRNA against RNF40 and UBE2B. β-Actin was used as the loading control. (f) Western blotting assay of Ggnbp2KO cells transfected with control scrambled siRNA, siRNA against RNF40, UBE2B, or Ggnbp2 overexpression plasmid. β-Actin was used as the loading control. All the values are represented as mean ± SEM; *p < 0.05, **p < 0.01.

Abbreviations: Ubi, ubiquitination; EV, empty vector; OE, overexpression; WT, wild type; siRNA, small interfering RNA.

Figure 6. GGNBP2 controlled patterns of histone modification.

(a-b) Western blotting analyses of various forms of histone methylation and acetylation on 18-day of WT and Ggnbp2KO mouse total testis, as well as immortalized WT and Ggnbp2KO cells. Quantitative data are from three experimental replicates. (c-d) Ggnbp2 knockout resulted in an increase in H3_K27me3 and a decrease in H3_K79me2 level in GC-2 cells. Ggnbp2 overexpression in Ggnbp2KO cells could restore H3_K27me3 and H3_K79me2 levels. β-Actin was used as the loading control. Quantitative data are from three experimental replicates (e-f) Western blotting analyses of PRC2 subunits (EZH1, EZH2, SUZ12, Rbap46/p48 and EED) in 18-day and 60-day WT and Ggnbp2KO mouse total testis. GAPDH was used as the loading control. Quantitative data are from three experimental replicates. (g-h) Western blotting analyses of histone H3 methyltransferase, DOT1L, in 18-day and 60-day WT and Ggnbp2KOmouse total testis. β-Actin was used as the loading control. Quantitative data are from three experimental replicates. All the values are represented as mean ± SEM; *p < 0.05, **p < 0.01

Abbreviations: me, EV empty vector; OE, overexpression; WT, wild type.

Figure 7. The mechanism of histone modifications regulated by GGNBP2.

We propose that GGNBP2 induced ASXL1 to activate the deubiquitinating enzyme BAP1 in deubiquitinating H2A, while GGNBP2 knockout disrupted the interaction between ASXL1 and BAP1. GGNBP2 deletion reduced H2B ubiquitination by affecting E2 enzymes and E3 ligase binding. GGNBP2 regulated H2A and H2B ubiquitination levels and controlled H3K27 and H3K79 methylation by PRC2 subunits and histone H3K79 methyltransferase. It is suggested that Ggnbp2 knockout increased DNA damage response by promoting H2A ubiquitination and H3K27 trimethylation (H3K27me3) and reduced nucleosome stability by decreasing H2B ubiquitination and H3K79 dimethylation (H3K79me2).

Data availability statement

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.