Phosphorylation of chromosome core components may serve as axis marks for the status of chromosomal events during mammalian meiosis

Abstract

Meiotic recombination and chromosome synapsis between homologous chromosomes are essential for proper chromosome segregation at the first meiotic division. While recombination and synapsis, as well as checkpoints that monitor these two events, take place in the context of a prophase I-specific axial chromosome structure, it remains unclear how chromosome axis components contribute to these processes. We show here that many protein components of the meiotic chromosome axis, including SYCP2, SYCP3, HORMAD1, HORMAD2, SMC3, STAG3, and REC8, become post-translationally modified by phosphorylation during the prophase I stage. We found that HORMAD1 and SMC3 are phosphorylated at a consensus site for the ATM/ATR checkpoint kinase and that the phosphorylated forms of HORMAD1 and SMC3 localize preferentially to unsynapsed chromosomal regions where synapsis has not yet occurred, but not to synapsed or desynapsed regions. We investigated the genetic requirements for the phosphorylation events and revealed that the phosphorylation levels of HORMAD1, HORMAD2, and SMC3 are dramatically reduced in the absence of initiation of meiotic recombination, whereas BRCA1 and SYCP3 are required for normal levels of phosphorylation of HORMAD1 and HORMAD2, but not of SMC3. Interestingly, reduced HORMAD1 and HORMAD2 phosphorylation is associated with impaired targeting of the MSUC (meiotic silencing of unsynapsed chromatin) machinery to unsynapsed chromosomes, suggesting that these post-translational events contribute to the regulation of the synapsis surveillance system. We propose that modifications of chromosome axis components serve as signals that facilitate chromosomal events including recombination, checkpoint control, transcription, and synapsis regulation.

Figure 1. Chromosome axis proteins are phosphorylated during prophase I.

(A) Testis nuclear extracts treated with (+) or without (−) phosphatase (PPase) and phosphatase inhibitors (Inhibitor) were probed with antibodies against meiotic chromosome axis components. Phosphatase-sensitive slow-migrating forms are indicated by black and gray arrowheads. (B) Testis nuclear extracts were fractionated into detergent-soluble and detergent-insoluble fractions and analyzed by immunoblotting using antibodies against meiotic chromosome axis components. Histone H3 was used as a control for chromosomal proteins. (C) Testis nuclear extracts from juvenile mice of each age were examined by immunoblotting. (D) Testis nuclear extracts were immunoprecipitated without (Mock) or with the antibody against the phosphorylated S/T-Q motif (pS/T-Q). The immunoprecipitates were electrophoresed on a gradient gel and examined by immunoblotting against chromosome axis proteins. Note that using a gradient gel did not enable separation of phosphorylated and non-phosphorylated forms of chromosome axis proteins.

Figure 2. HORMAD1 is phosphorylated at Ser³⁷⁵ on unsynapsed chromosomes.

Figure 3. SMC3 is phosphorylated at Ser¹⁰⁸³ during prophase I.

(A) Testis nuclear extracts were immunoprecipitated with the anti-SMC3 antibody, followed by treatment with (+) or without (−) phosphatase (PPase) and phosphatase inhibitors (Inhibitor). 80% of the immunoprecipitated SMC3 and the rest were separated on a gradient gel and immunoblotted with antibodies against the Ser¹⁰⁸³-phosphorylated form of SMC3 (pS1083) and normal SMC3, respectively. (B) Testis nuclear extracts were immunoprecipitated without (Mock) or with the anti-pS1083 antibody. The immunoprecipitates were probed with antibodies against meiotic chromosome axis components. The asterisk marks a non-specific band. (C) Nuclear spreads of spermatocytes were labeled with anti-pS1083, anti-SYCP3 and anti-HORMAD1 antibodies. Arrowheads indicate the XY bivalent. Bars, 10 µm.

Figure 4. Phosphorylation of chromosome axis proteins in the absence of a checkpoint protein.

(A and D) The insoluble fraction of testis nuclear extracts was prepared from Atm ^−/− (A) and Brca1 ^Δ11/Δ11 Trp53 ^+/− (Brca1^Δ) (D) males and probed with antibodies against meiotic chromosome axis components. (B and E) Testis nuclear extracts from Atm ^−/− (B) and Brca1 ^Δ11/Δ11 Trp53 ^+/− (E) males were immunoprecipitated with the anti-HORMAD1 antibody. 80% of the immunoprecipitated HORMAD1 and the rest were separated on a gradient gel and immunoblotted with anti-pS375 and anti-HORMAD1 antibodies, respectively. The asterisk marks a non-specific band. (C and F) Testis nuclear extracts from Atm ^−/− (C) and Brca1 ^Δ11/Δ11 Trp53 ^+/− (F) males were immunoprecipitated with the anti-SMC3 antibody. 80% of the immunoprecipitated SMC3 and the rest were separated on a gradient gel and immunoblotted with anti-pS1083 and anti-SMC3 antibodies, respectively. (G) Nuclear spreads of Brca1 ^Δ11/Δ11 Trp53 ^+/− pachytene spermatocytes were labeled with anti-pS375, anti-SYCP3 and anti-SYCP1 antibodies. Arrowheads indicate the XY bivalent. Bar, 10 µm.

Figure 5. Phosphorylation of HORMAD1, HORMAD2, and SMC3 is highly dependent on SPO11.

(A) The insoluble fraction of testis nuclear extracts was probed with antibodies against meiotic chromosome axis components. (B) The Ser³⁷⁵-phosphorylated form of HORMAD1 was examined as in Figure 4B. (C) The Ser¹⁰⁸³-phosphorylated form of SMC3 was examined as in Figure 4C. (D) Nuclear spreads of Spo11 ^−/− zygotene-like spermatocytes were labeled with anti-pS375, anti-SYCP3 and anti-HORMAD1 antibodies. (E) Nuclear spreads of Spo11 ^−/− zygotene-like spermatocytes were labeled with anti-pS1083, anti-SYCP3 and anti-HORMAD1 antibodies. Bars, 10 µm.

Figure 6. Phosphorylation of HORMAD1 is reduced in the absence of SYCP3.

(A) The insoluble fraction of testis nuclear extracts was probed with antibodies against meiotic chromosome axis components. (B) The Ser³⁷⁵-phosphorylated form of HORMAD1 was examined as in Figure 4B. (C) The Ser¹⁰⁸³-phosphorylated form of SMC3 was examined as in Figure 4C. (D) Nuclear spreads of Sycp3 ^−/− zygotene-like spermatocytes were labeled with anti-pS375, anti-SYCP1 and anti-HORMAD1 antibodies. (E) Nuclear spreads of Sycp3 ^−/− zygotene-like spermatocytes were labeled with anti-pS1083, anti-SYCP1 and anti-HORMAD1 antibodies. Bars, 10 µm.

Figure 7. Distribution of ATR at unsynapsed chromosomal regions is impaired in the absence of SYCP3.

(A–C) Nuclear spreads of wild-type (A), Sycp3 ^−/− (B) and Spo11 ^−/− (C) zygotene-like spermatocytes were labeled with anti-γH2AX, anti-HORMAD1 and anti-SYCP1 antibodies. (D–G) Nuclear spreads of wild-type (D), Sycp3 ^−/− (E), Sycp1 ^−/− (F) and Tex12 ^−/− (G) zygotene-like spermatocytes were labeled with anti-γH2AX, anti-REC8 and anti-ATR antibodies. Arrowheads indicate the position of the pseudo-sex body-like staining of γH2AX. Bars, 10 µm.

Figure 8. Chromosomal regions are marked by compositional differences and modification status of axis proteins.

(A) Schematic representation of the model for regulation of phosphorylation of meiotic chromosomal proteins at S/T-Q motifs. In response to SPO11-formed DSBs (arrow 1), ATM phosphorylates histone H2AX (arrow 2) and ATR phosphorylates HORMAD1/2 (arrow 3) and SMC3 (arrow 4). Phosphorylated HORMAD1/2 serves as a marker for unsynapsis and contributes to the correct localization of ATR at unsynapsed chromosomal regions (arrow 5). At the unsynapsed chromosomes, ATR phosphorylates H2AX to promote MSUC (arrow 6), as well as HORMAD1/2 (arrow 7) and SMC3 (arrow 8). Phosphorylated HORMAD1/2 further stabilizes ATR (arrow 9) at unsynapsed chromosomes and ATR further phosphorylates HORMAD1/2 (arrow 10), amplifying the unsynapsis signal via the positive feedback loop (arrow 9 and 10). (B) The status of chromosome synapsis can be indicated by the presence or absence of HORMAD1/2 and phosphorylation of HORMAD1 and SMC3. At unsynapsed chromosomal regions, the chromosome axis contains the S/T-Q motif-phosphorylated forms of HORMAD1/2 and SMC3. When homologs are synapsed, HORMAD1/2 and the Ser¹⁰⁸³-phosphorylated form of SMC3 are displaced from the chromosome axis. After desynapsis, HORMAD1/2 is again included in the chromosome axis but HORMAD1 (and possibly HORMAD2) is not phosphorylated at the S/T-Q motif. Distribution of the phosphorylated forms of other components of the chromosome axis remains to be determined.