UBR2 mediates transcriptional silencing during spermatogenesis via histone ubiquitination

Abstract

Ubiquitination of histones provides an important mechanism regulating chromatin remodeling and gene expression. Recent studies have revealed ubiquitin ligases involved in histone ubiquitination, yet the responsible enzymes and the function of histone ubiquitination in spermatogenesis remain unclear. We have previously shown that mice lacking the ubiquitin ligase UBR2, one of the recognition E3 components of the N-end rule proteolytic pathway, are infertile associated with meiotic arrest at prophase I. We here show that UBR2 localizes to meiotic chromatin regions, including unsynapsed axial elements linked to chromatin inactivation, and mediates transcriptional silencing via the ubiquitination of histone H2A. UBR2 interacts with the ubiquitin conjugating enzyme HR6B and its substrate H2A and promotes the HR6B-H2A interaction and the HR6B-to-H2A transfer of ubiquitin. UBR2 and ubiquitinated H2A (uH2A) spatiotemporally mark meiotic chromatin regions subject to transcriptional silencing, and UBR2-deficient spermatocytes fail to induce the ubiquitination of H2A during meiosis. UBR2-deficient spermatocytes are profoundly impaired in chromosome-wide transcriptional silencing of genes linked to unsynapsed axes of the X and Y chromosomes. Our findings suggest that insufficiency in UBR2-dependent histone ubiquitination triggers a pachytene checkpoint system, providing a new insight into chromatin remodeling and gene expression regulation.

Fig. 1.

Localization of UBR2 to meiotic chromosomes. (A) At zygotene, UBR2 staining is enriched on unsynapsed axial regions. (B) At diplotene, the number of UBR2 foci surge throughout the entire chromatin except for the XY chromatin domain (arrowhead). (C and D) Enlarged images for UBR2 localization to unsynapsed axial regions of autosomes at zygotene (C) and the sex chromosomes at early pachytene (D) Arrow, PAR. [Scale bar (A and B), 10 μm; (C and D), 5 μm.]

Fig. 2.

UBR2, in combination with HR6B, mediates ubiquitination of H2A. (A) Partial purification of UBR2 from rat testes by using degron-bearing peptides (a). UBR2 interacts with RAD6 in the testes (B–D). Protein samples prepared by Phe-peptide (E3-F) or Val-peptide (E3-V) were subjected to immunoblotting for UBR2 (B), RAD6 (C), and anti-UBR2 immunoprecipitation and anti-RAD6 immunoblotting (D). F, Phe-peptide; V, Val-peptide. (B) In vitro ubiquitination assay (20 μL) with 100 ng E3-F prepared from rat testes, 30 ng HR6B, and 1 μg H2A in the presence of Ub activating reagents, including 1 μg FLAG-Ub and 100 ng E1, followed by immunoblotting for uH2A. (C) Same as (B) except that H2A ubiquitination was detected by using anti-FLAG immunoblotting. (D) In vitro ubiquitination assay with 100 ng E3-F prepared from +/+ (wt) and UBR2^-/- mouse tissues (ko) and 2 μg free H2A. (E) Same as (B) except that 50 ng HR6B and 5 μg nucleosome (nucleo.) were used in the presence or absence of 0.05 mM Ub aldehyde (alde.).

Fig. 3.

Cooperative interactions between UBR2, HR6B, and H2A. (A) GST-pulldown assay (300 μl) with 200 ng GST-HR6B, 250 ng E3-F from rat testes, and 1 μg H2A, followed by immunoblotting for UBR2, H2A, and HR6B. Input: UBR2, 20%; H2A, 10%; HR6B, 10%. (B) Ubiquitination-coupled E3 binding assay. UBR2 (as a mixture with UBR1) immobilized on F-peptide-beads were mixed with HR6B and/or H2A in the presence of E1 and Ub activating reagents, followed by immunoblotting of proteins (HR6B, H2A, and uH2A) retained by X-peptide. (C) A model for H2A ubiquitination in the XY body by the UBR2-HR6B complex.

Fig. 4.

UBR2-deficient spermatocytes are impaired in chromosome-wide ubiquitination of H2A. Immunostaining of uH2A (red) was performed for meiotic chromosomes from control and UBR2^-/- spermatocytes at zygotene (A), early pachytene (B), and midpachytene (C). The X–Y pair is indicated by arrowheads. The majority of UBR2^-/- spermatocytes are arrested at or before early pachytene (5). The pachytene nuclei from UBR2^-/- mice shown in this study are typical for those surviving spermatocytes. (Scale bar, 10 μm.)

Fig. 5.

UBR2 is required for transcriptional silencing of the X and Y chromosomes in male meiosis. (A) Microarray analysis of control and UBR2^-/- testes at P16. (B) Chromosome painting for X (red, arrowhead) and Y (green, arrowhead) coupled with SCP3 immunostaining (green) in control and UBR2^-/- spermatocytes. Dense peripheral DAPI staining indicates the sex body (arrowheads). (Scale bar, 10 μm.)

Fig. 6.

The localization pattern of UBR2 to unsynapsed axial regions of autosomes is distinct from that of BRCA1. (A and C) Localization of UBR2 (green) and BRCA1 (red) on unsynapsed axes of the autosomes at zygotene (A) and the sex chromosomes at pachytene (C). (B) Enlarged images for chromosomal regions indicated by the insets in (A). [Scale bar (A and C), 5 μm; (B) 1 μm.]