Expression data from 17 day old testes of wild type and UBR2-/- mice

Ubiquitylation of histones provides an important mechanism regulating chromatin remodeling and gene expression. Recent studies have revealed ubiquitin ligases involved in histone ubiquitylation, yet the responsible enzymes and the function of histone ubiquitination in spermatogenesis remain unclear. Here we show that the ubiquitin ligase UBR2, one of the recognition E3 components of the N-end rule proteolytic pathway, localizes to meiotic chromatin regions, including unsynapsed axial elements linked to chromatin inactivation, and mediates, in combination with the ubiquitin-conjugating enzyme HR6B, the ubiquitination of histone H2A. UBR2 interacts with HR6B and 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 transcriptional silencing of genes linked to unsynapsed axes of the X and Y chromosomes. We propose a model, in which UBR2 on axial elements of the X-Y pair enables HR6B on the linked chromatin domain to repeat histone ubiquitination cycles while scanning a string of nucleosomes. Our results suggest that histone ubiquitination in germ cells may be mediated by E3-E2 pairs distinct from those in somatic cells, providing a new insight into chromatin remodeling and gene expression regulation. For each genotype, 2 testes were collected from different individual in same litter at 17 days old. Biotinylated cRNA was produced and hybridized on Affimetrix mouse genome 430A 2.0 array.组蛋白泛素化是调控染色质重塑与基因表达的重要机制。既往研究已鉴定出参与组蛋白泛素化的泛素连接酶，但精子发生过程中负责组蛋白泛素化的酶类及其功能仍未明确。本研究发现，作为N端规则蛋白水解途径的识别性E3组分之一的泛素连接酶UBR2，可定位于减数分裂染色质区域，包括与染色质失活相关的未联会轴元件；并可与泛素结合酶HR6B协同介导组蛋白H2A的泛素化。UBR2可与HR6B及H2A相互作用，促进HR6B与H2A的结合，以及泛素从HR6B向H2A的转移。UBR2与泛素化组蛋白H2A（uH2A）可在时空维度上标记发生转录沉默的减数分裂染色质区域；而UBR2缺陷的精母细胞无法在减数分裂过程中诱导H2A的泛素化。UBR2缺陷的精母细胞在X、Y染色体未联会轴相关基因的转录沉默过程中存在显著功能缺陷。我们提出如下工作模型：XY染色体配对的轴元件上的UBR2，可使相连染色质结构域内的HR6B在扫描一串核小体的过程中循环进行组蛋白泛素化反应。本研究结果提示，生殖细胞中的组蛋白泛素化可由不同于体细胞的E3-E2酶对介导，为染色质重塑与基因表达调控领域提供了全新研究视角。本实验中，我们于小鼠出生后17天时，从同窝不同个体各采集2枚睾丸。随后制备生物素标记的cRNA，将其与Affymetrix小鼠基因组430A 2.0芯片进行杂交。