The ubiquitin-conjugating enzyme HR6B is required for maintenance of X chromosome silencing in mouse spermatocytes and spermatids

Background: The ubiquitin-conjugating enzyme HR6B is required for spermatogenesis in mouse. Loss of HR6B results in aberrant histone modification patterns on the trancriptionally silenced X and Y chromosomes (XY body) and on centromeric chromatin in meiotic prophase. We studied the relationship between these chromatin modifications and their effects on global gene expression patterns, in spermatocytes and spermatids. Results: HR6B is enriched on the XY body and on centromeric regions in pachytene spermatocytes. Global gene expression analyses revealed that spermatid-specific single- and multicopy X-linked genes are prematurely expressed in Hr6b knockout spermatocytes. Very few other differences in gene expression were observed in these cells, except for upregulation of major satellite repeat transcription. In contrast, in Hr6b knockout spermatids, 7298 genes were differentially expressed; 65% of these genes was downregulated, but we observed a global upregulation of gene transcription from the X chromosome. In wildtype spermatids, approximately 20% of the single-copy X-linked genes reach an average expression level that is similar to the average expression from autosomes. Conclusions: Spermatids maintain an enrichment of repressive chromatin marks on the X chromosome, originating from meiotic prophase, but this does not interfere with transcription of the single-copy X-linked genes that are reactivated or specifically activated in spermatids. HR6B represses major satellite repeat transcription in spermatocytes, and functions in the maintenance of X chromosome silencing in spermatocytes and spermatids. It is discussed that these functions involve modification of chromatin structure, possibly including H2B ubiquitylation. 8 Affymetrix microarrays were generated: 4 from wildtype and HR6B knockout spermatocytes (2 replicates each), and 4 from wildtype and HR6B knockout spermatids (2 replicates each).背景：泛素结合酶HR6B（ubiquitin-conjugating enzyme HR6B）是小鼠精子发生（spermatogenesis）所必需的。HR6B缺失会导致减数分裂前期（meiotic prophase）中，转录沉默的X、Y染色体（XY小体，XY body）以及着丝粒染色质（centromeric chromatin）出现异常的组蛋白修饰（histone modification）模式。本研究在精母细胞（spermatocytes）与精子细胞（spermatids）中，探究了此类染色质修饰与全基因组基因表达模式之间的关联，以及该修饰对基因表达的影响。 结果：在粗线期精母细胞（pachytene spermatocytes）中，HR6B富集于XY小体与着丝粒区域。全基因组基因表达分析显示，在Hr6b敲除（Hr6b knockout）精母细胞中，精子细胞特异性单拷贝及多拷贝X连锁基因出现了早熟表达。除主要卫星重复转录（major satellite repeat transcription）上调外，此类细胞中几乎未观察到其他基因表达差异。与之相反，在Hr6b敲除精子细胞中，共有7298个基因呈现差异表达，其中65%的基因表达下调，同时我们观察到X染色体整体转录水平上调。在野生型（wildtype）精子细胞中，约20%的单拷贝X连锁基因的平均表达水平与常染色体（autosomes）的平均表达水平相当。 结论：精子细胞可维持源自减数分裂前期的X染色体抑制性染色质标记（repressive chromatin marks）富集，但这并不会干扰在精子细胞中被重新激活或特异性激活的单拷贝X连锁基因的转录。HR6B可抑制精母细胞中的主要卫星重复转录，并在精母细胞与精子细胞中维持X染色体的沉默状态。本研究讨论了此类功能可能通过修饰染色质结构实现，其中或包含H2B泛素化（H2B ubiquitylation）。本研究共制备了8张Affymetrix微阵列（Affymetrix microarrays）：其中4张来自野生型与Hr6b敲除精母细胞（每组设2次生物学重复（replicates）），另外4张来自野生型与Hr6b敲除精子细胞（每组设2次生物学重复）。