Gonen Nitzan, Quinn Alexander, O'Neill Helen C, Koopman Peter, Lovell-Badge Robin
The Francis Crick Institute, Midland Road, London, United Kingdom.
Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia.
PLoS Genet. 2017 Jan 3;13(1):e1006520. doi: 10.1371/journal.pgen.1006520. eCollection 2017 Jan.
During mouse sex determination, transient expression of the Y-linked gene Sry up-regulates its direct target gene Sox9, via a 3.2 kb testis specific enhancer of Sox9 (TES), which includes a core 1.4 kb element, TESCO. SOX9 activity leads to differentiation of Sertoli cells, rather than granulosa cells from the bipotential supporting cell precursor lineage. Here, we present functional analysis of TES/TESCO, using CRISPR/Cas9 genome editing in mice. Deletion of TESCO or TES reduced Sox9 expression levels in XY fetal gonads to 60 or 45% respectively relative to wild type gonads, and reduced expression of the SOX9 target Amh. Although human patients heterozygous for null mutations in SOX9, which are assumed to have 50% of normal expression, often show XY female sex reversal, mice deleted for one copy of Sox9 do not. Consistent with this, we did not observe sex reversal in either TESCO-/- or TES-/- XY embryos or adult mice. However, embryos carrying both a conditional Sox9 null allele and the TES deletion developed ovotestes. Quantitative analysis of these revealed levels of 23% expression of Sox9 compared to wild type, and a significant increase in the expression of the granulosa cell marker Foxl2. This indicates that the threshold in mice where sex reversal begins to be seen is about half that of the ~50% levels predicted in humans. Our results demonstrate that TES/TESCO is a crucial enhancer regulating Sox9 expression in the gonad, but point to the existence of additional enhancers that act redundantly.
在小鼠性别决定过程中，Y连锁基因Sry的瞬时表达通过Sox9的一个3.2 kb睾丸特异性增强子（TES）上调其直接靶基因Sox9，该增强子包括一个1.4 kb的核心元件TESCO。SOX9活性导致支持细胞前体双潜能谱系分化为支持细胞，而非颗粒细胞。在此，我们利用小鼠中的CRISPR/Cas9基因组编辑技术对TES/TESCO进行了功能分析。删除TESCO或TES后，XY胎儿性腺中Sox9的表达水平相对于野生型性腺分别降至60%或45%，并且SOX9靶基因Amh的表达也降低。尽管SOX9发生无效突变的杂合人类患者（假定其表达水平为正常的50%）常出现XY女性性反转，但删除一个Sox9拷贝的小鼠却不会。与此一致的是，我们在TESCO-/-或TES-/-的XY胚胎或成年小鼠中均未观察到性反转。然而，携带条件性Sox9无效等位基因和TES缺失的胚胎发育出了卵睾。对这些胚胎的定量分析显示，与野生型相比，Sox9的表达水平为23%，并且颗粒细胞标记物Foxl2的表达显著增加。这表明小鼠中开始出现性反转的阈值约为人类预测的~50%水平的一半。我们的结果表明，TES/TESCO是性腺中调节Sox9表达的关键增强子，但也表明存在其他起冗余作用的增强子。
