Sall4在早期胚胎发育和细胞重编程中的作用机制

Transcription factor Sall4, a member of the spalt (sal) gene family which encode proteins characterized by multiple double zinc finger motifs of the C2H2 type, is highly expressed both in the ES cells and iPS cells. Mutations in human SALL4 cause an autosomal dominant disorder known as Okihiro syndrome. Our preliminary data indicate that Sall4 has the DNA methylation/hydroxymethylation binding activity and is specifically enriched both in the male and female pronuclei in the fertilized egg. In the ES cells, depletion of Sall4 by shRNA induces ES cells differentiation. However, Sall4 overexpression during reprogramming can significantly improve the efficiency of iPS generation. Thus, we speculate that Sall4 must play an important role both in the early embryonic development and somatic cell reprogramming. In this project, we intend to generate Sall4 conditional knockout mice by CRISPR/Cas9 system to explore whether Sall4 involves in the active DNA demethylation of male pronucleus and to uncover its role in the early embryonic development. Moreover, through the somatic cell reprogramming system, we will reveal the functions of Sall4 in the establishment and maintenance of pluripotency via gene expression, epigenetic landscape and in vivo/in vitro differentiation potential analysis, thus providing a theoretical basis for the clinical application of embryonic stem cells and iPS cells.

转录因子Sall4在ES细胞和iPS细胞中高水平地表达，它是Spalt（sal）家族成员具有典型的C2H2双锌指结构。人SALL4基因的突变可引起常染色体显性遗传性疾病Okihiro综合症。我们前期的研究成果表明，Sall4蛋白具有DNA甲基化和羟甲基化结合活性；在受精卵中特异性地富集于雌雄原核内；其表达抑制可致ES细胞分化；而在体细胞重编程中，Sall4过表达可显著提高iPS效率。由此，我们推测Sall4在早期胚胎发育和体细胞重编程中扮演着重要角色。本课题拟通过CRISPR/Cas9技术建立Sall4条件性敲除小鼠模型，探索Sall4是否参与雄原核去甲基化并揭示其在早期胚胎发育中的作用。此外，我们将以体细胞重编程为研究平台，从基因表达、表观遗传和分化潜能等方面，揭示Sall4在细胞多能性建立和干细胞调控网络中的作用机制，为推进胚胎干细胞和诱导型重编程技术的临床应用提供理论基础。

在本项目的资助下，我们利用CRISPR/Cas9基因编辑系统，通过建立Sall4基因条件性敲除和基因标记小鼠模型，以早期胚胎发育为研究对象，揭示了Sall4参与调控早期卵母细胞发育的表观遗传机制。.我们首先成功建立了Sall4-mCherry荧光标记小鼠，揭示了Sall4在早期卵母细胞发育及受精后胚胎发育中的动态变化规律。随后，我们成功建立了Sall4loxp/loxp小鼠，并与Zp3-Cre和Gdf9-Cre小鼠交配，获得了Sall4母源性敲除小鼠，并证实该小鼠不育。进一步的研究发现，Sall4敲除的卵细胞都停滞在GV期，且几乎不能发生GVBD。单细胞的转录组分析证明了Sall4敲除的卵细胞转录组严重异常，且Sall4能够通过调控Kdm5b、Kdm6a和Kdm6b的表达，影响卵母细胞H3K4me3和H3K27me3水平，进一步造成转录组紊乱。此外，我们还利用RRBS等检测，发现Sall4敲除的卵细胞处于低甲基化状态，且对发育非常重要的gDMR区和ICR区域上几乎完全缺失了甲基化。总之，该研究揭示了Sall4作为重要的转录因子和表观遗传调控因子参与卵母细胞成熟的分子机制。此项目相关研究成果发表在Journal of Biological Chemistry和Molecular Cell等国际知名学术期刊上，提升了我国在该领域的影响力。