CRISPR editing of the GLI1 first intron abrogates GLI1 expression and differentially alters lineage commitment.
CRISPR editing of the GLI1 first intron abrogates GLI1 expression and differentially alters lineage commitment.
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DOI：
10.1002/stem.3341
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发表时间：
2021-05
期刊：
影响因子：
--
通讯作者：
Iannaccone PM
中科院分区：
文献类型：
--
作者：
Galat Y;Gu H;Perepitchka M;Taylor R;Yoon JW;Glukhova XA;Li XN;Beletsky IP;Walterhouse DO;Galat V;Iannaccone PM
GLI 1是介导Sonic Hedgehog信号传导的三个GLI家族转录因子之一，其在发育和细胞分化中起作用。GLI 1与GLI 2形成正反馈回路，并可能与其自身形成正反馈回路。为了确定GLI 1及其内含子调控位点对该转录环和人类干细胞分化的影响，我们使用CRISPR/Cas9编辑删除了人类GLI 1内含子中含有6个GLI结合位点的区域，以产生H1人类胚胎干细胞（hESC）GLI 1编辑的克隆。在不去除整个GLI 1基因的情况下编辑掉这个内含子区域，使我们能够研究这个高度复杂区域的影响，该区域结合了各种细胞中的转录因子。通过比较RNA测序、定量真实的时间PCR（q-rtPCR）和功能测定来研究GLI 1在人ESC分化中的作用。编辑该区域导致GLI 1转录敲低，延迟神经定型，并在自发和定向分化实验中抑制内胚层和中胚层分化。我们发现早期成骨标志物的出现延迟，形成粒细胞单位的造血潜能降低，以及癌症相关基因表达降低。此外，通过拮抗剂GANT-61抑制GLI 1具有相似的体外作用。这些结果表明，GLI 1内含子区域对于反馈回路至关重要，并且GLI 1对hESC分化具有谱系特异性作用。我们的工作是第一项记录GLI 1在人类发育早期阶段废除程度的研究，并表明GLI 1转录可以以治疗有用的方式改变。使用CRISPR/Cas9基因组编辑来删除含有六个高度保守的GLll结合位点的第一内含子区域。GL 11基因表达的降低影响了Sonic Hedgehog信号通路中GL 11的调节能力。结果是上游和下游GLl 1靶标的转录激活降低，其最终通过关键基因的下调影响内胚层、中胚层和外胚层分化。
GLI1 is one of three GLI family transcription factors that mediate Sonic Hedgehog signaling, which plays a role in development and cell differentiation. GLI1 forms a positive feedback loop with GLI2 and likely with itself. To determine the impact of GLI1 and its intronic regulatory locus on this transcriptional loop and human stem cell differentiation, we deleted the region containing six GLI binding sites in the human GLI1 intron using CRISPR/Cas9 editing to produce H1 human embryonic stem cell (hESC) GLI1‐edited clones. Editing out this intronic region, without removing the entire GLI1 gene, allowed us to study the effects of this highly complex region, which binds transcription factors in a variety of cells. The roles of GLI1 in human ESC differentiation were investigated by comparing RNA sequencing, quantitative‐real time PCR (q‐rtPCR), and functional assays. Editing this region resulted in GLI1 transcriptional knockdown, delayed neural commitment, and inhibition of endodermal and mesodermal differentiation during spontaneous and directed differentiation experiments. We found a delay in the onset of early osteogenic markers, a reduction in the hematopoietic potential to form granulocyte units, and a decrease in cancer‐related gene expression. Furthermore, inhibition of GLI1 via antagonist GANT‐61 had similar in vitro effects. These results indicate that the GLI1 intronic region is critical for the feedback loop and that GLI1 has lineage‐specific effects on hESC differentiation. Our work is the first study to document the extent of GLI1 abrogation on early stages of human development and to show that GLI1 transcription can be altered in a therapeutically useful way. CRISPR/Cas9 genomic editing was used to delete the first intronic region containing six highly conserved GLl1 binding sites. The decreased GLl1 gene expression impacted GLl1 regulatory capacity within the Sonic Hedgehog signaling pathway. The result was a reduced transcriptional activation of upstream and downstream GLl1 targets, which ultimately impacted endodermal, mesodermal, and ectodermal differentiation via downregulation of key genes.
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影响因子：
12.4
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Chen Q;Shou P;Zheng C;Jiang M;Cao G;Yang Q;Cao J;Xie N;Velletri T;Zhang X;Xu C;Zhang L;Yang H;Hou J;Wang Y;Shi Y
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