CRISPR-Cas9 Editing of Human Histone Deubiquitinase Gene USP16 in Human Monocytic Leukemia Cell Line THP-1.
CRISPR-Cas9 Editing of Human Histone Deubiquitinase Gene USP16 in Human Monocytic Leukemia Cell Line THP-1.
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DOI：
10.3389/fcell.2021.679544
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发表时间：
2021
影响因子：
5.5
通讯作者：
Summers KM
中科院分区：
文献类型：
--
作者：
Gažová I;Lefevre L;Bush SJ;Rojo R;Hume DA;Lengeling A;Summers KM
USP16是一种组蛋白去泛素酶，在细胞周期中促进G2/M转换，调节DNA损伤修复，并有助于诱导基因表达。我们利用CRISPR-Cas9系统对急性单核细胞白血病细胞系THP-1的高分化克隆中的Usp16基因进行了突变，并产生了4个纯合子敲除克隆。在佛波酯(PMA)处理下，所有细胞均能增殖和分化。有一株系在PMA处理前高度增殖，分化后停止增殖，与野生型一样。3个克隆的祖细胞标记MYB持续表达，表明分化并没有完全阻止这些克隆的增殖。对野生型、杂合子和纯合子之间转录差异的网络分析表明，在所有细胞系中，分化后的基因簇上调或下调。在PMA治疗之前，纯合子克隆的与代谢和线粒体相关的基因水平低于野生型，包括编码Usp16相互作用伙伴的SRPRB。干扰素信号也明显丢失。相比之下，与野生型相比，纯合子细胞中的一些基因在基线时上调，包括其他去泛素酶(USP12、BAP1和MYSM1)。然而，3个纯合子在分化过程中未能完全诱导USP3。在纯合子克隆中，其他网络簇在分化前或分化后表现出影响。因此，Usp16的去除影响了细胞的转录组，尽管所有这些株都能够存活，这表明Usp16的功能可能是多余的。我们的分析表明，白血病株可以通过不同的补偿途径来适应Usp16基因缺失所施加的极端选择压力，以及基因编辑和选择方案的苛刻条件。在体内癌症的进化过程中也存在类似的选择压力，我们的结果可以被视为白血病细胞适应的案例研究。Usp16一直被认为是癌症化疗的靶点，但我们的结果表明，治疗将选择对Usp16抑制剂具有耐药性的逃逸突变。
USP16 is a histone deubiquitinase which facilitates G2/M transition during the cell cycle, regulates DNA damage repair and contributes to inducible gene expression. We mutated the USP16 gene in a high differentiation clone of the acute monocytic leukemia cell line THP-1 using the CRISPR-Cas9 system and generated four homozygous knockout clones. All were able to proliferate and to differentiate in response to phorbol ester (PMA) treatment. One line was highly proliferative prior to PMA treatment and shut down proliferation upon differentiation, like wild type. Three clones showed sustained expression of the progenitor cell marker MYB, indicating that differentiation had not completely blocked proliferation in these clones. Network analysis of transcriptomic differences among wild type, heterozygotes and homozygotes showed clusters of genes that were up- or down-regulated after differentiation in all cell lines. Prior to PMA treatment, the homozygous clones had lower levels than wild type of genes relating to metabolism and mitochondria, including SRPRB, encoding an interaction partner of USP16. There was also apparent loss of interferon signaling. In contrast, a number of genes were up-regulated in the homozygous cells compared to wild type at baseline, including other deubiquitinases (USP12, BAP1, and MYSM1). However, three homozygotes failed to fully induce USP3 during differentiation. Other network clusters showed effects prior to or after differentiation in the homozygous clones. Thus the removal of USP16 affected the transcriptome of the cells, although all these lines were able to survive, which suggests that the functions attributed to USP16 may be redundant. Our analysis indicates that the leukemic line can adapt to the extreme selection pressure applied by the loss of USP16, and the harsh conditions of the gene editing and selection protocol, through different compensatory pathways. Similar selection pressures occur during the evolution of a cancer in vivo, and our results can be seen as a case study in leukemic cell adaptation. USP16 has been considered a target for cancer chemotherapy, but our results suggest that treatment would select for escape mutants that are resistant to USP16 inhibitors.
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影响因子：
3.5
作者：
Atanassov BS;Koutelou E;Dent SY
通讯作者：
Dent SY
DOI：
10.1038/nrd.2017.152
发表时间：
2018-01
期刊：
Nature reviews. Drug discovery
影响因子：
--
作者：
Harrigan JA;Jacq X;Martin NM;Jackson SP
通讯作者：
Jackson SP
影响因子：
50.3
作者：
Abdel-Wahab O;Adli M;LaFave LM;Gao J;Hricik T;Shih AH;Pandey S;Patel JP;Chung YR;Koche R;Perna F;Zhao X;Taylor JE;Park CY;Carroll M;Melnick A;Nimer SD;Jaffe JD;Aifantis I;Bernstein BE;Levine RL
通讯作者：
Levine RL
DOI：
10.1073/pnas.1517041113
发表时间：
2016-01-05
影响因子：
11.1
作者：
Gu, Yue;Jones, Amanda E.;Wang, Hengbin
通讯作者：
Wang, Hengbin
影响因子：
5.7
作者：
Fang CL;Lin CC;Chen HK;Hseu YC;Hung ST;Sun DP;Uen YH;Lin KY
通讯作者：
Lin KY