Department of Genetics, University of Alabama at Birmingham, 720 20th Street South, Birmingham, AL, 35294, USA.
Department of O'Neal Comprehensive Cancer Center, University of Alabama at Birmingham, 720 20th Street South, Birmingham, AL, 35294, USA.
Mol Cancer. 2022 Feb 7;21(1):38. doi: 10.1186/s12943-021-01472-x.
Unlike autosomal tumor suppressors, X-linked tumor suppressors can be inactivated by a single hit due to X-chromosome inactivation (XCI). Here, we argue that targeted reactivation of the non-mutated allele from XCI offers a potential therapy for female breast cancers.
Towards this goal, we developed a dual CRISPR interference and activation (CRISPRi/a) approach for simultaneously silencing and reactivating multiple X-linked genes using two orthogonal, nuclease-deficient CRISPR/Cas9 (dCas9) proteins.
Using Streptococcus pyogenes dCas9-KRAB for silencing XIST and Staphylococcus aureus dCas9-VPR for activating FOXP3, we achieved CRISPR activation of FOXP3 in various cell lines of human female breast cancers. In human breast cancer HCC202 cells, which express a synonymous heterozygous mutation in the coding region of FOXP3, simultaneous silencing of XIST from XCI led to enhanced and prolonged FOXP3 activation. Also, reactivation of endogenous FOXP3 in breast cancer cells by CRISPRi/a inhibited tumor growth in vitro and in vivo. We further optimized CRISPRa by fusing dCas9 to the demethylase TET1 and observed enhanced FOXP3 activation. Analysis of the conserved CpG-rich region of FOXP3 intron 1 confirmed that CRISPRi/a-mediated simultaneous FOXP3 activation and XIST silencing were accompanied by elevated H4 acetylation, including H4K5ac, H4K8ac, and H4K16ac, and H3K4me3 and lower DNA methylation. This indicates that CRISPRi/a targeting to XIST and FOXP3 loci alters their transcription and their nearby epigenetic modifications.
The simultaneous activation and repression of the X-linked, endogenous FOXP3 and XIST from XCI offers a useful research tool and a potential therapeutic for female breast cancers.
与常染色体肿瘤抑制因子不同，由于 X 染色体失活（XCI），X 连锁肿瘤抑制因子只需一次打击即可失活。在这里，我们认为从 XCI 靶向重新激活未突变的等位基因为女性乳腺癌提供了一种潜在的治疗方法。
为了实现这一目标，我们开发了一种双重 CRISPR 干扰和激活（CRISPRi/a）方法，使用两种正交的、无核酸酶的 CRISPR/Cas9（dCas9）蛋白同时沉默和激活多个 X 连锁基因。
使用酿脓链球菌 dCas9-KRAB 沉默 XIST 和金黄色葡萄球菌 dCas9-VPR 激活 FOXP3，我们在各种人乳腺癌细胞系中实现了 FOXP3 的 CRISPR 激活。在表达 FOXP3 编码区同义杂合突变的人乳腺癌 HCC202 细胞中，XCI 中 XIST 的同时沉默导致 FOXP3 的激活增强和延长。此外，CRISPRi/a 在内源性乳腺癌细胞中重新激活 FOXP3 可抑制体外和体内肿瘤生长。我们通过将 dCas9 融合到去甲基酶 TET1 上来进一步优化 CRISPRa，并观察到 FOXP3 的激活增强。对 FOXP3 内含子 1 的保守 CpG 丰富区进行分析证实，CRISPRi/a 介导的 FOXP3 激活和 XIST 沉默伴随着 H4 乙酰化的升高，包括 H4K5ac、H4K8ac 和 H4K16ac，以及 H3K4me3 和较低的 DNA 甲基化。这表明 CRISPRi/a 靶向 XIST 和 FOXP3 基因座改变了它们的转录及其附近的表观遗传修饰。
从 XCI 同时激活和抑制 X 连锁的、内源性的 FOXP3 和 XIST 为女性乳腺癌提供了一种有用的研究工具和潜在的治疗方法。
