IGF2BP1 stabilizes OTX2 mRNA in an m6A-dependent manner to restrict human germ cell fate by repressing TFAP2C [CUT&Tag]

N6-methyladenosine (m6A) is the most abundant and reversible modification on messenger RNAs. The differential m6A profiles in different cell types are installed by writers (such as METTL3) and removed by erasers (such as FTO). The m6A-modified RNAs are recognized by readers to trigger downstream regulation of RNA metabolism, eventually contributing to the regulation of cell fates. Primordial germ cells (PGCs) are specified early during embryogenesis and establish the germ cell lineage for transmitting genetic and epigenetic information from parents to offspring. Defects in the establishment of germline may lead to infertility, germ cell tumors, and birth defects. Despite extensive research, the m6A-mediated epigenetic regulation of the specification of PGCs remains elusive. In this study, we discovered that knock-out of m6A writer or over-expression of erasers leads to increased percentage of human PGC-like cells (hPGCLCs) induced from embryonic stem cells. We identified the m6A reader IGF2BP1 as the key factor for restricting hPGCLC fate induction by stabilizing OTX2 mRNAs in an m6A-dependent manner. Moreover, we discovered that OTX2 suppresses TFAP2C during germ cell lineage specification. In summary, we characterized the m6A-IGF2BP1-OTX2-TFAP2C axis in restricting the specification of human germ cell fate. Overall design: To study the function of m6A related components in the regulation of hPGCLCs induced from hESCs,we established H1 hESCs cell line in which each target gene has been knocked out by CRISPR Cas9 and overexpressed. CUT&TAG for OTX2 in iMeLC stage is performed. m6A-seq for detecting m6A profile in hESCs is performed.

N6-甲基腺嘌呤（N6-methyladenosine，m6A）是信使RNA（messenger RNAs）上含量最为丰富且可逆转的转录后修饰类型。不同细胞类型中的差异性m6A修饰谱由写入因子（如METTL3）催化建立，并由擦除因子（如FTO）介导移除。携带m6A修饰的RNA可被识别蛋白识别，进而触发RNA代谢的下游调控通路，最终参与细胞命运的调控。原始生殖细胞（Primordial germ cells，PGCs）在胚胎发育早期定向特化，并建立生殖细胞谱系，以传递亲代向子代的遗传与表观遗传信息。生殖细胞系建立缺陷可导致不育症、生殖细胞肿瘤以及出生缺陷。尽管已有大量研究，但m6A介导的表观遗传调控在PGC特化过程中的具体机制仍未明确。本研究发现，敲除m6A写入因子或过表达擦除因子，会增加由人类胚胎干细胞诱导得到的人类原始生殖细胞样细胞（human PGC-like cells，hPGCLCs）的占比。我们鉴定出m6A识别蛋白IGF2BP1是限制hPGCLC命运诱导的关键因子，其通过以m6A依赖的方式稳定OTX2 mRNA发挥作用。此外，我们还发现，在生殖细胞谱系特化过程中，OTX2会抑制TFAP2C的表达。综上，我们阐明了m6A-IGF2BP1-OTX2-TFAP2C调控轴在限制人类生殖细胞命运特化中的作用。总体实验设计：为研究m6A相关组分在调控人类胚胎干细胞（human embryonic stem cells，hESCs）诱导hPGCLCs过程中的功能，我们构建了通过CRISPR-Cas9对各靶基因进行敲除或过表达的H1 hESCs细胞系。我们开展了iMeLC阶段OTX2的CUT&TAG实验，并对hESCs中的m6A修饰谱进行m6A测序（m6A-seq）检测。