A critical requirement for IB in controlling dormancy in Hematopoietic stem cells via retinoic acid during embryonic development [RNA-seq]

Hematopoietic Stem Cells (HSCs) originate from the E11.5 aorta-gonads-and mesonephros (AGM) region during development before they migrate to the foetal liver for proliferation and maturation, and finally seed the bone marrow around birth, their final site of residence. In the AGM, HSCs reside within Intra-aortic hematopoietic clusters (IAHC) along with hematopoietic progenitors (HPC). Molecular pathways that determine HSC fate instead of HPCs are still unknown, although inflammatory signalling has been implicated in the development of all blood cells, including NF-B. Here, we describe a dormant phenotype of LT-HSCs in the IB KO. Although IB is critical for retaining inactive NF-B complexes in the cytoplasm, it can regulate stem cell related genes by interacting with the PRC2 complex in the nucleus. Accordingly, we find decreased PRC2 dependent H3K27me3 accumulation at the promoters of PI3K and retinoic acid signalling molecules by cut and tag assay in AGM derived CD31+ cells, which includes HE/IAHC derived from IB KO embryos. Furthermore, this regulation of the retinoic acid signalling by IB is further confirmed by cut and tag assay for IB itself in CD31+ cells of the AGM and more specifically also in sorted LT-HSCs from the E14.5 foetal liver. Over-activation of the retinoic acid/PI3K levels in LT-HSCs of the IB KO is evident in their dormant molecular profile. Functionally, IB KO LT-HSCs are less proliferative and respond with delayed activation upon transplantation. Overall, we identify nuclear IB as an essential player specifically for HSC specification/proliferation from the onset of HSPC emergence in the AGM. We performed bulk RNA seq with sorted E14.5 LT-HSCs (LSKCD48-CD150+) in order to define HSCs at a molecular level considering 3xWT, 2x IB KO and 3x IB HET independent biological replicates.造血干细胞（Hematopoietic Stem Cells, HSCs）在发育过程中起源于胚胎11.5天的主动脉-性腺-中肾区（aorta-gonads-and mesonephros, AGM），随后迁移至胎肝完成增殖与成熟，最终在围产期定植于骨髓这一最终居留位点。在AGM区中，HSCs与造血祖细胞（hematopoietic progenitors, HPCs）共同存在于主动脉内造血簇（Intra-aortic hematopoietic clusters, IAHC）内。目前，决定HSCs而非HPCs命运的分子通路仍未明确，尽管炎症信号已被证实参与所有血细胞的发育过程，其中包括NF-B。 本研究报道了IB敲除（IB KO）小鼠中长期造血干细胞（Long-term Hematopoietic Stem Cells, LT-HSCs）的休眠表型。尽管IB对于在细胞质中维持NF-B复合物的非活性状态至关重要，但它可通过与细胞核内的多梳抑制复合体2（Polycomb Repressive Complex 2, PRC2）相互作用，调控干细胞相关基因的表达。 据此，我们通过CUT&Tag测序（cut and tag assay）对AGM区来源的CD31阳性（CD31+）细胞（包含来自IB KO胚胎的造血内皮细胞/主动脉内造血簇HE/IAHC）进行检测，发现PI3K与维甲酸（retinoic acid）信号分子的启动子区域处，PRC2依赖的组蛋白H3赖氨酸27三甲基化（H3K27me3）沉积水平显著下降。 进一步地，我们通过对AGM区CD31+细胞，以及更特异性的胚胎14.5天胎肝中分选得到的LT-HSCs进行IB自身的CUT&Tag测序，证实了IB对维甲酸信号通路的上述调控作用。 在IB KO小鼠的LT-HSCs中，维甲酸/PI3K通路的过度激活可从其休眠分子特征中得到印证。功能层面上，IB KO小鼠的LT-HSCs增殖能力更弱，且在移植后表现出激活延迟的响应。 综上，我们证实核定位的IB是AGM区造血干祖细胞（Hematopoietic Stem and Progenitor Cells, HSPCs）出现伊始，HSC特化与增殖过程中的关键调控因子。 为从分子层面定义HSCs，我们对分选得到的胚胎14.5天LT-HSCs（LSK⁺CD48⁻CD150⁺）进行了批量RNA测序（bulk RNA-seq），实验设置了3组独立生物学重复的野生型（Wild Type, WT）、2组IB KO以及3组IB杂合子（IB Heterozygous, IB HET）样本。