Throughout life, hematopoietic stem cells (HSCs), residing in bone marrow (BM), continuously regenerate erythroid/megakaryocytic, myeloid, and lymphoid cell lineages. This steady-state hematopoiesis from HSC and multipotent progenitors (MPPs) in BM can be perturbed by stress. The molecular controls of how stress can impact hematopoietic output remain poorly understood. MicroRNAs (miRNAs) as posttranscriptional regulators of gene expression have been found to control various functions in hematopoiesis. We find that the miR-221/222 cluster, which is expressed in HSC and in MPPs differentiating from them, perturbs steady-state hematopoiesis in ways comparable to stress. We compare pool sizes and single-cell transcriptomes of HSC and MPPs in unperturbed or stress-perturbed, miR-221/222-proficient or miR-221/222-deficient states. MiR-221/222 deficiency in hematopoietic cells was induced in C57BL/6J mice by conditional vav-cre-mediated deletion of the floxed miR-221/222 gene cluster. Social stress as well as miR-221/222 deficiency, alone or in combination, reduced HSC pools 3-fold and increased MPPs 1.5-fold. It also enhanced granulopoisis in the spleen. Furthermore, combined stress and miR-221/222 deficiency increased the erythroid/myeloid/granulocytic precursor pools in BM. Differential expression analyses of single-cell RNAseq transcriptomes of unperturbed and stressed, proficient HSC and MPPs detected more than 80 genes, selectively up-regulated in stressed cells, among them immediate early genes (IEGs). The same differential single-cell transcriptome analyses of unperturbed, miR-221/222-proficient with deficient HSC and MPPs identified Fos, Jun, JunB, Klf6, Nr4a1, Ier2, Zfp36-all IEGs-as well as CD74 and Ly6a as potential miRNA targets. Three of them, Klf6, Nr4a1, and Zfp36, have previously been found to influence myelogranulopoiesis. Together with increased levels of Jun, Fos forms increased amounts of the heterodimeric activator protein-1 (AP-1), which is known to control the expression of the selectively up-regulated expression of the IEGs. The comparisons of single-cell mRNA-deep sequencing analyses of socially stressed with miR-221/222-deficient HSC identify 5 of the 7 Fos/AP-1-controlled IEGs, Ier2, Jun, Junb, Klf6, and Zfp36, as common activators of HSC from quiescence. Combined with stress, miR-221/222 deficiency enhanced the Fos/AP-1/IEG pathway, extended it to MPPs, and increased the number of granulocyte precursors in BM, inducing selective up-regulation of genes encoding heat shock proteins Hspa5 and Hspa8, tubulin-cytoskeleton-organizing proteins Tuba1b, Tubb 4b and 5, and chromatin remodeling proteins H3f3b, H2afx, H2afz, and Hmgb2. Up-regulated in HSC, MPP1, and/or MPP2, they appear as potential regulators of stress-induced, miR-221/222-dependent increased granulocyte differentiation. Finally, stress by serial transplantations of miR-221/222-deficient HSC selectively exhausted their lymphoid differentiation capacities, while retaining their ability to home to BM and to differentiate to granulocytes. Thus, miR-221/222 maintains HSC quiescence and multipotency by suppressing Fos/AP-1/IEG-mediated activation and by suppressing enhanced stress-like differentiation to granulocytes. Since miR-221/222 is also expressed in human HSC, controlled induction of miR-221/222 in HSC should improve BM transplantations.

中文翻译：

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miR-221/222 簇通过抑制 Fos/AP-1/IEG 通路激活和向粒细胞的应激样分化来调节造血干细胞静止和多能性。

在整个生命过程中，存在于骨髓 (BM) 中的造血干细胞 (HSC) 不断再生红细胞/巨核细胞、骨髓细胞和淋巴细胞谱系。这种来自 HSC 和 BM 多能祖细胞 (MPP) 的稳态造血作用可能会受到压力的干扰。压力如何影响造血输出的分子控制仍然知之甚少。人们发现 MicroRNA (miRNA) 作为基因表达的转录后调节因子可以控制造血过程中的各种功能。我们发现，在 HSC 和与之分化的 MPP 中表达的 miR-221/222 簇，以与应激类似的方式扰乱稳态造血功能。我们比较了未受干扰或应激干扰、miR-221/222 丰富或 miR-221/222 缺乏状态下 HSC 和 MPP 的库大小和单细胞转录组。通过条件性 vav-cre 介导的 floxed miR-221/222 基因簇删除，在 C57BL/6J 小鼠中诱导造血细胞中 miR-221/222 缺陷。社会压力以及 miR-221/222 缺陷，单独或组合，使 HSC 池减少 3 倍，MPP 增加 1.5 倍。它还增强了脾脏中的粒细胞生成。此外，应激和 miR-221/222 缺乏相结合增加了 BM 中的红细胞/髓细胞/粒细胞前体池。对未受干扰和应激的、熟练的 HSC 和 MPP 的单细胞 RNAseq 转录组进行差异表达分析，检测到 80 多个基因在应激细胞中选择性上调，其中包括立即早期基因 (IEG)。对未受干扰、精通 miR-221/222 并有缺陷的 HSC 和 MPP 进行相同的差异单细胞转录组分析，确定 Fos、Jun、JunB、Klf6、Nr4a1、Ier2、Zfp36（所有 IEG）以及 CD74 和 Ly6a 为潜在 miRNA目标。其中三个 Klf6、Nr4a1 和 Zfp36 先前已被发现影响骨髓粒细胞生成。随着 Jun 水平的增加，Fos 形成了异二聚体激活蛋白 1 (AP-1) 数量的增加，已知该蛋白可控制 IEG 选择性上调的表达。对社会压力与 miR-221/222 缺陷型 HSC 进行单细胞 mRNA 深度测序分析的比较，确定了 7 个 Fos/AP-1 控制的 IEG 中的 5 个，即 Ier2、Jun、Junb、Klf6 和 Zfp36，它们是常见的HSC 从静止状态的激活剂。与应激相结合，miR-221/222缺陷增强了Fos/AP-1/IEG通路，并将其延伸至MPP，并增加了BM中粒细胞前体的数量，诱导编码热休克蛋白Hspa5和Hspa8的基因选择性上调、微管蛋白细胞骨架组织蛋白 Tuba1b、Tubb 4b 和 5，以及染色质重塑蛋白 H3f3b、H2afx、H2afz 和 Hmgb2。它们在 HSC、MPP1 和/或 MPP2 中上调，似乎是应激诱导的 miR-221/222 依赖性粒细胞分化增加的潜在调节因子。最后，miR-221/222缺陷型HSC的连续移植造成的应激选择性地耗尽了它们的淋巴分化能力，同时保留它们归巢于 BM 和分化为粒细胞的能力。因此，miR-221/222通过抑制Fos/AP-1/IEG介导的激活和抑制向粒细胞的应激样分化增强来维持HSC静止和多能性。由于 miR-221/222 也在人 HSC 中表达，因此 HSC 中 miR-221/222 的受控诱导应可改善 BM 移植。