红细胞生成过程关键步骤确定

红细胞生成过程关键步骤确定 一个健康的成年人每天必须生成1千亿个新红血细胞，才能维持其血液循环中的红细胞数量。来自洛桑联邦理工学院(EPFL)的一个研究人员小组确定了红细胞生成过程中一个关键的步骤。这一研究发现可能不仅有助于阐明如贫血等血液疾病的病因，还使得医生们的梦想离现实更近了一步:在实验室能够制造出红血细胞，由此提供一个潜在的取之不竭的血液主要成分资源，用于输血。 红细胞，其本质就是一袋将氧气输送到全身的血红蛋白。其生命起始于骨髓中的造血干细胞，经历一个高度受控的增殖和分化过程后，获得其最终的身份。 在这一分化过程中的一个关键步骤就是线粒体自噬(mitophagy)。随着线粒体耗尽，细胞血红蛋白负载能力达到最大。然而直到现在，都还没有清楚了解控制线粒体自噬的机制。 在发表在本周《科学》(Science)杂志上的一篇中，洛桑联邦理工学院的Isabelle Barde及其同事通过试验证实，KRAB型锌指蛋白与KAP1辅因子协同作用，以精细且复杂的方式调节了线粒体自噬。 论文的资深作者、病毒学家Didier Trono多年来一直对KRAB/KAP1系统感兴趣。众所周知，其在“沉默”哺乳动物基因组反转录因子元件中发挥作用，已有3.5亿年历史。它们最初是可以整合到感染生物体遗传密码中的逆转录病毒。“它做着如此好的一份工作，以致在进化过程中它被指派完成了很多其他的事情，”Trono说。 KRAB/KAP1系统承担的之一就是调控线粒体自噬。研究人员发现，遗传改造缺失KAP1的小鼠迅速变得贫血，因为它们无法生成红血细胞。更特别的是，他们发现，干细胞分化过程在成红血细胞(erythroblast，红细胞前体)中线粒体降解的阶段停止。且在人类血细胞中敲除KAP1也会产生相似效应，表明其调控线粒体自噬的作用在从小鼠到人类的整个进化中是保守的。 研究人员进一步证明，KRAB/KAP1系统是通过抑制线粒体自噬阻遏物来发挥功能。换句话说，就像负负得正，它激活了这一靶过程。这表明，这一调控系统中的各种元件突变有可能导致了如贫血和某些类型白血病等血液疾病，从而反过来指出了这些疾病的未来治疗靶点。它还指出了有可能在实验室中模拟红血细胞合成的途径。 但这些研究发现还具有更广泛的意义。虽然线粒体对于许多细胞正常功能至关重要，但如果它们生成破坏性自由基(某些情况下细胞呼吸作用的副产物)对于细胞也会是致命的。这些自由基引起的氧化性应激与肝脏疾病、心脏病和肥胖有关联。因此，了解线粒体自噬受控机制，有可能促成更好地了解以及治疗这些疾病。 Trono认为这一多层次组合调控法则或许适应于广泛的生理系统。“它为自然完成生理活动赋予了极高水平的模块性。”他将之比喻为管风琴的运行方式。 每个风琴师都有一个键盘，以及受他掌控的脚踏板。他通过各种组合应用它们来调整乐器产生的声音。相似的，微调一个或几个控制元件可以在许多生物过程中产生显著的影响。尽管其中任何一个元件发生突变都可能导致故障，但由于每个的贡献很小，损害往往是有限的。反过来，这赋予了系统稳固性。Trono相信，这种稳固性是数亿年来进化一直在选择和改进的。(来源:生物通 何嫱) 更多阅读 《科学》发表论文摘要(英文) A KRAB/KAP1-miRNA Cascade Regulates Erythropoiesis Through Stage-Specific Control of Mitophagy 11. Isabelle Barde, 12. Benjamin Rauwel, 23. Ray Marcel Marin-Florez, 34. Andrea Corsinotti, 1,*5. Elisa Laurenti, 16. Sonia Verp, 17. Sandra Offner, 1,†8. Julien Marquis, 19. Adamandia Kapopoulou, 410. Jiri Vanicek, 1,‡11. Didier Trono DOI: 10.1126/science.1232398 Science , REPORT A KRAB/KAP1-miRNA Cascade Regulates Erythropoiesis Through Stage-Specific Control of Mitophagy 11. Isabelle Barde, 12. Benjamin Rauwel, 23. Ray Marcel Marin-Florez, 34. Andrea Corsinotti, 1,*5. Elisa Laurenti, 16. Sonia Verp, 17. Sandra Offner, 1,†8. Julien Marquis, 19. Adamandia Kapopoulou, 410. Jiri Vanicek, 1,‡11. Didier Trono +Author Affiliations 11. School of Life Sciences and Frontiers in Genetics Program, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland. 22. Center for Integrative Genomics, University of Lausanne, 1015 Lausanne, Switzerland. 33. Centre for Genomic Regulation, 08003 Barcelona, Spain. 44. School of Basic Sciences, EPFL, 1015 Lausanne, Switzerland. +Author Notes , ?* Present address: Campbell Family Institute for Cancer Research, Ontario Cancer Institute, Princess Margaret Cancer Centre, University Health Network and Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada. , ?† Present address: Functional Genomics Core, Nestlé Institute of Health Sciences, EPFL Campus, 1015 Lausanne, Switzerland. ‡1. ?Corresponding author. E-mail: didier.trono@epfl.ch , ABSTRACT During hematopoiesis, lineage- and stage-specific transcription factors work in concert with chromatin modifiers to direct the differentiation of all blood cells. Here, we explored the role of KRAB-containing zinc finger proteins (KRAB-ZFPs) and their cofactor KAP1 in this process. Hematopoietic-restricted deletion of Kap1 in the mouse resulted in severe hypoproliferative anemia. Kap1-deleted erythroblasts failed to induce mitophagy-associated genes and retained mitochondria. This was due to persistent expression of microRNAs targeting mitophagy transcripts, itself secondary to a lack of repression by stage-specific KRAB-ZFPs. The KRAB/KAP1-miRNA regulatory cascade is evolutionary conserved, as it also controls mitophagy during human erythropoiesis. Thus, a multilayered transcription regulatory system is present, where protein- and RNA-based repressors are superimposed in combinatorial fashion to govern the timely triggering of an important differentiation event. Through the process of erythropoiesis, about one hundred billion new red cells are generated every day in the human adult bone marrow. This process is initiated by the differentiation of hematopoietic stem cells (HSC) into the earliest erythroid progenitor, which was identified ex vivo as a slowly growing burst-forming unit-erythroid (BFU-E). This erythroid progenitor morphs into the rapidly dividing CFU-E (colony-forming unit-erythroid), the proliferation of which is stimulated by the hypoxia-induced hormone erythropoietin. Further differentiation occurs through a highly sophisticated program orchestrated by lineage- and stage-specific combinations of protein- and RNA-based transcription regulators (1–3). It culminates in the elimination of intracellular organelles including mitochondria and the nucleus to yield the fully mature erythrocyte, containing on the order of 250 million molecules of hemoglobin as almost sole cargo. Much is still to be learned about the molecular mechanisms of these events, not only to understand the cause of red cell disorders, but also to aid the in vitro manufacturing of the large supplies of oxygen-carrying cells for transfusion. Higher vertebrate genomes encode hundreds of KRAB-ZFPs that can bind DNA in a sequence-specific fashion through a C-terminal array of C2H2 zinc fingers and recruit the corepressor KAP1 via their N-terminal KRAB domain (4–7). KAP1, also known as TRIM28 (tripartite motif protein 28), TIF1β (transcription intermediary factor 1 beta) or KRIP-1 (KRAB-interacting protein 1), acts as a scaffold for a multi-molecular complex that silences transcription through the formation of heterochromatin (8–11). The KRAB/KAP1 system probably evolved initially to minimize retroelement-induced genome perturbations (12–14), but recent data indicate that it also regulates multiple aspects of mammalian physiology (15–24). The present study was undertaken to explore its role in hematopoiesis. The hemato-specific knockout of Kap1 in the mouse, whereby the hematopoietic system of otherwise wild type animals is reconstituted from Kap1-deleted hematopoietic stem cells and progenitors (fig. S1), resulted in a series of hematological abnormalities (table S1). Mutant mice displayed fatal hypo-regenerative anemia, characterized by the accumulation of transferrin receptor/CD71+ glycophorin-A-associated/Ter119- early erythroblasts and an almost complete absence of mature CD71-Ter119+ cells in the bone marrow (Fig. 1A). Electron microscopy and Mitotracker staining revealed that KO erythroblasts contain more mitochondria than their wild type counterparts (Fig. 1B), correlating with decreased expression of mitophagy genes such as Nix/Bnip3L, Ulk1, GABARAPl2, Sh3glb1, Atg12, Becn1 and Bcl2l1 (Fig. 2A). Since the KRAB/KAP1 pathway is mostly known to induce transcriptional repression (10, 11), it seemed likely that this effect was indirect. An examination of the miRNA expression profile of control and Kap1 KO CD71+Ter119+ cells revealed that, among 455 miRNAs tested, 5 were downregulated and 11 upregulated more than two-fold in KO cells (data are presented in the Gene Expression Omnibus dataset GSE44061). A recently described in silico approach (25,26) suggested that six of these upregulated miRNAs had mitophagy-associated deregulated transcripts as their targets, notably miR-351, predicted to act on Bnip3L (Fig. 2A). Consistent with this hypothesis, levels of miR-351 abruptly dropped in CD71+Ter119+ cells, compared to their CD71+Ter119- precursors, mirroring Bnip3L induction (Fig. 2B). Furthermore, transduction of mouse erythroleukemia (MEL) cells with a GFP-expressing lentiviral vector harboring, 3′ of GFP, the Bnip3L 3′UTR sequence predicted to be targeted by miR-351 resulted in miR-351-dependent downregulation of the reporter (Fig. 2C). Finally, similar to their KAP1-depleted counterparts, miR-351-overexpressing MEL cells were blocked in differentiation and accumulated mitochondria, and this phenotype was reversed by expression of a Bnip3L transcript devoid of this 3′UTR sequence (fig. S2). View larger version: , In this page , In a new window , Download PowerPoint Slide for Teaching Fig. 1 Blocked erythrocyte maturation and accumulation of mitochondria in Kap1-deleted erythroblasts. (A) FACS analysis of CD71 and Ter119 in bone marrow from control (Ctrl) and Kap1 KO mice 7 weeks after pIC injection. Percentage of each population from the total bone marrow is indicated. (B) Electron microscopy (left, stars indicate mitochondria; middle, average number of mitochondria visualized per cell; n = 10, *p < 0.05) and Mitotracker staining (right, n = 4, *p < 0.05). Decreased nuclear density was frequent in Kap1 KO cells, perhaps reflecting altered chromatin condensation. View larger version: , In this page , In a new window , Download PowerPoint Slide for Teaching Fig. 2 A KAP1-miRNA cascade controls red cell mitophagy. (A) Top, mitophagy-related transcripts in erythroblasts from control (Ctrl) and Kap1 KO mice (n = 4, *p < 0.05, **p < 0.01, ***p < 0.001). Bottom, indicated miRNAs expression in same samples; predicted miRNA-target pairs are indicated by X. (B) miR351 and Bnip3L expression in megakaryocyte/erythroid progenitors (MEP: Lin?Sca1?CD117+ CD34?CD16.32?, in which expression was set at 1) and indicated erythroblast subsets. (C) MiR-351 targets the Bnip3L 3′UTR. Ctrl, for which the normalized value was set at 1, was a combination of MEL cells not overexpressing miR-351 and transduced with a GFP-expressing lentiviral vector with the Bnip3L 3′UTR, and cells overexpressing miR-351 but transduced with the same vector without this sequence (n = 3, *p < 0.05). MiR-503 and miR-322*, which are located next to miR-351 on chromosome X, were also upregulated (2.46 and 2.17 fold, respectively) in Kap1 KO erythroblasts. Consistent with a role for KRAB/KAP1 in regulating this miRNA gene cluster, chromatin immunoprecipitation coupled to DNA sequencing (ChIPSeq) detected a strong KAP1 peak less than 4kb away (Fig. 3A). Because KAP1 is not a DNA binding protein, we postulated that it might be tethered to this and other relevant loci by stage-specific KRAB-ZFPs. Nine KRAB-ZFP genes were identified, which had human orthologs and were expressed exclusively in CD71+Ter119- and/or CD71+Ter119+ erythroblasts, but not in other hematopoietic cells. Six of these genes could be efficiently knocked down in MEL cells by lentivector-mediated RNA interference, and two of them, ZFP689 and ZFP13, emerged as potential Bnip3L regulators (fig. S3). Interestingly, ZFP689 is expressed in CD71+Ter119+ erythroblasts, whereas ZFP13 is expressed only in their CD71-Ter119+ counterparts (Fig. 3B). Both could repress reporter expression in MEL cells transduced with a lentiviral vector harboring the miR-351-close KAP1-binding site upstream of a human phosphoglycerate kinase promoter murine secreted alkaline phosphatase (mSEAP) cassette (Fig. 3C). We then validated these two candidates in vivo by transplanting CD45.2 hematopoietic stem cells (lineage-, Sca1+ and cKit+, or LSK) transduced with lentiviral vectors producing GFP and shRNAs against Zfp689, Zfp13, or Kap1 as a control, into irradiated CD45.1 mice, allowing the dual discrimination of donor vs. recipient and transduced vs. untransduced cells. Analyses of the red cell compartment in bone marrow harvested eight weeks after the graft revealed that knockdown of either Zfp689 or Zfp13 led to a decrease in CD71+Ter119- cells as pronounced as that observed with the Kap1 knockdown (Fig. 3D). Furthermore, RNA analyses of sorted transduced CD71+Ter119+ cells demonstrated that ZFP689-, ZFP13- and KAP1-depleted cells all exhibited an upregulation of miR-351 (Fig. 3E) and a marked downregulation of Bnip3L (Fig. 3F). View larger version: , In this page , In a new window , Download PowerPoint Slide for Teaching Fig. 3 Erythroblast-specific KRAB-ZFPs control the miR-351/Bnip3L/mitophagy axis. (A) Screen shots from the UCSC Genome Browser, with results of a KAP1 ChIPSeq analysis performed on CD71+Ter119+ bone marrow cells. (B) Zfp689 and Zfp13 are induced during erythroid differentiation. (C) ZFP689 and ZFP13 repress a lentiviral vector carrying a miR-351-close KAP1-binding site in transduced MEL cells (Ctrl is a combination of ZFP-overexpressing cells transduced with a vector without the KAP1-binding site and cells LacZ-overexpressing cells transduced with a vector carrying the KAP1-binding site; n = 3, *p < 0.05). (D) CD45.2+ LSK cells were transduced with GFP-expressing, empty or scramble (Ctrl), Kap1-, Zfp689- or Zfp13-directed shRNA lentiviral vectors, engrafted into irradiated CD45.1+ mice, and erythroid differentiation was evaluated by FACS 8 wks later. (E and F) The CD71+Ter119+, CD45.2+, eGFP+ population was then sorted and analyzed by RT-QPCR for miR351 (E) and Bnip3L (F) expression (n = 6, *p < 0.05). In a last series of experiments, we asked whether this erythropoiesis-regulating system has its equivalent in humans. We first found that Kap1 knockdown impaired the differentiation of human erythroleukemia (HEL) cells and increased their mitochondrial content (Fig. 4ABC), blocking several mitophagy effectors including Nix/Bnip3L (Fig. 4D). We further verified that KAP1-depleted HEL cells had increased levels of hsa-miR-125a-5p (Fig. 4D), which has the same seed as murine miR-351, and that overexpressing this miRNA triggered a downregulation of Nix and a rise in the mitochondrial content of these cells (Fig. 4E). Finally, when we knocked down Kap1 in human cord blood CD34+ cells, it resulted in decreasing their ability to undergo cytokine-induced ex vivo erythroid differentiation, which correlated with reduced Nix expression and elevated mitochondrial content (Fig. 4F), a phenotype that could be reproduced by hsa-miR-125a overexpression (Fig. 4G). View larger version: , In this page , In a new window , Download PowerPoint Slide for Teaching Fig. 4 KAP1-regulated RNA interference controls human red cell mitophagy. (A to C) HEL transduced with scramble or Kap1-specific shRNA-expressing lentiviral vectors and induced or not to differentiate were evaluated for Kap1 mRNA expression (A), and by benzidine (B) (n = 3, counting 100 cells for each condition) or Mitotracker (C) (n = 3) staining (*p < 0.05). (D) hsa-miR-125a-5p (miR125a) and Nix expression measured respectively by NanoString nCounter direct RNA quantification and RNA sequencing in HEL cells transduced with empty or Kap1 knockdown vectors. (E) Nix expression in Ctrl (setting normalized value at 1) or hsa-miR-125a-5p-overexpressing HEL cells, measuring their mitochondrial content by Mitotracker staining (n = 4, *p < 0.05). (F) Decreased erythroid differentiation of Kap1 knockdown human cord blood CD34+ cells, assessed by CD235a surface expression at seven (D7) and eleven (D11) days. At D7, sorted CD235a+eGFP+ cells were analyzed by RT-QPCR for Nix and hsa-miR125a expression, and for mitochondrial content by Mitotracker staining (n = 3, *p < 0.05). (G) Percentage of CD235a-expressing cells 7 days after inducing the differentiation of CD34+ cells transduced with empty or unrelated-miRNA- (Ctrl) or hsa-miR-125a-5p-overexpressing lentiviral vectors (n = 3, *p < 0.05). These results unveil a multilayered transcription regulatory system, where protein- and RNA-based repressors are super-imposed in combinatorial fashion to govern the timely triggering of a necessary step of erythropoiesis. miR-351 and several other microRNAs with predicted targets in the mitophagy pathway were upregulated in Kap1-deleted murine erythroblasts (Fig. 2). This apparent redundancy, or rather addition of parallel effects aimed at a same physiological process, is commonly observed with RNA interference (27). Our discovery that it can be further modulated by KRAB-ZFP-mediated repression, and that the latter can itself be multifactorial, adds a remarkable level of modularity to this type of regulation. In human erythroblasts, although KAP1 represses the Nix-targeting hsa-miR-125a-5p, downregulation of several other miRNAs, including hsa-miR-24, -221, -222, and -223, was previously found important for erythroid differentiation, which conversely requires the upregulation of hsa-miR-144/451 cluster (2, 3). Whether stage-specific KRAB-ZFPs are involved in controlling some of these other miRNAs remains to be determined. Even though KAP1 likely influences erythropoiesis by more than just allowing mitophagy, it is interesting to note that Znf205 and Znf689, the respective human orthologs of murine Zfp13 and Zfp689, are expressed in HEL cells and induced upon erythroid differentiation of CD34+ cells (fig. S4). Therefore, polymorphism or mutations in any genetic component of the pathway unveiled here, whether Znf205, Znf689, the genomic binding sites of their products, hsa-miR-125a-5p and other KAP1-regulated miRNA genes, or the sequences targeted by these RNA regulators, could underlie red cell-related pathologies such as anemia, polycythemia, or erythroleukemia. Supplementary Materials www.sciencemag.org/cgi/content/full/science.1232398/DC1 Materials and Methods Figs. S1 to S4 Table S1 References (28–38)