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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 super-imposed 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 multimolecular 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 hyporegenerative 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).
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).
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).
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.
We thank the staff of our core facilities for technical expertise, and P. Malik (Cincinnati Children’s Hospital) and G. Ferrari (San Raffaele Telethon Institute for Gene Therapy, Milano) for the kind gift of reagents. Supported by grants from the Swiss National Science Foundation and the European Research Council to DT. The cDNA and miRNA microarray and ChIP-seq data are deposited in the Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo) under the accession number GSE44061. DT and IB are inventors on a patent application submitted by EPFL for the control of mitophagy.