A KRAB/KAP1-miRNA cascade regulates erythropoiesis through stage-specific control of mitophagy

Abstract

During hematopoiesis, lineage- and stage-specific transcription factors work in concert with chromatin modifiers to direct the differentiation of all blood cells. We explored the role of KRAB-containing zinc finger proteins (KRAB-ZFPs) and their cofactor KAP1 in this process. In mice, hematopoietic-restricted deletion of Kap1 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 (miRNAs) targeting mitophagy transcripts, itself secondary to a lack of repression by stage-specific KRAB-ZFPs. The KRAB/KAP1-miRNA regulatory cascade is evolutionarily conserved, as it also controls mitophagy during human erythropoiesis. Thus, a multilayered transcription regulatory system is present, in which protein- and RNA-based repressors are superimposed in combinatorial fashion to govern the timely triggering of an important differentiation event.

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.

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).

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).

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).