Mark J. Koury, MD
Dr. Koury indicated no relevant conflicts of interest.
Barde I, Rauwel B, Marin-Florez RM, et al. A KRAB/KAP1-miRNA cascade regulates erythropoiesis through stage-specific control of mitophagy. Science. 2013;340:350-353.
Differentiation of eukaryotic cells is characterized by increased nuclear heterochromatin, cessation of cell division, and intracellular accumulation or secretion of highly specialized or unique proteins. The mammalian erythroblast goes even further than most differentiated cell types by enucleating and, then, ridding itself of intracellular organelles including endoplasmic reticulum, ribosomes, and mitochondria when it matures from a reticulocyte to an erythrocyte.1,2 This organelle clearance is achieved by proteasomal degradation, exocytosis of microvesicles, and autophagy. Most erythroid mitochondria are removed via autophagy in a process termed mitophagy. During mitophagy, mitochondria are engulfed by autophagosomes, which are double membrane structures that fuse with either lysosomes where proteolytic enzymes degrade the mitochondria or with the plasma membrane where the mitochondria are extruded from the reticulocyte. Reticulocyte mitophagy involves multiple proteins including: Nix/Bnip3L, a BH3-only member of the Bcl-2 family of apoptosis-related proteins that targets the outer mitochondrial membrane for autophagosome engulfment; ULK1, a serine-threonine kinase; and Atg-7, a conjugase that activates other components of the autophagy process. In mice, a genetic knockout of each of these genes leads to partially impaired mitophagy that causes mild-to-moderate anemia with retained erythrocyte mitochrondia.
In tetrapod vertebrate cells, hundreds of different Kruppel-associated box domain-containing zinc finger transcription proteins (KRAB-ZFPs) bind to specific DNA sites where they interact with a protein that does not directly bind DNA but has the major function of repressing gene transcription.3 This KRAB-associated protein 1 (KAP1) has thousands of potential locations throughout the genome where it can foster heterochromatin formation, most likely thorough its capacity to modify acetylation and methylation of local histones. KAP1 also has potential roles in DNA repair and suppression of DNA recombination. With such widespread activity and with such a large number of binding partners in the nucleus, KAP1 can affect many biologic events.
To discern the role(s) of KAP1 in hematopoiesis, Barde et al. created a conditional knockout mouse model. KAP1 knockout mice had thrombocytopenia and lethal anemia. The anemia was accompanied by increased percentages of early-stage erythroblasts, but greatly decreased numbers of late-stage erythroblasts and reticulocytes. The late-stage erythroblasts, in which mitophagy normally begins, had increased mitochondria, suggesting dysfunctional mitophagy. Erythroblasts from KAP1 knockout mice had decreased mRNA transcripts for seven mitophagyrelated genes, including Nix/Bnip3L and Ulk1. Since KRAB-ZFP/KAP1 complexes repress transcription, decreased mitophagy-related transcripts in KAP1 knockout erythroblasts suggested an indirect suppression of transcription. Analyses of microRNAs, which bind specific sequences in mRNA transcripts leading to their inhibited translation and enhanced degradation, revealed increased miR-351 that targets Nix/Bnip3L mRNAs. Because miR-351 and two other microRNAs clustered on the mouse X chromosome were all increased in KAP1 knockout erythroblasts, the authors investigated KRAB-ZFPs and identified two that could repress miR-351 expression, specifically in late-stage erythroblasts and reticulocytes. In vitro studies by Barde et al. showed that these two murine KRAB-ZFPs have human orthologs that repress the corresponding human microRNA, including hsa-miR-125a-5p, which regulates Nix/Bnip3L transcript levels, mitochondrial numbers, and terminal differentiation of human erythroblasts.
Barde et al. demonstrated that KRAB-ZFP/KAP1 complexes play a key role in the terminal differentiation of mammalian erythroblasts. These complexes mediate nuclear heterochromatin formation while regulating expression of multiple mitophagy components in late-stage erythroblasts and reticulocytes. Whether microRNA regulation of multiple gene transcripts or some other KAP1 function causes thrombocytopenia in KAP1 knockout mice is uncertain. However, the hundreds of specific KRABs in vertebrate cells suggest that microRNA-mediated regulation of a complex process, such as organelle degradation during terminal erythroid differentiation, has a role in the differentiation of many types of cells.
1. Mortensen M, Ferguson DJ, Simon AK. Mitochondrial clearance by autophagy in developing erythrocytes: clearly important, but just how much so? Cell Cycle. 2010;9:1901-1906.
2. Ney PA. Normal and disordered reticulocyte maturation. Curr Opin Hematol. 2011;18:152-157.
3. Iyengar S, Farnham PJ. KAP1 protein: an enigmatic master regulator ofthe genome. J Biol Chem. 2011;286:26267-26276.