(Background) In the context of erythroid differentiation, the importance of transcription factor GATA-1 has been unequivocally demonstrated through cell-based ex vivoassays, knockout mouse models, and rare patients with anemia. GATA-1regulates the expression of erythroid-related genes such as globins, and those involved in heme biosynthesis and an unrecognized function. A novel FAM210B (C20orf108) gene was recently identified as a novel GATA-1 target gene (Kondo et al. Int J Hematol. 2016). FAM210B gene is abundantly expressed in the later stage of erythroid differentiation and encodes a protein containing an N-terminal mitochondrial-targeting sequence, which was considered as a mitochondrial membrane protein. Although the FAM210B protein has been suggested to regulate mitochondrial metabolism (Sun et al. Cell Death Dis. 2017), its detailed function remains to be elucidated.
(Method) Endogenous FAM210B protein was deleted by CRISPR/Cas9 based on human iPS-derivederythroid progenitor (HiDEP) cells (Kurita et al. PLoS ONE 2103). To induce erythroid differentiation, HiDEP cells were co-cultured with OP9 stromal cells (ATCC) with IMDM medium supplemented with FBS, erythropoietin, dexamethasone, MTG, insulin-transferrin-selenium, ascorbic acid, and sodium ferrous citrate (Saito and Suzuki et al. MCB2019). Affinity purification of the FAM210B complex was conducted in K562 erythroid cell line (ATCC) stably expressing His/Biotin-tagged FAM210B. MitoXpress Xtra (Agilent) and Human Oligo chip 25K (Toray) were used to evaluate oxygen consumption rate and transcription profiling, respectively.
(Results) Clonal lines with HiDEP cells were established, which harbored deletion within coding exon 3 of FAM210B. Quantitative real-time polymerase chain reaction (RT-PCR) analysis confirmed a strong decrease in FAM210B expression. Microarray analysis revealed >1.5-fold up- and down-regulation of 104 and 53 genes caused by the FAM210B knockout, respectively. The down-regulated gene ensemble included TTC19, which is involved in mitochondrial respiratory chain complex III assembly, and genes encoding mitochondrial proteins (AARS2, YWHAE, CLIC1), whereas, the expressions for ALAS2, HBG, HBA, and HMOX1 were not significantly affected by FAM210B depletion. Intriguingly, when wild-type and FAM210B-depleted HiDEP cells were further induced to undergo erythroid differentiation, erythroid differentiation was more pronounced by FAM210B depletion. This was reflected by enhanced nuclear condensation and hemoglobinization, as well as upregulations for ALAS2, HBG, HBA, and HMOX1 in the FAM210B-depleted erythroblasts.
Recent report suggests that FAM210B functions as an adaptor protein in the mitochondria (Yien et al. JBC 2018). Thus, FAM210B-interacting protein was purified from K562 cells stably expressing His/Biotin-tagged FAM210B.Mass spectrometry analysis based on the His/Biotin-purified material showed that FAM210B may interact with multiple mitochondrial proteins, including multiple subunits of mitochondrial adenosine triphosphate synthases and mitochondrial heat shock proteins. Further functional analysis showed that oxygen consumption rates might be decreased in FAM210B-depleted HiDEP cells, indicating that FAM210B was possibly involved in mitochondrial energy metabolism in erythroblasts. At present, we are conducting detailed biological analyses to characterize the role of FAM210B during erythroid differentiation.
(Conclusion) Further characterization of FAM210B provides new insights in the study of erythroid differentiation, and this may lead to better understanding of the pathophysiology of certain anemias.
Disclosures
Fukuhara: Mundi: Honoraria; Janssen Pharma: Honoraria; Mochida: Honoraria; Gilead: Research Funding; Bayer: Research Funding; Takeda Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Eisai: Honoraria, Research Funding; Kyowa-Hakko Kirin: Honoraria; AbbVie: Research Funding; Solasia Pharma: Research Funding; Celgene Corporation: Honoraria, Research Funding; Nippon Shinkyaku: Honoraria; Zenyaku: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Ono Pharmaceutical Co., Ltd.: Honoraria. Onishi:MSD: Honoraria, Research Funding; Kyowa-Hakko Kirin: Honoraria; Sumitomo Dainippon Pharma: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria; Novartis Pharma: Honoraria; Celgene: Honoraria; Pfizer Japan Inc.: Honoraria; Astellas Pharma Inc.: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Nippon Shinyaku: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria. Yokoyama:Astellas: Other: Travel expenses.
ER-SURF: Riding the Endoplasmic Reticulum Surface to Mitochondria
