ALKBH1 deficiency leads to loss of homeostasis in human diploid somatic cells
- PDF / 1,286,166 Bytes
- 8 Pages / 595.276 x 785.197 pts Page_size
- 75 Downloads / 189 Views
Protein & Cell
LETTER
Dear Editor, As the most prevalent DNA methylation modification in prokaryotes, DNA N6-methyladenosine (6mA) in eukaryotic genomes has recently been observed in diverse species including Caenorhabditis elegans (Greer et al., 2015), Drosophila melanogaster (Zhang et al., 2015), mouse (Wu et al., 2016) and human (Xiao et al., 2018). 6mA has been reported to associate with multiple physiological processes including embryonic development and tumorigenesis (Greer et al., 2015; Zhang et al., 2015; Xie et al., 2018), yet some controversies exist. In contrast to the findings showing that ALKBH1 (alkB homolog 1) is a primary 6mA demethylase in mouse and human cells (Wu et al., 2016; Xiao et al., 2018; Xie et al., 2018), other studies indicate that ALKBH1 is prone to demethylate 6mA on bubbled or bulged DNAs that are often featured by a locally unpairing region with flanking duplex, such as D-loop, R-loop as well as DNA or RNA stemloop, and single-stranded DNAs at a lower efficiency, but not double-stranded DNAs (Tian et al., 2020; Zhang et al., 2020a). Stem cell exhaustion is a major causal and risk factor underlying the progressive disruption of physiological integrity during the development of aging-associated disorders, in which epigenetic alterations are closely implicated (Zhang et al., 2020b). Yet, the roles of 6mA and its putative regulators such as ALKBH1 in the homeostatic maintenance of human stem cells and their differentiated derivatives remain elusive. To investigate the role of ALKBH1 in regulating homeostatic maintenance in human diploid cells, we first generated ALKBH1-deficient human embryonic stem cells (hESCs) via clustered regularly interspaced short palindromic repeat/ CRISPR-associated protein 9 (CRISPR/Cas9)-mediated non-homologous end joining (NHEJ) (Fig. 1A, 1B and S1A). The absence of the ALKBH1 protein was verified by western blotting (Fig. 1C). Phenotypic analyses revealed that ALKBH1−/− hESCs expressed pluripotency markers including NANOG, SOX2 and OCT4 (Fig. 1D) and maintained a normal karyotype (Fig. 1E). Altogether, these data suggest that ALKBH1−/− hESCs maintain normal pluripotency. We next differentiated ALKBH1+/+ and ALKBH1−/− hESCs into human mesenchymal stem cells (hMSCs) and vascular smooth muscle cells (hVSMCs) (Fig. 1A). ALKBH1−/− © The Author(s) 2020
hMSCs expressed MSC-specific surface markers including CD73, CD90 and CD105 (Fig. 1F), and demonstrated a lack of ALKBH1 expression (Fig. 1G, 1H and S1B). Whole-genome sequencing (WGS) showed that ALKBH1 deficiency did not impair the genomic integrity in hMSCs (Fig. S1C). Earlyonset growth arrest of ALKBH1−/− hMSCs was observed through serial passaging, relative to that of ALKBH1+/+ hMSCs (Fig. 1I). Besides, ALKBH1−/− hMSCs exhibited higher percentage of senescence-associated β-galactosidase (SA-β-Gal)-positive cells and lower clonal expansion ability (Fig. 1J and 1K). Genome-wide RNA sequencing (RNA-seq) showed that ALKBH1 deficiency resulted in downregulation of some master genes promoting cell proliferation (e.g., FOS,
Data Loading...
