Persistent epigenetic memory impedes rescue of the telomeric phenotype in human ICF iPSCs following DNMT3B correction

Abstract

DNA methyltransferase 3B (DNMT3B) is the major DNMT that methylates mammalian genomes during early development. Mutations in human DNMT3B disrupt genome-wide DNA methylation patterns and result in ICF syndrome type 1 (ICF1). To study whether normal DNA methylation patterns may be restored in ICF1 cells, we corrected DNMT3B mutations in induced pluripotent stem cells from ICF1 patients. Focusing on repetitive regions, we show that in contrast to pericentromeric repeats, which reacquire normal methylation, the majority of subtelomeres acquire only partial DNA methylation and, accordingly, the ICF1 telomeric phenotype persists. Subtelomeres resistant to de novo methylation were characterized by abnormally high H3K4 trimethylation (H3K4me3), and short-term reduction of H3K4me3 by pharmacological intervention partially restored subtelomeric DNA methylation. These findings demonstrate that the abnormal epigenetic landscape established in ICF1 cells restricts the recruitment of DNMT3B, and suggest that rescue of epigenetic diseases with genome-wide disruptions will demand further manipulation beyond mutation correction.

Telomere dysfunction cooperates with epigenetic alterations to impair murine embryonic stem cell fate commitment

“…Knockdown of the TET enzymes Tet1 and Tet2 has been shown to impact telomere integrity and subtelomeric DNA methylation in addition to playing a key role in cell lineage commitment (Lu et al, 2014;Yang et al, 2016). Another example is the presence of unstable telomeres in humans harboring a DNMT3B deficiency (Sagie et al, 2017;Toubiana et al, 2019). Mutation of the TERT promoter, one of the mostly commonly found non-coding mutations in cancer (Mularoni et al, 2016;PCAWG Consortium, 2020), leads to telomerase upregulation via allelespecific epigenetic alterations involving PRC2 and DNA methylation (Leao et al, 2018;Lee et al, 2019;Stern et al, 2017).…”

Epigenetic Characteristics of Human Subtelomeres Vary in Cells Utilizing the Alternative Lengthening of Telomeres (ALT) Pathway

“…SK-LU-1 and H295R were cultured according to the conditions specified by ATCC. Control cell lines included the Immunodeficiency, Centromeric instability and Facial anomalies type 1 (ICF1) lymphoblastoid cell line (LCL), GM8714 (Coriell Institute) cultured in RPMI-1640 supplemented with 15 % FCS, glutamine and antibiotics, fibroblast-like cells (FLs) derived from induced pluripotent stem cells (iPSCs) of ICF1 patient pR [ 27 ], and wild-type primary fibroblasts FSE and UN [ 28 ]. All fibroblasts were cultured in DMEM supplemented with 10 % FCS, glutamine, and antibiotics.…”

“…Following conversion, DNA was amplified using FastStart Taq DNA Polymerase, dNTPack (04738314001, Sigma-Aldrich, St. Louis, MO, USA) using the followed amplification program: 95 °C for 5 min; 4 cycles of 95 °C for 1 min, 53 °C for 3 min, 72 °C for 3 min; 2 cycles of 95 °C for 30 s, 55 °C for 45 s, 72 °C for 45 s; 40 cycles of 95 °C for 30 s, 72 °C for 1.5 min; 72 °C for 10 min. Primers for amplification of satellite 2 and subtelomeric regions were described previously [ 27 , 28 ]. PCR products were purified by the QIAquick PCR purification kit (28104, Qiagen, Hilden, Germany), quantified, diluted to 10 pM, pooled into one tube, and subjected to emulsion PCR (E-PCR) on ion sphere particles (ISPs; Thermo Fisher Scientific, Waltham, MA, USA) using the Ion PGM TM Sequencing 400 Kit on the Ion OneTouch system (Thermo Fisher Scientific).…”

“…ChIP analyses were performed as described previously [ 27 ]. Antibodies used for immunoprecipitation included anti-H3K4me3 (ab8580, Abcam, Cambridge, UK) and anti-H3K9me3 (ab8898, Abcam).…”

Cardiac Fibroblasts and Myocardial Regeneration

“…However, cardiomyocytes appear to be more readily differentiated from iPSCs that have been reprogrammed from human CFs (hciPSCs) than from other cell types: the purity of hciPSC-derived cardiomyocytes (hciPSC-CMs) exceeded 92% without any subsequent selection procedures, whereas iPSCs reprogrammed from human dermal FBs (hdiPSCs) or blood mononuclear cells (h BMC iPSCs) yielded cardiomyocyte populations that were only 60–85% pure ( Ye et al, 2013 ; Zhang et al, 2015 ). This difference in differentiation efficiency can likely be attributed to the presence of epigenetic factors that the iPSCs retained from their tissues of origin ( Kim et al, 2010 ; Noguchi et al, 2018 ; Toubiana et al, 2019 ). Furthermore, the Ca 2+ -handling profile of the hciPSC-CMs was more cardiac-like than the profiles of cardiomyocytes differentiated from hdiPSCs or h BMC iPSCs, and when sheets of hciPSC-CMs were transplanted into infarcted mouse hearts, the rate of engraftment was exceptionally high (>30%) 28 days later; the treatment was also associated with significant improvements in cardiac contractile function.…”