Supplementary Figure 2: Analysis pipeline to identify ZSCAN10 and GSS.
From: ZSCAN10 expression corrects the genomic instability of iPSCs from aged donors
(a) ZSCAN10. Initially, 59 core pluripotency genes derived from a pluripotency network analysis were retrieved2 and filtered based on differential expression in A-iPSC and somatic cells (low) versus Y-iPSC or ESC (high) (96 genes with more than 2-fold difference), which narrowed down the candidates to eight genes (ZSCAN10, GBX2, SAL4, TCFAP2C, MYBL2, EVX1, OTX2, and MSH6). We cross-referenced each candidate gene that associated with the DNA damage response and genomic stability, and identified the top candidate, ZSCAN10, which was previously reported to have a regulatory link with ATM, GSS, p53, PARP, PLK1, and ZSCAN43,4,5,6. We confirmed differential expression of ZSCAN10 by qPCR in both mouse and human cells, and functionally assessed the recovery of the DNA damage response/genomic stability in this report. (b) Ontology analysis of genes with greater than two fold differential expression in A-iPSC versus Y-iPSC by Panther Go-Slin Ontology analysis tool (http://www.pantherdb.org). (c) Heatmap showing hierarchical clustering of differentially expressed genes between A-iPSC-ZSCAN10 and A-iPSC in the DNA damage response pathway. The gene list was extracted from Gene Ontology-GO (GO:0006974, http://www.geneontology.org/). Bar indicates the z-score scaled gene expression levels. Top genes include TLK1, PRPF19, APEX1, UNG, RAD52, BCCIP, GTF2H1, PRPF19, POLI, MSH3, RAD54L, BRE, FANCG, XRCC3, RAD51, DCLRE1C, and FANCL. Note that expression of ZSCAN10 in A-iPSC is sufficient to make A-iPSC-ZSCAN10 cluster together with ESC and Y-iPSC for a group of genes within the DNA damage response pathway. (d) GSS. To identify the ZSCAN10 targets involved in the DNA damage response defect in A-iPSC, we cross-referenced (1) differentially expressed genes in Y-iPSC/ESC and A-iPSC (634 genes with more than 2-fold difference), (2) genes with altered expression in A-iPSC compared with A-iPSC-ZSCAN10 (narrowed down to 464 genes), (3) the gene list of the ZSCAN10 targeted binding regions from those previously reported in ChIP-on-chip analysis in ESC5 (narrowed down to 39 genes), and (4) the list of genes extracted from Gene Ontology-GO involved in the DNA damage response-apoptosis-antioxidant-ROS-genomic stability-cell death pathway (GO:0006974, GO:0072593, GO:0048039, GO:0043295, GO:0034699, GO:0032275, GO:0031405, GO:0008431, GO:0008379, GO:0006776, GO:0006744, GO:0006915, GO:0001523, GO:0008219, GO:0012501, GO:0000302, GO:1903409, GO:0043066, GO:0097752). This narrowed down the list to eight candidate genes (BCCIP, PLAC8, DHODH, ESCO2, RASSF1, GSS, GDF15, MKNK2). Because cellular glutathione level is a well-established regulator of the DNA damage response and genomic stability30,31,32,33, we hypothesized that a glutathione imbalance contributes to the observed defects in A-iPSC. Among the differentially expressed glutathione regulatory genes, we identified glutathione synthetase (GSS), which is one of the eight genes directly bound by ZSCAN10, as the top candidate. The differential expression of GSS was confirmed by qPCR in both mouse and human cells, and the capacity to recovery of DNA damage response/genomic stability was functionally assessed in this report. (e) Heatmap shows hierarchical clustering analysis of differentially expressed genes between A-iPSC-ZSCAN10 and A-iPSC in the glutathione synthesis pathway. The gene list is extracted from Gene Ontology-GO (GO:0006749, http://www.geneontology.org). Color bar indicates the z-score scaled gene expression levels. Top genes include GSTP1, GSTP2, GSTT1, GSTT2, GSTT3, GSTA1, GSTK1, MGST1, GLRX2, and GSS. Note that expression of ZSCAN10 in A-iPSC is sufficient to make A-iPSC-ZSCAN10 cluster together with ESC and Y-iPSC for a group of the genes within the glutathione synthesis pathway, further supporting GSS as a top candidate.
