Stem Cell

Transcription factor-mediated direct cellular reprogramming yields cell-type specific DNA methylation signature – Scientific Reports

Time Stamp: December 14, 2023 7:00 PM
Source Node: 383767

iISC-BOs exhibit a CpG methylation signature that closely resembles ISC-BOs

Genomic DNA was extracted from MEFs, iISC-BOs, and ISC-BOs, and high-resolution methylome analysis was performed using the PBAT method (Fig. 1A). The genome-wide methylation states of cytosine-containing sequences such as CpG, CHH, and CHG were compared among the samples. The data showed that CpG methylation signatures were similar among replicates and between iISC-BOs and ISC-BOs (Pearson’s correlation coefficient [R] = 0.76–0.84), whereas substantial differences were found between iISC-BOs and MEFs, and between ISC-BOs and MEFs (R = 0.45–0.65; Fig. 1B,C, and Supplementary Fig. 1). The methylation states of CHH and CHG were highly correlated in comparison to iISC-BOs with their replicates and with ISC-BOs, and MEFs (R = 0.97–1.00; Fig. 1C and Supplementary Fig. 1).

In a genome-wide view, the total CpG methylation levels of MEFs were 69.6–70.1%, whereas those of ISC-BOs and iISC-BOs were 64.3–66.3% and 61.5–63.8%, respectively (Fig. 1D, the left panel). The CpG methylation levels of the introns and intergenic regions that occupy most of the genome showed the same tendencies as those of the total regions (Fig. 1D, right bottom panels). In contrast, the exons and CpG islands (CGIs) that may have an impact on transcriptional regulation exhibited slightly higher and higher levels of CpG methylation, respectively, in both iISC-BOs and ISC-BOs in comparison to that of MEFs (Fig. 1D, right upper panels). Moreover, the percentage of DMRs between iISC-BOs and ISC-BOs was one-tenth that between MEFs and iISC-BOs or ISC-BOs (Fig. 1E). Taken together, our data demonstrate that direct reprogramming from MEFs to iISCs is associated with variations in genome-wide CpG methylation and allows the generation of iISC-BOs that have a CpG methylation state similar to ISC-BOs. Additionally, we confirmed the gene expression of writers, readers, and erasers of the DNA methylation from previous microarray data²⁶. The expression of some of the genes was changed during the direct reprogramming from MEFs to iISC-BOs, however, no genes showed significant difference of expression between ISC-BOs and iISC-BOs (Supplementary Fig. 2).

Genome-wide similar distribution patterns of DMRs between MEFs and iISC-BOs or ISC-BOs result in a similar gene expression pattern of iISC-BOs and ISC-BOs

We sought to identify the genomic loci of DMRs between MEFs and iISC-BOs or ISC-BOs to investigate the transition of the CpG methylation state from MEFs to iISCs in more detail. The numbers of DMRs with hypermethylation in iISC-BOs (iISC-high DMRs) or ISC-BOs (ISC-high DMRs) and those with hypomethylation in iISC-BOs (iISC-low DMRs) or ISC-BOs (ISC-low DMRs) were not significantly different, whereas the numbers of iISC/ISC-low DMRs were slightly higher than those of iISC/ISC-high DMRs (Fig. 2A). Genome-wide distribution analysis revealed that the genomic loci of iISC-low and ISC-low DMRs and those of iISC-high and ISC-high DMRs were similarly distributed in the intergenic regions and regions of introns and exons, respectively (Fig. 2B). To examine how similar distribution patterns of iISC/ISC-low and iISC/ISC-high DMRs affected gene expression, we investigated the expression of DMR-associated genes by reanalyzing our previous transcriptome data obtained from MEFs, iISC-BOs, and ISC-BOs²⁶. To identify DMR-associated genes, we focused on iISC/ISC-low DMRs and iISC/ISC-high DMRs located within 10 kbp upstream and downstream of the TSS (Fig. 2C) because CpG methylation near the TSS affects transcription³⁰. We found that genes associated with iISC-low DMRs and ISC-low DMRs and those associated with iISC-high DMRs and ISC-high DMRs were similarly expressed between iISCs and ISCs compared with MEF (Fig. 2D and Supplementary Table 1). Thus, the similarity in the genome-wide distribution patterns of iISC-low and ISC-low DMRs and those of iISC-high and ISC-high DMRs may contribute to the similar gene expression patterns of iISC-BOs and ISC-BOs.

iISC-BOs have hyper- and hypo-methylated DNA, compared with ISC-BOs

As shown above, the CpG methylation state and the resulting gene expression pattern in iISC-BOs were similar to those in ISC-BOs. However, they were not the same. Thus, we examined the genomic loci of DMRs between iISC-BOs and ISC-BOs to explore the differences in CpG methylation states between these two samples. Our data showed that hypermethylation of DMRs in iISC-BOs (hyper DMRs) was more than twice as high as hypomethylation in iISC-BOs (hypo DMRs), suggesting that DNA methylation is induced more frequently than DNA demethylation during direct reprogramming from MEFs to iISCs (Fig. 3A). Heatmaps showing the average CpG methylation rates in the hyper-/hypo-DMRs among MEFs, iISC-BOs, and ISC-BOs revealed that these hyper-/hypo-DMRs could be mainly divided into two groups: those that maintain the levels of CpG methylation in the process of direct reprogramming from MEFs to iISCs, and those that represent insufficient or excessive CpG methylation in only iISC-BOs (Fig. 3B).

It has been reported that CpG methylation yielded around the TSS affects their transcription³⁰. Thus, we analyzed the genomic loci of the hyper-/hypo-DMRs around the TSS to examine the possibility that these hyper-/hypo-DMRs affect gene transcription. Our data demonstrated that the hyper-DMRs were located in regions distal and proximal to the TSS, whereas the hypo-DMRs were mainly located in regions distal to the TSS (Fig. 3C). Thus, hyper DMRs contribute more frequently to the misregulation of gene expression in iISCs than hypo DMRs.

iISC-BOs have a cellular reprogramming-associated aberrant DNA methylation signature

As shown in Fig. 3, an abnormal DNA methylation state may be induced during direct reprogramming. Reprogramming-associated aberrant DNA methylation signatures can be classified into at least two groups. One is an original cell type-specific DNA methylation signature that should be changed but are abnormally maintained in reprogrammed cells (maintained DNA methylation: mDNA methylation), and the other is a DNA methylation signature that is abnormally acquired or erased in only reprogrammed cells (specific DNA methylation: sDNA methylation). To identify regions with mDNA or sDNA methylation in iISC-BOs, DMRs between iISC-BOs and ISC-BOs were divided into two groups based on the overlap with DMRs between MEF and ISC-BOs (Fig. 4A). When there was an overlap, iISCs still had an MEF-specific DNA methylation signature, indicating an mDNA methylation signature in iISCs. When there was no overlap, iISCs had a DNA methylation signature that was distinct from that of MEFs and iISCs, indicating an sDNA methylation signature in iISCs. Our data demonstrated that both hyper- and hypo-DMRs could be divided into regions with mDNA or sDNA methylation signatures, and that DMRs with sDNA methylation signatures were found more frequently than those with mDNA methylation signatures (Fig. 4B). Genome-wide distribution analyses revealed that both mDNA and sDNA methylation was detected at higher frequencies in exons with hyper-DMRs than in those with hypo-DMRs (Fig. 4C). Because hypermethylation of gene body CGIs is considered a result of enhanced transcription³¹, our data suggest that the mDNA and sDNA methylation signatures found in exons with hypermethylated DMRs reflect errors in transcriptional activation during direct reprogramming from MEFs to iISCs.

Abnormal DNA methylation in iISC-BOs has little effect on gene expression

Our present data suggest that hyper-/hypo-DMRs affect the transcriptional levels of genes and lead to the misregulation of gene expression in iISC-BOs. Our previous study demonstrated that the transcriptional signature of iISC-BOs closely resembles that of ISC-BOs, although a small number of differentially expressed genes (DEGs) were found between iISC-BOs and ISC-BOs²⁶. Thus, in this study, we examined the relationship between hyper-/hypo-DMRs and DEGs, both of which were identified in iISC-BOs and ISC-BOs. Genes that contained hyper/hypo-DMRs within 10 kb upstream and downstream of the TSS were extracted and divided into DMRs with mDNA or sDNA methylation signatures (Fig. 5A). Among these hyper-/hypo-DMR-associated genes, hyper-DMRs were found at high frequencies downstream of the TSS, including the gene body, whereas only a few hypo-DMRs were found within 10 kbp upstream and downstream of the TSS (Fig. 5A). In addition, sDNA methylation was found more frequently than mDNA methylation in hyper-DMR-associated genes (Fig. 5A). These data suggest that hyper-DMRs are involved in the regulation of DEGs between iISC-BOs and ISC-BOs.

To examine this possibility, we classified DEGs into genes expressed at higher or lower levels in iISC-BOs than in ISC-BOs, designated iISC-high DEGs or iISC-low DEGs, respectively, and compared each of them with hyper-DMR-associated genes (Fig. 5B). Our data demonstrated that the number of genes contained in the hyper-DMR-associated genes and iISC-high DEGs or iISC-low DEGs were 9 and 12, respectively (Fig. 5B). These genes comprised only 6.5% and 8.6% of the hyper-DMR-associated genes, respectively. Scatter plots showing fold-differences in the expression of hyper-DMR-associated genes revealed that nine genes have hyper-DMRs with an sDNA methylation signature, while the 12 genes are divided into four and eight genes that have DMRs with an mDNA and sDNA methylation signature, respectively (Fig. 5C). Nevertheless, the expression levels of the majority of hyper-DMR-associated iISC-high and iISC-low DEGs (Foxc1, Gprc5b, Hoxa5, Satb2, H2-T3, Gata4, Lefty1, Foxa1, Tubb2b, Pbld1, and Proz) were significantly upregulated or downregulated in iISC-BOs compared to MEF, in the same manner as in ISC-BOs, while those of other DEGs (Hoxa13, Gsc, Hoxd9, Mkx, and Adprhl1) did not change significantly (Fig. 6).

In addition to the hyper-DMR-associated genes, the hypo-DMR-associated genes also overlapped with iISC-high and iISC-low DEGs, although the number of overlapping genes was only three and five, respectively (Fig. 7A). Five genes had hypo-DMRs with an sDNA methylation signature, while three genes were divided into one and two genes that had DMRs with mDNA and sDNA methylation signatures, respectively (Fig. 7B). Similar to the case of hyper-DMR-associated iISC-high and iISC-low DEGs, the hypo-DMR-associated iISC-high and iISC-low DEGs (Foxa1, Onecut1, Onecut2 and Ddr2), except for Hoxa9, were expressed at much higher or lower levels than in MEFs and ISC-BOs (Fig. 7C). Taken together, our data demonstrated that almost all abnormal DNA methylation states in iISC-BOs may not be involved in the dysregulation of genes that are normally expressed or silenced in ISC-BOs.