Genome architecture determines the function state of a cell. The conversion of MEFs into iPSCs by CIP should be one of the ideal systems to probe the reprogramming process at the chromatin level, which may provide a detailed understanding on how chemicals reorganize the overall architecture of a genome from a somatic to a pluripotent state. To answer this question, we performed assay for transposase-accessible chromatin sequencing (ATAC-seq) on MEFs, MEFs undergoing CIP at D6, 12, 18, 22, 30, 36, and finally 40 when CiPSC colonies can be picked up. We first performed a principal-component analysis (PCA) and show that the genome accessibility landscape as assessed by ATAC-seq undergoes gradual transitions that bridge between those of MEFs and ESCs ( Figure 3 A, left panel, blue arrow). We then compare it with similar dataset obtained from Yamanaka-factor-induced reprogramming (YIP) () and show that CIP and YIP follow quite distinct chromatin accessibility dynamics (CAD) toward pluripotency ( Figure 3 A, left panel, blue versus red arrows). As chromatin accessibility landscape determines the overall transcription program of a cell, we further compare the transcriptomes between CIP and YIP by PCA analysis and show that CIP and YIP also follow distinct transitions in their transcription programs to arrive at the same pluripotent state ( Figure 3 A, right panel, blue versus red arrows).

Li et al., 2017 Li D.

Liu J.

Yang X.

Zhou C.

Guo J.

Wu C.

Qin Y.

Guo L.

He J.

Yu S.

et al. Chromatin accessibility dynamics during iPSC reprogramming.

The apparent difference between CIP and YIP further inspired us to investigate in greater detail the CAD for CIP. In our earlier work, we have devised a simple close-open logic for YIP based reprogramming in terms of chromatin accessibility (). Accordingly, we first compare the peaks at each locus between MEFs and ESCs and divide the peaks into three categories, closed in MEFs but open in ESCs (CO), open in MEFs but closed in ESCs (OC), open in both MEFs and ESCs (PO). Then the CO and OC peaks of each reprogramming system were further divided into several subgroups based upon the day of reprograming to demonstrate the progression of chromatin accessibility (i.e., opening/closing) dynamics ( Figure 3 B). Under identical analytic conditions, despite the differences we observe for CIP and YIP in Figure 3 A, we arrive at a logic for CIP very similar to that for YIP, i.e., OC at the early stage followed by CO in the late stage ( Figure 3 B, left versus right panels). In terms of chromatin accessibility landscapes between MEFs and ESCs, CIP closes a great deal of loci that are ultimately closed in ESCs while opens only a small number that are open in ESCs at the first data point D6 ( Figure 3 B, left panel, OC1 versus CO1, and Figure 3 C). In fact, when peaks for all loci are quantified, it is apparent that successful closing of chromatin loci dominates the early part of CIP and this presumably prepares the genome to open the pluripotent loci at the end ( Figure 3 C). When compared to the same CAD, YIP opens more loci early ( Figures 3 C versus 3 D), perhaps reflecting the fact that O, K, and S can target the pluripotent loci directly. Indeed, when we compare the total CO and OC between CIP and YIP, the Venn diagram showed that CIP shared only 25% (4,265/16,983) in CO peaks, but 80% (34,524/43,049) in OC peaks, and 96% (15,800/16,500) in PO peaks with YIP ( Figure 3 E). This is quite unexpected as only 4,265 CO loci are shared between CIP and YIP. To further investigate whether the CO/OC or PO peaks are transcriptionally active, we performed H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) and show that the CO peaks undergo a slow and gradual gain of H3K27ac during reprogramming, in contrast to those OC and PO peaks nicely matched with H3K27ac ( Figure 3 F). These data indicate that CIP and YIP differ mainly in the opening of pluripotent loci while both close the open chromatin in MEFs (OC) and keep open loci open (PO) in a similar fashion.