Targeted Amplicon Sequencing: The Interferon ? Pathway Enhances Pluripotency and X-Chromosome Reactivation in iPSC Reprogramming
Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) requires activation of the pluripotency network and resetting of the epigenome by erasing the epigenetic memory of the somatic state. In female mouse cells, a critical epigenetic reprogramming step is the reactivation of the inactive X chromosome. Despite its importance, a systematic understanding of the regulatory networks linking pluripotency and X-reactivation is missing. Here we reveal important pathways for pluripotency acquisition and X-reactivation using a genome-wide CRISPR screen during neural precursor to iPSC reprogramming. In particular, we discover that activation of the interferon ? (IFN?) pathway early during reprogramming accelerates pluripotency acquisition and X-reactivation. IFN? stimulates STAT3 signaling and the pluripotency network and leads to enhanced TET-mediated DNA demethylation, which consequently boosts X-reactivation. We therefore gain a mechanistic understanding of the role of IFN? in reprogramming and X-reactivation and provide a comprehensive resource of the molecular networks involved in these processes. Overall design: A custom amplicon oxidative BS-sequencing protocol was developed to assess the allele-specific hydroxymethylation status of regions surrounding several Differential (hydroxy)Methylated CpGs -D(h)MP- in escapee and X-reactivating gene promoters identified at days 5 or 7 of reprogramming by DNA methylation arrays. The validation was done for day 5 reprogramming samples in the presence or absence of IFN? with the aim to calculate 5hmC percentages in an allele-specific manner. Regions to be analyzed were selected to include species-specific SNPs and at least a D(h)MP while maintaining an amplicon size of 200-500 bp. Paired oxidative Bisulfite (OxBS) and the mock-oxidation reaction (Bisulfite BS) treated DNA from day 5 reprogramming samples were used. Next Generation Sequencing was performed using MiSeq (300bp, single-end).