Epigenetic barriers to X chromosome reactivation during reprogramming to induced pluripotency & molecular evolution of myb genes in Hawaiian Silverswords and California Tarweeds

The differentiated state of somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs), leading to the reactivation of the inactive X chromosome (Xi) of female differentiated cells in the mouse. In somatic cells, the Xi is very stable however, when reprogramming mouse embryonic fibroblasts, the Xi reactivates upon completion to a pluripotent state. How somatic character is reversed as well as the sequence of epigenetic events leading to X chromosome reactivation during reprogramming to iPSCs, is not well understood. We found that, the non-coding RNA Xist and DNA methylation are present on the Xi in clonal late reprogramming intermediates, pre-iPSCs, suggesting that these are reversed late during reprogramming. To test whether these function in maintaining Xi repression at this stage of reprogramming we functionally interfered with Xist and DNA methylation using pre-iPSCs genetically deleted for Xist and/or treated with siDNMT1 (DNA methyltransferase 1) and DNA methyltransferase inhibitor 5AzadC (5-aza-2'-deoxycytidine). Reactivation of the X chromosome occurs upon deletion of Xist and both treatments. We used bisulfite sequencing to confirm the loss of DNA methylation of X-linked genes in pre-iPSC and found the loss of DNA methylation on the Xi occurs in the presence or absence of Xist with both 5AzadC and siDNMT1. This suggests DNA demethylation is a late event in Xi reactivation and occurs very late in reprogramming. To further test this, we purified the SSEA1+ population directly from a reprogramming culture that represents a state where the pluripotency marker Nanog is already active but the Xi is not yet reactivated. We show from these intermediates that the promoter region of Nanog is demethylated. However, promoter regions of Xi-linked genes from these intermediates are still completely methylated. Together, our results identify new late stages of reprogramming to iPSCs.