Female naive human pluripotent stem cells carry X chromosomes with Xa-like and Xi-like folding conformations

Three-dimensional (3D) genomics shows immense promise for studying X chromosome inactivation (XCI) by interrogating changes to the X chromosomes' 3D states. Here, we sought to characterize the 3D state of the X chromosome in naive and primed human pluripotent stem cells (hPSCs). Using chromatin tracing, we analyzed X chromosome folding conformations in these cells with megabase genomic resolution. X chromosomes in female naive hPSCs exhibit folding conformations similar to the active X chromosome (Xa) and the inactive X chromosome (Xi) in somatic cells. However, naive X chromosomes do not exhibit the chromatin compaction typically associated with these somatic X chromosome states. In H7 naive human embryonic stem cells, XIST accumulation observed on damaged X chromosomes demonstrates the potential for naive hPSCs to activate XCI-related mechanisms. Overall, our findings provide insight into the X chromosome status of naive hPSCs with a single-chromosome resolution and are critical in understanding the unique epigenetic regulation in early embryonic cells.