Mitotic chromosomes harbor cell type- and species-specific structural features within a universal loop array conformation.

Mitotic chromosomes are considered to be universally folded as loop arrays across species and cell types. However, some studies suggest that features of mitotic chromosomes might be cell type- or species-specific. We previously reported that CTCF binding in human differentiated cell lines is lost in mitosis, whereas mitotic mouse embryonic stem cells (mESC) display prominent binding at a subset of CTCF sites. Here, we perform footprint ATAC-seq analyses of mESCs, and somatic mouse and human cells, confirming these findings. We then investigate roles of mitotically bookmarked CTCF in prometaphase chromosome organization by Hi-C. We do not find any remaining interphase structures, such as TADs or loops, at bookmarked CTCF sites in mESCs. This suggests that mitotic loop extruders condensin I and II are not blocked by CTCF and, thus, that maintained CTCF binding does not alter mitotic chromosome folding. Lastly, we compare mitotic Hi-C data generated in this study in mouse with public data in human and chicken. We do not find any cell type-specific differences; however, we find a difference between species. The average genomic size of mitotic loops is smaller in chicken (200-300 kb) compared to human (400-600 kb) and especially mouse (1-1.5 Mb). Interestingly, we find that this difference is correlated with the genomic length of q-arms in these species, a finding we confirm by microscopy measurements of chromosome compaction. This suggests that the dimensions of mitotic chromosomes can be modulated through control of loop size by condensins to facilitate species-appropriate shortening of chromosome arms.