Chromatin states at homeoprotein loci distinguish axolotl limb segments prior to regeneration [Cut&Tag]

The salamander limb regenerates only the missing portion. Each limb segment can only form segments equivalent to- or more distal to their own identity, relying on a property termed “positional information”. How positional information is encoded in limb cells has been unknown. By cell-type-specific chromatin profiling of upper arm, lower arm, and hand, we found segment-specific levels of histone H3K27me3 at limb homeoprotein gene loci but not their upstream regulators, constituting an intrinsic segment information code. During regeneration, regeneration-specific regulatory elements became active prior to the re-appearance of developmental regulatory elements. This means that, in the hand segment, the permissive chromatin state of the hand homeoprotein gene HoxA13 engages with regeneration regulatory elements, bypassing the upper limb program. Profiling histone marks of Axoltol limb connective tissue cells, and comparing among limb different limb segments and mature vs regeneration time points. Overall design: We performed Histone mark profiling analysis using data obtained from CUT&Tag of different mature limb segments (mature UA vs LA vs hand). Therefore, we also performed libraries from the mature arm and 5, 9, 13 dpa regenerating arm blastema. Histone mark profiling analysis using IgG (negative control), H3K4me1 (Enhancer associate), H3K4me3 (Active promoter associate), and H3K27me3 (Repressive region associate). .