Many amphibians can regenerate limbs, even in adulthood. If a limb is amputated, the stump generates a blastema that makes a complete, new limb in a process similar to developmental morphogenesis. The blastema is thought to inherit its limb-patterning properties from cells in the stump, and it retains the information despite changes in morphology, gene expression, and differentiation states required by limb regeneration. We hypothesized that these cellular properties are maintained as epigenetic memory through histone modifications. To test this hypothesis, we analyzed genome-wide histone modifications in Xenopus limb bud regeneration. The trimethylation of histone H3 at lysine 4 (H3K4me3) is closely related to an open chromatin structure that allows transcription factors access to genes, whereas the trimethylation of histone H3 at lysine 27 (H3K27me3) is related to a closed chromatin state that blocks the access of transcription factors. We compared these two modification profiles by high-throughput sequencing of samples prepared from the intact limb bud and the regenerative blastema by chromatin immunoprecipitation. For many developmental genes, histone modifications at the transcription start site were the same in the limb bud and the blastema, were stable during regeneration, and corresponded well to limb properties. These results support our hypothesis that histone modifications function as a heritable cellular memory to maintain limb cell properties, despite dynamic changes in gene expression during limb bud regeneration in Xenopus.
Bibliographical noteFunding Information:
We thank Drs. Makoto Asashima, Shuji Takahashi and Akihiko Kashiwagi for providing X. tropicalis . This work was supported by the National Bio-Resource Project (NBRP) of the MEXT , Japan. We thank Dr. Jose F. de Celis for providing mkp3 (dusp6) cDNA, and Dr. Hirohumi Huruhashi for valuable advice on anti-H3K4me3 and H3K27me3 antibodies. We thank Dr. David L. Stocum for valuable discussion about positional memory. We thank Drs. Masakado Kawata, Takashi Maruyama and Tatsushi Muta for cooperation in use of a sonicator for ChIP. We thank Yoshiko Yoshizawa-Ohuchi for excellent care of the experimental animals and Natsume Sagawa for maintaining the frog facility. Genome-wide analysis with a next-generation sequencer in this study was supported by the Great East Japan Earthquake Reconstruction Support Project of the RIKEN Omics Science Center (currently RIKEN Center for Life Science Technologies ), the RIKEN Genome Analysis Service (GeNAS) , Illumina Co. and CLC Bio Co . This study was carried out under the NIBB Cooperative Research Program (12-386, 13-361, 14-360). Computational resources were provided by the Data Integration and Analysis Facility, National Institute for Basic Biology, Okazaki, Japan. This work was supported by MEXT and JSPS KAKENHI Grant nos. 22124005 to HY, 22124002 to KA, JSPS KAKENHI Grant nos. 25870058 to HY, 15K07082 to HO, “ Funding Program for Next Generation World-Leading Researchers ” [ LS007 ] from the Cabinet Office, Government of Japan to KT, the Kurata Memorial Hitachi Science and Technology Foundation to HY, and the Asahi Glass Foundation to HY.
- Cellular memory
- Epigenetic modification
- Limb regeneration