TY - JOUR
T1 - Heterochromatin dynamics during the differentiation process revealed by the DNA methylation reporter mouse, methylRO
AU - Ueda, Jun
AU - Maehara, Kazumitsu
AU - Mashiko, Daisuke
AU - Ichinose, Takako
AU - Yao, Tatsuma
AU - Hori, Mayuko
AU - Sato, Yuko
AU - Kimura, Hiroshi
AU - Ohkawa, Yasuyuki
AU - Yamagata, Kazuo
N1 - Funding Information:
We would like to thank Drs. Frank Costantini and Junji Takeda for providing the pBigT and ROSA26-targeting vector plasmids, Drs. Jan Ellenberg, Tomoya Kitajima, and Miho Ohsugi for the EGFP-CENP-C expression plasmid, Dr. Cristina M. Cardoso for the EGFP-PCNA expression plasmid, and Dr. Hitoshi Niwa for the Oct4-EGFP knockin-targeting vector. We would also like to thank Ms. Masumi Fujikawa for the MethylRO mouse illustration and Mr. Masanaga Muto for help with cryosectioning. Finally, we express our greatest gratitude to Drs. Masaru Okabe and Masahito Ikawa for their encouragement, guidance, and support throughout this work. This study was supported in part by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
PY - 2014/6/3
Y1 - 2014/6/3
N2 - In mammals, DNA is methylated at CpG sites, which play pivotal roles in gene silencing and chromatin organization. Furthermore, DNA methylation undergoes dynamic changes during development, differentiation, and in pathological processes. The conventional methods represent snapshots; therefore, the dynamics of this marker within living organisms remains unclear. To track this dynamics, we made a knockin mouse that expresses a red fluorescent protein (RFP)-fused methyl-CpG-binding domain (MBD) protein from the ROSA26 locus ubiquitously; we named it MethylRO (methylation probe in ROSA26 locus). Using this mouse, we performed RFP-mediated methylated DNA immunoprecipitation sequencing (MeDIP-seq), whole-body section analysis, and live-cell imaging. We discovered that mobility and pattern of heterochromatin as well as DNA methylation signal intensity inside the nuclei can be markers for cellular differentiation status. Thus, the MethylRO mouse represents a powerful bioresource and technique for DNA methylation dynamics studies in developmental biology, stem cell biology, as well as in disease states.
AB - In mammals, DNA is methylated at CpG sites, which play pivotal roles in gene silencing and chromatin organization. Furthermore, DNA methylation undergoes dynamic changes during development, differentiation, and in pathological processes. The conventional methods represent snapshots; therefore, the dynamics of this marker within living organisms remains unclear. To track this dynamics, we made a knockin mouse that expresses a red fluorescent protein (RFP)-fused methyl-CpG-binding domain (MBD) protein from the ROSA26 locus ubiquitously; we named it MethylRO (methylation probe in ROSA26 locus). Using this mouse, we performed RFP-mediated methylated DNA immunoprecipitation sequencing (MeDIP-seq), whole-body section analysis, and live-cell imaging. We discovered that mobility and pattern of heterochromatin as well as DNA methylation signal intensity inside the nuclei can be markers for cellular differentiation status. Thus, the MethylRO mouse represents a powerful bioresource and technique for DNA methylation dynamics studies in developmental biology, stem cell biology, as well as in disease states.
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U2 - 10.1016/j.stemcr.2014.05.008
DO - 10.1016/j.stemcr.2014.05.008
M3 - Article
C2 - 24936475
AN - SCOPUS:84902209239
SN - 2213-6711
VL - 2
SP - 910
EP - 924
JO - Stem Cell Reports
JF - Stem Cell Reports
IS - 6
ER -