RRC ID 28643
Author Kobayakawa S, Miike K, Nakao M, Abe K.
Title Dynamic changes in the epigenomic state and nuclear organization of differentiating mouse embryonic stem cells.
Journal Genes Cells
Abstract Changes in nuclear organization and the epigenetic state of the genome are important driving forces for developmental gene expression. However, a strategy that allows simultaneous visualization of the dynamics of the epigenomic state and nuclear structure has been lacking to date. We established an experimental system to observe global DNA methylation in living mouse embryonic stem (ES) cells. The methylated DNA binding domain (MBD) and the nuclear localization signal (nls) sequence coding for human methyl CpG-binding domain protein 1 (MBD1) were fused to the enhanced green fluorescent protein (EGFP) reporter gene, and ES cell lines carrying the construct (EGFP-MBD-nls) were established. The EGFP-MBD-nls protein was used to follow DNA methylation in situ under physiological conditions. We also monitored the formation and rearrangement of methylated heterochromatin using EGFP-MBD-nls. Pluripotent mouse ES cells showed unique nuclear organization in that methylated centromeric heterochromatin coalesced to form large clusters around the nucleoli. Upon differentiation, the organization of these heterochromatin clusters changed dramatically. Time-lapse microscopy successfully captured a moment of dramatic change in chromosome positioning during the transition between two differentiation stages. Thus, this experimental system should facilitate studies focusing on relationships between nuclear organization, epigenetic status and cell differentiation.
Volume 12(4)
Pages 447-60
Published 2007-4-1
DOI 10.1111/j.1365-2443.2007.01063.x
PMID 17397393
MeSH Animals Cell Differentiation / physiology* Cell Nucleus* / metabolism Cell Nucleus* / ultrastructure Cells, Cultured Chromosomes / metabolism Chromosomes / ultrastructure DNA (Cytosine-5-)-Methyltransferase 1 DNA (Cytosine-5-)-Methyltransferases / genetics DNA (Cytosine-5-)-Methyltransferases / metabolism DNA Methylation* DNA-Binding Proteins / chemistry DNA-Binding Proteins / genetics DNA-Binding Proteins / metabolism Embryonic Stem Cells* / cytology Embryonic Stem Cells* / physiology Epigenesis, Genetic* Fluorescence Recovery After Photobleaching Genes, Reporter Humans Mice Mice, Knockout Nuclear Localization Signals Recombinant Fusion Proteins / chemistry Recombinant Fusion Proteins / genetics Recombinant Fusion Proteins / metabolism Transcription Factors / chemistry Transcription Factors / genetics Transcription Factors / metabolism
IF 1.655
Times Cited 64
DNA material pEGFP-MBD-nls (RDB08344)