98 HETEROCHROMATIN REPROGRAMMING IN MOUSE EARLY DEVELOPMENT

Abstract

Heterochromatin is essential for epigenetic gene silencing and nuclear chromatin architecture. Early mouse development is accompanied by dynamic epigenetic changes and heterochromatin restructuring in the cell nuclei of cleavage stage embryos. We have previously shown that disruption of heterochromatin markers such as DNA methylation following somatic nuclear transfer causes developmental failure (Beaujean N et al. 2004 Biol. Reprod. 71, 185–193). The aim of the present study was to investigate the transitions and maturation of heterochromatin during normal development to improve nuclear reprogramming technology and understand developmental abnormalities caused by epigenetic alterations in somatic nuclear transfer and assisted reproductive procedures. Mouse pre- and post-implantation embryos and ES cells from B6CBAF1 mice were used in experiments employing the following methodologies. Embryos and ES cells were stained by immunofluorescence to detect heterochromatin proteins and epigenetic markers. Images were captured using laser confocal microscopy. Relative quantification of fluorescent intensity was performed using Zeiss LSM-meta and WCIF-ImageJ software. mRNA of Histone methyltransferase (HMTase), suv39h, suv420h, ehmt, eset, and demethyltransferase (DHMTase), jmjd1a, jmjd2a, jmjd2c were quantified by real-time PCR. siRNA and specific inhibitors were used to study the function of suv39h, ehmt2, jmjd2c genes that may be responsible for heterochromatin reprogramming. Our results demonstrated that H3K9me3, H3K9me2, H4K20me2, H4K20me3, HP1α and HP1β are reprogrammed during early development. The gene expression results showed the dynamic and temporal gene expression of HMTases and DHMTase during development. Inhibition of Ehmt2 and jmjd2c caused preimplantation developmental arrest. Furthermore, we found chromatin modification differences in the heterochromatin of ES cells, ICM and epiblast. We conclude that heterochromatin reprogramming might be essential for development because it may contribute to chromatin architecture, thus influencing gene expression. HMTases and DHTMases could be implicated in the mechanism of heterochromatin reprogramming. It is possible that Ehmt2 and jmjd2c play an important role in preimplantation development by modifying chromatin globally and at the local gene level. This work was supported by a The Royal Thai Studentship. We acknowledge the contributions of technical assistance from T. O’Connor, B. Wongtawan and P. Travers.