Setdb1/Eset, a histone lysine methyltransferase, is recruited by various transcription factors to modify local chromatin. The observation that Setdb1-null blastocysts fail to produce epiblast-lineage cells suggests a role for Setdb1 in generating mouse embryonic stem cells (mESCs). When examined in mouse zygotes, Setdb1 proteins appeared as dots at perinucleolar rims of pronuclei, with the dot-shaped signals more prominent in male pronuclei. Setdb1 signals were observed diffusely in the nucleus from the two-cell stage onward and, by the blastocyst, took a punctate form, away from nucleolus. Such varying expression patterns suggest its involvement in diverse biological processes at preimplantation stage. Setdb1 appeared in Oct4-positive cells of inner-cell-mass origin but not in trophectoderm-lineage cells in blastocyst outgrowths. Setdb1 co- immunoprecipitated with Oct4 in mESCs, and Setdb1 expression was markedly reduced upon retinoic acid- induced differentiation. These observations suggest that Setdb1 has an important role in maintaining the self-renewal of mESCs through collaboration with Oct4.

Covalent modifications of histone tails have fundamental roles in chromatin structure and transcriptional activity of a target locus. One of such modifications, Methylation at Lysine 9 of histone H3 (H3-K9) causes several epigenetic phenomena including heterochromatin formation, transcriptional regulation and DNA methylation. Setdb1, H3-K9 specific histone methyltransferase, functions in gene silencing, heterochromatin formation and essential role for early development. Here, we demonstrate that Setdb1 associates with promyelocytic leukemia (Pml) protein from the early stage of mouse development and is a constitutive member of PML nuclear bodies (PML-NBs) that have been linked to many cellular processes such as apoptosis, DNA damage responses, and transcriptional regulation. Immunostaining of mouse blastocyst showed that Setdb1 and Pml signals were scattered in nucleus as a few speckles and microinjected Pmlmyc signals colocalize with Setdb1 signals. This colocalization was observed in mEF and the punctate signals of Setdb1 were observed to be present in every nucleus of mEFs and dividing cells with condensed chromosomes. Arsenic treatment, which induces Pml degradation, also caused Setdb1 signals to disappear. Setdb1 knockdown resulted in disassembly of PML-NBs and immunoprecipitation results demonstrated physical interactions between Setdb1 and Pml. These data suggest that Setdb1 was associated in PML-NB and Setdb1 has important function in maintenance of PML-NB structure.

본 연구는 소 배반포의 내부 세포괴로부터 다능성(pluripotency)을 지닌 배아 줄기 세포(embryonic stem cell) 또는 그 유사 세포를 분리 및 배양함으로써 줄기 세포 관련 분야의 기반 기술을 확립하고자 하였다. 소 체외수정란을 10～12일간 체외배양하여 생산된 부화 배반포를 세포분열이 불활성화된 생쥐 태아 섬유아 세포(mouse embryonic fibroblast, MEF) 위에서 배양하여 콜로니 형성을 유도하였으며, 이들로부터 내부 세포괴 유래의 형태를 지닌 것만을 광학현미경 하에서 물리적으로 분리하여 약 5～7일 간격으로 계대배양을 실시하였다. 이러한 방법을 통하여 배아 줄기 유사 세포의 특성을 40계대 이상 유지하는 2개의 세포주를 확립하였다. 각각의 세포주들은 높은 alkaline phosphatase(AP) 활성을 지니고 있었으며, 형광 면역 염색법과 PCR 기법을 사용하여 Oct-4, Nanog, STAT3, SSEA3 및 SSEA4의 발현을 관찰할 수 있었다. 이러한 결과를 종합하여 볼 때 ,본 연구에서는 소 배반포로부터 배아 줄기 세포주를 확립하는 제반 기술이 확립되었다고 판단되며, 향후 관련 분야 연구에 활용될 수 있을 것으로 기대된다.