Analysis of Intranuclear Communication of Homologous Alleles of Imprinted Genes and Transgenes

对印迹区域的研究提供了线索，增加了我们对高阶染色体结构域基因功能调控的理解。由于先前在淋巴细胞和淋巴母细胞中观察到这种印迹染色体结构域的同源关联，因此我们使用多色FISH比较了人髓性白血病HL60细胞间期细胞核中15号染色体着丝粒和印迹SNRPN基因的相对位置。尽管SNRPN基因表现出异步复制和单等位基因表达，但在细胞周期中不会发生SNRPN间同源物的优先关联。发现SNRPN定位于染色体区域的外围，并优先面向核膜。SNRPN基因和着丝粒在S期后期位置靠得很近，这反映了随着S期的进展，这些DNA片段可能被压缩成相同的核内亚室。我们的研究结果表明，印迹区相互识别的特征是由一定的细胞调控决定的，而不是由一个印迹基因的等位基因特异性特征决定的。我们采用FISH方法检测了人类HL60和WI38细胞细胞核中初始转录位点印迹SNRPN基因的新生RNA分子。使用cosmid探针同时检测SNRPN的RNA和DNA，证实其在这些细胞系中的单等位基因表达。用DNA甲基化和组蛋白去乙酰化抑制剂处理这两种细胞，导致SNRPN双等位基因表达的细胞群时间依赖性增加。我们通过与异染色质的关联研究了印迹区等位基因特异性基因的沉默。我们的结果提示了DNA复制的可能性和依赖性。我们利用印迹基因进一步研究了基因表达与核基质的关系，表明表达的等位基因与核基质相关。少

Investigations of imprinted regions provide clues that increase our understanding of the regulation of gene functions at higher order chromosomal domains. The relative positions of the chromosome 15 centromere and the imprinted SNRPN gene in interphase nuclei of human myeloid leukemia HL60 cells were compared by multicolor FISH, because the homologous association of this imprinted chromosomal domain was previously observed in lymphocytes and lymphoblasts. Preferential association of SNRPN interhomologues does not occur during the cell cycle, although this gene exhibited asynchronous replication and monoallelic expression. SNRPN was found to localize at the periphery of the chromosome territories, and it preferentially faces the nuclear membrane. The SNRPN gene and the centromere are located close to each other late in S phase, reflecting that these DNA segments may be compacted into the same intranuclear subcompartments with the progress of S phase. Our results suggest that the charact … More eristic of mutual recognition of imprinted regions is determined by certain cellular regulation, and it is not necessary for the allele-specific features of an imprinted gene. We have adapted a FISH method to detect nascent RNA molecules of the imprinted SNRPN gene at the initial transcription sites in nuclei of human HL60 and WI38 cells. Simultaneous detection of RNA and DNA of SNRPN by FISH using the cosmid probe confirmed its monoallelic expression in these cell lines. Treatment of both cells by inhibitors of DNA methylation and histone deacetylation resulted in time-dependent increase of the cell population with the biallelic expression of SNRPN.We investigated the allele-specific gene silencing of imprinted regions by their association with heterochromatin. Our results suggested the possibility and dependency on DNA replication. We further examined the relationship between gene expression and nuclear matrix by using imprinted genes, suggesting that the expressed allele associated with the nuclear matrix. Less