FUNCTION OF SEX SPECIFIC EXONS IN DNMT1 GENE

基因组甲基化模式在配子发生期间建立， 通过克隆遗传在体细胞中早期发育和繁殖; 甲基化模式的破坏导致 内源性逆转录病毒，异位X染色体失活，双等位基因表达 以及分化细胞的死亡。 Dnmt 1， 哺乳动物的主要DNA（胞嘧啶-5）-甲基转移酶，涉及 在甲基化模式的建立和维持中。 的 Dnmt 1基因具有性别特异的5 '外显子和启动子， 仅在生殖细胞中;这些替代外显子可能参与生殖细胞的基因表达。 建立性别特异性甲基化模式。 外显子1 o（卵母细胞） 仅存在于出生后生长的卵母细胞中， 大量截短但有活性的Dnmt 1蛋白存在于 细胞质，而外显子1 p（粗线期精母细胞）阻断翻译 并在雄性减数分裂粗线期消除所有Dnmt 1蛋白 尽管存在大量的mRNA。 外显子1 s（体细胞）是 在所有的体细胞中使用。 提出了Dnmt 1基因的性别特异性外显子 在重新设置表观遗传程序中发挥作用， 生殖系和性别特异性DNA甲基化，这是 基因组印记现象。 我们将决定 卵子发生和精子发生，在这些印记位点获得性别， 特定的甲基化模式，并将检查原始生殖细胞， 从它们衍生的细胞系（EG细胞），用于5'的选择性剪接， Dnmt 1蛋白的外显子和细胞质定位可以解释 体细胞中EG细胞的优势脱甲基化特性 混血儿 我们将确定性别特异性外显子的作用， 靶向Cre-loxP缺失，并通过强制表达体细胞 Dnmt 1的形式在替代形式通常表达的阶段。 我们对控制这种形成的机制知之甚少 在生殖细胞和早期胚胎中的甲基化模式; 这里描述的实验将提供重要的见解， 基因组组织的重要调节因子， 基因组生物：脊椎动物和开花植物。

Genomic methylation patterns are established during gametogenesis and early development and propogated in somatic cells by clonal inheritance; disruption of methylation patterns causes fulminating expression of endogenous retroviruses, ectopic X inactivation, biallelic expression of imprinted genes, and death of differentiating cells. Dnmt1, the predominant DNA (cytosine-5)-methyltransferase of mammals, is involved in both the establishment and maintenance of methylation patterns. The Dnmt1 gene has sex-specific 5'exons and promoters that are expressed only in germ cells; these alternative exons may be involved in the establishment of sex-specific methylation patterns. Exon 1o (oocyte) is present only in postnatal growing oocytes and causes the accumulation of enourmous amounts of truncated but active Dnmt1 protein in the cytoplasm, whereas exon 1p (pachytene spermatocyte) blocks translation and eliminates all Dnmt1 protein at the pachytene stage of male meiosis even though large amounts of mRNA are present. Exon 1s (somatic) is used in all somatic cells. The sex-specific exons in Dnmt1 are proposed to play a role in the re-setting of the epigenetic program in the germline and in sex-specific DNA methylation that underlies the phenomenon of genomic imprinting. We will determine the stage of oogenesis and spermatogenesis at which imprinted loci acquire sex- specific methylation patterns and will examine primordial germ cells and cell lines derived from them (EG cells) for alternative splicing of 5' exons and cytoplasmic localization of Dnmt1 protein that could explain the dominant demethylating properties of EG cells in somatic cell hybrids. We will determine the roles of the sex-specific exons by targeted Cre-loxP deletion, and by forcing expression of the somatic form of Dnmt1 at stages where alternative forms are normally expressed. Remarkably little is known of the mechanisms that control the formation of methylation patterns in germ cells and the early embryo; the experiments described here will give important insights into an important regulator of genome organization that is unique to large- genome organisms: vertebrates and flowering plants.