Pfeifer K, Leighton P A, Tilghman S M
Howard Hughes Medical Institute, Princeton University, NJ 08544, USA.
Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):13876-83. doi: 10.1073/pnas.93.24.13876.
The product of the H19 gene is an untranslated RNA that is expressed exclusively from the maternal chromosome during mammalian development. The H19 gene and its 5'-flanking sequence are required for the genomic imprinting of two paternally expressed genes, Ins-2 (encodes insulin-2) and Igf-2 (encodes insulin-like growth factor-2), that lie 90 and 115 kb 5' to the H19 gene, respectively. In this report, the role of the H19 gene in its own imprinting is investigated by introducing a Mus spretus H19 gene into heterologous locations in the mouse genome. Multiple copies of the transgene were sufficient for its paternal silencing and DNA methylation. Replacing the H19 structural gene with a luciferase reporter gene resulted in loss of imprinting of the transgene. That is, high expression and low levels of DNA methylation were observed upon both paternal and maternal inheritance. The removal of 701 bp at the 5' end of the structural gene resulted in a similar loss of paternal-specific DNA methylation, arguing that those sequences are required for both the establishment and maintenance of the sperm-specific gametic mark. The M. spretus H19 transgene could not rescue the loss of Igf-2 imprinting in trans in H19 deletion mice, implying a cis requirement for the H19 gene. In contrast to a previous report in which overexpression of a marked H19 gene was a prenatal lethal, expression of the M. spretus transgene had no deleterious effect, leading to the conclusion that the 20-base insertion in the marked gene created a neomorphic mutation.
H19基因的产物是一种未翻译的RNA，在哺乳动物发育过程中仅从母本染色体表达。H19基因及其5'侧翼序列是两个父本表达基因Ins-2（编码胰岛素-2）和Igf-2（编码胰岛素样生长因子-2）基因组印记所必需的，它们分别位于H19基因5'端90和115 kb处。在本报告中，通过将一只家鼠H19基因引入小鼠基因组的异源位置，研究了H19基因在其自身印记中的作用。转基因的多个拷贝足以使其父本沉默和DNA甲基化。用荧光素酶报告基因取代H19结构基因导致转基因印记丢失。也就是说，在父本和母本遗传时均观察到高表达和低水平的DNA甲基化。在结构基因5'端去除701 bp导致父本特异性DNA甲基化类似地丢失，这表明这些序列对于精子特异性配子标记的建立和维持都是必需的。家鼠H19转基因不能挽救H19缺失小鼠中Igf-2印记的反式丢失，这意味着对H19基因有顺式需求。与之前一份报告中标记的H19基因过表达是产前致死的情况相反，家鼠转基因的表达没有有害影响，从而得出结论，标记基因中的20个碱基插入产生了一个新形态突变。
