Aronow B J, Ebert C A, Valerius M T, Potter S S, Wiginton D A, Witte D P, Hutton J J
Department of Pediatrics, Children's Hospital Medical Center, University of Cincinnati College of Medicine, Ohio 45229.
Mol Cell Biol. 1995 Feb;15(2):1123-35. doi: 10.1128/MCB.15.2.1123.
Using transgenic mice, we have defined novel gene regulatory elements, termed "facilitators." These elements bilaterally flank, by up to 1 kb, a 200-bp T-cell-specific enhancer domain in the human adenosine deaminase (ADA) gene. Facilitators were essential for gene copy-proportional and integration site-independent reporter expression in transgenic thymocytes, but they had no effect on the enhancer in transfected T cells. Both segments were required. Individual segments had no activity. A lack of facilitator function caused positional susceptibility and prevented DNase I-hypersensitive site formation at the enhancer. The segments were required to be at opposed ends of the enhancer, and they could not be grouped together. Reversing the orientation of a facilitator segment caused a partial loss of function, suggesting involvement of a stereospecific chromatin structure. trans-acting factor access to enhancer elements was modeled by exposing nuclei to a restriction endonuclease. The enhancer domain was accessible to the 4-cutter DpnII in a tissue- and cell-type-specific fashion. However, unlike DNase I hypersensitivity and gene expression, accessibility to the endonuclease could occur without the facilitator segments, suggesting that an accessible chromatin domain is an intermediate state in the activational pathway. These results suggest that facilitators (i) are distinct from yet positionally constrained to the enhancer, (ii) participate in a chromatin structure transition that is necessary for the DNase I hypersensitivity and the transcriptional activating function of the enhancer, and (iii) act after cell-type-specific accessibility to the enhancer sequences is established by factors that do not require the facilitators to be present.
利用转基因小鼠，我们定义了一种新型基因调控元件，称为“促进子”。这些元件在人类腺苷脱氨酶（ADA）基因中，以长达1 kb的距离双侧侧翼于一个200 bp的T细胞特异性增强子结构域。促进子对于转基因胸腺细胞中基因拷贝比例和整合位点无关的报告基因表达至关重要，但对转染T细胞中的增强子没有影响。两个片段都是必需的。单个片段没有活性。促进子功能的缺失导致位置易感性，并阻止增强子处DNase I超敏感位点的形成。这些片段必须位于增强子的相对两端，且不能聚集在一起。颠倒一个促进子片段的方向会导致部分功能丧失，这表明涉及立体特异性染色质结构。通过将细胞核暴露于限制性内切酶来模拟反式作用因子对增强子元件的作用。增强子结构域以组织和细胞类型特异性方式可被四碱基切割酶DpnII作用。然而，与DNase I超敏感性和基因表达不同，内切酶的可及性在没有促进子片段的情况下也会发生，这表明可及的染色质结构域是激活途径中的一个中间状态。这些结果表明，促进子（i）与增强子不同但在位置上受其限制，（ii）参与染色质结构转变，这对于增强子的DNase I超敏感性和转录激活功能是必需的，并且（iii）在细胞类型特异性可及性通过不需要促进子存在的因子建立到增强子序列之后起作用。
