Maier Elizabeth A, Dusing Mary R, Wiginton Dan A
Department of Pediatrics, Division of Developmental Biology, University of Cincinnati College of Medicine and Cincinnati Children's Hospital Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229, USA.
J Biol Chem. 2006 Oct 27;281(43):32263-71. doi: 10.1074/jbc.M606699200. Epub 2006 Aug 31.
An intestine-specific gene regulatory region was previously identified near the second exon of the human adenosine deaminase (ADA) gene. In mammalian intestine, ADA is expressed at high levels only along the villi of the duodenal epithelium, principally if not exclusively in enterocytes. Within the ADA intestinal regulatory region, a potent duodenum-specific enhancer was identified that controls this pattern of expression. This enhancer has been shown to rely on PDX-1, GATA factors, and Cdx factors for its function. Upstream of the enhancer, a separate temporal regulatory region was identified that has no independent enhancer capability but controls the timing of enhancer activation. DNase I footprinting and electrophoretic mobility shift assays were used to begin to characterize temporal region function at the molecular level. In this manner, binding sites for the Onecut (OC) family of factors, YY1, and NFI family members were identified. Identification of the OC site was especially interesting, because almost nothing is known about the function of OC factors in intestine. In transgenic mice, mutation of the OC site to ablate binding resulted in a delay of 2-3 weeks in enhancer activation in the developing postnatal intestine, a result very similar to that observed previously when the entire temporal region was deleted. In mammals, the OC family is comprised of OC-1/HNF-6, OC-2, and OC-3. An examination of intestinal expression patterns showed that all three OC factors are expressed at detectable levels in adult mouse duodenum, with OC-2 predominant. In postnatal day 2 mice only OC-2 and OC-3 were detected in intestine, with OC-2 again predominant.
先前在人类腺苷脱氨酶（ADA）基因的第二个外显子附近鉴定出一个肠道特异性基因调控区域。在哺乳动物肠道中，ADA仅在十二指肠上皮绒毛处高水平表达，主要（如果不是唯一的话）在肠细胞中表达。在ADA肠道调控区域内，鉴定出一个强大的十二指肠特异性增强子，它控制着这种表达模式。已证明该增强子的功能依赖于PDX-1、GATA因子和Cdx因子。在增强子上游，鉴定出一个单独的时间调控区域，它没有独立的增强子能力，但控制着增强子激活的时间。使用DNA酶I足迹法和电泳迁移率变动分析开始在分子水平上表征时间区域的功能。通过这种方式，鉴定出了Onecut（OC）家族因子、YY1和NFI家族成员的结合位点。OC位点的鉴定特别有趣，因为几乎对OC因子在肠道中的功能一无所知。在转基因小鼠中，将OC位点突变以消除结合导致出生后发育中的肠道中增强子激活延迟2至3周，这一结果与之前删除整个时间区域时观察到的结果非常相似。在哺乳动物中，OC家族由OC-1/HNF-6、OC-2和OC-3组成。对肠道表达模式的检查表明，所有三种OC因子在成年小鼠十二指肠中均以可检测水平表达，其中OC-2占主导。在出生后第2天的小鼠中，仅在肠道中检测到OC-2和OC-3，OC-2再次占主导。
