Roberson M S, Schoderbek W E, Tremml G, Maurer R A
Department of Cell Biology and Anatomy, Oregon Health Sciences University, Portland 97201.
Mol Cell Biol. 1994 May;14(5):2985-93. doi: 10.1128/mcb.14.5.2985-2993.1994.
Recently, a pituitary-specific enhancer was identified within the 5' flanking region of the mouse glycoprotein hormone alpha-subunit gene. This enhancer is active in pituitary cells of the gonadotrope and thyrotrope lineages and has been designated the pituitary glycoprotein hormone basal element (PGBE). In the present studies, we sought to isolate and characterize proteins which interact with the PGBE. Mutagenesis experiments identified a 14-bp imperfect palindrome that is required for binding of a factor which is present in cells of gonadotrope and thyrotrope lineages but not in other cells. Screening of a mouse cDNA library with a DNA probe containing the imperfect palindrome resulted in the isolation of a LIM-homeodomain transcription factor. The cDNA predicts a mouse protein which is 94% identical to the recently described rat LIM-homeodomain protein LH-2. LH-2 contains two zinc fingers (LIM domain) and a consensus homeodomain. Hybridization analysis revealed relatively high expression of LH-2 mRNA in the central nervous system and in pituitary cells of the gonadotrope and thyrotrope lineages. Lower or nondetectable levels of LH-2 mRNA were found in other pituitary cells and tissues, including placental cells. Recombinant LH-2 homeodomain was found to selectively bind to the previously identified imperfect palindrome in the PGBE. Point mutations in the PGBE resulted in parallel losses in the binding of a nuclear factor from a cell line of the gonadotrope lineage and recombinant LH-2-binding activity. Use of an antibody to LH-2 provided evidence that endogenous PGBE-binding activity from cells of the gonadotrope lineage involves a protein which is immunologically related to LH-2. Expression of LH-2 in two heterologous cell types resulted in activation of a reporter gene containing the mouse alpha promoter. These data suggest that the LIM-homeodomain factor LH-2 plays a role in stimulating tissue-specific expression of the mouse glycoprotein hormone alpha subunit. The finding that a LIM-homeodomain protein can stimulate expression of one of the earliest markers of pituitary differentiation raises the possibility that this factor plays a role in cell lineage determination in the pituitary.
最近，在小鼠糖蛋白激素α亚基基因的5'侧翼区域内鉴定出一种垂体特异性增强子。该增强子在促性腺激素细胞和促甲状腺激素细胞系的垂体细胞中具有活性，并被命名为垂体糖蛋白激素基础元件（PGBE）。在本研究中，我们试图分离和鉴定与PGBE相互作用的蛋白质。诱变实验确定了一个14bp的不完全回文序列，它是促性腺激素细胞和促甲状腺激素细胞系细胞中存在的一种因子结合所必需的，但在其他细胞中不存在。用含有不完全回文序列的DNA探针筛选小鼠cDNA文库，结果分离出一种LIM-同源结构域转录因子。该cDNA预测的小鼠蛋白与最近描述的大鼠LIM-同源结构域蛋白LH-2有94%的同源性。LH-2含有两个锌指（LIM结构域）和一个共有同源结构域。杂交分析显示，LH-2 mRNA在中枢神经系统以及促性腺激素细胞和促甲状腺激素细胞系的垂体细胞中表达相对较高。在其他垂体细胞和组织，包括胎盘细胞中，发现LH-2 mRNA水平较低或无法检测到。发现重组LH-2同源结构域能选择性地结合PGBE中先前鉴定的不完全回文序列。PGBE中的点突变导致促性腺激素细胞系细胞系核因子结合和重组LH-2结合活性平行丧失。使用抗LH-2抗体提供了证据，表明促性腺激素细胞系细胞的内源性PGBE结合活性涉及一种与LH-2免疫相关的蛋白质。LH-2在两种异源细胞类型中的表达导致含有小鼠α启动子的报告基因激活。这些数据表明，LIM-同源结构域因子LH-2在刺激小鼠糖蛋白激素α亚基的组织特异性表达中起作用。LIM-同源结构域蛋白能刺激垂体分化最早标志物之一的表达，这一发现增加了该因子在垂体细胞谱系决定中起作用的可能性。
