Boothby M, Liou H C, Glimcher L H
Department of Cancer Biology, Harvard School of Public Health, Boston, MA 02155.
J Immunol. 1989 Feb 1;142(3):1005-14.
The class II (Ia) MHC Ag are integral membrane proteins whose expression is limited to specific cell types. A pair of consensus sequences, X and Y, is found upstream from all class II genes and deletion of each of these sequences eliminates expression of transfected genes. Cells that express Ia demonstrate a coordinate response to lymphokines and other stimuli. These conserved sequences might, therefore, play a role in tissue specificity or lymphokine inducibility of Ia gene expression. The X box sequence of the murine class II A alpha gene diverges much more substantially from the X consensus than does the Y box motif of this gene. We demonstrate that this X box motif is nonetheless recognized by sequence-specific DNA-binding proteins, as is the more closely conserved Y box. Gel retardation assays and DNase I footprints were compared for a panel of Ia+ and Ia- cells as well as for cells stimulated with the Ia-inducing lymphokines IL-4 and IFN-gamma. The level, retardation pattern and region of DNA contact were comparable in all instances. Thus the availability of active DNA-binding X and Y box factors cannot alone account for the regulation of A alpha expression. To test whether the same set of proteins binds all class II MHC conserved motifs, oligonucleotide probe binding and cross-competition experiments with X box sequences from A alpha, E alpha, and E beta genes were performed. These studies demonstrated A alpha, E alpha, and E beta DNA-protein complexes with unique mobilities and specificities. In addition, all three X box oligonucleotide probes generated one faint complex with an affinity profile of E beta greater than E alpha much greater than A alpha. These three complexes comigrated and thus may represent a communal binding protein. The data are most consistent with the conclusion that multiple proteins bind class II MHC X boxes. For A alpha, the predominant complexes represent different specificities from the predominant E alpha and E beta X box binding proteins.
II类（Ia）主要组织相容性复合体（MHC）抗原是整合膜蛋白，其表达仅限于特定细胞类型。在所有II类基因的上游发现了一对共有序列，即X和Y，缺失这些序列中的任何一个都会消除转染基因的表达。表达Ia的细胞对淋巴因子和其他刺激表现出协同反应。因此，这些保守序列可能在Ia基因表达的组织特异性或淋巴因子诱导性中发挥作用。小鼠II类Aα基因的X框序列与X共有序列的差异比该基因的Y框基序大得多。我们证明，尽管如此，该X框基序仍能被序列特异性DNA结合蛋白识别，就像更保守的Y框一样。对一组Ia+和Ia-细胞以及用诱导Ia的淋巴因子IL-4和IFN-γ刺激的细胞进行了凝胶阻滞试验和DNase I足迹分析。在所有情况下，DNA结合的水平、阻滞模式和区域都是可比的。因此，活性DNA结合X和Y框因子的可用性不能单独解释Aα表达的调控。为了测试是否同一组蛋白质结合所有II类MHC保守基序，进行了寡核苷酸探针结合以及与Aα、Eα和Eβ基因的X框序列的交叉竞争实验。这些研究证明了Aα、Eα和Eβ的DNA-蛋白质复合物具有独特的迁移率和特异性。此外，所有三种X框寡核苷酸探针都产生了一种微弱的复合物，其亲和力谱为Eβ大于Eα远大于Aα。这三种复合物迁移在一起，因此可能代表一种共同的结合蛋白。数据最符合的结论是，多种蛋白质结合II类MHC X框。对于Aα，主要复合物代表与主要的Eα和Eβ X框结合蛋白不同的特异性。
