Goodglick L, Felsher D W, Neshat M S, Braun J
Department of Pathology and Laboratory Medicine, Jonsson Comprehensive Cancer Center, University of California Los Angeles School of Medicine, USA.
J Immunol. 1995 May 1;154(9):4546-56.
B-1 cells (CD5 B cells) represent an initial fetal wave of B cell lymphopoiesis. B-1 cells have fundamental properties that are unique from conventional B cells, including a restricted Ab repertoire. We investigated the mechanism for the overrepresentation of one such Ig H chain variable-region gene, VH11, by murine B-1 cells. We postulated that a cis-regulatory element contributed to the use of VH11. We observed that the DNA encoding the leader peptide of VH11 was atypically A/T rich and thus was a candidate for nuclear protein binding. By electrophoretic mobility shift analysis, we found that the VH11 leader DNA specifically bound to three protein complexes present in the nucleus of the B-1 cell line AJ9. Of these bands, one was ubiquitous for all cells examined (lymphoid and nonlymphoid); another band was present only in B cells, and the third band was specific for B-1 cells that expressed VH11 or VH12. In addition to its binding properties, the VH11 leader sequence also displayed modest tissue-specific enhancer activity. By DNA footprint analysis, all three protein complexes were found to bind to an octamer motif embedded within the VH11 leader DNA. To identify the octamer-binding proteins, a panel of octamer-specific Abs was used. We found that the ubiquitous band was Oct-1, and the B cell-specific band was Oct-2. The B-1 cell-specific nuclear binding protein was neither Oct-1 nor Oct-2, but may be a novel POU domain protein. We hypothesize that the VH11 leader octamer site may target this gene for preferential rearrangement and/or expression and therefore would be a contributing factor in the increased use of this gene by B-1 cells.
B-1细胞（CD5 B细胞）代表了B细胞淋巴细胞生成的初始胎儿波。B-1细胞具有与传统B细胞不同的基本特性，包括受限的抗体库。我们研究了小鼠B-1细胞对一种这样的Ig H链可变区基因VH11过度表达的机制。我们推测一个顺式调节元件促成了VH11的使用。我们观察到编码VH11前导肽的DNA非典型地富含A/T，因此是核蛋白结合的候选者。通过电泳迁移率变动分析，我们发现VH11前导DNA特异性结合到B-1细胞系AJ9细胞核中存在的三种蛋白质复合物上。在这些条带中，一条在所有检测的细胞（淋巴细胞和非淋巴细胞）中普遍存在；另一条条带仅存在于B细胞中，第三条条带对表达VH11或VH12的B-1细胞具有特异性。除了其结合特性外，VH11前导序列还表现出适度的组织特异性增强子活性。通过DNA足迹分析，发现所有三种蛋白质复合物都结合到VH11前导DNA中嵌入的一个八聚体基序上。为了鉴定八聚体结合蛋白，使用了一组八聚体特异性抗体。我们发现普遍存在的条带是Oct-1，B细胞特异性条带是Oct-2。B-1细胞特异性核结合蛋白既不是Oct-1也不是Oct-2，可能是一种新型的POU结构域蛋白。我们假设VH11前导八聚体位点可能将该基因靶向优先重排和/或表达，因此将是B-1细胞增加使用该基因的一个促成因素。
