Toth C R, Hostutler R F, Baldwin A S, Bender T P
Department of Microbiology and Immunology, University of Virginia, Charlottesville 22903, USA.
J Biol Chem. 1995 Mar 31;270(13):7661-71. doi: 10.1074/jbc.270.13.7661.
Expression of the c-myb proto-oncogene is primarily detected in normal tissue and tumor cell lines of immature hematopoietic origin, and the down-regulation of c-myb expression is associated with hematopoietic maturation. Cell lines that represent mature, differentiated hematopoietic cell types contain 10-100-fold less c-myb mRNA than immature hematopoietic cell types. Differences in steady-state c-myb mRNA levels appear to be primarily maintained by a conditional block to transcription elongation that occurs in the first intron of the gene. The block to transcription elongation has been mapped, using nuclear run-on analysis, to a region of DNA sequence that is highly conserved between mouse and man. Two sets of DNA-protein interactions, flanking the site of the block to transcription elongation, were detected that exhibited DNA-binding activities that strongly correlated with low steady-state c-myb mRNA levels. Several criteria demonstrated that members of the nuclear factor kappa B (NF-kappa B) family of transcription factors were involved in the DNA-protein interactions identified in these two sets. Surprisingly, cotransfection experiments demonstrated that coexpression of members of the NF-kappa B family, specifically p50 with p65 and p65 with c-Rel, transactivated a c-myb/chloramphenicol acetyltransferase reporter construct that contained 5'-flanking sequences, exon I, intron I, and exon II of the c-myb gene. Transactivation by these heterodimer combinations was dependent on regions of the c-myb first intron containing the NF-kappa B-binding sites. These findings suggest that NF-kappa B family members may be involved in either modifying the efficiency of transcription attenuation or acting as an enhancer-like activity to increase transcription initiation. Thus, the regulation of c-myb transcription may be quite complex, and members of the NF-kappa B family likely play an important role in this regulation.
c-myb原癌基因的表达主要在起源于未成熟造血组织的正常组织和肿瘤细胞系中检测到，而c-myb表达的下调与造血成熟相关。代表成熟、分化造血细胞类型的细胞系所含的c-myb mRNA比未成熟造血细胞类型少10至100倍。稳态c-myb mRNA水平的差异似乎主要通过基因第一内含子中发生的转录延伸条件性阻滞来维持。利用核转录分析，已将转录延伸阻滞定位到小鼠与人之间高度保守的一段DNA序列区域。在转录延伸阻滞位点两侧检测到两组DNA-蛋白质相互作用，它们表现出与低稳态c-myb mRNA水平密切相关的DNA结合活性。多项标准表明，转录因子核因子κB（NF-κB）家族成员参与了这两组中鉴定出的DNA-蛋白质相互作用。令人惊讶的是，共转染实验表明，NF-κB家族成员的共表达，特别是p50与p65以及p65与c-Rel的共表达，可激活一个c-myb/氯霉素乙酰转移酶报告构建体，该构建体包含c-myb基因的5'侧翼序列、外显子I、内含子I和外显子II。这些异二聚体组合的反式激活依赖于c-myb第一内含子中包含NF-κB结合位点的区域。这些发现表明，NF-κB家族成员可能参与改变转录衰减效率或作为类似增强子的活性来增加转录起始。因此，c-myb转录的调控可能相当复杂，NF-κB家族成员可能在这种调控中发挥重要作用。
