Role of myb gene family in the proliferation control of hematopoietic cells

目前获得的结果都表明c-myb在维持造血祖细胞增殖状态中起作用。位点定向诱变对亮氨酸拉链的破坏显著增加了反式激活和转化能力。由于亮氨酸拉链基序最初被鉴定为介导几种dna结合蛋白的二聚化，这些结果表明c-Myb活性通过亮氨酸拉链受到负调控，并暗示存在c-Myb抑制剂。胚胎干细胞同源重组产生的纯合子c-myb突变小鼠由于胎儿肝造血的特异性失败而在胚胎期死亡。因此，该突变体不能用于分析成人组织中c-myb的功能。我们尝试在胚胎干细胞中采用两步同源重组法制备c-myb点突变小鼠。在这些点突变体中，c-Myb活性预计是野生型的1/10或10倍，突变小鼠在胚胎期不致死。我们制作了杂合点突变体，期望得到的纯合点突变体对myb功能的分析很有帮助。我们发现c-Myb以磷酸化不依赖的方式与CBP结合。由于CBP与TFIIB相互作用，CBP介导CREB或c-Myb依赖的转录激活，作为磷酸化的CREB或c-Myb与RNA聚合酶II复合物之间的桥梁。最近，CBP被证明是许多其他转录激活因子（包括c-Jun和c-Fos）的转录辅激活因子。有趣的是，人类CBP基因的突变通过单倍功能不全导致Rubinstein-Taybi综合征，这表明细胞中CBP的含量并不过量，其含量减少50%会影响正常发育。因此，c-Myb、c-Myb、cAMP和AP-1通路之间可能通过竞争结合CBP而发生串扰。

The reults obtained so far all indicate a role for c-myb in maintaining the proliferative state of hematopoietic prgenitor cells. Disruption of this leucine zipper by site-directed mutagenesis markedly increases both trans-activating and transforming capacities. Since the leucine zipper motif was originallyn identified as mediating dimerization of several DNA-binding proteins, these results indicate that c-Myb activity is negatively regulated through the leucine zipper and imply the presence of an inhibitor (s) of c-Myb.Homozygous c-myb mutant mice generated by homologous recombination in embryonic stem cells die at the embryonic stage due to a specific failure of fetal hepatic haematopoiesis. Therefore, this mutant cannot used for the analyzes of c-myb function in the adult tissues. We have tried to make the point mutant mice of c-myb by two step homologous recombination in ES cells. In these point mutants, the c-Myb activity is expected to be 1/10 or 10-fold compared with the wild type, and the mutant mice is not lethal at embryonic stage. We have made the heterozygous point mutants, so that the expected homozgous mutants will be very useful for the analyzes of myb function.We have found that c-Myb binds to CBP in a phosphorylation-independent manner. Since CBP interacts with TFIIB,CBP mediates CREB-or c-Myb-dependent transcriptional activation as a bridge between phosphorylated CREB or c-Myb and the RNA polymerase II complex. Recently, CBP was demonstated to function as a transcriptional coactivator for many other transcriptional activators including c-Jun and c-Fos. Interestingly, mutations in the human CBP gene cause Rubinstein-Taybi syndrome through haploinsufficiency, suggesting that theamount of CBP in cells is not excessive and a 50% decrease in the amount affects normal development. Therefore, cross talk between the c-Myb, c-Myb, cAMP,and AP-1 pathways through competition for binding to CBP is possible.