Kennedy B K, Liu O W, Dick F A, Dyson N, Harlow E, Vidal M
Massachusetts General Hospital Cancer Center, Building 149, 13th Street, Charlestown, MA 02129, USA.
Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8720-5. doi: 10.1073/pnas.151240898. Epub 2001 Jul 10.
We have developed a yeast model system to address transcriptional repression by the retinoblastoma protein (pRB). When fused to the DNA-binding domain of Gal4p (DB-pRB), pRB can repress transcription of reporter genes containing Gal4p binding sites; the histone deacetylase activity encoded by yeast RPD3 is required for DB-pRB repression. Mutation of the LXCXE binding cleft in pRB, a region reported to be required for histone deacetylase recruitment, does not interfere with pRB-mediated repression. From these findings based on yeast experiments, we surmise that the small pocket region of pRB must contain an additional domain that confers histone deacetylase-dependent transcriptional repression. This hypothesis was verified by experiments examining pRB-dependent histone deacetylase association in mammalian cells. In addition to RPD3, repression by pRB in yeast requires MSI1, an ortholog of RbAp48, but not SIN3 or SAP30. By comparing the genetic requirements of DB-pRB repression in yeast to those of other DB-repressor fusions, we can suggest a mechanism by which pRB recruits histone deacetylase activity.
我们开发了一种酵母模型系统，以研究视网膜母细胞瘤蛋白（pRB）介导的转录抑制作用。当与Gal4p的DNA结合结构域（DB-pRB）融合时，pRB能够抑制含有Gal4p结合位点的报告基因的转录；酵母RPD3编码的组蛋白脱乙酰酶活性是DB-pRB抑制作用所必需的。pRB中LXCXE结合裂隙的突变（据报道该区域是募集组蛋白脱乙酰酶所必需的）并不干扰pRB介导的抑制作用。基于酵母实验的这些发现，我们推测pRB的小口袋区域必定包含一个额外的结构域，该结构域赋予组蛋白脱乙酰酶依赖性的转录抑制作用。通过检测哺乳动物细胞中pRB依赖性组蛋白脱乙酰酶结合的实验，这一假设得到了验证。除了RPD3之外，pRB在酵母中的抑制作用还需要MSI1（RbAp48的直系同源物），但不需要SIN3或SAP30。通过比较酵母中DB-pRB抑制作用与其他DB-阻遏物融合体的遗传需求，我们可以提出一种pRB募集组蛋白脱乙酰酶活性的机制。
