Marzio G, Tyagi M, Gutierrez M I, Giacca M
Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 34012 Trieste, Italy.
Proc Natl Acad Sci U S A. 1998 Nov 10;95(23):13519-24. doi: 10.1073/pnas.95.23.13519.
In cells infected with HIV type 1 (HIV-1), the integrated viral promoter is present in a chromatin-bound conformation and is transcriptionally silent in the absence of stimulation. The HIV-1 Tat protein binds to a stem-loop structure at the 5' end of viral mRNA and relieves this inhibition by inducing a remodeling of the nucleosome arrangement downstream of the transcription-initiation site. Here we show that Tat performs this activity by recruiting to the viral long terminal repeat (LTR) the transcriptional coactivator p300 and the closely related CREB-binding protein (CBP), having histone acetyltransferase (HAT) activity. Tat associates with HAT activity in human nuclear extracts and binds to p300 and CBP both in vitro and in vivo. Integrity of the basic domain of Tat is essential for this interaction. By a quantitative chromatin immunoprecipitation assay we show that the delivery of recombinant Tat induces the association of p300 and CBP with the chromosomally integrated LTR promoter. Expression of human p300 in both human and rodent cells increases the levels of Tat transactivation of the integrated LTR. These results reinforce the evidence that p300 and CBP have a pivotal function at both cellular and viral promoters and demonstrate that they also can be recruited by an RNA-targeted activator. Additionally, these findings have important implications for the understanding of the mechanisms of HIV-1 latency and reactivation.
在感染了1型人类免疫缺陷病毒（HIV-1）的细胞中，整合后的病毒启动子以染色质结合的构象存在，在没有刺激的情况下转录沉默。HIV-1反式激活因子（Tat）蛋白与病毒mRNA 5'端的茎环结构结合，并通过诱导转录起始位点下游核小体排列的重塑来解除这种抑制。我们在此表明，Tat通过招募具有组蛋白乙酰转移酶（HAT）活性的转录共激活因子p300和密切相关的CREB结合蛋白（CBP）到病毒长末端重复序列（LTR）来发挥此活性。Tat在人核提取物中与HAT活性相关联，并且在体外和体内均与p300和CBP结合。Tat碱性结构域的完整性对于这种相互作用至关重要。通过定量染色质免疫沉淀试验，我们表明重组Tat的传递诱导p300和CBP与染色体整合的LTR启动子结合。人p300在人和啮齿动物细胞中的表达均增加了整合LTR的Tat反式激活水平。这些结果强化了p300和CBP在细胞和病毒启动子上均具有关键功能的证据，并证明它们也可以被RNA靶向激活剂招募。此外，这些发现对于理解HIV-1潜伏和重新激活的机制具有重要意义。
