Activation of HIV Transcription by the Viral Tat Protein Requires a Demethylation Step Mediated by Lysine-specific Demethylase 1 (LSD1/KDM1)

The essential transactivator function of the HIV Tat protein is regulated by multiple posttranslational modifications. Although individual modifications are well characterized, their crosstalk and dynamics of occurrence during the HIV transcription cycle remain unclear.
We examine interactions between two critical modifications within the RNA-binding domain of Tat: monomethylation of lysine 51 (K51) mediated by Set7/9/KMT7, an early event in the Tat transactivation cycle that strengthens the interaction of Tat with TAR RNA, and acetylation of lysine 50 (K50) mediated by p300/KAT3B, a later process that dissociates the complex formed by Tat, TAR RNA and the cyclin T1 subunit of the positive transcription elongation factor b (P-TEFb). We find K51 monomethylation inhibited in synthetic Tat peptides carrying an acetyl group at K50 while acetylation can occur in methylated peptides, albeit at a reduced rate. To examine whether Tat is subject to sequential monomethylation and acetylation in cells, we performed mass spectrometry on immunoprecipitated Tat proteins and generated new modification-specific Tat antibodies against monomethylated/acetylated Tat. No bimodified Tat protein was detected in cells pointing to a demethylation step during the Tat transactivation cycle. We identify lysine-specific demethylase 1 (LSD1/KDM1) as a Tat K51-specific demethylase, which is required for the activation of HIV transcription in latently infected T cells. LSD1/KDM1 and its cofactor CoREST associates with the HIV promoter in vivo and activate Tat transcriptional activity in a K51-dependent manner. In addition, small hairpin RNAs directed against LSD1/KDM1 or inhibition of its activity with the monoamine oxidase inhibitor phenelzine suppresses the activation of HIV transcription in latently infected T cells.
Our data support the model that a LSD1/KDM1/CoREST complex, normally known as a transcriptional suppressor, acts as a novel activator of HIV transcription through demethylation of K51 in Tat. Small molecule inhibitors of LSD1/KDM1 show therapeutic promise by enforcing HIV latency in infected T cells.

HIV Tat蛋白的核心反式激活功能受多种翻译后修饰调控。尽管单一修饰的特征已被充分阐明，但它们之间的串扰以及在HIV转录周期中的发生动态仍未明确。
我们探究了Tat RNA结合结构域内两种关键修饰之间的相互作用：由Set7/9/KMT7介导的赖氨酸51（K51）单甲基化——这是Tat反式激活周期中的早期事件，可增强Tat与TAR RNA的结合；以及由p300/KAT3B介导的赖氨酸50（K50）乙酰化——这是后续过程，可解离Tat、TAR RNA与正性转录延伸因子b（P-TEFb）的细胞周期蛋白T1亚基形成的复合物。我们发现，携带K50乙酰基团的合成Tat肽会抑制K51单甲基化；而甲基化肽虽乙酰化速率降低，但仍可发生乙酰化。为探究Tat在细胞内是否会依次经历单甲基化与乙酰化，我们对免疫沉淀获取的Tat蛋白进行了质谱分析，并制备了针对单甲基化/乙酰化Tat的新型修饰特异性Tat抗体。未在细胞中检测到双修饰Tat蛋白，这表明Tat反式激活周期中存在去甲基化步骤。我们鉴定出赖氨酸特异性去甲基化酶1（LSD1/KDM1）为Tat K51特异性去甲基化酶，其对潜伏感染T细胞中HIV转录的激活是必需的。LSD1/KDM1及其辅助因子CoREST可在体内结合HIV启动子，并以K51依赖的方式激活Tat的转录活性。此外，靶向LSD1/KDM1的小发夹RNA，或使用单胺氧化酶抑制剂苯乙肼（phenelzine）抑制其活性，均可抑制潜伏感染T细胞中HIV转录的激活。
我们的数据支持如下模型：通常被认为是转录抑制因子的LSD1/KDM1/CoREST复合物，可通过对Tat的K51进行去甲基化，成为HIV转录的新型激活因子。LSD1/KDM1的小分子抑制剂可通过维持感染T细胞的HIV潜伏状态，展现出治疗潜力。