RNA Seq analysis of 2.5 hour embryos of Drosophila melanogaster in the absence of Ataxin-7 or non-stop.

The Spt-Ada-Gcn5-acetyltransferase (SAGA) chromatin-modifying complex is a transcriptional coactivator that contains four different modules of subunits. The intact SAGA complex has been well characterized for its function in transcription regulation and development. However, little is known about the roles of individual modules within SAGA and if they have any SAGA independent functions. Here we demonstrate that the two enzymatic modules of Drosophila SAGA are differently required in oogenesis. Ada2b is part of the HAT module (Histone Acetyl Transferase), whereas Ataxin-7 and non-stop are members of the DUB module (Deubiquitinase). Loss of the HAT activity blocks oogenesis, while loss of the DUB activity does not. However, the DUB module regulates a subset of genes in early embryogenesis and loss of the DUB subunits causes defects in embryogenesis. ChIP-seq analysis of ada2b, spt3, nonstop, and sgf11revealed that both the DUB and HAT modules bind most SAGA target genes even though many of these targets do not require the DUB module for expression. Furthermore, we found that the DUB module can bind to chromatin and regulate transcription independently of the rest of SAGA. Our results suggest that the DUB module has functions within SAGA as well as independent functions. Overall design: Gene expression was assessed by RNA Seq analysis in embryos mutant for either Ataxin-7, or non-stop, relative to wt in separate experiments, each in triplicate.

Spt-Ada-Gcn5-乙酰转移酶（Spt-Ada-Gcn5-acetyltransferase，SAGA）染色质修饰复合物是一类转录共激活因子，包含四种不同的亚基模块。完整的SAGA复合物在转录调控与发育过程中的功能已得到充分阐释。然而，学界对于SAGA内部各单个模块的功能，以及它们是否具备不依赖于SAGA的独立功能，仍知之甚少。本研究证实，果蝇（Drosophila）SAGA的两个酶活模块在卵子发生过程中的需求存在差异。Ada2b属于组蛋白乙酰转移酶（Histone Acetyl Transferase，HAT）模块，而Ataxin-7与non-stop则是去泛素化酶（Deubiquitinase，DUB）模块的组成成员。HAT活性缺失会阻断卵子发生，而DUB活性缺失则不会产生该效应。不过，DUB模块可调控早期胚胎发生中的特定基因子集，DUB亚基的缺失会导致胚胎发生缺陷。对ada2b、spt3、non-stop及sgf11进行的染色质免疫沉淀测序（ChIP-seq）分析显示，尽管众多靶基因的表达并不依赖DUB模块，但DUB与HAT模块均可结合绝大多数SAGA靶基因。此外，本研究发现DUB模块能够结合染色质，并独立于SAGA其余组分调控转录。上述结果表明，DUB模块不仅在SAGA复合物内部发挥功能，同时也具备独立的生物学功能。本研究的实验设计如下：分别以野生型（wt）胚胎为对照，对Ataxin-7突变体与non-stop突变体胚胎进行RNA测序（RNA Seq）分析，以评估基因表达情况，每组实验均设置三次生物学重复。