Institute of Molecular BioSciences, Massey University, Private Bag 11222, Palmerston North, New Zealand.
BMC Mol Biol. 2010 Nov 9;11:80. doi: 10.1186/1471-2199-11-80.
In male Drosophila melanogaster, the male specific lethal (MSL) complex is somehow responsible for a two-fold increase in transcription of most X-linked genes, which are enriched for histone H4 acetylated at lysine 16 (H4K16ac). This acetylation requires MOF, a histone acetyltransferase that is a component of the MSL complex. MOF also associates with the non-specific lethal or NSL complex. The MSL complex is bound within active genes on the male X chromosome with a 3' bias. In contrast, the NSL complex is enriched at promoter regions of many autosomal and X-linked genes in both sexes. In this study we have investigated the role of MOF as a transcriptional activator.
MOF was fused to the DNA binding domain of Gal4 and targeted to the promoter region of UAS-reporter genes in Drosophila. We found that expression of a UAS-red fluorescent protein (DsRed) reporter gene was strongly induced by Gal4-MOF. However, DsRed RNA levels were about seven times higher in female than male larvae. Immunostaining of polytene chromosomes showed that Gal4-MOF co-localized with MSL1 to many sites on the X chromosome in male but not female nuclei. However, in female nuclei that express MSL2, Gal4-MOF co-localized with MSL1 to many sites on polytene chromosomes but DsRed expression was reduced. Mutation of conserved active site residues in MOF (Glu714 and Cys680) reduced HAT activity in vitro and UAS-DsRed activation in Drosophila. In the presence of Gal4-MOF, H4K16ac levels were enriched over UAS-lacZ and UAS-arm-lacZ reporter genes. The latter utilizes the constitutive promoter from the arm gene to drive lacZ expression. In contrast to the strong induction of UAS-DsRed expression, UAS-arm-lacZ expression increased by about 2-fold in both sexes.
Targeting MOF to reporter genes led to transcription enhancement and acetylation of histone H4 at lysine 16. Histone acetyltransferase activity was required for the full transcriptional response. Incorporation of Gal4-MOF into the MSL complex in males led to a lower transcription enhancement of UAS-DsRed but not UAS-arm-lacZ genes. We discuss how association of Gal4-MOF with the MSL or NSL proteins could explain our results.
在雄性黑腹果蝇中，雄性特异致死（MSL）复合物在某种程度上负责使大多数 X 连锁基因的转录增加两倍，这些基因富含组蛋白 H4 赖氨酸 16 乙酰化（H4K16ac）。这种乙酰化需要 MOF，一种组蛋白乙酰转移酶，是 MSL 复合物的组成部分。MOF 还与非特异性致死或 NSL 复合物相关联。MSL 复合物结合在雄性 X 染色体上的活性基因上，具有 3' 偏向性。相比之下，NSL 复合物在两性的许多常染色体和 X 连锁基因的启动子区域富集。在这项研究中，我们研究了 MOF 作为转录激活剂的作用。
MOF 与 Gal4 的 DNA 结合域融合，并靶向果蝇 UAS-报告基因的启动子区域。我们发现，Gal4-MOF 强烈诱导 UAS-红色荧光蛋白（DsRed）报告基因的表达。然而，在雌性幼虫中，DsRed RNA 水平比雄性幼虫高约 7 倍。多线染色体的免疫染色显示，Gal4-MOF 在雄性核中与 MSL1 共定位到 X 染色体上的许多位点，但在雌性核中不与 MSL1 共定位。然而，在表达 MSL2 的雌性核中，Gal4-MOF 与 MSL1 共定位到多线染色体上的许多位点，但 DsRed 的表达减少。MOF 中保守活性位点残基的突变（Glu714 和 Cys680）降低了体外的 HAT 活性和果蝇中的 UAS-DsRed 激活。在 Gal4-MOF 的存在下，H4K16ac 水平在 UAS-lacZ 和 UAS-arm-lacZ 报告基因上富集。后者利用臂基因的组成型启动子驱动 lacZ 表达。与 UAS-DsRed 表达的强烈诱导相比，UAS-arm-lacZ 的表达在两性中增加了约 2 倍。
将 MOF 靶向报告基因导致组蛋白 H4 赖氨酸 16 的乙酰化和转录增强。组蛋白乙酰转移酶活性是完全转录反应所必需的。Gal4-MOF 整合到雄性 MSL 复合物中导致 UAS-DsRed 基因的转录增强降低，但 UAS-arm-lacZ 基因的转录增强不变。我们讨论了 Gal4-MOF 与 MSL 或 NSL 蛋白的结合如何解释我们的结果。
