Histone H4K16 acetylation modulates the dynamics of linker H1 and heterochromatin protein HP1 in male X chromosome dosage compensation. Drosophila melanogaster

Drosophila dosage compensation is an epigenetic phenomenon in which the transcription of most genes on X-chromosome is enhanced by approximately two folds in males to equalize that in females. In this study, we provide evidence that transcriptional repressors, such as HP1 and linker histone H1, are globally reduced on male X chromosome. We further investigated the role of HP1 and linker H1 on X chromosome dosage compensation using flies with overexpression of HP1 and H1, we demonstrate that these repressors surpress H4K16 acetylation, presence of the elongating RNA Pol II and the transcription of the genes on male X chromosomes. To understand how H1 and HP1 are regulated on male X chromosome, we next explored the relationship between MOF and the two repressors of chromatin. We show that the hyperacetylation of H4K16 induced by MOF resulted in the loss of H1 and HP1 on chromatin and global chromatin decondensation. Our biochemical analysis further shows that the presence of acetylation on histone H4 at lysine 16 directly inhibits the interaction between histone H4 and linker H1. This study therefore provides novel clues on understanding the dynamic regulation of chromatin regulators on male X chromosome dosage compensation. Overall design: In this study, we used ChIP-Seq to accurately map HP1 binding profiles on whole drosophila genome in drosophila S2 and Kc cells. Chromatin components do not work solely; they cooperate with each other to regulate the transcription activity. So besides HP1, we also generated ChIP maps of histone H1 and the histone marks H3K4me2, H3K9me2, H3K36me3 and H4K16Ac in drosophila S2 cells. Affinity-purified polyclonal antibody of HP1 was used in this experiment, 5ul for each ChIP. H4K16ac (07-329) and H3K4me2 (07-030) antibodies purchased from Upstate were also used in this experiment, 5ul for each ChIP. H3K36me3 (ab9050) and H3K9me2 (ab1220) antibodies were purchased from Abcam, 5ul for each ChIP. The polyclonal antibody for Sample H1_S2 was made by our lab (detailed information can be found in Ni et al.,2006 Genes Dev.).

果蝇剂量补偿（Drosophila dosage compensation）是一种表观遗传现象，即雄性果蝇X染色体上多数基因的转录水平被提升约两倍，以实现与雌性的转录水平均衡。本研究提供证据表明，雄性X染色体上的转录抑制因子（如异染色质蛋白HP1和连接组蛋白H1）整体水平显著降低。我们进一步通过过表达HP1与H1的果蝇，探究了二者在X染色体剂量补偿中的调控作用，实验证实这些抑制因子会抑制雄性X染色体上的H4K16乙酰化、延伸型RNA聚合酶II的富集以及相关基因的转录。为阐明雄性X染色体上H1与HP1的调控机制，我们后续探索了组蛋白乙酰转移酶MOF与这两种染色质抑制因子之间的关联。结果显示，MOF诱导的H4K16高度乙酰化会导致染色质上H1与HP1的解离丢失，并引发整体染色质解凝聚。生化分析进一步表明，组蛋白H4第16位赖氨酸的乙酰化修饰会直接阻断组蛋白H4与连接组蛋白H1的相互作用。因此，本研究为理解染色质调控因子在雄性X染色体剂量补偿中的动态调控提供了全新线索。

总体实验设计：本研究采用染色质免疫共沉淀测序（ChIP-Seq, Chromatin Immunoprecipitation Sequencing）技术，在果蝇S2与Kc细胞中精准绘制全基因组范围内HP1的结合图谱。染色质调控因子并非单独发挥功能，而是通过相互协同调控基因转录活性。因此除HP1外，我们还在果蝇S2细胞中生成了组蛋白H1以及组蛋白修饰标记H3K4me2、H3K9me2、H3K36me3和H4K16Ac的ChIP图谱。本实验使用亲和纯化的HP1多克隆抗体，每次ChIP实验加入5μl。购自Upstate公司的H4K16ac（货号07-329）与H3K4me2（货号07-030）抗体同样用于本实验，每次ChIP加入5μl。购自Abcam公司的H3K36me3（货号ab9050）与H3K9me2（货号ab1220）抗体亦用于本实验，每次ChIP加入5μl。针对样本H1_S2的多克隆抗体由本实验室制备（详细信息可参见Ni等人2006年发表于《Genes Dev.》的研究）。