Genes for embryo development are packaged in blocks of multivalent chromatin in zebrafish sperm. Danio rerio

In mature human sperm, genes of importance for embryo development (i.e. transcription factors) lack DNA methylation and bear nucleosomes with distinctive histone modifications, suggesting the specialized packaging of these developmental genes in the germline. Here, we explored the tractable zebrafish model and found conceptual conservation as well as several new features. Biochemical and mass spectrometric approaches reveal the zebrafish sperm genome packaged in nucleosomes and histone variants (and not protamine), and we find linker histones high and H4K16ac absent - key factors which may contribute to genome condensation. We examined several activating (H3K4me2/3, H3K14ac, H2AFV) and repressing (H3K27me3, H3K36me3, H3K9me3, hypoacetylation) modifications/compositions genome-wide, and find developmental genes packaged in large blocks of chromatin with coincident activating and repressing marks and DNA hypomethylation, revealing complex ‘multivalent’ chromatin. Notably, genes that acquire DNA methylation in the soma (muscle) are enriched in transcription factors for alternative cell fates. Remarkably, we find H3K36me3 located in ‘silent’ developmental gene promoters, and not present at the 3’ ends of coding regions of genes heavily transcribed during sperm maturation, suggesting different rules for H3K36me3 in the germline and soma. We also reveal the chromatin patterns of transposons, rDNA, and tRNAs. Finally, high levels of H3K4me3 and H3K14ac in sperm are correlated with genes activated in embryos prior to the mid-blastula transition (MBT), whereas multivalent genes are correlated with activation at or after MBT. Taken together, gene sets with particular functions in the embryo are packaged by distinctive types of complex and often atypical chromatin in sperm. Overall design: [DNA profiling]: H3K4me3, H3K4me2, H3K14ac, H3K36me3, H3K27me3, H3K9me3, and H2AFV ChIP-chip in zebrafish sperm; DNA methylation in zebrafish sperm and muscle by MeDIP-chip. (two replicates and dye-swaps for all experiments). Antibodies: H3K4me3 (Abcam ab8580 and Active Motif 39159), H3K4me2 (Abcam ab7766), H3K14Ac (Upstate 07-353), H2AZ (Abcam ab4174), H3K27me3 (Upstate 07-449), H3K9me3 (Active Motif 39161), H3K36me3 (Abcam ab9050), 5-methylcytidine (Eurogentec BI-MECY-1000). Supplementary files: Processed data files reporting calculated enrichment log2 ratio (mean ChIP/mean Input in the log2 ratio). Note: For analysis, "mean Input" from H3K4me3-rep1 ch1, H3K4me2-rep1 ch2, and H3K36me3-rep2 ch1 for all ChIP eluates. [mRNA profiling]: Transcripts in zebrafish mature sperm using zebrafish expression microarray from Agilent. (two replicates)

在成熟人类精子中，参与胚胎发育的重要基因（即转录因子（transcription factors））未发生DNA甲基化，且携带有独特组蛋白修饰的核小体，提示这类发育基因在生殖系中存在特殊的包装模式。本研究以可操作性优异的斑马鱼为模型开展探索，不仅发现了概念层面的保守性，还揭示了若干新颖特征。

通过生化与质谱分析手段，我们发现斑马鱼精子基因组由核小体与组蛋白变体（histone variants）包装，而非鱼精蛋白（protamine）；同时观察到连接组蛋白含量较高，且不存在H4K16ac修饰——上述关键因素可能参与基因组浓缩过程。

我们在全基因组范围内检测了多种激活型修饰/组分（H3K4me2、H3K4me3、H3K14ac、H2AFV）与抑制型修饰/组分（H3K27me3、H3K36me3、H3K9me3、低乙酰化状态），结果显示发育基因以大块染色质区域的形式存在，同时兼具激活与抑制型标记及DNA低甲基化特征，揭示了复杂的“多价染色质（multivalent chromatin）”结构。值得注意的是，在体细胞（肌肉）中发生DNA甲基化的基因，富集了参与其他细胞命运决定的转录因子。

尤为关键的是，我们发现H3K36me3定位于“沉默”的发育基因启动子区域，而非精子成熟过程中高转录基因的编码区3'端，这表明生殖系与体细胞中H3K36me3的调控规则存在差异。本研究还解析了转座子（transposons）、核糖体DNA（rDNA）及转运RNA（tRNAs）的染色质分布模式。

最后，精子中高丰度的H3K4me3与H3K14ac与胚胎在中囊胚转换（mid-blastula transition, MBT）前激活的基因相关，而多价染色质相关基因则与MBT时期及之后的基因激活相关。综上，在胚胎发育中发挥特定功能的基因集，在精子中由独特类型的复杂且通常非典型的染色质结构进行包装。

总体实验设计：
[DNA谱型分析]：对斑马鱼精子开展H3K4me3、H3K4me2、H3K14ac、H3K36me3、H3K27me3、H3K9me3及H2AFV的染色质免疫沉淀芯片（ChIP-chip, chromatin immunoprecipitation-chip）实验；通过甲基化DNA免疫沉淀芯片（MeDIP-chip, methylated DNA immunoprecipitation-chip）检测斑马鱼精子与肌肉组织的DNA甲基化水平。所有实验均设置两次生物学重复，并进行染料互换（dye-swaps）。

所用抗体信息：H3K4me3（Abcam ab8580与Active Motif 39159）、H3K4me2（Abcam ab7766）、H3K14Ac（Upstate 07-353）、H2AZ（Abcam ab4174）、H3K27me3（Upstate 07-449）、H3K9me3（Active Motif 39161）、H3K36me3（Abcam ab9050）、5-甲基胞苷（Eurogentec BI-MECY-1000）。

补充文件：包含计算得到的富集log2比值（即染色质免疫沉淀信号均值/输入样本信号均值的log2转换值）的处理后数据文件。注：分析过程中，所有染色质免疫沉淀洗脱样本均采用H3K4me3重复1通道1、H3K4me2重复1通道2以及H3K36me3重复2通道1的“平均输入样本”作为对照。

[mRNA谱型分析]：采用安捷伦（Agilent）斑马鱼表达芯片，检测斑马鱼成熟精子中的转录本。设置两次生物学重复。