Genome-wide localisation of histone variants in Toxoplasma gondii implicates variant exchange in transcriptional control by demarcation of functional chromatin regions (ChIP-seq)

Chromatin is composed of DNA wrapped around nucleosomes, complexes consisting of highly basic proteins called histones. Histone variants are non-canonical variants of histones that have specific functions. The Apicomplexan parasite Toxoplasma gondii possesses conserved histone variants CenH3, H3.3, H2A.Z, H2A.X, as well as the parasite-specific H2B variant, H2BV (or H2B.Z). We surveyed the genome-wide distribution of T. gondii histone variants by chromatin immunoprecipitation coupled with microarray (ChIP-chip) and next generation sequencing (ChIP-seq). Histone variants have specific localisations in chromosomes and in relation to genomic features. Histones H2BV and H2AZ localise to promoter regions with the transcriptional activation mark H3K4me3. Sites where H2BV, H2AZ and H3K4me3 colocalise are enriched in motifs recognised by AP2 transcription factors, particularly in the nucleosome-free region just upstream of the transcription start site. In contrast, histone variants H3.3 and H2AX are largely absent from promoters but enriched in gene bodies. Consistent with previous biochemical work, the majority of H2AX and H2AZ loci do not overlap, suggesting that they are present in different nucleosomes. In addition, the T. gondii homologue of the centromeric histone CenH3 demarcates centromeric chromatin and appears to be part of a unique centromeric histone complex including H3.3 that localises to centromeric chromatin as detected in proteomic analysis of co-immunoprecipitated CenH3 complexes. Together, these data suggest that the position of variant histones demarcates specific functional regions of chromatin and that exchange of histone variants is an important transcriptional regulatory mechanism in T. gondii. Localization of histone variants in Toxoplasma gondii by ChIP-chip and ChIP-seq

染色质（Chromatin）由缠绕在核小体（nucleosomes）上的DNA构成，核小体是由高度碱性的组蛋白（histones）组成的复合物。组蛋白变体（histone variants）是组蛋白的非经典亚型，具有特定的生物学功能。顶复门（Apicomplexan）寄生虫刚地弓形虫（Toxoplasma gondii）携带保守的组蛋白变体着丝粒组蛋白CenH3、H3.3、H2A.Z、H2A.X，以及该寄生虫特异性的H2B变体H2BV（或称H2B.Z）。
我们通过染色质免疫共沉淀结合芯片（ChIP-chip）与下一代测序（ChIP-seq）技术，对刚地弓形虫组蛋白变体的全基因组分布进行了系统性分析。
组蛋白变体在染色体上以及与基因组特征相关的位置呈现特异性定位。组蛋白H2BV与H2AZ定位于带有转录激活标记H3K4me3的启动子区域。H2BV、H2AZ与H3K4me3共定位的位点富集AP2转录因子（AP2 transcription factors）识别的基序，尤其集中在转录起始位点上游的无核小体区域。
与之相反，组蛋白变体H3.3与H2AX在启动子区域基本缺失，但在基因体中显著富集。与此前的生化研究结果一致，绝大多数H2AX与H2AZ的定位位点并无重叠，提示二者分别存在于不同的核小体中。
此外，刚地弓形虫的着丝粒组蛋白CenH3同源物可界定着丝粒染色质（centromeric chromatin），通过对免疫共沉淀获取的CenH3复合物进行蛋白质组学分析发现，其属于包含H3.3的独特着丝粒组蛋白复合物的组分，并定位于着丝粒染色质区域。
综上，本研究数据表明，组蛋白变体的定位可界定染色质的特定功能区域，而组蛋白变体的替换是刚地弓形虫中一种关键的转录调控机制。基于ChIP-chip与ChIP-seq的刚地弓形虫组蛋白变体定位