Department of Chemistry and Biology, Toronto Metropolitan University, 350 Victoria St, Toronto, M5B 2K3, Canada.
Donnelly Centre, University of Toronto, Toronto, M5S 3E1, Canada.
Epigenetics Chromatin. 2023 Apr 7;16(1):10. doi: 10.1186/s13072-023-00484-9.
Eukaryotic cells can rapidly adjust their transcriptional profile in response to molecular needs. Such dynamic regulation is, in part, achieved through epigenetic modifications and selective incorporation of histone variants into chromatin. H3.3 is the ancestral H3 variant with key roles in regulating chromatin states and transcription. Although H3.3 has been well studied in metazoans, information regarding the assembly of H3.3 onto chromatin and its possible role in transcription regulation remain poorly documented outside of Opisthokonts.
We used the nuclear dimorphic ciliate protozoan, Tetrahymena thermophila, to investigate the dynamics of H3 variant function in evolutionarily divergent eukaryotes. Functional proteomics and immunofluorescence analyses of H3.1 and H3.3 revealed a highly conserved role for Nrp1 and Asf1 histone chaperones in nuclear influx of histones. Cac2, a putative subunit of H3.1 deposition complex CAF1, is not required for growth, whereas the expression of the putative ortholog of the H3.3-specific chaperone Hir1 is essential in Tetrahymena. Our results indicate that Cac2 and Hir1 have distinct localization patterns during different stages of the Tetrahymena life cycle and suggest that Cac2 might be dispensable for chromatin assembly. ChIP-seq experiments in growing Tetrahymena show H3.3 enrichment over the promoters, gene bodies, and transcription termination sites of highly transcribed genes. H3.3 knockout followed by RNA-seq reveals large-scale transcriptional alterations in functionally important genes.
Our results provide an evolutionary perspective on H3.3's conserved role in maintaining the transcriptional landscape of cells and on the emergence of specialized chromatin assembly pathways.
真核细胞可以快速响应分子需求来调整其转录谱。这种动态调节部分是通过表观遗传修饰和组蛋白变体选择性地掺入染色质来实现的。H3.3 是原始的 H3 变体，在调节染色质状态和转录方面具有关键作用。尽管 H3.3 在后生动物中得到了很好的研究，但关于 H3.3 组装到染色质上及其在转录调节中的可能作用的信息在后生动物之外的真核生物中仍然记录甚少。
我们使用核二态纤毛虫原生动物嗜热四膜虫来研究 H3 变体在进化上不同的真核生物中的功能动态。对 H3.1 和 H3.3 的功能蛋白质组学和免疫荧光分析表明，Nrpl 和 Asf1 组蛋白伴侣在核内输入组蛋白中具有高度保守的作用。Cac2，H3.1 沉积复合物 CAF1 的假定亚基，对于生长不是必需的，而 H3.3 特异性伴侣 Hir1 的假定同源物的表达在嗜热四膜虫中是必不可少的。我们的结果表明，Cac2 和 Hir1 在嗜热四膜虫生命周期的不同阶段具有不同的定位模式，并表明 Cac2 可能对染色质组装不是必需的。在生长中的嗜热四膜虫中进行的 ChIP-seq 实验表明，H3.3 在高度转录基因的启动子、基因体和转录终止位点上富集。H3.3 敲除后进行 RNA-seq 揭示了功能重要基因的大规模转录改变。
我们的结果提供了一个进化的视角，即 H3.3 在维持细胞转录景观方面的保守作用，以及专门的染色质组装途径的出现。
