Department of Chemistry and Biology, Ryerson University, 350 Victoria St., Toronto, M5B 2K3, Canada.
Department of Biology, York University, 4700 Keele St., Toronto, M3J 1P3, Canada.
Epigenetics Chromatin. 2018 Mar 9;11(1):10. doi: 10.1186/s13072-018-0180-6.
The chromatin remodelers of the SWI/SNF family are critical transcriptional regulators. Recognition of lysine acetylation through a bromodomain (BRD) component is key to SWI/SNF function; in most eukaryotes, this function is attributed to SNF2/Brg1.
Using affinity purification coupled to mass spectrometry (AP-MS) we identified members of a SWI/SNF complex (SWI/SNF) in Tetrahymena thermophila. SWI/SNF is composed of 11 proteins, Snf5, Swi1, Swi3, Snf12, Brg1, two proteins with potential chromatin-interacting domains and four proteins without orthologs to SWI/SNF proteins in yeast or mammals. SWI/SNF subunits localize exclusively to the transcriptionally active macronucleus during growth and development, consistent with a role in transcription. While Tetrahymena Brg1 does not contain a BRD, our AP-MS results identified a BRD-containing SWI/SNF component, Ibd1 that associates with SWI/SNF during growth but not development. AP-MS analysis of epitope-tagged Ibd1 revealed it to be a subunit of several additional protein complexes, including putative SWR, and SAGA complexes as well as a putative H3K4-specific histone methyl transferase complex. Recombinant Ibd1 recognizes acetyl-lysine marks on histones correlated with active transcription. Consistent with our AP-MS and histone array data suggesting a role in regulation of gene expression, ChIP-Seq analysis of Ibd1 indicated that it primarily binds near promoters and within gene bodies of highly expressed genes during growth.
Our results suggest that through recognizing specific histones marks, Ibd1 targets active chromatin regions of highly expressed genes in Tetrahymena where it subsequently might coordinate the recruitment of several chromatin-remodeling complexes to regulate the transcriptional landscape of vegetatively growing Tetrahymena cells.
SWI/SNF 家族的染色质重塑因子是关键的转录调控因子。通过溴结构域（BRD）组件识别赖氨酸乙酰化对于 SWI/SNF 功能至关重要；在大多数真核生物中，这种功能归因于 SNF2/Brg1。
我们使用亲和纯化结合质谱（AP-MS）在嗜热四膜虫中鉴定了 SWI/SNF 复合物（SWI/SNF）的成员。SWI/SNF 由 11 种蛋白质组成，包括 Snf5、Swi1、Swi3、Snf12、Brg1、两种具有潜在染色质相互作用结构域的蛋白质和四种在酵母或哺乳动物中没有与 SWI/SNF 蛋白同源的蛋白质。SWI/SNF 亚基仅在生长和发育过程中定位于转录活跃的大核，与转录功能一致。虽然嗜热四膜虫 Brg1 不含 BRD，但我们的 AP-MS 结果鉴定出一个含有 BRD 的 SWI/SNF 成分 Ibd1，它在生长过程中与 SWI/SNF 相关，但在发育过程中不相关。Ibd1 的表位标记 AP-MS 分析表明，它是几个额外蛋白质复合物的一个亚基，包括假定的 SWR 和 SAGA 复合物以及假定的 H3K4 特异性组蛋白甲基转移酶复合物。重组 Ibd1 识别与活跃转录相关的组蛋白上的乙酰-赖氨酸标记。与我们的 AP-MS 和组蛋白阵列数据表明其在基因表达调控中的作用一致，Ibd1 的 ChIP-Seq 分析表明，它主要结合在生长过程中高度表达基因的启动子附近和基因体中。
我们的结果表明，通过识别特定的组蛋白标记，Ibd1 靶向嗜热四膜虫中高度表达基因的活性染色质区域，随后可能协调几个染色质重塑复合物的募集，以调节营养生长的四膜虫细胞的转录景观。
