Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal.
Nucleic Acids Res. 2013 Mar 1;41(5):2881-93. doi: 10.1093/nar/gks1472. Epub 2013 Jan 15.
Histone H3 of nucleosomes positioned on active genes is trimethylated at Lys36 (H3K36me3) by the SETD2 (also termed KMT3A/SET2 or HYPB) methyltransferase. Previous studies in yeast indicated that H3K36me3 prevents spurious intragenic transcription initiation through recruitment of a histone deacetylase complex, a mechanism that is not conserved in mammals. Here, we report that downregulation of SETD2 in human cells leads to intragenic transcription initiation in at least 11% of active genes. Reduction of SETD2 prevents normal loading of the FACT (FAcilitates Chromatin Transcription) complex subunits SPT16 and SSRP1, and decreases nucleosome occupancy in active genes. Moreover, co-immunoprecipitation experiments suggest that SPT16 is recruited to active chromatin templates, which contain H3K36me3-modified nucleosomes. Our results further show that within minutes after transcriptional activation, there is a SETD2-dependent reduction in gene body occupancy of histone H2B, but not of histone H3, suggesting that SETD2 coordinates FACT-mediated exchange of histone H2B during transcription-coupled nucleosome displacement. After inhibition of transcription, we observe a SETD2-dependent recruitment of FACT and increased histone H2B occupancy. These data suggest that SETD2 activity modulates FACT recruitment and nucleosome dynamics, thereby repressing cryptic transcription initiation.
核小体上定位在活性基因的组蛋白 H3 在赖氨酸 36 处被 SETD2（也称为 KMT3A/SET2 或 HYPB）甲基转移酶三甲基化（H3K36me3）。酵母中的先前研究表明，H3K36me3 通过募集组蛋白去乙酰化酶复合物来防止基因内转录起始的错误，这种机制在哺乳动物中并不保守。在这里，我们报告在人类细胞中下调 SETD2 会导致至少 11%的活性基因发生基因内转录起始。SETD2 的减少会阻止 FACT（促进染色质转录）复合物亚基 SPT16 和 SSRP1 的正常加载，并降低活性基因中的核小体占有率。此外，共免疫沉淀实验表明，SPT16 被招募到含有 H3K36me3 修饰核小体的活性染色质模板上。我们的结果进一步表明，在转录激活后的几分钟内，基因体内组蛋白 H2B 的占有率会出现 SETD2 依赖性降低，但组蛋白 H3 则不会，这表明 SETD2 在转录偶联核小体位移过程中协调 FACT 介导的组蛋白 H2B 交换。在转录抑制后，我们观察到 FACT 的 SETD2 依赖性募集和组蛋白 H2B 占有率的增加。这些数据表明，SETD2 活性调节 FACT 的募集和核小体动力学，从而抑制隐匿性转录起始。
