Laboratory of Eukaryotic Gene Regulation, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
FEBS J. 2018 Apr;285(8):1491-1510. doi: 10.1111/febs.14423. Epub 2018 Mar 23.
TAF6, bearing the histone H4-like histone-fold domain (HFD), is a subunit of the core TAF module in TFIID and SAGA transcriptional regulatory complexes. We isolated and characterized several yeast TAF6 mutants bearing amino acid substitutions in the HFD, the middle region or the HEAT repeat domain. The TAF6 mutants were highly defective for transcriptional activation by the Gcn4 and Gal4 activators. CHIP assays showed that the TAF6-HFD and the TAF6-HEAT domain mutations independently abrogated the promoter occupancy of TFIID and SAGA complex in vivo. We employed genetic and biochemical assays to identify the relative contributions of the TAF6 HFD and HEAT domains. First, the temperature-sensitive phenotype of the HEAT domain mutant was suppressed by overexpression of the core TAF subunits TAF9 and TAF12, as well as TBP. The HFD mutant defect, however, was suppressed by TAF5 but not by TAF9, TAF12 or TBP. Second, the HEAT mutant but not the HFD mutant was defective for growth in the presence of transcription elongation inhibitors. Third, coimmunoprecipitation assays using yeast cell extracts indicated that the specific TAF6 HEAT domain residues are critical for the interaction of core TAF subunits with the SAGA complex but not with TFIID. The specific HFD residues in TAF6, although required for heterodimerization between TAF6 and TAF9 recombinant proteins, were dispensable for association of the core TAF subunits with TFIID and SAGA in yeast cell extracts. Taken together, the results of our studies have uncovered the non-overlapping requirement of the evolutionarily conserved HEAT domain and the HFD in TAF6 for transcriptional activation.
TAF6 含有组蛋白 H4 样结构域（HFD），是 TFIID 和 SAGA 转录调控复合物核心 TAF 模块的一个亚基。我们分离并鉴定了几种酵母 TAF6 突变体，这些突变体在 HFD、中间区域或 HEAT 重复结构域中具有氨基酸取代。TAF6 突变体在 Gcn4 和 Gal4 激活子的转录激活中高度缺陷。CHIP 分析表明，TAF6-HFD 和 TAF6-HEAT 结构域突变独立地消除了 TFIID 和 SAGA 复合物在体内的启动子占据。我们采用遗传和生化分析来确定 TAF6 HFD 和 HEAT 结构域的相对贡献。首先，HEAT 结构域突变体的温度敏感表型被核心 TAF 亚基 TAF9 和 TAF12 以及 TBP 的过表达所抑制。然而，HFD 突变体的缺陷被 TAF5 而非 TAF9、TAF12 或 TBP 所抑制。其次，HEAT 突变体而非 HFD 突变体在转录延伸抑制剂存在的情况下生长缺陷。第三，使用酵母细胞提取物的共免疫沉淀分析表明，特定的 TAF6 HEAT 结构域残基对于核心 TAF 亚基与 SAGA 复合物的相互作用是关键的，但不是与 TFIID 的相互作用。TAF6 中的特定 HFD 残基，尽管对于 TAF6 和 TAF9 重组蛋白之间的异二聚化是必需的，但在酵母细胞提取物中对于核心 TAF 亚基与 TFIID 和 SAGA 的结合是可有可无的。总之，我们的研究结果揭示了进化上保守的 HEAT 结构域和 TAF6 中的 HFD 在转录激活中不重叠的需求。
