Erkine A M, Adams C C, Diken T, Gross D S
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport 71130, USA.
Mol Cell Biol. 1996 Dec;16(12):7004-17. doi: 10.1128/MCB.16.12.7004.
Transcription in eukaryotic cells occurs in the context of chromatin. Binding of sequence-specific regulatory factors must contend with the presence of nucleosomes for establishment of a committed preinitiation complex. Here we demonstrate that the high-affinity binding site for heat shock transcription factor (HSF) is occupied independently of other cis-regulatory elements and is critically required for preventing nucleosomal assembly over the yeast HSC82 core promoter under both noninducing (basal) and inducing conditions. Chromosomal mutation of this sequence, termed HSE1, erases the HSF footprint and abolishes both transcription and in vivo occupancy of the TATA box. Moreover, it dramatically reduces promoter chromatin accessibility to DNase I and TaqI, as the nuclease-hypersensitive region is replaced by a localized nucleosome. By comparison, in situ mutagenesis of two other promoter elements engaged in stable protein-DNA interactions in vivo, the GRF2/REB1 site and the TATA box, despite reducing transcription three- to fivefold, does not compromise the nucleosome-free state of the promoter. The GRF2-binding factor appears to facilitate the binding of proteins to both HSE1 and TATA, as these sequences, while still occupied, are less protected from in vivo dimethyl sulfate methylation in a deltaGRF2 strain. Finally, deletion of a consensus upstream repressor sequence (URS1), positioned immediately upstream of the GRF2-HSE1 region and only weakly occupied in chromatin, has no expression phenotype, even under meiotic conditions. However, deletion of URS1, like mutation of GRF2, shifts the translational setting of an upstream nucleosomal array flanking the promoter region. Taken together, our results argue that HSF, independent of and dominant among sequence-specific factors binding to the HSC82 upstream region, antagonizes nucleosomal repression and creates an accessible chromatin structure conducive to preinitiation complex assembly and transcriptional activation.
真核细胞中的转录发生在染色质环境中。序列特异性调节因子的结合必须与核小体的存在相抗衡，以建立一个稳定的转录起始前复合物。在这里，我们证明热休克转录因子（HSF）的高亲和力结合位点独立于其他顺式调节元件而被占据，并且在非诱导（基础）和诱导条件下，对于防止酵母HSC82核心启动子上的核小体组装至关重要。这个被称为HSE1的序列发生染色体突变会消除HSF足迹，并消除TATA框的转录和体内占据。此外，它显著降低了启动子染色质对DNase I和TaqI的可及性，因为核酸酶超敏区域被一个局部核小体所取代。相比之下，体内参与稳定蛋白质-DNA相互作用的另外两个启动子元件，即GRF2/REB1位点和TATA框，尽管转录降低了三到五倍，但并不损害启动子的无核小体状态。GRF2结合因子似乎促进了蛋白质与HSE1和TATA的结合，因为在deltaGRF2菌株中，这些序列虽然仍然被占据，但对体内硫酸二甲酯甲基化的保护作用较弱。最后，位于GRF2-HSE1区域上游且在染色质中仅被微弱占据的共有上游抑制序列（URS1）的缺失，即使在减数分裂条件下也没有表达表型。然而，URS1的缺失，与GRF2的突变一样，改变了启动子区域侧翼上游核小体阵列的翻译状态。综上所述，我们的结果表明，HSF独立于并在与HSC82上游区域结合的序列特异性因子中占主导地位，它拮抗核小体抑制作用，并创造了一种有利于转录起始前复合物组装和转录激活的可及染色质结构。
