Patient R, Leonard M, Brewer A, Enver T, Wilson A, Walmsley M
Department of Biophysics, Cell and Molecular Biology, University of London, King's College, UK.
Prog Clin Biol Res. 1989;316A:105-16.
Comparative protein binding studies have been performed on the Xenopus beta globin gene promoter. Erythroblast nuclear extracts 'footprint' over the erythroid-specific consensus sequence, AGGATAAG, which is located immediately upstream of the CCAAT footprint. Nonerythroid cell extracts do not give rise to an AGGATAAG footprint but rather to an extended CCAAT footprint reminiscent of the CCAAT displacement protein (CDP). Erythroblast extracts also protect a sequence similar to the chicken stage selector element (SSE) immediately downstream of the CCAAT box footprint. In contrast to these discrete footprints observed using erythroblast extracts, Xenopus erythrocyte nuclear extracts give rise to more extensive promoter protection. We have previously reported that this promoter is active in transfected HeLa cells when linked to the SV40 enhancer and that transcriptional activation is accompanied by the formation in the chromatin of a nuclease hypersensitive site (HS) in this region. As a first step towards defining the roles of the various promoter-binding proteins in transcriptional activation and HS formation, we transfected deletion mutants of the promoter into HeLa cells. Deletion of the sequences upstream of -116 had no effect on transcription or HS formation. Indeed the upstream boundary of the HS remained unchanged (at around-170) even though plasmid sequences had replaced Xenopus sequences. If the HS boundary reflects resumption of nucleosomal structure, then sequences downstream of -116 must be able to position a nucleosome from at least 50 bp away. beta globin gene activation in a number of transfected cell lines is absolutely dependent on DNA replication. The replication requirement is not a consequence of template copy number or methylation, nor is it dependent on the direction in which the replication fork passes through the gene. We conclude that replication facilitates active transcription complex formation by disrupting a stable association of the template with negative factors, which could include histones. About 200 bp upstream of the Xenopus beta globin gene promoter is a tract of alternating A and T residues which adopts cruciform geometry at low levels of supercoiling. Because of this sensitivity to torsional stress, we have probed the structure of the (AT)n sequence in microinjected Xenopus oocytes, where the Xenopus beta globin gene is transcribed very efficiently. We find that S1 nuclease cleaves specifically in the middle of the (AT)n tract, suggesting that the gene is under torsional stress.
已对非洲爪蟾β珠蛋白基因启动子进行了蛋白质结合比较研究。成红细胞核提取物在位于CCAAT足迹上游紧邻的红系特异性共有序列AGGATAAG上形成“足迹”。非红系细胞提取物不会产生AGGATAAG足迹，而是产生一个延伸的CCAAT足迹，类似于CCAAT置换蛋白（CDP）。成红细胞提取物还能保护与鸡阶段选择元件（SSE）相似的序列，该序列位于CCAAT框足迹紧邻的下游。与使用成红细胞提取物观察到的这些离散足迹不同，非洲爪蟾红细胞核提取物能使启动子受到更广泛的保护。我们之前报道过，当该启动子与SV40增强子相连时，在转染的HeLa细胞中具有活性，并且转录激活伴随着该区域染色质中核酸酶超敏位点（HS）的形成。作为确定各种启动子结合蛋白在转录激活和HS形成中作用的第一步，我们将启动子的缺失突变体转染到HeLa细胞中。缺失-116上游的序列对转录或HS形成没有影响。实际上，即使质粒序列取代了非洲爪蟾序列，HS的上游边界仍保持不变（在约-170处）。如果HS边界反映了核小体结构的恢复，那么-116下游的序列必须能够从至少50 bp外定位一个核小体。许多转染细胞系中的β珠蛋白基因激活绝对依赖于DNA复制。复制需求不是模板拷贝数或甲基化的结果，也不依赖于复制叉穿过基因的方向。我们得出结论，复制通过破坏模板与负因子（可能包括组蛋白）的稳定结合来促进活性转录复合物的形成。在非洲爪蟾β珠蛋白基因启动子上游约200 bp处是一段交替的A和T残基序列，在低水平超螺旋时呈现十字形结构。由于对扭转应力敏感，我们在显微注射的非洲爪蟾卵母细胞中探究了（AT）n序列的结构，在该卵母细胞中非洲爪蟾β珠蛋白基因转录非常高效。我们发现S1核酸酶在（AT）n序列的中间特异性切割，这表明该基因处于扭转应力之下。
