Tomaszewski R, Jerzmanowski A
1 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
Nucleic Acids Res. 1997 Feb 1;25(3):458-66. doi: 10.1093/nar/25.3.458.
In vivo, histone H1 plays an active role in establishing the transcriptionally repressed chromatin state of the oocyte-type 5S RNA genes in the early stages of Xenopus development. By using fully defined in vitro system of chromatin assembly on plasmids with cloned oocyte- or somatic-type 5S gene repeats we found that the oocyte repeat which comprises a 120 bp oocyte-type 5S RNA gene placed within the few hundred bp long native AT-rich flanks, but not the somatic repeat (a similar 120 bp somatic-type 5S RNA gene placed within native GC-rich flanks) enables histone H1 to realign the nucleosomal core particles densely packed on plasmid DNA. The realignment results in creation of the repeat unit of approximately 240 bp and is achieved through complete removal of several core histone complexes from plasmid template with the oocyte-type repeat. This effect of H1 is independent on the plasmid sequences and seems to be solely due to the presence in the oocyte-repeat of the AT-rich flanks. The effects of H1 are completely suppressed by distamycin A, a drug that specifically recognizes and binds oligo(dA).oligo(dT) runs in DNA. The binding of H1 results in increased protection of DNA sites within the AT-rich oocyte-type 5S repeat. In an in vitro transcription assay performed with reconstituted chromatin templates containing plasmids with the oocyte- or somatic-type repeats only the transcription of the oocyte-type 5S RNA gene was repressed in the presence of physiological concentration of histone H1. These results support the view that the AT-rich flanks of the oocyte-type 5S RNA gene are involved in histone H1-mediated chromatin reorganization that results in the transcriptional repression observed in vivo.
在体内，组蛋白H1在非洲爪蟾发育早期建立卵母细胞型5S RNA基因的转录抑制染色质状态中发挥积极作用。通过使用在含有克隆的卵母细胞型或体细胞型5S基因重复序列的质粒上进行染色质组装的完全确定的体外系统，我们发现，包含一个位于几百碱基对长的天然富含AT侧翼内的120碱基对卵母细胞型5S RNA基因的卵母细胞重复序列，而非体细胞重复序列（一个位于天然富含GC侧翼内的类似120碱基对体细胞型5S RNA基因），能使组蛋白H1重新排列紧密堆积在质粒DNA上的核小体核心颗粒。这种重新排列导致形成约240碱基对的重复单元，并且是通过从含有卵母细胞型重复序列的质粒模板中完全去除几个核心组蛋白复合物来实现的。H1的这种作用不依赖于质粒序列，似乎完全是由于卵母细胞重复序列中存在富含AT的侧翼。H1的作用被Distamycin A完全抑制，Distamycin A是一种能特异性识别并结合DNA中寡聚（dA）·寡聚（dT）序列的药物。H1的结合导致富含AT的卵母细胞型5S重复序列内的DNA位点受到更强的保护。在用含有卵母细胞型或体细胞型重复序列质粒的重组染色质模板进行的体外转录试验中，只有在生理浓度的组蛋白H1存在时，卵母细胞型5S RNA基因的转录才受到抑制。这些结果支持这样一种观点，即卵母细胞型5S RNA基因富含AT的侧翼参与了组蛋白H1介导的染色质重组，这种重组导致了在体内观察到的转录抑制。
