van der Houven van Oordt C W, van Wijnen A J, Carter R, Soprano K, Lian J B, Stein G S, Stein J L
Department of Cell Biology, University of Massachusetts Medical School, Worcester 01655.
J Cell Biochem. 1992 May;49(1):93-110. doi: 10.1002/jcb.240490115.
Upstream sequences of the H4 histone gene FO108 located between nt -418 to -213 are stimulatory for in vivo transcription. This domain contains one protein/DNA interaction site (H4-Site III) that binds factor H4UA-1. Based on methylation interference, copper-phenanthroline protection, and competition assays, we show that H4UA-1 interacts with sequences between nt -345 to -332 containing an element displaying sequence-similarity with the thyroid hormone response element (TRE). Using gel retardation assays, we also demonstrate that H4UA-1 binding activity is abolished at low concentrations of Zn2+ (0.75 mM), a characteristic shared with the thyroid hormone (TH) receptor DNA binding protein. Interestingly, phosphatase-treatment of nuclear proteins inhibits formation of the H4UA-1 protein/DNA complex, although a complex with higher mobility (H4UA-1b) can be detected; both complexes share identical protein-DNA contacts and competition behaviors. These findings suggest that phosphorylation may be involved in the regulation of H4-Site III protein/DNA interactions by directly altering protein/protein associations. H4-Site III interactions were examined in several cell culture systems during cell growth and differentiation. We find that H4UA-1 binding activity is present during the cell cycle of both normal diploid and transformed cells. However, during differentiation of normal diploid rat calvarial osteoblasts, we observe a selective loss of the H4UA-1/H4-Site III interaction, concomitant with an increase of the H4UA-1b/H4-Site III complex, indicating modifications in the heteromeric nature of protein/DNA interactions during downregulation of transcription at the cessation of proliferation. Transformed cells have elevated levels of H4UA-1, whereas H4UA-1b is predominantly present in normal diploid cells; this alteration in the ratio of H4UA-1 and H4UA-1b binding activities may reflect deregulation of H4-Site III interactions in transformed cells. We propose that H4-Site III interactions may contribute, together with protein/DNA interactions at proximal regulatory sequences, in determining the level of H4-FO108 histone gene transcription.
位于核苷酸-418至-213之间的H4组蛋白基因FO108的上游序列对体内转录具有刺激作用。该结构域包含一个与因子H4UA-1结合的蛋白质/DNA相互作用位点（H4-位点III）。基于甲基化干扰、铜菲咯啉保护和竞争分析，我们发现H4UA-1与核苷酸-345至-332之间的序列相互作用，该序列包含一个与甲状腺激素反应元件（TRE）具有序列相似性的元件。使用凝胶阻滞分析，我们还证明在低浓度的Zn2+（0.75 mM）下，H4UA-1的结合活性被消除，这是甲状腺激素（TH）受体DNA结合蛋白的一个共同特征。有趣的是，对核蛋白进行磷酸酶处理会抑制H4UA-1蛋白/DNA复合物的形成，尽管可以检测到一种迁移率更高的复合物（H4UA-1b）；这两种复合物具有相同的蛋白质-DNA接触和竞争行为。这些发现表明磷酸化可能通过直接改变蛋白质/蛋白质相互作用参与对H4-位点III蛋白/DNA相互作用的调节。在几种细胞培养系统中，在细胞生长和分化过程中检测了H4-位点III的相互作用。我们发现在正常二倍体细胞和转化细胞的细胞周期中都存在H4UA-1结合活性。然而，在正常二倍体大鼠颅骨成骨细胞的分化过程中，我们观察到H4UA-1/H4-位点III相互作用选择性丧失，同时H4UA-1b/H4-位点III复合物增加，这表明在增殖停止时转录下调期间蛋白质/DNA相互作用的异聚性质发生了改变。转化细胞中H4UA-1水平升高，而H4UA-1b主要存在于正常二倍体细胞中；H4UA-1和H4UA-1b结合活性比例的这种改变可能反映了转化细胞中H4-位点III相互作用的失调。我们提出H4-位点III相互作用可能与近端调控序列处的蛋白质/DNA相互作用一起，共同决定H4-FO108组蛋白基因的转录水平。
