Aziz F, van Wijnen A J, Vaughan P S, Wu S, Shakoori A R, Lian J B, Soprano K J, Stein J L, Stein G S
Department of Cell Biology, University of Massachusetts Medical Center, Worcester 01655, USA.
Mol Biol Rep. 1998 Jan;25(1):1-12. doi: 10.1023/a:1006888731301.
Maximal transcription of a prototypical cell cycle controlled histone H4 gene requires a proliferation-specific in vivo genomic protein/DNA interaction element, Site II. Three sequence-specific transcription factors interact with overlapping recognition motifs within Site II: interferon regulatory factor IRF-2 (HiNF-M), the putative H4 subtype-specific protein H4TF-2 (HiNF-P), and HiNF-D which represents a complex of the homeodomain protein CDP/cut, CDC2, cyclin A and pRB. However, natural sequence variation in the Site II sequences of different human H4 genes abolishes binding of specific trans-acting factors; the functional consequences of these variations have not been investigated. To address the precise contribution of H4 promoter factors to the level of H4 gene transcription, we performed a systematic mutational analysis of Site II transcriptional motifs. These mutants were tested for ability to bind each of the Site II cognate proteins, and subsequently evaluated for ability to confer H4 transcriptional activity using chimeric H4 promoter/CAT fusion constructs in different cell types. We also analyzed the effect of over-expressing IRF-2 on CAT reporter gene expression driven by mutant H4 promoters and assessed H4 transcriptional control in cells nullizygous for IRF-1 and IRF-2. Our results show that the recognition sequence for IRF-2 (HiNF-M) is the dominant component of Site II and modulates H4 gene transcription levels by 3 fold. However, the overlapping recognition sequences for IRF-2 (HiNF-M), H4TF-2 (HiNF-P) and CDP/cut (HiNF-D) together modulate H4 gene transcription levels by at least an order of magnitude. Thus, maximal activation of H4 gene transcription during the cell cycle in vivo requires the integrated activities of multiple transcription factors at Site II. We postulate that the composite organization of Site II supports responsiveness to multiple signalling pathways modulating the activities of H4 gene transcription factors during the cell cycle. Variations in Site II sequences among different H4 genes may accommodate differential regulation of H4 gene expression in cells and tissues with unique phenotypic properties.
典型的细胞周期调控组蛋白H4基因的最大转录需要一个增殖特异性的体内基因组蛋白/DNA相互作用元件，即位点II。三种序列特异性转录因子与位点II内重叠的识别基序相互作用：干扰素调节因子IRF-2（HiNF-M）、假定的H4亚型特异性蛋白H4TF-2（HiNF-P）以及HiNF-D，后者代表同源结构域蛋白CDP/cut、CDC2、细胞周期蛋白A和pRB的复合物。然而，不同人类H4基因位点II序列中的自然序列变异消除了特定反式作用因子的结合；这些变异的功能后果尚未得到研究。为了确定H4启动子因子对H4基因转录水平的确切贡献，我们对位点II转录基序进行了系统的突变分析。测试这些突变体与每个位点II同源蛋白结合的能力，随后使用嵌合H4启动子/CAT融合构建体在不同细胞类型中评估其赋予H4转录活性的能力。我们还分析了过表达IRF-2对由突变H4启动子驱动的CAT报告基因表达的影响，并评估了IRF-1和IRF-2纯合缺失细胞中的H4转录调控。我们的结果表明，IRF-2（HiNF-M）的识别序列是位点II的主要成分，可将H4基因转录水平调节3倍。然而，IRF-2（HiNF-M）、H4TF-2（HiNF-P）和CDP/cut（HiNF-D）的重叠识别序列共同调节H4基因转录水平至少一个数量级。因此，体内细胞周期中H4基因转录的最大激活需要多个转录因子在位点II的整合活性。我们推测，位点II的复合组织支持对多种信号通路的响应，这些信号通路在细胞周期中调节H4基因转录因子的活性。不同H4基因之间位点II序列的变异可能适应具有独特表型特性的细胞和组织中H4基因表达的差异调节。
