Kontaraki J, Chen H H, Riggs A, Bonifer C
University of Leeds, Molecular Medicine Unit, St. James's University Hospital, Leeds LS9 7TF, UK.
Genes Dev. 2000 Aug 15;14(16):2106-22.
The chicken lysozyme locus is activated in a stepwise fashion during myeloid differentiation. We have used this locus as a model to study at high resolution changes in chromatin structure both in chicken cell lines representing various stages of macrophage differentiation and in primary cells from transgenic mice. In this study we have addressed the question of whether chromatin rearrangements can be detected in myeloid precursor cells at a stage well before overt transcription of the lysozyme gene begins. In addition to restriction enzyme accessibility assays and DMS footprinting, we have applied new, very sensitive techniques to assay for chromatin changes. Particularly informative was UV photofootprinting, using terminal transferase-dependent PCR and nonradioactive detection. We find that the basic chromatin structure in lysozyme nonexpressing hematopoietic precursor cells is highly similar to the pattern found in fully differentiated lysozyme-expressing cells. In addition, we find that only in nonexpressing cells are dimethylsulfate footprints and UV photofootprints affected by trichostatin, an inhibitor of histone deacetylation. These results are interpreted to mean that most chromatin pattern formation is complete before the binding of end-stage trans-activators, supporting the notion that heritable chromatin structure is central to the stable epigenetic programs that guide development.
鸡溶菌酶基因座在髓系分化过程中以逐步的方式被激活。我们已将该基因座作为模型，在代表巨噬细胞分化不同阶段的鸡细胞系以及转基因小鼠的原代细胞中，以高分辨率研究染色质结构的变化。在本研究中，我们探讨了在溶菌酶基因开始明显转录之前的阶段，髓系前体细胞中是否能检测到染色质重排这一问题。除了限制性内切酶可及性分析和二甲基亚砜足迹分析外，我们还应用了新的、非常灵敏的技术来检测染色质变化。特别有信息量的是利用依赖末端转移酶的聚合酶链反应和非放射性检测的紫外线光足迹分析。我们发现，在不表达溶菌酶的造血前体细胞中，基本的染色质结构与在完全分化的表达溶菌酶的细胞中发现的模式高度相似。此外，我们发现只有在不表达的细胞中，二甲基亚砜足迹和紫外线光足迹会受到组蛋白去乙酰化抑制剂曲古抑菌素的影响。这些结果被解释为意味着大多数染色质模式的形成在终末期反式激活因子结合之前就已完成，这支持了可遗传的染色质结构对于指导发育的稳定表观遗传程序至关重要这一观点。
