Turner B M, Franchi L, Wallace H
School of Basic Medical Sciences, Department of Anatomy, University of Birmingham Medical School, UK.
J Cell Sci. 1990 Jun;96 ( Pt 2):335-46. doi: 10.1242/jcs.96.2.335.
The four histones of the nucleosome core particle are all subject to enzyme-catalysed, post-translational acetylation at defined lysine residues in their amino-terminal domains. Much circumstantial evidence suggests a role for this process in modifying chromatin structure and function, but detailed mechanisms have not been defined. To facilitate studies on the functional significance of histone acetylation, we have prepared antibodies specific for the acetylated isoforms of histone H4. Because of the extreme evolutionary conservation of H4, these antisera can be applied to a wide variety of organisms and experimental systems. In the present study we have used polytene chromosomes from the salivary glands of larvae of the midge Chironomus to examine the distribution of acetylated H4 in interphase chromatin. By indirect immunofluorescence, antisera to acetylated H4 labeled the four Chironomus chromosomes with reproducible patterns of sharply defined, fluorescent bands. An antiserum to non-acetylated H4 gave a completely different, more-diffuse labelling pattern. Thus, there are defined regions, or islands, in the interphase genome that are enriched in acetylated H4. Double-labelling experiments with two antisera specific for H4 molecules acetylated at different sites, showed that each antiserum gave the same banding pattern. Immunolabelling patterns were not dependent on the pattern of phase-dense bands characteristic of these chromosomes; strongly labelled regions could correspond to phase-dense bands (i.e. condensed chromatin), to interbands or, frequently, to band-interband junctions. Immunogold electron microscopy confirmed the immunofluorescence results and showed further that regions of relatively high labelling could be either transcriptionally active or quiescent, as judged by the presence or absence of ribonucleoprotein particles. Two rapidly transcribed genes on chromosome 4 of Chironomus form characteristic 'puffs', the Balbiani rings BRb and BRc. The antiserum to non-acetylated H4 gave diffuse labelling throughout these puffs, demonstrating the continued presence of this histone in these transcriptionally active regions. Antisera to acetylated H4 strongly labelled the boundaries of BRb and BRc, and revealed clearly defined islands of increased H4 acetylation just within the expanded chromatin of the puffs. Labelling within the central region of each puff was much less intense. A similar pattern was observed in puffs on other chromosomes. Thus, increased H4 acetylation is not found throughout actively transcribed chromatin but occurs only at defined sites, possibly in the non-transcribed flanking regions. H4 acetylation is clearly not required for the passage of RNA polymerase through the nucleosome and we speculate that its role may be to facilitate the binding to DNA of polymerases and other proteins prior to the onset of transcription and possibly replication.
核小体核心颗粒的四种组蛋白在其氨基末端结构域中特定的赖氨酸残基上均会发生酶催化的翻译后乙酰化。大量间接证据表明该过程在染色质结构和功能的修饰中起作用，但具体机制尚未明确。为便于研究组蛋白乙酰化的功能意义，我们制备了针对组蛋白H4乙酰化异构体的特异性抗体。由于H4具有高度的进化保守性，这些抗血清可应用于多种生物体和实验系统。在本研究中，我们使用摇蚊幼虫唾液腺的多线染色体来检测乙酰化H4在间期染色质中的分布。通过间接免疫荧光法，针对乙酰化H4的抗血清以可重复的、清晰界定的荧光带模式标记了摇蚊的四条染色体。针对非乙酰化H4的抗血清给出了完全不同的、更弥散的标记模式。因此，在间期基因组中存在特定区域或岛，其中富含乙酰化H4。用两种针对不同位点乙酰化的H4分子的特异性抗血清进行的双标记实验表明，每种抗血清都给出相同的条带模式。免疫标记模式并不依赖于这些染色体特有的相致密带模式；强标记区域可能对应于相致密带（即浓缩染色质）、带间区，或经常对应于带 - 带间区交界处。免疫金电子显微镜证实了免疫荧光结果，并进一步表明，根据核糖核蛋白颗粒的有无判断，标记相对较高的区域可能是转录活跃的或静止的。摇蚊第4号染色体上的两个快速转录基因形成特征性的“胀泡”，即巴尔比亚尼环BRb和BRc。针对非乙酰化H4的抗血清在这些胀泡中给出弥散标记，表明该组蛋白在这些转录活跃区域持续存在。针对乙酰化H4的抗血清强烈标记了BRb和BRc的边界，并在胀泡扩展的染色质内清晰地显示出H4乙酰化增加的明确岛状区域。每个胀泡中心区域的标记强度要低得多。在其他染色体上的胀泡中也观察到类似模式。因此，在活跃转录的染色质中并非处处都有H4乙酰化增加，而是仅发生在特定位点，可能在非转录的侧翼区域。显然，RNA聚合酶通过核小体并不需要H4乙酰化，我们推测其作用可能是在转录和可能的复制开始之前促进聚合酶和其他蛋白质与DNA的结合。
