一个正在分裂的细胞必须准确复制其基因组，以使每个子细胞都能接受一个完整的配套基因。复制中所出现的缺陷会导致基因组不稳定和癌症。令人吃惊的是，人们对某些染色体区域是怎样被选择来启动基因复制的知之甚少。基因复制被认为从特定位置开始，这些特定位置即所谓的“起点”，但能代表这些位置特征的DNA序列一直没有发现，这使得人们想到其他染色体结构可能影响基因复制从哪里开始和什么时候开始。现在，Aggarawal和Calvi用果蝇中一个人们已经非常了解的模型起点研究表明，情况正是这样。在发育过程中变动起点上的组蛋白对它们的调节来说很重要。调节这一变动的蛋白中有一种是Rpd3，人们知道它在人体中帮助 Retinoblastoma (Rb)和其他蛋白抑制肿瘤形成。于是，研究人员便想到这样一个可能性：因消除Rb的抑制作用所产生的致癌效应部分原因可能是由于基因组的不稳定性。
Nature 430, 372 - 376 (15 July 2004); doi:10.1038/nature02694
Chromatin regulates origin activity in Drosophila follicle cells
It is widely believed that DNA replication in multicellular animals (metazoa) begins at specific origins to which a pre-replicative complex (pre-RC) binds1. Nevertheless, a consensus sequence for origins has yet to be identified in metazoa. Origin identity can change during development, suggesting that there are epigenetic influences. A notable example of developmental specificity occurs in Drosophila, where somatic follicle cells of the ovary transition from genomic replication to exclusive re-replication at origins that control amplification of the eggshell (chorion) protein genes2. Here we show that chromatin acetylation is critical for this developmental transition in origin specificity. We find that histones at the active origins are hyperacetylated, coincident with binding of the origin recognition complex (ORC). Mutation of the histone deacetylase (HDAC) Rpd3 induced genome-wide hyperacetylation, genomic replication and a redistribution of the origin-binding protein ORC2 in amplification-stage cells, independent of effects on transcription. Tethering Rpd3 or Polycomb proteins to the origin decreased its activity, whereas tethering the Chameau acetyltransferase increased origin activity. These results suggest that nucleosome acetylation and other epigenetic changes are important modulators of origin activity in metazoa.
Figure 1 Histone hyperacetylation and ORC2 co-localize at chorion origins. a, Organization of the 3rd chromosome chorion locus. Arrows represent the four chorion genes, grey boxes represent the five regions that contribute to amplification. The two most important, ACE3 and Ori- are binding sites for ORC. SalI sites (S) define the 3.8-kb fragment used in subsequent experiments. b–d, Labelling of stage-10B follicle cells with poly-acetylated histone H4 (AcH4) antibody (green; b), ORC2 (red; c), and merge (yellow; d). The two brightest foci represent the chorion locus on the 3rd and X chromosome. e, Representation of ORC (red) binding to ACE3/Ori- on amplified chorion DNA fibres based on previous reports6,10,13. f, High magnification showing co-localization of ORC2 (red) and AcH4 (green) at the 3rd chorion origin in stage 11. g, AcH4 (green) does not co-localize with Dup protein (red) which labels replication forks that migrate bi-directionally outward from the 3rd chorion origin13. Scale bars represent 10 µm (b–d) and 3 µm (f, g).
Figure 3 Rpd3 loss-of-function clones show hyperacetylation, increased replication and altered ORC2 distribution. Homozygous mutant Rpd3m5-5-follicle-cell clones were generated using the FLP/FRT technique. a, c, e, g, i, Low magnification of stage-10B egg chambers. b, d, f, h, j, Higher magnification images of clones designated by arrows. a, b, Mutant clones were identified in stage-10B egg chambers by the absence of green fluorescent protein (GFP) fluorescence (green), and labelling for AcH4 indicated that most Rpd3m5-5 mutant cells within small clones have nucleus-wide hyperacetylation (red). Rare regional hyperacetylation can be seen for one cell in the lower part of the clone in (b). c, d, Many cells in small clones also had inappropriate nucleus-wide incorporation of BrdU, instead of the focal staining at chorion loci seen in neighbouring wild-type cells (see Supplementary Fig. S7). e–j, Orc2, AcH4 double labelling. Acetylation (red; e, f), ORC2 (green; g, h), merge (yellow; i, j). ORC2 was distributed throughout the nucleus in 51% of clones comprised of ten or fewer cells (n = 242 clones; see Supplementary Fig. S8). Note that outside the clones the green colour is from ORC2 and GFP. The asterisk in f, h and j indicates a nucleus that contains twice the DNA content of its neighbours, as measured by total DAPI fluorescence. Scale bars represent 20 µm (a, c, e, g, i) and 10 µm (b, d, f, h, j).
Figure 4 Sodium butyrate induces extra replication, which is not blocked by -amanitin. a–c, BrdU incorporation in stage-10B follicle cells (untreated, a; sodium butyrate, b; -amanitin and sodium butyrate, c). The arrow in b points to intense focal incorporation in one chromocentre. d–f, Anti-Myc labelling as a control for the effectiveness of -amanitin to inhibit transcription of a strong hsp70:Myc reporter in follicle cells (no heat induction, d; heat induction without -amanitin, e; heat induction with -amanitin, f). g, The percentage of total egg chambers that had genomic BrdU incorporation (grey bars) or Myc labelling (black bars) after the indicated treatments. Bars represent average and standard deviation of three experiments; scale bars represent 10 µm.
Figure 5 Tethering chromatin modifiers to chorion origins alters their activity. a, Method to tether chromatin modifiers to the chorion origin. TT1 contains the 3.8-kb minimal origin from the 3rd chromosome chorion locus, which contains ACE3 and Ori- (dark-grey boxes; see Fig. 1a), adjacent to GAL4-binding sites (UAS, striped box), and is marked with the mini-white gene (w + ). GAL4 fusions contain the heat-inducible hsp70 promoter (light-grey box) driving expression of the GAL4-DNA-binding domain (DBD) to Rpd3 (white boxes), or other chromatin modifiers. Boxes with arrowheads are the inverted repeats of the P element transformation vector. Heat-induced expression in progeny with both P elements results in binding of GAL4:Rpd3 to the UAS sites next to the origin in TT1. b–i, Single nuclei with representative FISH labelling of the endogenous 3rd chromosome chorion locus (arrow) and TT1 (arrowhead). b, c, GAL4DBD only. d, e, Rpd3 fusion.f, g, HAT1 fusion. h, i, Polycomb fusion. Uninduced (b, d, f, h), induced (c, e, g, i) GAL4 fusion. The variable nuclear morphology results from the squashing technique for FISH. j, Percentage of amplification for TT1 relative to uninduced sibling controls that were normalized to 100%. Females contained GAL4DBD alone or the indicated fusions. TT1 levels were measured by: the average ratio of TT1 to endogenous fluorescence intensity (white bars); the fraction of nuclei with endogenous 3rd chorion labelling that had a detectable TT1 spot (black bars); and Southern blotting (striped bars). Each bar represents the average and standard error of the mean for three to four independent experiments. ***P < 0.0001 for mean of TT1/3rd locus ratio between induced and uninduced controls by t test. Scale bar represents 5 µm (b–i).
