Histone H1 binding is restricted by histone variant H3.3 (FAIRE). Drosophila melanogaster

Linker histones are involved in the formation of higher-order chromatin structure. Although linker histones have been implicated in the regulation of specific genes, it still remains unclear what their principal binding determinants are and how their repressive function in vitro can be reconciled with presumed broad binding in vivo. We generated a full genome, high resolution binding map of linker histone H1 in Drosophila Kc cells, using DamID. H1 binds at similar levels across much of the genome, both in classical euchromatin and heterochromatin. Strikingly, there are pronounced dips of low H1 occupancy around transcription start sites of active genes and at many distant cis-regulatory sites. H1 dips are not due to lack of nucleosomes. Rather, all regions with low binding of H1 show enrichment of the histone variant H3.3 which itself has been linked to high nucleosome turnover. Upon knockdown of H3.3, we find that H1 levels increase at sites previously not covered with H1 with a concomitant increase in nucleosome repeat length. These changes are independent of transcriptional changes. Our results show that the H3.3 protein counteracts association of H1 at genomic sites with high rates of histone turnover. This antagonism provides a mechanism to keep diverse genomic sites in an open chromatin conformation. For this study, we generated DamID profiles of histone H1 and RpII18 and a FAIRE profile in Drosophila Kc167 cells. Additionally, we generated H1 profiles in cells treated with RNAi against white, H3.3B, or H3.3A and H3.3B. Nucleosome positioning profiles were generated in untreated cells and cells treated with RNAi against white, H3.3B, or H3.3A and H3.3B. Profiles of expression changes were generated for H3.3B RNAi and H3.3A and H3.3B RNAi. Overall design: DamID experiments for H1 and RpII18 were performed in Drosophila cell cultures. Samples were hybridized to 380k NimbleGen arrays with 300 bp probe spacing. Formaldehyde-assisted isolation of regulatotry elements (FAIRE) was performed in Drosophila Kc167 cells. Samples were hybridized to 380k NimbleGen arrays with 300 bp probe spacing over non-crosslinked genomic DNA. Nucleosome positioning profiles were made by hybridizing mononucleosomal DNA over MNase digested purified genomic DNA on 380k NimbleGen arrays with 10 bp probe spacing. Expression profiles were made as H3.3 RNAi over white RNAi cohybridizations on spotted INDAC long oligo arrays. Every experiment was done in duplicate in the reverse dye orientation.

连接组蛋白（linker histones）参与高阶染色质结构的形成。尽管连接组蛋白已被证实参与特定基因的调控，但其核心结合决定因素究竟是什么，以及如何协调其体外的抑制功能与体内推测的广泛结合特性，目前仍未明确。

我们利用DamID技术，构建了果蝇Kc细胞中连接组蛋白H1的全基因组高分辨率结合图谱。H1在基因组大部分区域的结合水平相近，既分布于经典常染色质区域，也存在于异染色质区域。值得注意的是，在活性基因的转录起始位点周围以及诸多远端顺式调控位点处，存在显著的H1结合占有率降低的凹陷区域。此类H1结合凹陷并非源于核小体缺失；相反，所有H1结合水平较低的区域均富集组蛋白变体H3.3，而H3.3本身已被证实与高核小体周转率相关。在敲低H3.3后，我们发现原本未被H1结合的位点处H1水平显著升高，同时核小体重复长度也随之增加，且此类变化与转录水平改变无关。

本研究结果证实，H3.3可在组蛋白周转率较高的基因组位点上拮抗H1的结合，这种拮抗作用为维持多样化基因组位点处于开放染色质构象提供了潜在机制。

本研究中，我们在果蝇Kc167细胞内生成了组蛋白H1与RpII18的DamID图谱，以及甲醛辅助调控元件分离（FAIRE, Formaldehyde-assisted isolation of regulatory elements）图谱。此外，我们还在经RNA干扰（RNAi, RNA interference）靶向white、H3.3B或H3.3A与H3.3B的细胞中构建了H1结合图谱。核小体定位图谱分别在未处理细胞，以及经RNAi靶向white、H3.3B或H3.3A与H3.3B的细胞中生成。针对H3.3B RNAi以及H3.3A与H3.3B RNAi的样本，我们还生成了表达变化图谱。

整体实验设计如下：针对H1和RpII18的DamID实验在果蝇细胞培养体系中完成，样本与探针间距为300 bp的380k NimbleGen阵列进行杂交。甲醛辅助调控元件分离实验在果蝇Kc167细胞中开展，样本与探针间距为300 bp的380k NimbleGen阵列杂交，对照为未交联的基因组DNA。核小体定位图谱通过将微球菌核酸酶（MNase, Micrococcal nuclease）消化纯化的单核小体DNA，与探针间距为10 bp的380k NimbleGen阵列杂交获得。表达谱分析通过将H3.3 RNAi样本与white RNAi样本进行反向染料标记的双色杂交，使用点样的INDAC长寡核苷酸阵列完成。所有实验均采用反向染料方向进行重复实验。