Histone H1 is the most mobile histone in the cell nucleus. Defining the positions of H1 on chromatin in situ, therefore, represents a challenge. Immunoprecipitation of formaldehyde-fixed and sonicated chromatin, followed by DNA sequencing (xChIP-seq), is traditionally the method for mapping histones onto DNA elements. But since sonication fragmentation precedes ChIP, there is a consequent loss of information about chromatin higher-order structure. Here, we present a new method, xxChIP-seq, employing antibody binding to fixed intact in situ chromatin, followed by extensive washing, a second fixation, sonication and immunoprecipitation. The second fixation is intended to prevent the loss of specifically bound antibody during washing and subsequent sonication and to prevent antibody shifting to epitopes revealed by the sonication process. In many respects, xxChIP-seq is comparable to immunostaining microscopy, which also involves interaction of the primary antibody with fixed and permeabilized intact cells. The only epitopes displayed after immunostaining are the “exposed” epitopes, not “hidden” by the fixation of chromatin higher-order structure. Comparison of immunoprecipitated fragments between xChIP-seq versus xxChIP-seq should indicate which epitopes become inaccessible with fixation and identify their associated DNA elements. We determined the genomic distribution of histone variants H1.2 and H1.5 in human myeloid leukemia cells HL-60/S4 and compared their epitope exposure by both xChIP-seq and xxChIP-seq, as well as high-resolution microscopy, illustrating the influences of preserved chromatin higher-order structure in situ. We found that xChIP and xxChIP H1 signals are in general negatively correlated, with differences being more pronounced near active regulatory regions. Among the intriguing observations, we find that transcription-related regions and histone PTMs (i.e., enhancers, promoters, CpG islands, H3K4me1, H3K4me3, H3K9ac, H3K27ac and H3K36me3) exhibit significant deficiencies (depletions) in H1.2 and H1.5 xxChIP-seq reads, compared to xChIP-seq. These observations suggest the existence of in situ transcription-related chromatin higher-order structures stabilized by formaldehyde. Comparison of H1 xxChIP-seq to H1 xChIP-seq allows the development of hypotheses on the chromosomal localization of (stabilized) higher-order structure, indicated by the generation of “hidden” H1 epitopes following formaldehyde crosslinking. Changes in H1 epitope exposure surrounding averaged chromosomal binding sites or epigenetic modifications can also indicate whether these sites have chromatin higher-order structure. For example, comparison between averaged active or inactive promoter regions suggests that both regions can acquire stabilized higher-order structure with hidden H1 epitopes. However, the H1 xChIP-seq comparison cannot define their differences. Application of the xxChIP-seq versus H1 xChIP-seq method is particularly relevant to chromatin-associated proteins, such as linker histones, that play dynamic roles in establishing chromatin higher-order structure.

中文翻译：

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接头组蛋白表位被原位高阶染色质结构隐藏。

组蛋白H1是细胞核中最易移动的组蛋白。因此，在染色质上原位确定H1的位置是一个挑战。甲醛固定和超声处理的染色质的免疫沉淀，然后进行DNA测序（xChIP-seq），传统上是将组蛋白定位到DNA元件上的方法。但是，由于超声破碎先于ChIP，因此会丢失有关染色质高阶结构的信息。在这里，我们提出了一种新方法，即xxChIP-seq，它采用与固定的完整原位染色质结合的抗体，然后进行大量洗涤，第二次固定，超声处理和免疫沉淀。第二种固定是为了防止在洗涤和随后的超声处理期间丢失特异性结合的抗体，并防止抗体转移到超声处理过程中揭示的表位。在许多方面，xxChIP-seq可与免疫染色显微镜相媲美，后者还涉及一抗与固定和透化的完整细胞的相互作用。免疫染色后显示的唯一表位是“暴露的”表位，而不是被染色质高阶结构固定的“隐藏”表位。比较xChIP-seq和xxChIP-seq之间的免疫沉淀片段，应表明哪些表位固定不易进入并鉴定其相关的DNA元件。我们确定了人类髓样白血病细胞HL-60 / S4中组蛋白变体H1.2和H1.5的基因组分布，并通过xChIP-seq和xxChIP-seq以及高分辨率显微镜对它们的表位暴露进行了比较，从而说明了染色质高阶结构原位的影响 我们发现xChIP和xxChIP H1信号通常呈负相关，差异在活跃的调节区域附近更为明显。在有趣的观察中，我们发现与转录相关的区域和组蛋白PTM（即增强子，启动子，CpG岛，H3K4me1，H3K4me3，H3K9ac，H3K27ac和H3K36me3）在H1.2和H1.5 xxChIPIP中表现出明显的缺陷（耗竭）。 -seq读取，与xChIP-seq相比。这些观察结果表明存在由甲醛稳定的原位转录相关的染色质高阶结构。H1 xxChIP-seq与H1 xChIP-seq的比较允许对（稳定的）高阶结构的染色体定位进行假说发展，这表明甲醛交联后“隐藏”的H1表位的产生。H1表位暴露在平均染色体结合位点周围或表观遗传修饰周围的变化也可以表明这些位点是否具有染色质高阶结构。例如，平均活性启动子区域或非活性启动子区域之间的比较表明，两个区域均可获得具有隐藏的H1表位的稳定的高阶结构。但是，H1 xChIP-seq比较无法定义它们之间的差异。xxChIP-seq与H1 xChIP-seq方法的应用与染色质相关蛋白（如接头组蛋白）特别相关，它们在建立染色质高阶结构中起动态作用。平均启动子区域或非启动子区域之间的比较表明，两个区域均可获得具有隐藏的H1表位的稳定的高阶结构。但是，H1 xChIP-seq比较无法定义它们之间的差异。xxChIP-seq与H1 xChIP-seq方法的应用与染色质相关蛋白（如接头组蛋白）特别相关，它们在建立染色质高阶结构中起动态作用。平均启动子区域或非启动子区域之间的比较表明，两个区域均可获得具有隐藏的H1表位的稳定的高阶结构。但是，H1 xChIP-seq比较无法定义它们之间的差异。xxChIP-seq与H1 xChIP-seq方法的应用与染色质相关蛋白（如接头组蛋白）特别相关，它们在建立染色质高阶结构中起动态作用。