Medrzycki Magdalena, Zhang Yunzhe, Cao Kaixiang, Fan Yuhong
School of Biology and the Parker H. Petit Institute of Bioengineering and Biosciences, Georgia Institute of Technology, Georgia, USA.
J Vis Exp. 2012 Mar 19(61):3577. doi: 10.3791/3577.
Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure. H1 is essential for mammalian development and regulates specific gene expression in vivo. Among the highly conserved histone proteins, the family of H1 linker histones is the most heterogeneous group. There are 11 H1 subtypes in mammals that are differentially regulated during development and in different cell types. These H1 subtypes include 5 somatic H1s (H1a-e), the replacement H1(0), 4 germ cell specific H1 subtypes, and H1x. The presence of multiple H1 subtypes that differ in DNA binding affinity and chromatin compaction ability provides an additional level of modulation of chromatin function. Thus, quantitative expression analysis of individual H1 subtypes, both of mRNA and proteins, is necessary for better understanding of the regulation of higher order chromatin structure and function. Here we describe a set of assays designed for analyzing the expression levels of individual H1 subtypes. mRNA expression of various H1 variant genes is measured by a set of highly sensitive and quantitative reverse transcription-PCR (qRT-PCR) assays, which are faster, more accurate and require much less samples compared with the alternative approach of Northern blot analysis. Unlike most other cellular mRNA messages, mRNAs for most histone genes, including the majority of H1 genes, lack a long polyA tail, but contain a stem-loop structure at the 3' untranslated region (UTR). Therefore, cDNAs are prepared from total RNA by reverse transcription using random primers instead of oligo-dT primers. Realtime PCR assays with primers specific to each H1 subtypes are performed to obtain highly quantitative measurement of mRNA levels of individual H1 subtypes. Expression of housekeeping genes are analyzed as controls for normalization. The relative abundance of proteins of each H1 subtype and core histones is obtained through reverse phase high-performance liquid chromatography (RP-HPLC) analysis of total histones extracted from mammalian cells. The HPLC method and elution conditions described here give optimum separations of mouse H1 subtypes. By quantifying the HPLC profile, we calculate the relative proportion of individual H1 subtypes within H1 family, as well as determine the H1 to nucleosome ratio in the cells.
连接组蛋白H1与核小体核心颗粒和连接DNA结合，促进染色质折叠成更高阶结构。H1对哺乳动物发育至关重要，并在体内调节特定基因表达。在高度保守的组蛋白中，H1连接组蛋白家族是最具异质性的群体。哺乳动物中有11种H1亚型，它们在发育过程中和不同细胞类型中受到差异调节。这些H1亚型包括5种体细胞H1（H1a - e）、替代型H1（0）、4种生殖细胞特异性H1亚型和H1x。多种H1亚型在DNA结合亲和力和染色质压缩能力上存在差异，这为染色质功能的调节提供了额外层次。因此，对单个H1亚型的mRNA和蛋白质进行定量表达分析，对于更好地理解高阶染色质结构和功能的调节是必要的。在此，我们描述了一组用于分析单个H1亚型表达水平的检测方法。通过一组高度灵敏且定量的逆转录 - PCR（qRT - PCR）检测方法来测量各种H1变体基因的mRNA表达，与Northern印迹分析这种替代方法相比，qRT - PCR检测速度更快、更准确且所需样本量少得多。与大多数其他细胞mRNA不同，大多数组蛋白基因的mRNA，包括大多数H1基因，缺乏长的多聚A尾，但在3'非翻译区（UTR）含有茎环结构。因此，使用随机引物而非寡聚dT引物通过逆转录从总RNA制备cDNA。使用针对每种H1亚型的特异性引物进行实时PCR检测，以获得单个H1亚型mRNA水平的高度定量测量。分析管家基因的表达作为标准化对照。通过对从哺乳动物细胞中提取的总组蛋白进行反相高效液相色谱（RP - HPLC）分析，获得每种H1亚型和核心组蛋白的相对丰度。此处描述的HPLC方法和洗脱条件能对小鼠H1亚型进行最佳分离。通过对HPLC图谱进行定量，我们计算H1家族中单个H1亚型的相对比例，并确定细胞中H1与核小体的比例。
