Abstract
Chromatin structure, a key contributor to the regulation of gene expression, is modulated by a broad array of histone post-translational modifications (PTMs). Taken together, these “histone marks” comprise what is often referred to as the “histone code”. The quantitative analysis of histone PTMs by mass spectrometry (MS) offers the ability to examine the response of the histone code to physiological signals. However, few studies have examined the stability of histone PTMs through the process of isolating and culturing primary cells. To address this, we used bottom-up, MS-based analysis of histone PTMs in liver, freshly isolated hepatocytes, and cultured hepatocytes from adult male Fisher F344 rats. Correlations between liver, freshly isolated cells, and primary cultures were generally high, with R2 values exceeding 0.9. However, a number of acetylation marks, including those on H2A K9, H2A1 K13, H3 K4, H3 K14, H4 K8, H4 K12 and H4 K16 differed significantly among the three sources. Inducing proliferation of primary adult hepatocytes in culture affected several marks on histones H3.1/3.2 and H4. We conclude that hepatocyte isolation, culturing and cell cycle status all contribute to steady-state changes in the levels of a number of histone PTMs, indicating changes in histone marks that are rapidly induced in response to alterations in the cellular milieu. This has implications for studies aimed at assigning biological significance to histone modifications in tumors versus cancer cells, the developmental behavior of stem cells, and the attribution of changes in histone PTMs to altered cell metabolism.
Highlights
Nucleosomes, the basic repeating units of eukaryotic chromatin, are formed by the wrapping of DNA around histone octamers composed of two copies of each of the four core histones: H2A, H2B, H3 and H4 [1]
We hypothesized that changes in the cellular environment that occur during hepatocyte isolation and culture could result in changes in histone post-translational modifications (PTMs)
Histones prepared from liver tissue were studied as a condition reflecting the in vivo metabolic and hormonal milieu and the microenvironment in which hepatocytes reside in the intact liver
Summary
Nucleosomes, the basic repeating units of eukaryotic chromatin, are formed by the wrapping of DNA around histone octamers composed of two copies of each of the four core histones: H2A, H2B, H3 and H4 [1]. The structure of chromatin plays an important role in the regulation of gene expression by determining the accessibility of specific regions of DNA [2]. Critical to this relationship between chromatin structure and gene expression is a broad array of post-translational modifications (PTMs) to which histones are subject [3]. The modifications at specific histone sites are often referred to as the “histone code”. As per a recent survey of the literature, known histone modifications occur at over two hundred and thirty sites, and total more than five hundred [4], offering the potential for extraordinary combinatorial modification and highly complex regulation of gene expression
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