Simian Virus 40 Chromatin Interaction with the Capsid Proteins

Abstract

It has been established that both in virions and in infected cells, the cellular core histones fold the SV40 DNA into nucleosomes to form the SV40 chromosome or chromatin. We and others have begun to examine how the capsid proteins assemble the SV40 chromatin into virions and to investigate whether these proteins interact with the encapsidated chromatin. To follow the pathway of virus assembly, we have analyzed the nucleoproteins which accumulate in cells infected with the SV40 mutants temperature-sensitive in assembly: tsC, tsBC, and tsB. (The temperature-sensitivity of these mutants result from alterations in the amino acid sequence of the major capsid protein VP1). We have found that mutants belonging to the same class accumulate similar types of nucleoproteins at the nonpermissive temperature (40°C) and thus, share characteristics in common. For example, the tsC mutants accumulate only the 75 S chromatin. Both tsBC and tsB mutants produce in addition to chromatin, nucleoprotein complexes which sediment broadly from 100–160 S and contain all the three capsid proteins VP1, VP2, and VP3. These nucleoproteins can be distinguished morphologically, however. Under the electron microscope, the tsBC 100–160 S nucleoproteins appear as chromatin to which a small cluster of the capsid proteins is attached; the tsB nucleoproteins appear as partially assembled virions. In addition, we find that the 220 S virions are assembled in cells coinfected with tsB and tsC mutants at 40°C, in agreement with genetic analysis. Our observations favor the hypothesis that the VP1 protein contains three discrete domains. We speculate that each domain may play a specific function in SV40 assembly. To gain more insight into VP1-VP1 interactions, we have examined the nucleoproteins which result from treatment of the mature wild-type virions with increasing concentrations of the reducing agent DTT. In the presence of as low a concentration of DTT as 0.1 mM, the virion shell can be penetrated by micrococcal nuclease, which then cleaves the viral DNA. This result indicates that some of the disulfide bonds bridging the VP1 proteins are on the virion surface.     

Simian Virus 40 Gene A Regulates the Association Between a Highly Phosphorylated Protein and Chromatin and Ribosomes in Simian Virus 40-Transformed Cells

The species of proteins associated with chromatin and ribosomes of simian virus 40 (SV40)-transformed and untransformed monkey, mouse, and rat cells have been compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after phosphorylation of these proteins either in vivo or in vitro. In vitro phosphorylation was carried out by protein kinase associated with these organelles and [gamma-(32) P]ATP as the phosphoryl donor. The reaction products contained both phosphoserine and phosphothreonine in approximately equal amounts. The electrophoretic analysis of the phosphorylated proteins revealed that the highly phosphorylated protein with a molecular weight of approximately 90,000 (90K protein) was associated with chromatin and ribosomes from transformed cells but not from untransformed cells. The 90K protein could be extracted from chromatin and ribosomes with 0.5 to 1.0 M NaCl or KCl. The 90K protein was still associated with the runoff ribosomes prepared by the puromycin reaction of the post-mitochondrial supernatant in the protein-synthesizing system. In vitro phosphorylation of chromatin and ribosomes from SV40 tsA-transformed mouse and rat cells indicated that the amounts of 90K protein associated with these organelles decreased greatly when the cells were cultivated at the restrictive temperature. A similar temperature-dependent decrease in the amount of (32)P-labeled 90K protein was observed in nonhistone chromosomal and ribosome-associated protein fractions prepared from SV40 tsA-transformed cells labeled with [(3)H]leucine and [(32)P]orthophosphate in vivo. In vitro phosphorylated 90K protein in nonhistone chromosomal and ribosome-associated proteins extracted with high salt was not immunoprecipitated with anti-SV40 T sera.     

染色质结构之美——染色质可及性

染色质可及性(chromatin accessibility)作为一种衡量染色质结合因子与染色质DNA结合能力高低的染色质属性,是评价染色质结构稳态的重要指标之一,在多种细胞核进程中扮演重要角色,包括基因转录调控以及DNA损伤修复等。该属性的异常调控与多种疾病的发生发展密切相关,包括肿瘤以及神经退行性疾病等。对于该属性探究已经成为生命科学与疾病领域的热点。伴随越来越多的新技术应运而生,例如染色质构象捕获技术、高通量测序技术以及两种技术的结合等。随着技术的进步,多种参与调控染色质可及性的因素被发现和总结,包括核小体占位、组蛋白修饰以及非编码RNA等。多项大规模的染色质组学数据绘制了多种疾病的染色质可及性图谱,为揭示疾病的发生发展与染色质可及性之间的关系提供了数据支持。同时,随着单细胞染色质可及性测序技术的发展,实现了对细胞类型染色质层面的划分,弥补了单纯依赖基因表达划分细胞类型的不足。本文将从染色质的组成与可及性、影响染色质可及性的因素、染色质可及性的检测方法,以及染色质可及性与癌症的关系等方面简要阐述染色质可及性的研究进展。     

Chromatin remodelling

Epigenetic mechanisms are heritable traits that are mediated by changes in a genetic locus that do not involve a modification at the nucleotide level. As eukaryotic DNA is organised in chromatin units, epigenetic modifications can be mediated by chromatin remodelling. Although there are a number of well-characterised chromatin remodelling factors to which we can allocate a defined molecular function, we need to understand chromatin remodelling processes as the combined effects of such factors in higher order complexes.     

Chromatin Hydrodynamics

Following recent observations of large scale correlated motion of chromatin inside the nuclei of live differentiated cells, we present a hydrodynamic theory—the two-fluid model—in which the content of a nucleus is described as a chromatin solution with the nucleoplasm playing the role of the solvent and the chromatin fiber that of a solute. This system is subject to both passive thermal fluctuations and active scalar and vector events that are associated with free energy consumption, such as ATP hydrolysis. Scalar events drive the longitudinal viscoelastic modes (where the chromatin fiber moves relative to the solvent) while vector events generate the transverse modes (where the chromatin fiber moves together with the solvent). Using linear response methods, we derive explicit expressions for the response functions that connect the chromatin density and velocity correlation functions to the corresponding correlation functions of the active sources and the complex viscoelastic moduli of the chromatin solution. We then derive general expressions for the flow spectral density of the chromatin velocity field. We use the theory to analyze experimental results recently obtained by one of the present authors and her co-workers. We find that the time dependence of the experimental data for both native and ATP-depleted chromatin can be well-fitted using a simple model—the Maxwell fluid—for the complex modulus, although there is some discrepancy in terms of the wavevector dependence. Thermal fluctuations of ATP-depleted cells are predominantly longitudinal. ATP-active cells exhibit intense transverse long wavelength velocity fluctuations driven by force dipoles. Fluctuations with wavenumbers larger than a few inverse microns are dominated by concentration fluctuations with the same spectrum as thermal fluctuations but with increased intensity.     

Chromatin architecture

A complete understanding of the structure-function relationships of chromatin requires extending primarily one dimensional information, obtained from molecular genetic techniques and based on the underlying linear DNA sequence, to the three dimensional conformation. Recent progress in this endeavor has included the examination of fully defined nucleosomes and nucleosomal arrays assembled in vitro using X-ray diffraction, NMR spectroscopy, electron microscopy and atomic force microscopy. These studies have provided valuable insights into the structural roles of histone variants, the impact of histone mutations and the compaction of nucleosomal arrays. In addition, the diverse structural consequences of the binding of specific chromatin 'architectural' proteins are becoming apparent. These approaches provide an essential basis for understanding the conformation of the 'epigenome'.     

Chromatin Hydrodynamics

Following recent observations of large scale correlated motion of chromatin inside the nuclei of live differentiated cells, we present a hydrodynamic theory—the two-fluid model—in which the content of a nucleus is described as a chromatin solution with the nucleoplasm playing the role of the solvent and the chromatin fiber that of a solute. This system is subject to both passive thermal fluctuations and active scalar and vector events that are associated with free energy consumption, such as ATP hydrolysis. Scalar events drive the longitudinal viscoelastic modes (where the chromatin fiber moves relative to the solvent) while vector events generate the transverse modes (where the chromatin fiber moves together with the solvent). Using linear response methods, we derive explicit expressions for the response functions that connect the chromatin density and velocity correlation functions to the corresponding correlation functions of the active sources and the complex viscoelastic moduli of the chromatin solution. We then derive general expressions for the flow spectral density of the chromatin velocity field. We use the theory to analyze experimental results recently obtained by one of the present authors and her co-workers. We find that the time dependence of the experimental data for both native and ATP-depleted chromatin can be well-fitted using a simple model—the Maxwell fluid—for the complex modulus, although there is some discrepancy in terms of the wavevector dependence. Thermal fluctuations of ATP-depleted cells are predominantly longitudinal. ATP-active cells exhibit intense transverse long wavelength velocity fluctuations driven by force dipoles. Fluctuations with wavenumbers larger than a few inverse microns are dominated by concentration fluctuations with the same spectrum as thermal fluctuations but with increased intensity.