Quantitative proteomic analysis of yeast DNA replication proteins

Chromatin is dynamically regulated, and proteomic analysis of its composition can provide important information about chromatin functional components. Many DNA replication proteins for example bind chromatin at specific times during the cell cycle. Proteomic investigation can also be used to characterize changes in chromatin composition in response to perturbations such as DNA damage, while useful information is obtained by testing the effects on chromatin composition of mutations in chromosome stability pathways. We have successfully used the method of stable isotope labeling by amino acids in cell culture (SILAC) for quantitative proteomic analysis of normal and pathological changes to yeast chromatin. Here we describe this proteomic method for analyzing changes to Saccharomyces cerevisiae chromatin, illustrating the procedure with an analysis of the changes that occur in chromatin composition as cells progress from a G1 phase block (induced by alpha factor) into S phase (in the presence of DNA replication inhibitor hydroxyurea).     

Isolation of nuclei for chromatin analysis in fission yeast.

The methods available for analysis of the chromatin of Schizosaccharomyces pombe are time consuming (>8 h) and/or result in some degradation of the chromatin. Here we report an optimised method for the preparation of spheroplasts and the isolation of nuclei which takes <25 min and is suitable for analysis of chromatin structure by micrococcal nuclease, restriction endonuclease or by immunoprecipitation.     

Rapid isolation of yeast plasmids as native chromatin.

Many regions of chromatin are subject to dynamic changes. We have developed a rapid method for isolation of small chromatin templates from yeast which will facilitate biochemical analysis of chromatin composition. Using the PHO5 promoter we show that templates prepared from cells grown in inducing or repressing conditions show native chromatin structures. This method may be widely applicable as the chromatin structures at a centromere, at ARS1 and at part of the lacZ region on two other plasmids are preserved after chromatin isolation.     

Easy detection of chromatin binding proteins by the histone association assay

The Histone Association Assay provides an easy approach for detecting proteins that bind chromatin in vivo. This technique is based on a chromatin immunoprecipitation protocol using histone H3-specific antibodies to precipitate bulk chromatin from crosslinked whole cell extracts. Proteins that co-precipitate with chromatin are subsequently detected by conventional SDS-PAGE and Western blot analysis. Unlike techniques that separate chromatin and nonchromatin interacting proteins by centrifugation, this method can be used to delineate whether a protein is chromatin associated regardless of its innate solubility. Moreover, the relative amount of protein bound to DNA can be ascertained under quantitative conditions. Therefore, this technique may be utilized for analyzing the chromatin association of proteins involved in diverse cellular processes.     

荧光晕法在细胞染色质结构状态检测中的应用

荧光晕法是近年来发展起来的定量检测细胞染色质结构状态的方法,由于其简易、直观、灵敏、快速,已被广泛用于放射生物学及肿瘤细胞生物学研究中。我们依据文献报道建立了此法,并对其进行了改良,进一步优化了操作步骤。并用该法检测了两种相同来源(具有相似遗传背景)、具不同辐射敏感性的细胞株的染色质结构状态,发现辐射敏感株SX-9具有更松散的染色质结构,可能与两者的辐射敏感性差异有关,为研究染色质结构状态与辐射敏感性的关系提供了佐证。     

染色质互作相关转录因子的挖掘及功能分析

染色质互作是真核生物基因组组装的基础,并且在调控真核基因细胞特异性表达中发挥重要作用.染色质互作的发生与特定的蛋白质有关,目前已经发现CTCF (CCCTC binding facor,转录阻抑物)和黏连蛋白与染色质互作相关,然而并不清楚是否还有其他蛋白质参与染色质互作.我们将整合高通量染色体构象捕获(Hi-C)和染色质免疫沉淀-测序(ChIP-seq)数据,在GM12878和K562细胞系中挖掘与染色质互作相关的转录因子,并对发现的转录因子做功能分析.我们在频繁发生互作的染色质位点中发现RUNX3、SPI1等转录因子也可能参与染色质互作.另外,通过FP-growth的数据挖掘方法还发现多个转录因子可能协同作用参与染色质互作.研究结果将为染色质互作相关实验的开展提供先验知识.     

Fractionation of L-cell chromatin into DNA, RNA, and protein fractions on Cs2SO4 equilibruim density gradients

A new procedure is described for fractionation of chromatin into DNA, RNA, and total chromatin protein. By isopycnic gradient centrifugation of chromatin preparations in Cs₂SO₄ solutions containing dimethylsulfoxide and sodium sarcosyl it is possible to obtain highly purified fractions of these components. The method gives a very high yield of these chromatin fractions unlike some other methods, where irreversible binding to columns occurs. Also with this method it is possible to obtain highly concentrated fractions, which after a simple dialysis step, can be conveniently analyzed by polyacrylamide gel electrophoresis.Nuclei from L-929 cells were isolated by a method involving citric acid or by a method using a nonionic detergent. The yields of DNA obtained by both methods was compared. Chromatin was isolated from purified nuclei (prepared in either of the above ways) in two different ways also. In one method, chromatin was extracted from nuclei with 1 m NaCl. A second method involving fractionation of lysed nuclei in sucrose and metrizamide solutions was also used. The yields of DNA obtained by both methods was compared. There appears to be little nuclear membrane contamination of any of these chromatin preparations.A preliminary analysis of L-929 cell chromatin total RNA and protein fractions on polyacrylamide and agarose gels has been made. Both fractions appear to be quite complex with a wide spectrum of subcomponents of differing S values.     

Chromatin affinity purification and quantitative mass spectrometry defining the interactome of histone modification patterns

DNA and histone modifications direct the functional state of chromatin and thereby the readout of the genome. Candidate approaches and histone peptide affinity purification experiments have identified several proteins that bind to chromatin marks. However, the complement of factors that is recruited by individual and combinations of DNA and histone modifications has not yet been defined. Here, we present a strategy based on recombinant, uniformly modified chromatin templates used in affinity purification experiments in conjunction with SILAC-based quantitative mass spectrometry for this purpose. On the prototypic H3K4me3 and H3K9me3 histone modification marks we compare our method with a histone N-terminal peptide affinity purification approach. Our analysis shows that only some factors associate with both, chromatin and peptide matrices but that a surprisingly large number of proteins differ in their association with these templates. Global analysis of the proteins identified implies specific domains mediating recruitment to the chromatin marks. Our proof-of-principle studies show that chromatin templates with defined modification patterns can be used to decipher how the histone code is read and translated.     

The organization of oligonucleosomes in yeast

We have developed a method of preparing yeast chromatin that facilitates the analysis of nucleoprotein organization. Yeast chromatin, isolated as an insoluble complex, is digested with micrococcal nuclease and fractionated into major insoluble and soluble fractions. No nucleosomal repeat is seen early in digestion for the insoluble fraction. Nucleosomal complexes of the soluble fraction are excised by nuclease in a distinctive non-random pattern; they are markedly depleted in mononucleosomes. When we analyze the soluble material by high resolution native electrophoresis, we find that the nucleoproteins resolve into two bands for each DNA multimer of the nucleosomal repeat. Our results suggest that there are structural similarities between bulk yeast chromatin and chromatin configurations found in transcribing genes of complex eukaryotes.     

Texture analysis of cervical cell nuclei by segmentation of chromatin patterns.

Texture parameters of the nuclear chromatin pattern can contribute to the automated classification of specimens on the basis of single cell analysis in cervical cytology. Current texture parameters are abstract and therefore hamper understanding. In this paper texture parameters are described that can be derived from the chromatin pattern after segmentation of the nuclear image. These texture parameters are more directly related to the visual properties of the chromatin pattern. The image segmentation procedure is based on a region grow algorithm which specifically isolates high chromatin density. The texture analysis method has been tested on a data set of images of 112 cervical nuclei on photographic negatives digitized with a step size of 0.125 micron. The preliminary results of a classification trial indicate that these visually interpretable parameters have promising discriminatory power for the distinction between negative and positive specimens.     

Advances in computational ChIA-PET data analysis

Genome-wide chromatin interaction analysis has become important for understanding 3D topological structure of a genome as well as for linking distal cis-regulatory elements to their target genes. Compared to the Hi-C method, chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) is unique, in that one can interrogate thousands of chromatin interactions (in a genome) mediated by a specific protein of interest at high resolution and reasonable cost. However, because of the noisy nature of the data, efficient analytical tools have become necessary. Here, we review some new computational methods recently developed by us and compare them with other existing methods. Our intention is to help readers to better understand ChIA-PET results and to guide the users on selection of the most appropriate tools for their own projects.     

Image preprocessing improves Fourier-based texture analysis of nuclear chromatin

OBJECTIVE: To investigate whether preprocessing of digitized images can improve the image analysis of chromatin of cytologic preparations using Fast Fourier Transformation (FFT). STUDY DESIGN: In a preprocessing step the nuclear borders of the segmented nuclei were smoothed, thus avoiding the Airy ring artifact. We tested this method, comparing the inertia values of digitalized cardiomyocyte nuclei of rats of different ages. Furthermore, we created in silicio nuclear images with chromatin alterations at or nearby the nuclear edge in order to investigate the robustness of our method. RESULTS: After preprocessing, the FFT-derived variable inertia discriminated significantly better the chromatin structure of the nuclei at different ages in every frequency range. The investigation on simulated nuclei revealed that within the frequency ranges from 1.8 microm to 0.72 microm smoothing of the borders does not interfere with the detection of chromatin changes at the nuclear border. CONCLUSION: Smoothing of borders in segmented images can improve the analysis of Fourier-derived variables of the chromatin texture.     

In vitro dynamics of chromatin organization and migration

The organization of eukaryotic chromatin is not static but changes as a function of cell status during processes such as proliferation, differentiation, and migration. DNA quantification has not been used extensively to investigate chromatin dynamics in combination with cellular migration. In this context, an optimized DNA-specific, nonperturbant method has been developed for studying chromatin organization, using the fluorescent vital bisbenzimidazole probe Hoechst 33342: this property has been described by Hamori et al. (1980). Computer-assisted image analysis was used to follow migratory activity and chromatin organization of L929 fibroblasts during in vitro wound healing. Cell movements were analyzed using an optical flow technique, which consists in the calculation of the velocity field of cells and nuclear movements in the frame. This system allows the correlation of cell migration and position in the cell cycle. It makes it possible to study chromatin dynamics using a quantitative analysis of nuclear differentiation reorganization (nuclear texture) and to correlate this with migration characteristics. The present system would be of interest for studying cell-extracellular matrix interactions using differing substrates, and also the migratory response to chemotactic factors. Such a model is a prerequisite for gaining better understanding of drug action.     

On the fidelity of deoxyribonucleic acid synthesis directed by chromatin-associated deoxyribonucleic acid polymerase beta

Accuracy of poly[d(A-T)] synthesis catalyzed by chromatin-bound deoxyribonucleic acid (DNA) polymerase beta was measured with several types. A new procedure was developed for the isolation of copied poly[d(A-T)] from chromatin DNA. This method involved in vitro copying of poly[d(A-T)] by native chromatin and subsequent selective fragmentation of chromatin by restriction nucleases, proteinase K, and heat denaturation. The fragmented natural DNA is then separated from the high molecular weight poly[d(A-T)] by gel filtration. The efficacy of DNA removal by this procedure was validated by cesium chloride gradient and nearest-neighbor analysis of the product of the reaction and by measurement of the fidelity of poly[d(A-T)] synthesis by Escherichia coli DNA Pol I contaminated with increasing amounts of DNA. Also, DNA polymerases dissociated from chromatin retain the same accuracy as that of native chromatin. Synthesis of poly[d(A-T)] by chromatin is catalyzed mainly by DNA polymerase-beta. By use of the described technique, we find that the fidelity of this reaction is exceptionally low; approximately one dGTP was incorporated for every thousand complementary nucleotides polymerized.     

The sequencing bias relaxed characteristics of Hi-C derived data and implications for chromatin 3D modeling

The 3D chromatin structure modeling by chromatin interactions derived from Hi-C experiments is significantly challenged by the intrinsic sequencing biases in these experiments. Conventional modeling methods only focus on the bias among different chromatin regions within the same experiment but neglect the bias arising from different experimental sequencing depth. We now show that the regional interaction bias is tightly coupled with the sequencing depth, and we further identify a chromatin structure parameter as the inherent characteristics of Hi-C derived data for chromatin regions. Then we present an approach for chromatin structure prediction capable of relaxing both kinds of sequencing biases by using this identified parameter. This method is validated by intra and inter cell-line comparisons among various chromatin regions for four human cell-lines (K562, GM12878, IMR90 and H1hESC), which shows that the openness of chromatin region is well correlated with chromatin function. This method has been executed by an automatic pipeline (AutoChrom3D) and thus can be conveniently used.     

Nucleases from Brevibacterium ammoniagenes differing in their effects on chromatin]

A method of isolation of three different, partially purified deoxyribonucleases from the cells of Brevibacterium ammoniagenes is descrirbed. The enzyme preparations were activated by various bivalent metal ions: 50mM MgCl2 (I), 5 mM CaCl2+5 mM MgCl2 (II), 10 mM CaCl2 (III), and had different pH optima -- 8.8 (I), 7.2 (II) and 8.2 (III). In the isolated nuclei of rat brain the first and third fractions split chromatin at the internucleosomal sites with a formation of nucleosomes -- structural subunits of chromatin. The second fraction exhibited no structural specificity for chromatin. A possible use of the enzymes for the analysis of chromatin structure is discussed.