Modifications Of Chromosomal Proteins Research Articles

DNA activates the Nse2/Mms21 SUMO E3 ligase in the Smc5/6 complex.

Modification of chromosomal proteins by conjugation to SUMO is a key step to cope with DNA damage and to maintain the integrity of the genome. The recruitment of SUMO E3 ligases to chromatin may represent one layer of control on protein sumoylation. However, we currently do not understand how cells upregulate the activity of E3 ligases on chromatin. Here we show that the Nse2 SUMO E3 in the Smc5/6 complex, a critical player during recombinational DNA repair, is directly stimulated by binding to DNA Activation of sumoylation requires the electrostatic interaction between DNA and a positively charged patch in the ARM domain of Smc5, which acts as a DNA sensor that subsequently promotes a stimulatory activation of the E3 activity in Nse2. Specific disruption of the interaction between the ARM of Smc5 and DNA sensitizes cells to DNA damage, indicating that this mechanism contributes to DNA repair. These results reveal a mechanism to enhance a SUMO E3 ligase activity by direct DNA binding and to restrict sumoylation in the vicinity of those Smc5/6-Nse2 molecules engaged on DNA.

Reversal of cell differentiation in eggs and embryos

Oocytes and eggs of mammals and amphibia have a remarkable capacity to reprogram the nuclei of adult differentiated cells towards embryonic gene expression. They achieve this with natural components and more efficiently than by transcription factor overexpression or cell fusion. The success with which reprogramming is achieved by all procedures declines dramatically as the nuclei of increasingly differentiated cells are used. Current work aims to (1) identify the reprogramming components of eggs and oocytes, and (2) to identify the components of differentiated cells that restrict the responsiveness of their nuclei to reprogramming. The mechanisms used by eggs and oocytes to reprogram somatic cell nuclei include major changes to the composition of chromatin, and involve the exchange and modification of chromosomal proteins. These changes result in a global transcription of genes. Progress in identifying reprogramming factors as well as components of chromatin that resist reprogramming will be reviewed. Source of support: Wellcome Trust/Cancer Research UK

Rod/Zw10 Complex Is Required for PIASy-dependent Centromeric SUMOylation

SUMO conjugation of cellular proteins is essential for proper progression of mitosis. PIASy, a SUMO E3 ligase, is required for mitotic SUMOylation of chromosomal proteins, yet the regulatory mechanism behind the PIASy-dependent SUMOylation during mitosis has not been determined. Using a series of truncated PIASy proteins, we have found that the N terminus of PIASy is not required for SUMO modification in vitro but is essential for mitotic SUMOylation in Xenopus egg extracts. We demonstrate that swapping the N terminus of PIASy protein with the corresponding region of other PIAS family members abolishes chromosomal binding and mitotic SUMOylation. We further show that the N-terminal domain of PIASy is sufficient for centromeric localization. We identified that the N-terminal domain of PIASy interacts with the Rod/Zw10 complex, and immunofluorescence further reveals that PIASy colocalizes with Rod/Zw10 in the centromeric region. We show that the Rod/Zw10 complex interacts with the first 47 residues of PIASy which were particularly important for mitotic SUMOylation. Finally, we show that depletion of Rod compromises the centromeric localization of PIASy and SUMO2/3 in mitosis. Together, we demonstrate a fundamental mechanism of PIASy to localize in the centromeric region of chromosome to execute centromeric SUMOylation during mitosis.

Study of Nuclear Receptor-Induced Transcription Complex Assembly and Histone Modification by Chromatin Immunoprecipitation Assays

This chapter focuses on the study of nuclear receptor-induced transcription complex assembly and histone modification by chromatin immunoprecipitation (ChIP) assays. Nuclear receptor-mediated transcriptional regulation is a complex process involving the dynamic assembly of multiprotein complexes and modifications of chromosomal proteins, such as histones, at the target gene promoter. An increasingly used method to study this process in the context of natural chromosome structure is the chromatin immunoprecipitation assay. The ChIP assay employs a combination of formaldehyde induced in vivo cross-linking, immunoprecipitation, and sequence-specific DNA detection methods to observe the specific proteins associated with specific gene promoter regions in tissues or cultured cells. During the procedure of the ChIP assay a nuclear lysate is prepared from formaldehyde cross-linked cells that have been previously treated with or without hormones. ChIP assays allow the accurate assessment of the protein complexes associated with a given promoter during transcriptional regulation, spatially and temporally. The proteins of interest that can be examined by the use of specific antibodies include transcription factors that bind directly to the DNA, coactivators, corepressors, RNA polymerase and its associated proteins, and modified histones. ChIP assays have been applied to study nuclear receptor-mediated cofactor complex assembly and histone modifications at target gene promoters. The amount of antibody used in the immunoprecipitation depends on the affinity of the specific antibody, as well as the abundance of the chromatin-associated proteins and protein modifications.

Chromosomal imprinting in plants

Chromosomal imprints in the broadest sense can arise in somatic as well as germline cells. They can be imposed through the modification of chromosomal proteins or by the modification of chromosomal DNA, and they typically effect the expression of nearby genes. Modification enzymes — such as histone deacetylases and cytosine methyltransferases, as well as chromatin components — are known to play this role in animals and many of these same enzymes and components have been found in plants. Transposable elements are subject to chromosomal imprinting and may play a fundamental role in this process in plant and other eukaryotic genomes.

Inhibition of condensation in human chromosomes induced by the thymidine analogue 5-iododeoxyuridine.

Human lymphocyte cultures were treated with iododeoxyuridine. This 5-halogenated thymidine analogue induces distinct undercondensations of the heterochromatin of human chromosome 9. The condensation of the other heterochromatic regions on chromosomes 1, 15, 16 and Y is also inhibited, but to a lesser extent. Optimum cell culture conditions required for the induction of undercondensations were determined. Up to 90% of mitotic cells reveal chromosome 9 heterochromatin decondensations when the substance is present in quantities of 1 x 10(-4) M during the last 7 h before cell fixation. In addition, examples of the usefulness of 5-IUDR in the analysis of chromosome aberrations involving chromosome 9 are presented. The interaction between 5-IUDR and chromosomal DNA, the modification of chromosomal proteins and factors inducing chromosomal decondensations are discussed.

Selective modification of nuclear proteins by polycyclic aromatic hydrocarbons and by benzo[a]pyrene diol epoxides.

Metabolites of benzo[a]pyrene (B[a]P) have been shown to modify chromosomal proteins with great specificity. Using the (+) and (-) enantiomers of anti-B[a]P diol epoxide to label isolated nuclei we found a remarkable difference in the capacity of these two compounds to modify histones H2A and H3. The (+) enantiomer modified histones H2A and H3, while the (-) enantiomer, which was shown to modify mainly histone H2A, had a much lower affinity for histone H3. We have also examined the selective, modification of chromosomal proteins by different polycyclic aromatic hydrocarbons and it was observed that 7,12-dimethylbenz[a]-anthracene (DMBA), 3-methylcholanthrene (3-MC) and B[a]P showed qualitative similarities in terms of their protein binding. This suggests that stereospecific interactions leading to binding of reactive metabolites of DMBA, B[a]P and 3-MC to chromosomal proteins share common features.

Evidence for the involvement of H1 histone phosphorylation in chromosome condensation.

Changes in the structural organisation of chromatin are necessary for the progression of the cell cycle. These changes are thought to be regulated mainly by the modification of chromosomal proteins by reactions such as phosphorylation1–8, acetylation9–12, methylation13,14 and poly(ADP-ribosyl)-ation15,16. Among these modifications, phosphorylation of histones, especially that of the H1 histone, is the most likely candidate for a factor which regulates chromosome condensation1–8. It has been proposed that the phosphorylated form of H1 histone may be involved in the initiation of chromosome condensation1–4 or in the maintenance of the condensed state of chromatin8. The latter possibility is unlikely because zinc chloride, which inhibits phosphatase in vivo and preserves the highly phosphorylated form of H1 histone at metaphase, does not prevent the dispersion of chromosomes at the end of mitosis17. As for the former possibility, Bradbury et al. reported2 that the activity of H1 histone phosphorylation is closely correlated with mitotic triggering in the cell cycle and suggest3,4 that H1 histone phosphokinase is involved in the initiation of mitosis. However, no causal relationship has been established. To analyse the mechanism of the progression of the cell cycle, we have isolated several temperature-sensitive (ts) growth mutants from FM3A, a cell line derived from C3H mouse mammary carcinoma18. We report here on one of these ts mutants, designated the ts85 strain, which shows an abnormal chromosome condensation as well as deficiency in H1 histone phosphorylation at the non-permissive temperature. Our results support the hypothesis that H1 histone phosphorylation controls the initiation step of mitosis through chromosome condensation.

Phosphorylation of chromosomal proteins as a function of age and its modulation by calcium

In vitro phosphorylation of histones and non histone chromosomal proteins (NHCP) and its modulation by calcium have been studied using slices of cerebral hemisphere of rats of various ages. Phosphorylation of histones decreases, and that of NHCP increases with increasing age. Calcium inhibits phosphorylation of histones of young and adult rats, but stimulates phosphorylation of NHCP. Phosphorylation of H1 and H4 histones is greater than that of other histones, and calcium inhibits their phosphorylation more markedly than of other histones. The significance of such modifications of chromosomal proteins in the aging process is discussed.

Overview: Molecular changes associated with large bowel cancer and their potential as markers and chemotherapeutic agents

The search for molecular changes that may be diagnostic of malignancy in the colonic epithelium is complicated by the diversity of cell types and complex cell kinetics of a tissue in which most of the cells are destined to leave within hours or days. Methods for cell separation and nuclear fractionation now permit biochemical studies of those cells that retain or regain the capacity for DNA synthesis and that are likely to include the transformed cell population. Among the changes associated with malignant transformation to be described are alterations in nuclear protein composition and metabolism, qualitative and quantitative differences in adenosine deaminase activities, activation of the guanylate/cyclic GMP system, and modification of both DNA and chromosomal proteins by alkylating carcinogens. DNA modification to produce O6-methylguanine correlates well with the incidence of tumor induction by methylazoxymethanol. Modifications of chromosomal proteins to produce methylated derivatives of lysine and arginine have been observed after the administration of 1,2-dimethylhydrazine. Such changes are likely to lead to aberrant interactions between DNA and regulatory elements in chromatin, and may not be subject to repair.

Studies on the Reversible Effects of Concanavalin A on CHO Cells in Culture

Plant lectins have been useful probes to test the concept that the plasma membrane plays an important role in the regulation of mitotic activity. The carbohydrate specificity of the proteins provides a means of identification of the residues available to the external environment of the cell surface as well as a capacity for the rapid reversal of its effect.As part of a series of studies on the post-synthetic modifications of chromosomal proteins during mitosis, we tested the effect of the lectin Concanaval- in A (ConA) on Chinese hamster ovary (CHO) cells (clone, K1, Pro-). The generation time for untreated cells was 11.9 hrs. Preliminary observations demonstrate that 30 μg/ml of ConA inhibits the cell cycle at a stage immediately prior to mitosis. The ConA treated cells assume a spherical configuration, which is maximal at the end of 10-11 hrs, with thymidine uptake evident at 4 hrs but not at 10 hrs.

Amino-terminal arginylation of chromosomal proteins by arginyl-tRNA.

Arginine was transferred from arginyl-tRNA to the amino-terminal end of chromatin proteins by L-arginyl-transferase. The reaction was dependent on the presence of potassium ion and beta-mercaptoethanol and was sensitive to RNase and trypsin. Treatment with DNase partially inhibited the transfer of arginine from arginyl-tRNA suggesting that intact chromatin structure is necessary for modification of chromatin. The radioactivity incorporated into chromatin was sensitive to trypsin but not to DNase or RNase. Most of the incorporated radioactivity was recovered in the phenol fraction, supporting the notion that modification of chromatin takes place in proteins but not in nucleic acids of chromatin. Modification of the proteins by transfer of arginine from arginyl-tRNA takes place mainly in the nonhistone fraction of chromatin. Major portions of chromosomal proteins modified in this manner appear to be released from chromatin. Incubation of incorporated radioactive product with [12C]arginyl-tRNA did not alter the product, showing that incorporated arginine is stable and does not exchange with added arginine or arginyl-tRNA. These observations suggest that aminoacyl-transferase may function in the modification of chromosomal proteins and that modification of chromatin may alter the regulatory mechanisms of cellular functions.