Histone hyperacetylation facilitates chromatin remodelling in a Drosophila embryo cell-free system

We found that Drosophila embryo extract contains a protein activity (or activities) that can destabilize nucleosomes, resulting in increased sensitivity to DNase I, release of nucleosomal supercoiling, high levels of conformational flexibility of DNA and more diffuse micrococcal nuclease digestion patterns. Incorporation of histone H1 did not significantly affect this nucleosome remodelling. Remodelling occurs more efficiently in hyperacetylated chromatin. It was shown previously that hyperacetylated chromatin, reconstituted in a Drosophila embryo cell-free system, exhibits increased DNase I sensitivity and a high degree of conformational flexibility of DNA. The present data suggest that the more diffuse structure of acetylated chromatin is a result of chromatin remodelling by protein activities in the Drosophila embryo extract. Received: 4 November 1998 / Accepted: 10 May 1999     

The effect of a high mobility group protein (HMG 17) on the structure of acetylated and control core HeLa cell chromatin

The effect of binding a high mobility group protein (HMG 17) on the stability and conformation of acetylated and control HeLa high molecular weight core chromatin (stripped of H1 and non-histone chromosomal proteins) was studied by circular dichroism and thermal-denaturation measurements. Previously it had been shown that conformational differences exist between native whole chromatin derived from butyrate-treated (acetylated) and control HeLa cells and that these conformational differences disappear by removing H1 and non-histone chromosomal proteins (Reczek, P.R., Weissman, D., Huvos, P.E. and Fasman, G.D. (1982) Biochemistry 21, 993–1002). The circular dichroism spectra and the thermal denaturation profiles of control and acetylated core chromatin were found to be similar. The circular dichroism properties of HMG 17 reconstituted highly acetylated and control core chromatin indicated the same alteration of chromatin structure at low ionic strength (1 mM sodium phosphate/0.25 mM EDTA, pH 7.0). The magnitudes of the decrease in ellipticity were proportional to the amount of HMG 17 bound and were found to be the same for both the acetylated and control core chromatin. Thermal denaturation profiles confirmed this change in structure induced by HMG 17 on control and highly acetylated core chromatin. The thermal denaturation profiles, which were resolved into three component transitions, exhibited a shifting of hyperchromicity from the lower melting transitions to the higher melting transitions, with a concomitant rise in T_m, on HMG 17 binding to both control and acetylated chromatin. The natures of the interactions of HMG 17 at higher ionic strength (50 mM NaCl/0.25 mM EDTA/1 mM sodium phosphate, pH 7.0) with acetylated and control core chromatin were slightly different, as measured by circular dichroism; however, a decrease in ellipticity was observed for both samples upon binding of HMG 17. These observations suggest that acetylation coupled with HMG 17 binding to core chromatin does not loosen chromatin structure. HMG 17 binding to control and acetylated core chromatin produces an overall stabilization and compaction of chromatin structure.     

Enrichment of transcribed and newly replicated DNA in soluble chromatin released from nuclei by mild micrococcal nuclease digestion

A chromatin fraction solubilized from mouse myeloma nuclei under near-physiological ionic conditions by very mild micrococcal nuclease digestion at 0°C is enriched at least 7-fold in DNA complementary to total myeloma polyadenylated mRNA, and 15-fold in DNA originating near the replication fork (labeled within 30 s). Newly replicated DNA recovered in solubilized chromatin after brief labeling was incorporated mainly into particles sedimenting with, or faster than, mononucleosomes. A rapid decrease in enrichment of newly replicated DNA in readily released, soluble chromatin with increasing labeling times indicated that newly replicated chromatin matured within 90 s to a form that was partitioned similarly to bulk chromatin by this fractionation method. Previous studies showed that chromatin readily solubilized from myeloma nuclei is enriched in high-mobility-group (HMG) and other non-histone proteins, RNA and single-stranded DNA; and depleted in H1 and 5-methylcytosine, relative to bulk chromatin (Jackson, J.B., Pollock, J.M., Jr., and Rill, R.L. (1979) Biochemistry 18, 3739–3748). Mild digestion of chicken erythrocyte nuclei with micrococcal nuclease yielded a soluble chromatin fraction (1–2% of the total DNA) with similar properties. This fraction was enriched at least 6-fold in DNA complementary to chicken globin mRNA, relative to total erythrocyte DNA.     

大鼠老化过程小脑神经元染色质核小体线性和空间排布特征分析

阐明真核细胞染色质核小体线性和空间排布特征及其机制是染色质结构和功能研究的核心内容．近年来随着染色质分子生物学研究的深入,人们发现染色质核小体不仅作为真核基因组三维结构的基本结构单元,而且在细胞核内线性和空间排布（ｌｉｎ－ｅａｒａｎｄｓｐａｃｉａｌａ...     

Generation of an antibody recognizing a set of acetylated proteins,including subunits of BAF complexes

The aim of this study was to generate an antibody specific to Ki-67 acetylated at lysine 3180, whose existence was reported in an acetylome study (Scholz, C., B.T. Weinert, S.A. Wagner, P. Beli, Y. Miyake, J. Qi, L.J. Jensen, W. Streicher, A.R. McCarthy, N.J. Westwood, S. Lain, J. Cox, P. Matthias, M. Mann, J.E. Bradner, and C. Choudhary. 2015). Rabbits were immunized with a synthetic acetylated peptide corresponding to acetylated lysine 3180 of Ki-67 and the residues flanking it. The obtained antibody, referred to as Ab3180 in this study, was affinity purified with the antigen peptide and characterized. Immunoblot analysis of cell extracts using Ab3180 revealed that this antibody unexpectedly recognized a set of acetylated proteins unrelated to Ki-67. Ab3180-recognizable proteins were immunoprecipitated from cell extracts in a stringent condition and identified by mass-spec analysis as subunits of BAF (mammalian SWI/SNF) chromatin remodeling complexes. The unique specificity of Ab3180 will allow this antibody to be a useful tool for analyzing the acetylation of BAF complexes and its significance to the formation/function of BAF complexes.     

Structure of adenovirus chromatin

The structure of adenovirus type 2 chromatin isolated from wild-type and ts1 virions was investigated by micrococcal nuclease digestion and electron microscopy. Partial digestion of wild-type and ts1 chromatin with micrococcal nuclease generated a multimeric DNA smear devoid of the 200 basepair nucleosome repeating pattern characteristic of cellular chromatin. However, 11 S monomer cores of 150 basepairs were detectable. The chromatin of ts1 (39°C) was more resistant to digestion by micrococcal nuclease. Two-dimensional electrophoresis of the monomer core showed that wild-type core contained protein VII while ts1 (39°C) core contained PVI and PVII. Protein V appears to be located on the variable-length intermonomer region. Crosslinking studies suggest that proteins PVII and VII exist in dimeric form within the monomer core. Electron microscopy revealed a 5.5-fold-condensed two-micron-long beaded structure with about 200 monomer particles spaced irregularly. Based on these observations, a model for adenovirus prochromatin and chromatin is proposed that differs in important aspects from the model proposed previously (Corden, J., Engelking, H.M. and Pearson, G.D. (1976) Proc. Natl. Acad. Sci. USA 73, 401–404).     

正常及白血病小鼠白细胞染色质和DNA的酶切分析

 本文应用的核酸酶为DNaseⅡ、微球菌核酸酶与限制性内切核酸酶BstNI、EcoRⅡ、HpaⅡ和MspⅠ,将它们作用于正常小鼠615和可移植性白血病小鼠L7712脾脏白细胞染包质及其DNA,根据酶切电泳谱及水解动力学分析表明:1.白血病小鼠染色质相对正常小鼠染色质易被DNaseⅡ微球菌核酸酶水解;2.白血病小鼠染色质比正常小鼠者易被MspⅠ水解,但其DNA的MspⅠ酶切电泳谱无明显差别;3.白血病小鼠染色质及其DNA较正常小鼠染色质及其DNA易被EcoRⅡ水解。这些观察说明,白血病小鼠脾脏白细胞染色质有较活跃的构象状态;其染色质DNA的CCATGC区段内有较低的甲基化程度。     

Insights into role of bromodomain, testis-specific (Brdt) in acetylated histone H4-dependent chromatin remodeling in mammalian spermiogenesis

Mammalian spermiogenesis is of considerable biological interest especially due to the unique chromatin remodeling events that take place during spermatid maturation. Here, we have studied the expression of chromatin remodeling factors in different spermatogenic stages and narrowed it down to bromodomain, testis-specific (Brdt) as a key molecule participating in chromatin remodeling during rat spermiogenesis. Our immunocytochemistry experiments reveal that Brdt colocalizes with acetylated H4 in elongating spermatids. Remodeling assays showed an acetylation-dependent but ATP-independent chromatin reorganization property of Brdt in haploid round spermatids. Furthermore, Brdt interacts with Smarce1, a member of the SWI/SNF family. We have studied the genomic organization of smarce1 and identified that it has two splice variants expressed during spermatogenesis. The N terminus of Brdt is involved in the recognition of Smarce1 as well as in the reorganization of hyperacetylated round spermatid chromatin. Interestingly, the interaction between Smarce1 and Brdt increases dramatically upon histone hyperacetylation both in vitro and in vivo. Thus, our results indicate this interaction to be a vital step in the chromatin remodeling process during mammalian spermiogenesis.     

A Low Repeat Length in Oligodendrocyte Chromatin

The behavior of oligodendrocyte chromatin after micrococcal nuclease digestion of nuclei was assayed in brains of rats of four different ages. During oligodendrocyte differentiation, a decreasing sensitivity of the chromatin to enzymatic attack was observed. On the other hand, the nucleosomal repeat length showed a slight tendency to increase during development. It is worth noting that even the highest values reported here for "oligodendrocyte" chromatin repeat lengths are significantly lower than 200 base pairs, the value previously reported by others for "non-astrocytic glia."     

Histone hyperacetylation has little effect on the higher order folding of chromatin

HeLa cells were grown in the presence of 10 mM sodium butyrate and soluble chromatin containing hyperacetylated histones was prepared by mild micrococcal nuclease digestion and sucrose gradient fractionation. Sedimentation and electric dichroism were used to study the cation-induced folding of this acetylated chromatin from the 10 nm filament to the 30 nm solenoid conformation. Although under some conditions acetylated chromatin appears slightly less condensed than control chromatin, the major conclusion is that hyperacetylation of histones does not in itself prevent the formation of the higher order chromatin solenoid.     

Chromatin organization during mouse oocyte growth

We investigated the changes in the organization of oocyte nuclear chromatin and nucleolar-associated chromatin throughout folliculogenesis. Zona-free oocytes were isolated from ovaries, grouped into seven classes according to size and chromatin organization, and analyzed after staining with Hoechst 33342. We show that oocyte differentiation from the dictyate stage to the conclusion of maturation is associated with either of two chromatin configurations. Initially, all oocytes are in the NSN configuration (nonsurrounded nucleolus oocytes; characterized by a Hoechst positive-chromatin pattern of small clumps forming a network on the nuclear surface, with a nucleolus nonsurrounded by chromatin). While growing, some of these NSN oocytes continue their development in the NSN configuration, whereas others shift (from class IV on) into the SN configuration (surrounded nucleolus oocytes; characterized by a threadlike chromatin organization that may partially surround the nucleolus or project towards the nuclear periphery). The percentage of SN oocytes increases both with increasing size of the oocyte (class I–III, 10–40 μm in diameter: 100% NSN vs. 0% SN; class VII 70–80 μm in diameter: 47.3% NSN vs. 52.3 SN, in 4–6-week-old females), and with aging (class VII: 94.1% NSN vs. 5.9% SN in 2-week-old females; 11.8% NSN vs. 8.2% SN in 56-week-old females). Further, we suggest as a working hypothesis that those oocytes that switch to the SN chromatin organization early in maturation may not be ovulated, even though this particular chromatin structure normally occurs just prior to ovulation. © 1995 Wiley-Liss, Inc.     

Identification of a replication-independent replacement histone H3 in the basidiomycete Ustilago maydis

Ustilago maydis is a haploid basidiomycete with single genes for two distinct histone H3 variants. The solitary U1 gene codes for H3.1, predicted to be a replication-independent replacement histone. The U2 gene is paired with histone H4 and produces a putative replication-coupled H3.2 variant. These predictions were evaluated experimentally. U2 was confirmed to be highly expressed in the S phase and had reduced expression in hydroxyurea, and H3.2 protein was not incorporated into transcribed chromatin of stationary phase cells. Constitutive expression of U1 during growth produced ~25% of H3 as H3.1 protein, more highly acetylated than H3.2. The level of H3.1 increased when cell proliferation slowed, a hallmark of replacement histones. Half of new H3.1 incorporated into highly acetylated chromatin was lost with a half-life of 2.5 h, the fastest rate of replacement H3 turnover reported to date. This response reflects the characteristic incorporation of replacement H3 into transcribed chromatin, subject to continued nucleosome displacement and a loss of H3 as in animals and plants. Although the two H3 variants are functionally distinct, neither appears to be essential for vegetative growth. KO gene disruption transformants of the U1 and U2 loci produced viable cell lines. The structural and functional similarities of the Ustilago replication-coupled and replication-independent H3 variants with those in animals, in plants, and in ciliates are remarkable because these distinct histone H3 pairs of variants arose independently in each of these clades and in basidiomycetes.     

Parameters influencing the flow cytometric analysis of DNA sensitivity to nuclease S1

Some parameters that influence the analysis in situ of DNA sensitivity to digestion with nuclease S1 have been studied in isolated HeLa nuclei with flow cytometry. DNA staining with the intercalating fluorochrome propidium iodide allowed the nucleolytic activity on double-stranded (ds) DNA to be determined by monitoring the relative reduction in nuclear fluorescence intensity. Nuclei isolated in buffer at low ionic strength in order to decondense chromatin fibres, showed a lower fluorescence intensity than nuclei with native chromatin, after digestion with nuclease S1 under identical conditions. Nuclei prepared with dispersed chromatin and digested with increasing amounts of enzyme showed a decrease in fluorescence intensity that reached a limit value at about 50% of the value of undigested control samples. On the other hand, in nuclei with native chromatin, fluorescence intensity decreased only about 18%. The NaCl concentration in the reaction buffer strongly influenced the DNA sensitivity to S1 nuclease. By increasing salt molarity from 5 mM to 200 mM, the digestion of dsDNA was significantly reduced as also shown by the amount of released nucleotides from purified calf thymus DNA. The detection of DNA sensitivity to nuclease S1, as assessed by the cytometric method, was shown to be more sensitive than a biochemical technique involving hydrolysis of purines. These results indicate that both the procedure for nuclei isolation and the digestion conditions have to be carefully controlled when evaluating in situ the presence of S1-sensitive sites.     

Parameters influencing the flow cytometric analysis of DNA sensitivity to nuclease S1

Summary Some parameters that influence the analysis in situ of DNA sensitivity to digestion with nuclease S1 have been studied in isolated HeLa nuclei with flow cytometry. DNA staining with the intercalating fluorochrome propidium iodide allowed the nucleolytic activity on double-stranded (ds) DNA to be determined by monitoring the relative reduction in nuclear fluorescence intensity. Nuclei isolated in buffer at low ionic strength in order to decondense chromatin fibres, showed a lower fluorescence intensity than nuclei with native chromatin, after digestion with nuclease S1 under identical conditions. Nuclei prepared with dispersed chromatin and digested with increasing amounts of enzyme showed a decrease in fluorescence intensity that reached a limit value at about 50% of the value of undigested control samples. On the other hand, in nuclei with native chromatin, fluorescence intensity decreased only about 18%. The NaCl concentration in the reaction buffer strongly influenced the DNA sensitivity to S1 nuclease. By increasing salt molarity from 5 mM to 200 mM, the digestion of dsDNA was significantly reduced as also shown by the amount of released nucleotides from purified calf thymus DNA. The detection of DNA sensitivity to nuclease S1, as assessed by the cytometric method, was shown to be more sensitive than a biochemical technique involving hydrolysis of purines. These results indicate that both the procedure for nuclei isolation and the digestion conditions have to be carefully controlled when evaluating in situ the presence of S1-sensitive sites.     

Histone H3 lysine 14 acetylation is required for activation of a DNA damage checkpoint in fission yeast

Histone lysine acetylation has emerged as a key regulator of genome organization. However, with a few exceptions, the contribution of each acetylated lysine to cellular functions is not well understood because of the limited specificity of most histone acetyltransferases and histone deacetylases. Here we show that the Mst2 complex in Schizosaccharomyces pombe is a highly specific H3 lysine 14 (H3K14) acetyltransferase that functions together with Gcn5 to regulate global levels of H3K14 acetylation (H3K14ac). By analyzing the effect of H3K14ac loss through both enzymatic inactivation and histone mutations, we found that H3K14ac is critical for DNA damage checkpoint activation by directly regulating the compaction of chromatin and by recruiting chromatin remodeling protein complex RSC.     

Anti-bromodeoxyuridine monoclonal antibody: an alternative tool for the identification of replicated DNA at the electron microscope level

A new method for identifying the replicated DNA at the electron microscope level is described. Cells were first exposed in vitro to 5-bromodeoxyuridine (BUdR) in conjunction with 5-fluorodeoxyuridine (FUdR) and BUdR incorporated into DNA was then detected on Lowicryl-embedded sections by immunogold technique using a monoclonal anti-BUdR antibody. After using this method, chromatin and chromosomes are strongly labelled.     

Organization of the thyroid hormone receptor in the chromatin of C6 glial cells: Evidence that changes in receptor levels are not associated with changes in receptor distribution

The association of [¹²⁵I]T3-receptor complexes with C6 cell chromatin was analysed after a limited digestion with micrococcal nuclease (MN) or DNase I. Both nucleases solubilized up to 60–70% of receptor and 0.4 M KCl extracted 70%, of the non-digested receptor, thus showing that only a residual fraction of receptor is associated with the nuclear matrix. With DNase I the receptor was released 2–3-fold faster than the bulk of chromatin, whereas a preferential release of receptor over total chromatin was not observed with MN. The digestion of receptor with DNase I and MN occurred 14- and 6-fold faster, respectively, than the appearance of PCA-soluble chromatin. Preincubation for 48 h with 4 nM T3 of 2 mM butyrate significantly altered receptor levels but did not change sensitivity to the nucleases. These results suggest that the thyroid hormone receptor is associated with chromatin highly sensitive to nuclease digestion, and that changes in receptor number are not associated with changes in its distribution in chromatin.     

Estimation of domain length of chicken erythrocyte chromatin

Chromatin of chicken erythrocyte nuclei was extracted by digestion with micrococcal nuclease. The length distribution of the soluble chromatin was determined by gel electrophoresis and electron microscopy. These results were fitted with a theoretical distribution which was an outcome of the domain model proposed by Igo-Kemenes and Zachau (Igo-Kemenes, T. and H.G. Zachau (1977) Cold Spring Harbour Symp. Quant. Biol. 42, 109–118). A domain length of 45 kbp was obtained.     

DNAase I and cellular factors that affect chromatin structure

The use of DNAase I as a probe of chromatin structure is frequently fraught with problems of irreproducibility. We have recently evaluated this procedure, documented the sources of the problems, and standardized the method for reproducible results (Prentice and Gurley (1983) Biochim. Biophys. Acta 740, 134–144). We have now used this probe to detect differences in chromatin structure between cells blocked (1) in G₁ phase by isoleucine deprivation, or (2) in early S phase by sequential use of isoleucine deprivation followed by release into the presence of hydroxyurea. The cells blocked in G₁ phase have easily-digestible chromatin, while cells blocked in early S phase have chromatin which is much more resistant to DNAase I. These differences were found to be the result of diffusible factors found in the cytoplasm and nuclei of G₁- and S-phase cells, respectively. The G₁ cells contained a cytoplasmic factor which modulates the chromatin structure of S-phase nuclei to a more easily digestible state, while cells blocked in S phase contain a nuclear factor which modulates the chromatin structure of G₁ nuclei to a state more resistant to digestion. DNAase I is much more sensitive to these cell cycle-specific chromatin changes than is micrococcal nuclease. The results indicate that, under controlled conditions, DNAase I should be a valuable probe for detecting chromatin structural changes associated with cell cycle traverse, differentiation, development, hormone action and chemical toxicity.