Preparation of Chromatin Fragments From Human Cells for Cryo-EM Analysis

Chromatin structure organization is crucial for regulating many fundamental cellular processes. However, the molecular mechanism that regulates the assembly of higher-order chromatin structure remains poorly understood. In this study, we demonstrate that Brd4 (bromodomain-containing protein 4) protein participates in the maintenance of the higher-order chromatin structure. Brd4, a member of the BET family of proteins, has been shown to play important roles in cellular growth control, cell cycle progression, and cancer development. We apply in situ single cell chromatin imaging and micrococcal nuclease (MNase) assay to show that Brd4 depletion leads to a large scale chromatin unfolding. A dominant-negative inhibitor encoding the double bromodomains (BDI/II) of Brd4 can competitively dissociate endogenous Brd4 from chromatin to trigger severely fragmented chromatin morphology. Mechanistic studies using Brd4 truncation mutants reveal that the Brd4 C-terminal domain is crucial for maintaining normal chromatin structure. Using bimolecular fluorescence complementation technology, we demonstrate that Brd4 molecules interact intermolecularly on chromatin and that replacing Brd4 molecules by BDI/II causes abnormal nucleosome aggregation and chromatin fragmentation. These studies establish a novel structural role of Brd4 in supporting the higher chromatin architecture.

Trophoblast cell lineage is established through the first cellular differentiation in mammalian embryogenesis, and its developmental potential is restricted to the extraembryonic tissues contributing solely to the placenta. Several lines of evidence suggest a relative lack of importance of DNA methylation in gene regulation in the extraembryonic tissues when compared with embryonic ones. Here we analyzed the dynamics of epigenetic status in the upstream region of mouse Ddah2 gene, which was found to be specifically repressed in a stem cell population of trophoblast cell lineage. We found a tissue-dependent differentially methylated region in the regulatory region of the Ddah2 gene. This region was hypermethylated in trophoblast stem cells and was hypomethylated in differentiated cells both in vivo and in vitro. This change was well correlated with Ddah2 expression. In addition, in vitro methylation confined to the differentially methylated region was sufficient to repress promoter activity in the reporter assay. Furthermore, a repressive pattern of histone modifications was formed around the differentially methylated region in undifferentiated trophoblast stem cells with repressed Ddah2. Our data suggest that DNA methylation-mediated chromatin remodeling is involved in the regulation of the Ddah2 gene expression and thus is important even in trophoblast cell lineage.

In eukaryotic cells, genomic DNA is compacted by nucleosomes, as basic repeating units, into chromatin. The nucleosome arrangement in chromatin fibers could be an important determinant for chromatin folding, by which genomic DNA is regulated in the nucleus. To study the structures of chromatin units in cells, we have established a method for the structural analysis of native mono- and poly-nucleosomes prepared from HeLa cells. In this method, the chromatin in isolated nuclei was crosslinked to preserve the proximity information between nucleosomes, followed by chromatin fragmentation by micrococcal nuclease treatment. The mono- and poly-nucleosomes were then fractionated by sucrose gradient ultracentrifugation, and their structures were analyzed by cryo-electron microscopy. Cryo-electron microscopy single particle analysis and cryo-electron tomography visualized a native nucleosome structure and secondary nucleosome arrangements in cellular chromatin. This method provides a complementary strategy to fill the gap between invitro and insitu analyses of chromatin structure.

IBD Genetics: Focus on (Dys) Regulation in Immune Cells and the Epithelium

Rat liver nuclei were isolated in low-ionic-strength buffer in the absence of bi- and multi-valent cations. Digestion of these nuclei by endogenous nuclease, micrococcal nuclease and DNase I revealed that a minor chromatin fraction was preferentially digested into poly- and oligo-nucleosomes. Southern blot hybridization with various active gene probes confirmed that these chromatin fragments represent coding and 5' upstream regions of transcriptionally active chromatin. Active chromatin fragments were released selectively into the medium, with inactive chromatin remaining inside the nuclei, under the above ionic conditions. The inclusion of bivalent cations during the digestion of nuclei reversed the solubility behaviour of active chromatin. Rearrangement and exchange of histone H1 between chromatin fragments was prevented by using low-salt conditions in all steps in the absence of bivalent cations. All histones, including H1, were present in stoichiometric amounts in this active chromatin fraction. Active nucleosomes showed a lower electrophoretic mobility than bulk nucleosomes in an acrylamide/agarose composite gel in the absence of Mg2+, but were selectively bound to the gel in the presence of this ion.

Positions of Potential:Nuclear Organization and Gene Expression

Focus

Chromatin and Transcription: Merging Package and Process

The basic chromatin fibre (the 10 nm diameter fibre) is a linear repeating array of nucleosomes, or nucleosome filament. The core of each disk-like nucleosome is a wedge-shaped protein octamer containing two molecules of each of the four core histones (H3, H4, H2A and H2B) around which two turns of DNA are wound in a left-handed superhelix with about 80 base-pairs per turn. The two turns are sealed by a molecule of the fifth histone H1 (or H5 in nucleated erythrocytes). The linker DNA that connects one two-turn particle to the next varies from essentially zero to about 80 base-pairs in chromatins from different sources. The exact significance of this variation is unclear. Interphase chromatin exists largely in the form of 30 nm fibres. Folding of the nucleosome filament into very similar 30 nm fibres, which is H1-dependent, occurs in vitro in the presence of monovalent cations or much lower concentrations of divalent cations. These higher-order structures probably arise by helical coiling of the nucleosome filament into a solenoid. Systematic studies of chromatin folding in solution, for a range of chromatin fragment sizes and ionic strengths, reveal two discontinuities in behaviour that reflect two structural transitions. One is interpreted as the formation of a turn of a solenoid with about six nucleosomes, at ionic strength 25 mM, and is a common feature of chromatin from three different sources, which differ in DNA repeat length, and type and amount of H1. The other transition is interpreted in terms of hydrodynamic shearing of long solenoids at low ionic strengths (below approximately 45 mM). It suggests a more stable higher-order structure for chicken erythrocyte chromatin than for rat liver chromatin (attributed largely to the presence of H5), and may prove to be a useful general assay for the relative stabilities of different chromatins, which might be relevant to their ease of unravelling for transcription. A study of short (165 base-pair) repeat chromatin from cerebral cortex neurons has led to the suggestion that in the general case the linker DNA might be located, perhaps with H1, in the central hole in the solenoid. H1 molecules in both extended and condensed chromatin (although not when dissociated from it) are close enough to be chemically cross-linked with reagents of span 2-12A.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylation of specific lysines within the core histone tail domains plays a critical role in regulating chromatin-based activities. However, the structures and interactions of the tail domains and the molecular mechanisms by which acetylation directly alters chromatin structures are not well understood. To address these issues we developed a chemical method to quantitatively determine binding affinities of specific regions within the individual tail domains in model chromatin complexes. Examinations of specific sites within the H2B tail domain indicate that this tail contains distinct structural elements and binds within nucleosomes with affinities that would reduce the activity of tail-binding proteins 10-50-fold from that deduced from peptide binding studies. Moreover, we find that mutations mimicking lysine acetylation do not cause a global weakening of tail-DNA interactions but rather the results suggest that acetylation leads to a much more subtle and specific alteration in tail interactions than has been assumed. In addition, we provide evidence that acetylation at specific sites in the tail is not additive with several events resulting in similar, localized changes in tail binding.

Lactogenic hormone regulation of beta-casein gene expression in mammary epithelial cells provides an excellent model in which to study the mechanisms by which steroid and peptide hormone signaling control gene expression. Prolactin- and glucocorticoid-mediated induction of beta-casein gene expression involves two principal regulatory regions, a proximal promoter and a distal enhancer located in the mouse approximately -6 kb upstream of the transcription start site. Using a chromosome conformation capture assay and quantitative real time PCR, we demonstrate that a chromatin loop is created in conjunction with the recruitment of specific transcription factors and p300 in HC11 mammary epithelial cells. Stimulation with both prolactin and hydrocortisone is required for the induction of these long range interactions between the promoter and enhancer, and no DNA looping was observed in nontreated cells or cells treated with each of the hormones separately. The lactogenic hormone-induced interaction between the proximal promoter and distal enhancer was confirmed in hormone-treated primary three-dimensional mammary acini cultures. In addition, the developmental regulation of DNA looping between the beta-casein regulatory regions was observed in lactating but not in virgin mouse mammary glands. Furthermore, beta-casein mRNA induction and long range interactions between these regulatory regions were inhibited in a progestin-dependent manner following stimulation with prolactin and hydrocortisone in HC11 cells expressing human PR-B. Collectively, these data suggest that the communication between these regulatory regions with intervening DNA looping is a crucial step required to both create and maintain active chromatin domains and regulate transcription.

Histones H1 and H5 can exchange between an H1-containing chromatin fragment from rat liver and an H1, H5-containing fragment from chicken erythrocytes at ionic strengths from about 35 mM to 105 mM. The redistribution has reached equilibrium by ionic strength 75 mM in 1 h or less at 4 degrees C. After exchange at ionic strength 75 mM, long fragments, whether of rat liver or chicken erythrocyte chromatin, are recovered with a higher H5:H1 ratio than short fragments, suggesting a stronger preference of H5 than of H1 for higher-order structures which exist for long fragments at ionic strength 75 mM. Competition experiments between occupied H1 or H5 binding sites on chromatin fragments from rat liver or chicken erythrocytes and empty sites on H1-depleted rat chromatin show that rat H1 does not distinguish between the two types of site, whereas H5 discriminates in favour of sites on native chromatin, even when the chicken fragments are too short to form higher-order structures. (The behavior of the chicken H1, which may be bound less tightly than rat H1, depends on the length of the chicken fragment, in a manner suggesting that fragments of 15 nucleosomes and longer can form stable higher-order structures which have high-affinity binding sites for both H5 and H1.) We conclude that the affinity of sites for H5 is in the order:higher-order structures greater than nucleosome filament much greater than H1-depleted chromatin. The same relative order of affinities may well apply for H1 but the discrimination is much lower. This difference between H1 and H5 seems likely to be relevant to the greater stability of H5-containing chromatin, and in turn its transcriptional inactivity, and indeed to the mechanism of replacement of H1 by H5 during the terminal stages of erythropoiesis.

Digestion of chromatin in nuclei by micrococcal nuclease, measured as the change in the concentration of monomer-length DNA with time, displays Michaelis-Menten kinetics. Redigestion of soluble chromatin prepared from nuclei by micrococcal nuclease treatment, however, is apparently first order in enzyme and independent of chromatin concentration. This qualitative difference results from an increase in the apparent second order rate constant, kcat/Km, for liberation of monomer DNA: the apparent Km for soluble chromatin is lower by close to 3 orders of magnitude than that for chromatin in nuclei, whereas kcat decreases by less than 1 order of magnitude. Neither the integrity of the nuclear membrane nor the presence of histone H1 contributes to the high Michaelis constant characteristic of chromatin in nuclei. Moreover, differences due to the buffers used for digestion and redigestion are minimal. Low catalytic efficiency is, however, correlated with the presence of higher order chromatin superstructure. Micrococcal nuclease added to soluble chromatin under nondigesting conditions at low ionic strength (I = 0.002) co-sediments with chromatin in sucrose gradients. In 0.15 M NaCl, added nuclease no longer sediments with chromatin and redigestion kinetics become first order in both enzyme and substrate. Kinetic analysis of this type may afford an assay for native, higher order structures in chromatin. Our results suggest that micrococcal nuclease binds to soluble chromatin through additional interactions not present in nuclei, which may be partly ionic in nature.

The organization of the higher order structure of chromatin has been examined in chicken erythrocyte. Chromatin solubilized during the time course of a gentle micrococcal nuclease digestion of nuclei shows a continuous variation in the distribution of molecular weights. Electron microscopy studies of large chromatin fragments solubilized at physiological ionic strength (0.14 M NaCl or KCl) suggest that the polynucleosome chain is folded in continuous compact structures of an average diameter of 23 nm in which the individual nucleosomes are difficult to distinguish. This compact structure is destabilized even at intermediate ionic strengths (e.g., 40 mM NaCl), resulting in looser fibers of similar diameter. At 5 mM NaCl the fiber is unraveled into a continuous filament of 10-nm diameter. These conformational changes are reversible as determined by hydrodynamic and biochemical parameters. The 10-nm leads to 23-nm transition of chromatin appears to be a cooperative process requiring the full complement of histones H1 and H5. Micrococcal nuclease cleaves the DNA in the compact chromatin structure to an apparent limit of digestion corresponding to an average of eight to nine nucleosomes with little effect on the size of the fiber. Thus, the continuity of the DNA is not required for the stability of the folded chromatin fiber. Histones H1 and H5 exhibit a binding preference to larger chromatin fragments regardless of the length of the DNA. This behavior is not observed with relaxed chromatin, suggesting that multiple stabilizing interactions involving H1 (H5) are possible only in the compact configuration.

Transcriptionally silent chromatin in Saccharomyces cerevisiae is associated with histone hypoacetylation and is formed through the action of the Sir histone deacetylase complex. A histone acetyltransferase (HAT) targeted near silent chromatin can overcome silencing at a distance by increasing histone acetylation in a sizable region. However, how a tethered HAT acetylates distant nucleosomes has not been resolved. We demonstrate here that targeting the histone H3-specific HAT Gcn5p promotes acetylation of not only histone H3 but also histone H4 in a broad region. We also show that long range anti-silencing and histone acetylation by targeted HATs can be blocked by nucleosome-excluding sequences. These results are consistent with the contention that a tethered HAT promotes stepwise propagation of histone acetylation along the chromatin. Because histone hypoacetylation is key to the formation and maintenance of transcriptionally silent chromatin, it is believed that acetylation promoted by a targeted HAT disrupts silent chromatin thereby overcoming silencing. However, we show that the acetylated and transcriptionally active region created by a tethered HAT retains structural hallmarks of Sir-dependent silent chromatin and remains associated with Sir proteins indicating that tethered HATs overcome silencing without completely dismantling silent chromatin.