Glucocorticoid receptor structure as probed by endogenous proteases

Transformation of the glucocorticoid-receptor complex by heating the cytosol in the presence of calcium is accompanied by formation of a series of truncated complexes, of which DI and DIIc are the major members. Formation of DIIc (but not of DI) is inhibited by leupeptin, and the intact transformed complex DIIa appears instead. Estimation of the molecular weights and Stokes' radii of all major complexes revealed that forms DI and DIIc have the same Mr, 48 kDa, but differ in shape, and appear to be digestion products generated by cleavage at the same site. Proteolysis of glucocorticoid receptor, covalently labelled with [3H]dexamethasone mesylate in rat thymus and brain cytosol, corroborated these findings and further implied that DI is the product of digestion of the non-transformed form of the receptor. Covalently labelled receptor fragments, related to the products formed when cytosol is heated, are detected in the nuclei of thymocytes, implying that the same proteolytic cleavages sites are involved in receptor turnover. Cleavage sites in the non-transformed covalently labelled receptor were identified in the "stepladder" of fragments of Mr, 85, 65, 49, 35, 27-30 kDa, generated in the absence of calcium, with an additional 78 kDa fragment in its presence. In the transformed conformation, two of the cleavage sites giving rise to the 65 and 35 kDa fragments, appear to be protected. It is speculated that the change in the proteolytic susceptibility of the cleavage site for the 35 kDa fragment relates to the "unmasking" of enhancer-activating and/or DNA-binding receptor functions previously postulated.     

Nucleosomal structure as probed by H3 histone thiol reactivity

Two H3 histone variants are found in equal amount in HeLa cells, and they have been characterized by two-dimensional gel electrophoresis followed by reaction with specific antibodies. These molecules are the only cysteine-containing histones, and they have been used as the target for thiol-specific reagents, in intact nuclei, isolated nucleosomes, histone complexes, and purified histones. Cysteine residues are available toN-ethylmaleimide only when histones are disassembled from the core particles. Upon reaction with these reagents, one of the H3 variants undergoes profound conformational changes, as revealed by an altered electrophoretic mobility.     

Chromatin structure of Drosophila nasutoides satellites as probed by cross-linking DNA in situ

Chromatin structure can be probed by cross-linking DNA in situ using trioxsalen and irradiation with UV light. Presumably DNA within a nucleosome is protected from cross-linking so that this region appears as a single-strand loop in the electron microscope under a condition in which single-strands and double-strands are distinguished. Unprotected regions appear as duplex due to cross-linking.We have used this approach to investigate the structure of chromatins containing satellite DNAs of Drosophila nasutoides. We have previously shown that D. nasutoides has an unusually large autosome pair which is almost entirely heterochromatic. Its nuclear DNA reveals four major satellite components amounting up to 60% of the total genome. All of them are localized in this large heterochromatic chromosome. We wish to ask whether chromatins containing different satellite sequences have different arrangements of nucleosomes. Our results from cross-linking experiments show that all DNA components including main band DNA have different patterns of protected and unprotected regions: (a) The length distributions of protected regions show multiple peaks with the smallest unit lengths being 200 nucleotides for main band DNA, 180 for satellites I, II and III, and 160 for satellite IV. (b) The amounts of unprotected regions, presumably internucleosome DNA, vary from 16% for main band DNA to 60% for satellite IV, suggesting that satellite chromatins have fewer nucleosomes per given length of chromatin than main band DNA chromatin. The spacings between nucleosomes appear to be random in satellite chromatins.     

Chromatin core particle obtained by selective cleavage of histones H3 and H4 by clostripain

A new chromatin core particle characterized by a half-proteolyzed octamer is obtained by controlled digestion of the native core particle by clostripain. The proteolyzed histones correspond to four polypeptide fragments which are tentatively assigned to H2A[4-129], H2B[1-125], H3[27-135] and H4[18-102] on the basis of electrophoretic evidence and the known specificity of clostripain for arginyl residues. Despite the loss of the N-terminal regions of histones H3 and H4, the partially proteolyzed core particle retains the structural conformation of the native one as shown by circular dichroism. As expected, this half-proteolyzed core particle presents an intermediate accessibility to polycations, such as spermidine, in comparison with that observed with the native core particle and a fully proteolyzed core particle. The latter includes the polypeptide fragments H2A[12-129], H2B[21-125], H3[27-135] and H4[20-102].     

Nucleosomal structure as probed by H3 histone thiol reactivity. Conformation of H3 histone variants is differently affected by thiol group reagents

Two H3 histone variants are found in equal amount in HeLa cells, and they have been characterized by two-dimensional gel electrophoresis followed by reaction with specific antibodies. These molecules are the only cysteine-containing histones, and they have been used as the target for thiol-specific reagents, in intact nuclei, isolated nucleosomes, histone complexes, and purified histones. Cysteine residues are available to N-ethylmaleimide only when histones are disassembled from the core particles. Upon reaction with these reagents, one of the H3 variants undergoes profound conformational changes, as revealed by an altered electrophoretic mobility.     

Sea urchin sperm chromatin structure as probed by pancreatic DNase I: Evidence for a novel cutting periodicity

Chromatin from mature sea urchin spermatozoa is highly compacted and composed almost entirely of DNA and the five histones, although sperm H1, H2A, and H2b histones differ from those found in embryo or somatic cell nuclei. Release of acid-soluble DNA during pancreatic DNase I digestion is 20-fold slower from sperm nuclei than from embryonic nuclei. Following DNase I digestion, most sperm nuclear DNA remains at high molecular weight, although there appears to be some release of 10 base oligomer fragments. Size analysis of the higher molecular weight DNA reveals a series of fragments that indicate a cutting periodicity of approximately 500 base pairs. This pattern remains when electrophoretic separation is carried out under denaturing conditions. The 500 base pair cleavage pattern was not detected in digestions of embryonic nuclei. Nucleosomes reconstituted with fractionated core histones from sperm gave, upon digestion, a characteristic 10 base “ladder,” with no resistant high molecular weight DNA. Micrococcal nuclease and DNase II digested sperm nuclei to produce DNA fragments with a calculated repeat length of 248 ± 3 and 246 ± 6 base pairs, respectively. The structural basis for the 500 base pair cutting periodicity in sperm nuclei may reside in the unique sperm H1 histone.     

Involvement of histone H1 in the structure of the linker DNA in nucleosomes as revealed by nucleases

This report describes experiments designed to study the organization of the linker DNA in nucleosomes. When rat liver nucleosomes (145 to 188 base pairs in length) were digested by Exonuclease III and then by nuclease S1 a series of bands of sizes 90-102-112-125-135-142-154-166-172-181-bases was observed in denaturing electrophoretic gels. Digestion of H1-depleted nucleosomes under the same conditions results in a series of products of sizes (10.4) xn in base (n14) only. This result is interpreted as reflecting a particular arrangement of linker DNA under the influence of histone H1.     

H3 and H4 histone tails play a central role in the interactions of recombinant NCPs

Using small-angle x-ray scattering, we probe the effect of histone tails on both internucleosomal interactions and nucleosome conformation. To get insight into the specific role of H3 and H4 histone tails, perfectly monodisperse recombinant nucleosome core particles were reconstituted, either intact or deprived of both H3 and H4 histone tails (gH3gH4). The main result is that H3 and H4 histone tails are necessary to induce attractive interactions between NCPs. A pair potential model was used to describe interactions between NCPs. At all salt concentrations, interactions between gH3gH4 NCPs are best described by repulsive interactions exclusively. For intact NCPs, an additional attractive term, with a 5–10 kT magnitude and 20 Å range, is required to account for interparticle interactions above 50 mM monovalent salt. Regarding conformation, intact NCPs in solution are similar to NCPs in 3D crystals. gH3gH4 NCPs instead give rise to slightly different small-angle x-ray scattering curves that can be understood as a more opened conformation of the particle, where DNA ends are slightly detached from the core.     

Evidence for the nuclear import of histones H3.1 and H4 as monomers

Newly synthesised histones are thought to dimerise in the cytosol and undergo nuclear import in complex with histone chaperones. Here, we provide evidence that human H3.1 and H4 are imported into the nucleus as monomers. Using a tether‐and‐release system to study the import dynamics of newly synthesised histones, we find that cytosolic H3.1 and H4 can be maintained as stable monomeric units. Cytosolically tethered histones are bound to importin‐alpha proteins (predominantly IPO4), but not to histone‐specific chaperones NASP, ASF1a, RbAp46 (RBBP7) or HAT1, which reside in the nucleus in interphase cells. Release of monomeric histones from their cytosolic tether results in rapid nuclear translocation, IPO4 dissociation and incorporation into chromatin at sites of replication. Quantitative analysis of histones bound to individual chaperones reveals an excess of H3 specifically associated with sNASP, suggesting that NASP maintains a soluble, monomeric pool of H3 within the nucleus and may act as a nuclear receptor for newly imported histone. In summary, we propose that histones H3 and H4 are rapidly imported as monomeric units, forming heterodimers in the nucleus rather than the cytosol.     

Chromatin fiber folding: requirement for the histone H4 N-terminal tail

We have developed a self-assembly system for nucleosome arrays in which recombinant, post-translationally unmodified histone proteins are combined with DNA of defined-sequence to form chromatin higher-order structure. The nucleosome arrays obtained are highly homogeneous and sediment at 53S when maximally folded in 1mM or 100mM MgCl(2). The folding properties are comparable to established systems. Analytical ultracentrifugation is used to determine the consequence of individual histone tail domain deletions on array folding. Fully compacted chromatin fibers are obtained with any one of the histone tails deleted with the exception of the H4 N terminus. The region of the H4 tail, which mediates compaction, resides in the stretch of amino acids 14-19.     

A Role for H3K4 Monomethylation in Gene Repression and Partitioning of Chromatin Readers

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An octamer of core histones in solution: central role of the H3-H4 tetramer in the self-assembly.

The association of histones H2A, H2B, H3, and H4 in solution has been studied. In 2 M NaCl and at neutral pH they can assemble in a complex in which each histone is present in equimolar amounts. The complex has a weight average molecular weight of 98,000 (+/- 3700) and a sedimentation coefficient (so20,w) of 4.8. The value of the weight average molecular weight and the histone stoichiometry indicate that the complex is an octamer. The pairs of histones H2A,H2B and H3,H4 studied separately under identical conditions only associated as equimolar complexes consistent with dimeric and tetrameric structures, respectively. The stability of the core histone octamer is a function of the ionic strength, pH, and concentration of protein. The octamer dissociates by losing dimers of H2A,H2B until the main complexes existing in solution are the H3.H4 tetramer and the H2A.H2B dimer. This process is reversible upon reestablishing the original conditions.     

Chromatin on a membrane: accessibility of histone H5 for antibodies in the supernucleosomal structure

Histone H5 accessibility for the antibodies in chromatin was studied. Chromatin was immobilised on the nitrocellulose membrane in conditions which provide different levels of its compactization. Antiserum specific to the globular domain of histone H5 was used. It was shown, that for establishing real protection of histone H5 in the supernucleosomal structure it is necessary to use long fibers of chromatin. Their linking to the membrane must occur by a minimum quantity of points. It was established, that histone H5 is 5 times more accessive in the preparations of dispersed chromatin (low ionic strength) then in chromatin with the supernucleosomal organization (physiological ionic strength). We suppose that the small level of accessibility of histone H5 for the antibodies in the compact chromatin can be explained by some disruptions in the supernucleosomal organization. On the contrary, the long equable solenoid of nucleosomes provides complete protection of histone H5. In accordance with the results obtained, the model of ordered packaging of nucleosomes in the solenoid is discussed. In this model the point of entrance and exit of DNA on the nucleosomes, fixed by globular region of histone H5, is localized inside the solenoid.     

Protein structure probed by polarization spectroscopy. I. Evidence for fibrinogen rigidity from stationary fluorescence

Two fluorescent derivatives of human fibrinogen have been synthesized, by the covalent bonding of 1-dimethylaminoaphthalene-5-sulphonyl and methylpyrene chromophores, to investigate the internal molecular dynamics of the protein in solution. The stationary fluorescence depolarization of these derivatives under isothermal conditions is described here while in an accompanying paper (part II) a time-resolved study is reported. From the static fluorescence data it is concluded that reorientational processes in the subnanosecond and microsecond time ranges account for all the observed depolarization. The faster motion was assigned to the restricted, localized oscillations of the label while the slow motion was ascribed to the overall rotation of the protein molecule. Consequently, the protein in solution appears considerably rigid in the 10-1000 ns range, in contrast with a previous conception of a flexible fibrinogen based on non-isothermal depolarization experiments. These previous experiments are, in fact, concordant with the rigid fibrinogen proposed here if they are reinterpreted using Weber's early ideas on thermally activated depolarization (G. Weber, J. Biochem. 51 (1952) 145).     

Recognition of sequence-directed structure of the ssDNA backbone by nucleases

Escherichia coli endonuclease I and exonuclease VII appear to recognize sequence-dependent conformations in the ssDNA backbone. ssDNAs, containing either A- and/or T-tract or a CAP binding region, were digested with these nucleases under conditions which minimize the formation of secondary structures. The digestion patterns were examined in relation to previous results of biochemical and crystallographic studies on dsDNA, and showed broad agreement. Endonuclease I cleaved ssDNA at sites corresponding to bent sites in dsDNA.     

Evidence for a central role for electro-osmosis in fluid transport by corneal endothelium

The mechanism of transepithelial fluid transport remains unclear. The prevailing explanation is that transport of electrolytes across cell membranes results in local concentration gradients and transcellular osmosis. However, when transporting fluid, the corneal endothelium spontaneously generates a locally circulating current of approximately 25 microA cm(-2), and we report here that electrical currents (0 to +/-15 microA cm(-2)) imposed across this layer induce fluid movements linear with the currents. As the imposed currents must be approximately 98% paracellular, the direction of induced fluid movements and the rapidity with which they follow current imposition (rise time < or =3 sec) is consistent with electro-osmosis driven by sodium movement across the paracellular pathway. The value of the coupling coefficient between current and fluid movements found here (2.37 +/- 0.11 microm cm(2) hr(-1) microA (-1), suggests that: 1) the local endothelial current accounts for spontaneous transendothelial fluid transport; 2) the fluid transported becomes isotonically equilibrated. Ca(++)-free solutions or endothelial damage eliminate the coupling, pointing to the cells and particularly their intercellular junctions as a main site of electro-osmosis. The polycation polylysine, which is expected to affect surface charges, reverses the direction of current-induced fluid movements. Fluid transport is proportional to the electrical resistance of the ambient medium. Taken together, the results suggest that electro-osmosis through the intercellular junctions is the primary process in a sequence of events that results in fluid transport across this preparation.     

Chromatin state analysis of the barley epigenome reveals a higher‐order structure defined by H3K27me1 and H3K27me3 abundance

Combinations of histones carrying different covalent modifications are a major component of epigenetic variation. We have mapped nine modified histones in the barley seedling epigenome by chromatin immunoprecipitation next‐generation sequencing (ChIP‐seq). The chromosomal distributions of the modifications group them into four different classes, and members of a given class also tend to coincide at the local DNA level, suggesting that global distribution patterns reflect local epigenetic environments. We used this peak sharing to define 10 chromatin states representing local epigenetic environments in the barley genome. Five states map mainly to genes and five to intergenic regions. Two genic states involving H3K36me3 are preferentially associated with constitutive gene expression, while an H3K27me3‐containing genic state is associated with differentially expressed genes. The 10 states display striking distribution patterns that divide barley chromosomes into three distinct global environments. First, telomere‐proximal regions contain high densities of H3K27me3 covering both genes and intergenic DNA, together with very low levels of the repressive H3K27me1 modification. Flanking these are gene‐rich interior regions that are rich in active chromatin states and have greatly decreased levels of H3K27me3 and increasing amounts of H3K27me1 and H3K9me2. Lastly, H3K27me3‐depleted pericentromeric regions contain gene islands with active chromatin states separated by extensive retrotransposon‐rich regions that are associated with abundant H3K27me1 and H3K9me2 modifications. We propose an epigenomic framework for barley whereby intergenic H3K27me3 specifies facultative heterochromatin in the telomere‐proximal regions and H3K27me1 is diagnostic for constitutive heterochromatin elsewhere in the barley genome.     

The role of the central globular domain of histone H5 in chromatin structure

Histone H5 contains three tyrosines in the central, apolar region of the molecule. All three tyrosines can be spin labeled at low ionic strength. When the central globular domain is folded at high ionic strength, only one tyrosine becomes accessible to the imidazole spin label. Spin labeling the buried tyrosines prevents the folding of the globular structure, which, in turn, affects the proper binding of the H5 molecule to stripped chromatin. Chromatin complexes reconstituted from such an extensively modified H5 molecule show a weaker protection of the 168 base pair chromatosome during nuclease digestion. However, when only the surface tyrosine of the H5 molecule is labeled, such a molecule can still bind correctly to stripped chromatin, yielding a complex very similar to that of native chromatin. Our data supports the idea that not just the presence of the linker histone H5, but the presence of an intact H5 molecule with a folded, globular central domain in essential in the recognition of its specific binding sites on the nucleosomes. Our data also show that during the chromatin condensation process, the tumbling environment of the spin label attached to the surface tyrosine in the H5 molecule is not greatly hindered but remains partially mobile. This suggests that either the labeled domain of the H5 molecule is not directly involved in the condensation process or the formation of the higher-order chromatin structure does not result is a more viscous or tighter environment around the spin label. The folded globular domain of H5 molecule serves in stabilizing the nucleosome structure, as well as the higher-order chromatin structure.