Linker histone H1 plays an important role in chromatin folding in vitro. To study the role of H1 in vivo, mouse embryonic stem cells null for three H1 genes were derived and were found to have 50% of the normal level of H1. H1 depletion caused dramatic chromatin structure changes, including decreased global nucleosome spacing, reduced local chromatin compaction, and decreases in certain core histone modifications. Surprisingly, however, microarray analysis revealed that expression of only a small number of genes is affected. Many of the affected genes are imprinted or are on the X chromosome and are therefore normally regulated by DNA methylation. Although global DNA methylation is not changed, methylation of specific CpGs within the regulatory regions of some of the H1 regulated genes is reduced. These results indicate that linker histones can participate in epigenetic regulation of gene expression by contributing to the maintenance or establishment of specific DNA methylation patterns.
Magnesium ions added to tRNAfMET1 selectively stabilize the dihydrouridine helix-tertiary structural region. Low Mg2+ levels have little direct effect on the remaining three cloverleaf helices, but these are prevented from melting independently when their intrinsic Tm is surpassed by the Tm of the tertiary structure. At high Mg2+ concentration the thermal unfolding of tRNAfMet1 is approximately a two-state, concerted transition from the globular native structure to the random coil, in contrast to the sequential unfolding observed without Mg2+. We interpret the kinetics of refolding to mean that the D helix serves as a required nucleus for the rate-limiting step of tertiary structure formation. We found that unfolding of the tertiary structure leads to loss of the tightly bound Mg2+ ions, and showed with a Mn2+-sensitive fluorescent indicator that the rate of Mn2+ release is the same as the rate of unfolding the tertiary structure. Hence the tightly bound divalent ion must be located in a site formed by the tertiary structure-D helix region of the molecule.
We provide evidence that nucleosomes can assemble in vitro at physiological ionic strength (0.1-0.2 M NaCl/10 mM Tris'HCI, pH 8.0) in the absence of "assembly factors" and that poly(gfutamic acid) greatly facilitates chromatin assembly under these conditions. We also show that in the presence of either poly(glutamic acid) or poly(aspartic acid), core histones assemble into octamers at physiological ionic strength. We suggest that it is a property of histones to assemble into octamers upon their interaction with macromolecules containing regions of high negative charge density, and we discuss several implications of this property. In eukaryotic cells, nuclbar DNA is wrapped around histone octamers, forming nucleosomes (1-3). Knowledge of the mechanisms by which DNA and histones are assembled into nucleosomes will be required for an understanding of chromatin replication and, possibly, gene regulation. It is possible to reconstitute nucleosomes in vitro from DNA and histones by using lengthy dialysis procedures starting from 2 M NaCl in the presence or absence of urea (4-7); in contrast, direct mixing of DNA and histones at physiological ionic strength results in precipitation of the nucleoprotein, and the nature of the interactions that occur is largely obscured. Recently, Laskey et al. (8) wert-able to assemble chromatin in vitro at physiological ionic strength by using an extract from the eggs of Xenopus laevis. Furthermore, Laskey et al. (9) purified from this extract an acidic "assembly protein" that binds histones and transfers them to the DNA. In 0.6 M NaCI, nucleosomes form rapidly when DNA and histones are mixed in the absence of any "assembly factors"; however, a competing assembly pathway exists in which newly formed nucleosomes bind additional histones as octamers, which subsequently are transferred to protein-free DNA (10). Thus, the possibility exists that this octamer transfer mechanism observed in 0.6 M salt and the mechanism at lower ionic strength, which appears to require an assembly factor, may be related.In this paper, we have investigated in vitro nucleosome assembly at "physiological" ionic strength (0.1-0.2 M NaCl/10 mM Tris-HCl, pH 8.0). We provide evidence that (i) nucleosomes can assemble in the absence of assembly factors, (ii) histones interact as octamers with acidic polypeptides, and (iii) poly(glutamic acid) greatly facilitates nucleosome assembly. MATERIALS AND METHODSPreparative Procedures. Chromatin core particles were prepared by micrococcal nuclease (Worthington) digestion of chicken erythrocyte nuclei as described (10). Salt-extracted core histones were prepared from chromatin that had been washed with 0.6 M NaCl as described (10). DNA was extracted from purified core particles with 3 M NaCI/0.05 M sodium phosphate buffer, pH 7.0, and purified with hydroxylapatite by a batch procedure. Simian virus 40 (SV40) DNA component Ir was prepared as described (11). SV40 DNA I was obtained from Bethesda Research Laboratories (Rockville, MD).Histone Crosslinking. Samples were cr...
The chromatin structures of a variety of plasmids and plasmid constructions, transiently transfected into mouse Ltk- cells using the DEAE-dextran procedure, were studied by micrococcal nuclease digestion of nuclei and Southern hybridization. Although regularly arranged nucleosome-like particles clearly were formed on the transfected DNA, the nucleosome ladders, in some cases with 13-14 bands, were anomalous. Most often, a ladder of DNA fragments with lengths of approximately 300, 500, 700, 900 bp, etc. was generated. In contrast, typical 180-190 bp multiples were generated from bulk cellular or endogenous beta-actin gene chromatin. Very similar results were obtained with all DNA's transfected, and in a variety of cell lines, provided that plasmid replication did not occur. Additionally, after digestion of nuclei, about 90% of the chromatin fragments that contained transfected DNA sequences could not be solubilized at low ionic strength, in contrast with bulk cellular chromatin, suggesting association with nuclear structures or nuclear matrix. The remaining 10% of transfected DNA sequences, arising from soluble chromatin fragments, generated a typical nucleosome ladder. These results are consistent with the idea that assembly of atypical chromatin structures might be induced by proximity to elements of the nuclear pore complex or by nuclear compartmentalization.
Equilibrium dialysis measurements show that tRNAfMet1 in 0.17 M Na+ has one strong Mg2+ binding site, K = 3 X 10(4) M-1, and approximately 26 weak binding sites with K = 4 X 10(2) M-1, with RNA concentration measured in moles of tRNA per liter and T = 4 degrees C. The data fit significantly less well to a model with two strong sites and a large class of weak sites. Binding is noncooperative. Our results differ from previous experiments showing cooperative binding because the binding equilibrium is not coupled to a cooperative conformational change of the macromolecule. Measurements at relatively high Na+ concentrations and low temperature ensure that the tRNA is in the "native" region of the conformational phase diagram for all Mg2+ concentrations.
A purine repressor (PurR) mediates adenine nucleotide-dependent regulation of transcription initiation of the Bacillus subtilis pur operon. This repressor has been purified for the first time, and binding to control site DNA was characterized. PurR binds in vitro to four operons. Apparent K d values for binding were 7 nM for the pur operon, 8 nM for purA, 13 nM for purR, and 44 nM for the pyr operon. In each case, DNase I footprints exhibited a pattern of protected and hypersensitive sites that extended over more than 60 bp. A GAAC-N 24 -GTTC sequence in the pur operon was necessary but not sufficient for the PurR-DNA interaction. However, this motif, which is conserved in the four binding sites, was not required for binding of PurR to purA. Thus, the common DNA recognition element for binding of PurR to the four operons is not known. Multiple PurR-pur operon DNA complexes having a binding stoichiometry that was either approximately two or six repressor molecules per DNA fragment were detected. The results of a torsional constraint experiment suggest that control site DNA forms one right-handed turn around PurR.The genes required for de novo synthesis of IMP are clustered in a 12-gene polycistronic operon in Bacillus subtilis (10). Transcription of the pur operon is subject to dual regulation. The addition of adenine to cells results in the repression of transcription initiation, whereas the addition of guanine signals premature transcription termination in the 242-nucleotide (nt) mRNA leader region preceding the first gene of the operon. A purine repressor (PurR) is required for the regulation of transcription initiation (11). The repressor is a 62-kDa homodimer containing 285 amino acid subunits (31) encoded by a purR gene at about 6°in a sequenced region (19) of the B. subtilis chromosome. The pur operon DNA site to which the crude repressor bound was mapped to a position corresponding to approximately Ϫ136 to Ϫ26 relative to the start of transcription (11). This site is contiguous with and overlaps the Ϫ35 promoter element at nts Ϫ33 to Ϫ28. Binding of PurR to the pur operon control site was blocked by phosphoribosylpyrophosphate (PRPP) leading to a model in which the excess adenine signal is transmitted to PurR by the PRPP pool (31). It was proposed that upon uptake, adenine is converted to adenine 5Ј nucleotides and that the resulting allosteric inhibition of PRPP synthetase by ADP (1) lowers the PRPP pool levels (26), permitting PurR to bind to and repress the transcription of the pur operon. B. subtilis PurR bears no amino acid sequence similarity to Escherichia coli PurR. Furthermore, there is no similarity in the DNA control sites for these two repressors or in the purine or purine nucleotide signals that modulate binding to the DNA control sites.There is presently no information about what determines B. subtilis PurR-pur operon DNA binding specificity. It has been noted, however, that PurR also binds to purR and to purA (31), but the common recognition determinant is unknown. The purA gene encodes the...
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