Modeling Studies of Chromatin Fiber Structure as a Function of DNA Linker Length

Chromatin fibers encountered in various species and tissues are characterized by different nucleosome repeat lengths (NRLs) of the linker DNA connecting the nucleosomes. While single cellular organisms and rapidly growing cells with high protein production have short NRL ranging from 160 to 189 bp, mature cells usually have longer NRLs ranging between 190 and 220 bp. Recently, various experimental studies have examined the effect of NRL on the internal organization of chromatin fiber. Here, we investigate by mesoscale modeling of oligonucleosomes the folding patterns for different NRL, with and without linker histone (LH), under typical monovalent salt conditions using both one-start solenoid and two-start zigzag starting configurations. We find that short to medium NRL chromatin fibers (173 to 209 bp) with LH condense into zigzag structures and that solenoid-like features are viable only for longer NRLs (226 bp). We suggest that medium NRLs are more advantageous for packing and various levels of chromatin compaction throughout the cell cycle than their shortest and longest brethren; the former (short NRLs) fold into narrow fibers, while the latter (long NRLs) arrays do not easily lead to high packing ratios due to possible linker DNA bending. Moreover, we show that the LH has a small effect on the condensation of short-NRL arrays but has an important condensation effect on medium-NRL arrays, which have linker lengths similar to the LH lengths. Finally, we suggest that the medium-NRL species, with densely packed fiber arrangements, may be advantageous for epigenetic control because their histone tail modifications can have a greater effect compared to other fibers due to their more extensive nucleosome interaction network.     

A critical role for linker DNA in higher-order folding of chromatin fibers

Nucleosome-nucleosome interactions drive the folding of nucleosomal arrays into dense chromatin fibers. A better physical account of the folding of chromatin fibers is necessary to understand the role of chromatin in regulating DNA transactions. Here, we studied the unfolding pathway of regular chromatin fibers as a function of single base pair increments in linker length, using both rigid base-pair Monte Carlo simulations and single-molecule force spectroscopy. Both computational and experimental results reveal a periodic variation of the folding energies due to the limited flexibility of the linker DNA. We show that twist is more restrictive for nucleosome stacking than bend, and find the most stable stacking interactions for linker lengths of multiples of 10 bp. We analyzed nucleosomes stacking in both 1- and 2-start topologies and show that stacking preferences are determined by the length of the linker DNA. Moreover, we present evidence that the sequence of the linker DNA also modulates nucleosome stacking and that the effect of the deletion of the H4 tail depends on the linker length. Importantly, these results imply that nucleosome positioning in vivo not only affects the phasing of nucleosomes relative to DNA but also directs the higher-order structure of chromatin.     

Organization of 5S genes in chromatin of Xenopus laevis.

The chromatin organization of the genes coding for 5S RNA in Xenopus laevis has been investigated with restriction endonucleases and micrococcal nuclease. Digestion of nuclei from liver, kidney, blood and kidney cells maintained in culture with micrococcal nuclease reveals that these Xenopus cells and tissues have shorter nucleosome repeat lengths than the corresponding cells and tissues from other higher organisms. 5S genes are organized in nucleosomes with repeat lengths similar to those of the bulk chromatin in liver (178 bp) and cultured cells (165 bp); however, 5S gene chromatin in blood cells has a shorter nucleosome repeat (176 bp) than the bulk of the genome in these cells (184 bp). From an analysis of the 5S DNA fragments produced by extensive restriction endonuclease cleavage of chromatin in situ, no special arrangement of the nucleosomes with respect to the sequence of 5S DNA can be detected. The relative abundance of 5S gene multimers follows a Kuhn distribution, with about 57% of all HindIII sites cleaved. This suggests that HindIII sites can be cleaved both in the nucleosome core and linker regions.     

Novel nucleosomal particles containing core histones and linker DNA but no histone H1

Eukaryotic chromosomal DNA is assembled into regularly spaced nucleosomes, which play a central role in gene regulation by determining accessibility of control regions. The nucleosome contains ∼147 bp of DNA wrapped ∼1.7 times around a central core histone octamer. The linker histone, H1, binds both to the nucleosome, sealing the DNA coils, and to the linker DNA between nucleosomes, directing chromatin folding. Micrococcal nuclease (MNase) digests the linker to yield the chromatosome, containing H1 and ∼160 bp, and then converts it to a core particle, containing ∼147 bp and no H1. Sequencing of nucleosomal DNA obtained after MNase digestion (MNase-seq) generates genome-wide nucleosome maps that are important for understanding gene regulation. We present an improved MNase-seq method involving simultaneous digestion with exonuclease III, which removes linker DNA. Remarkably, we discovered two novel intermediate particles containing 154 or 161 bp, corresponding to 7 bp protruding from one or both sides of the nucleosome core. These particles are detected in yeast lacking H1 and in H1-depleted mouse chromatin. They can be reconstituted in vitro using purified core histones and DNA. We propose that these ‘proto-chromatosomes’ are fundamental chromatin subunits, which include the H1 binding site and influence nucleosome spacing independently of H1.     

Relationship Between Chromatin High-order Folding and Nucleosomal Linker Twist in Nuclei of Human HeLa S3 Cells

Abstract

We have used the intercalative agent ethidium bromide to examine the association between chromatin high-order folding and the twist of internucleosomal DNA regions. The analysis was carried out on intact nuclei isolated from human HeLa S3 cells. Our data shows that alterations in the nucleosomal linker twist significantly influence the way in which a chain of nucleosomes folds to form different higher-order structures. The assay used allowed us to identify the existence of two chromatin fractions differing in their extent of high-order folding. We have also found that active gene sequences are preferentially associated with the chromatin fraction corresponding to the more extended conformation. A model is proposed to account for the effect of variations in the nucleosome linker twist on the state of chromatin folding.     

Linker histone subtypes differ in their effect on nucleosomal spacing in vivo

Linker histone H1 is located on the surface of the nucleosome where it interacts with the linker DNA region and stabilizes the 30-nm chromatin fiber. Vertebrates have several different, relatively conserved subtypes of H1; however, the functional reason for this is unclear. We have previously shown that H1 can be reconstituted in Xenopus oocytes, cells that lack somatic H1, by cytosolic mRNA injection and incorporated into in vivo assembled chromatin. Using this assay, we have expressed individual H1 subtypes in the oocytes to study their effect on chromatin structure using nucleosomal repeat length (NRL) as readout. We have compared chicken differentiation-specific histone H5, Xenopus differentiation-specific xH1(0) and the somatic variant xH1A as well as the ubiquitously expressed human somatic subtypes hH1.2, hH1.3, hH1.4 and hH1.5. This shows that all subtypes, except for human H1.5, result in a saturable increase in NRL. hH1.4 results in an increase of approximately 13-20 bp as does xH1(0) and xH1A. chH5 gives rise to the same or slightly longer increase compared to hH1.4. Interestingly, both hH1.2 and hH1.3 show a less extensive increase of only 4.5-7 bp in the NRL, thus yielding the shortest increase of the studied subtypes. We show for the first time in an in vivo system lacking H1 background that ubiquitously expressed and redundant H1 subtypes that coexist in most types of cells of higher eukaryotes differ in their effects on the nucleosomal spacing in vivo. This suggests that H1 subtypes have different roles in the organization and functioning of the chromatin fiber.     

An all-atom model of the chromatin fiber containing linker histones reveals a versatile structure tuned by the nucleosomal repeat length

In the nucleus of eukaryotic cells, histone proteins organize the linear genome into a functional and hierarchical architecture. In this paper, we use the crystal structures of the nucleosome core particle, B-DNA and the globular domain of H5 linker histone to build the first all-atom model of compact chromatin fibers. In this 3D jigsaw puzzle, DNA bending is achieved by solving an inverse kinematics problem. Our model is based on recent electron microscopy measurements of reconstituted fiber dimensions. Strikingly, we find that the chromatin fiber containing linker histones is a polymorphic structure. We show that different fiber conformations are obtained by tuning the linker histone orientation at the nucleosomes entry/exit according to the nucleosomal repeat length. We propose that the observed in vivo quantization of nucleosomal repeat length could reflect nature's ability to use the DNA molecule's helical geometry in order to give chromatin versatile topological and mechanical properties.     

Chromatin structure of Schizosaccharomyces pombe. A nucleosome repeat length that is shorter than the chromatosomal DNA length.

We have used new methods for chromatin isolation, together with conventional methods for measuring the nucleosome repeat length, to determine the repeat length of Schizosaccharomyces pombe chromatin. We obtain a result of 156(+/- 2) bp. Equivalent results are obtained using a psoralen crosslinking method for measuring the repeat length in viable spheroplasts. That result, together with other control experiments, rules out many possible artifacts. The measured value of 156(+/- 2) bp is smaller than the length of DNA found in the chromatosome. Thus, the chromatosome cannot be the fundamental unit of chromatin structure in all eukaryotes. The crossed linker model of chromatin higher order structure is incompatible with a nucleosome repeat length of 156 bp, and thus cannot apply to all eukaryotes. The solenoid model of higher order structure is compatible with this repeat length only if the solenoid is right-handed. We note two other properties of this chromatin. (1) Early in digestion, the DNA length of mononucleosomes from S. pombe and Aspergillus nidulans exceeds the nucleosome repeat length. (2) Many methods for isolating chromatin from S. pombe yield an apparent nucleosome repeat length of less than or equal to 140 bp; this result is found to be an artifactual consequence of nucleosome sliding.     

Nucleosome repeat lengths and columnar chromatin structure

Thorough quantitative study of nucleosome repeat length (NRL) distributions, conducted in 1992 by J. Widom, resulted in a striking observation that the linker lengths between the nucleosomes are quantized. Comparison of the NRL average values with the MNase cut distances predicted from the hypothetical columnar structure of chromatin (this work) shows a close correspondence between the two. This strongly suggests that the NRL distribution, actually, reflects the dominant role of columnar chromatin structure common for all eukaryotes.     

Preferentially quantized linker DNA lengths in Saccharomyces cerevisiae

The exact lengths of linker DNAs connecting adjacent nucleosomes specify the intrinsic three-dimensional structures of eukaryotic chromatin fibers. Some studies suggest that linker DNA lengths preferentially occur at certain quantized values, differing one from another by integral multiples of the DNA helical repeat, approximately 10 bp; however, studies in the literature are inconsistent. Here, we investigate linker DNA length distributions in the yeast Saccharomyces cerevisiae genome, using two novel methods: a Fourier analysis of genomic dinucleotide periodicities adjacent to experimentally mapped nucleosomes and a duration hidden Markov model applied to experimentally defined dinucleosomes. Both methods reveal that linker DNA lengths in yeast are preferentially periodic at the DNA helical repeat ( approximately 10 bp), obeying the forms 10n+5 bp (integer n). This 10 bp periodicity implies an ordered superhelical intrinsic structure for the average chromatin fiber in yeast.     

Effect of salt on the binding of the linker histone H1 to DNA and nucleosomes

The linker histones are involved in the salt-dependent folding of the nucleosomes into higher-order chromatin structures. To better understand the mechanism of action of these histones in chromatin, we studied the interactions of the linker histone H1 with DNA at various histone/DNA ratios and at different ionic strengths. In direct competition experiments, we have confirmed the binding of H1 to superhelical DNA in preference to linear or nicked circular DNA forms. We show that the electrophoretic mobility of the H1/supercoiled DNA complex decreases with increasing H1 concentrations and increases with ionic strengths. These results indicate that the interaction of the linker histone H1 with supercoiled DNA results in a soluble binding of H1 with DNA at low H1 or salt concentrations and aggregation at higher H1 concentrations. Moreover, we show that H1 dissociates from the DNA or nucleosomes at high salt concentrations. By the immobilized template pull-down assay, we confirm these data using the physiologically relevant nucleosome array template.     

A protein binds to a satellite DNA repeat at three specific sites that would be brought into mutual proximity by DNA folding in the nucleosome

Using a generally applicable assay for specific DNA-binding proteins in crude extracts, we have detected and purified an HMG-like nuclear protein from African green monkey cells that preferentially binds to the 172 bp repeat of alpha-satellite DNA (alpha-DNA). DNAase I footprinting with the purified protein detects three specific binding sites (I-III) per alpha-DNA repeat. Site II is 145 bp (one core nucleosome length) from site III on the adjacent alpha-DNA repeat, while site I lies midway between sites II and III. In the alpha-nucleosome phasing frame corresponding with this arrangement, sites I-III would be brought into mutual proximity by DNA folding in the nucleosome. This phasing frame is identical with the preferred frame detected previously in isolated chromatin. Our results suggest that this new and abundant protein recognizes a family of short, related nucleotide sequences found not only in alpha-DNA but also throughout the genome, and that functions of this protein are mediated through its nucleosome-positioning activity. Such nucleosome-positioning proteins may underlie the sequence specificity of both nucleosome arrangements and higher order chromatin structures.