Cryoelectron tomography reveals the multiplex anatomy of condensed native chromatin and its unfolding by histone citrullination.

Nucleosome chains fold and self-associate to form higher-order structures whose internal organization is unknown. Here, cryoelectron tomography (cryo-ET) of native human chromatin reveals intrinsic folding motifs such as (1) non-uniform nucleosome stacking, (2) intermittent parallel and perpendicular orientations of adjacent nucleosome planes, and (3) a regressive nucleosome chain path, which deviates from the direct zigzag topology seen in reconstituted nucleosomal arrays. By examining the self-associated structures, we observed prominent nucleosome stacking in cis and anti-parallel nucleosome interactions, which are consistent with partial nucleosome interdigitation in trans. Histone citrullination strongly inhibits nucleosome stacking and self-association with a modest effect on chromatin folding, whereas the reconstituted arrays undergo a dramatic unfolding into open zigzag chains induced by histone citrullination. This study sheds light on the internal structure of compact chromatin nanoparticles and suggests a mechanism for how epigenetic changes in chromatin folding are retained across both open and condensed forms.

Unraveling the multiplex folding of nucleosome chains in higher order chromatin.

The DNA of eukaryotic chromatin and chromosomes is repeatedly supercoiled around histone octamers forming 'beads-on-a-string' chains of nucleosomes. The extent of nucleosome chain folding and DNA accessibility vary between different functional and epigenetic states of nuclear chromatin and change dramatically upon cell differentiation, but the molecular mechanisms that direct 3D folding of the nucleosome chain in vivo are still enigmatic. Recent advances in cell imaging and chromosome capture techniques have radically challenged the established paradigm of regular and hierarchical chromatin fibers by highlighting irregular chromatin organization and the importance of the nuclear skeletal structures hoisting the nucleosome chains. Here, we argue that, by analyzing individual structural elements of the nucleosome chain - nucleosome spacing, linker DNA conformations, internucleosomal interactions, and nucleosome chain flexibility - and integrating these elements in multiplex 3D structural models, we can predict the features of the multiplex chromatin folding assemblies underlying distinct developmental and epigenetic states in living cells. Furthermore, partial disassembly of the nuclear structures suspending chromatin fibers may reveal the intrinsic mechanisms of nucleosome chain folding. These mechanisms and structures are expected to provide molecular cues to modify chromatin structure and functions related to developmental and disease processes.

Nucleosome spacing periodically modulates nucleosome chain folding and DNA topology in circular nucleosome arrays.

The length of linker DNA that separates nucleosomes is highly variable, but its mechanistic role in modulating chromatin structure and functions remains unknown. Here, we established an experimental system using circular arrays of positioned nucleosomes to investigate whether variations in nucleosome linker length could affect nucleosome folding, self-association, and interactions. We conducted EM, DNA topology, native electrophoretic assays, and Mg2+-dependent self-association assays to study intrinsic folding of linear and circular nucleosome arrays with linker DNA length of 36 bp and 41 bp (3.5 turns and 4 turns of DNA double helix, respectively). These experiments revealed that potential artifacts arising from open DNA ends and full DNA relaxation in the linear arrays do not significantly affect overall chromatin compaction and self-association. We observed that the 0.5 DNA helical turn difference between the two DNA linker lengths significantly affects DNA topology and nucleosome interactions. In particular, the 41-bp linkers promoted interactions between any two nucleosome beads separated by one bead as expected for a zigzag fiber, whereas the 36-bp linkers promoted interactions between two nucleosome beads separated by two other beads and also reduced negative superhelicity. Monte Carlo simulations accurately reproduce periodic modulations of chromatin compaction, DNA topology, and internucleosomal interactions with a 10-bp periodicity. We propose that the nucleosome spacing and associated chromatin structure modulations may play an important role in formation of different chromatin epigenetic states, thus suggesting implications for how chromatin accessibility to DNA-binding factors and the RNA transcription machinery is regulated.

Chromatin Higher-Order Folding: A Perspective with Linker DNA Angles.

The mechanism by which the "beads-on-a-string" nucleosome chain folds into various higher-order chromatin structures in eukaryotic cell nuclei is still poorly understood. The various models depicting higher-order chromatin as regular helical fibers and the very opposite "polymer melt" theory imply that interactions between nucleosome "beads" make the main contribution to the chromatin compaction. Other models in which the geometry of linker DNA "strings" entering and exiting the nucleosome define the three-dimensional structure predict that small changes in the linker DNA configuration may strongly affect nucleosome chain folding and chromatin higher-order structure. Among those studies, the cross-disciplinary approach pioneered by Jörg Langowski that combines computational modeling with biophysical and biochemical experiments was most instrumental for understanding chromatin higher-order structure in vitro. Strikingly, many recent studies, including genome-wide nucleosome interaction mapping and chromatin imaging, show an excellent agreement with the results of three-dimensional computational modeling based on the primary role of linker DNA geometry in chromatin compaction. This perspective relates nucleosome array models with experimental studies of nucleosome array folding in vitro and in situ. I argue that linker DNA configuration plays a key role in determining nucleosome chain flexibility, topology, and propensity for self-association, thus providing new implications for regulation of chromatin accessibility to DNA binding factors and RNA transcription machinery as well as long-range communications between distant genomic sites.

DNA topology in chromatin is defined by nucleosome spacing.

In eukaryotic nucleosomes, DNA makes ~1.7 superhelical turns around histone octamer. However, there is a long-standing discrepancy between the nucleosome core structure determined by x-ray crystallography and measurements of DNA topology in circular minichromosomes, indicating that there is only ~1.0 superhelical turn per nucleosome. Although several theoretical assumptions were put forward to explain this paradox by conformational variability of the nucleosome linker, none was tested experimentally. We analyzed topological properties of DNA in circular nucleosome arrays with precisely positioned nucleosomes. Using topological electrophoretic assays and electron microscopy, we demonstrate that the DNA linking number per nucleosome strongly depends on the nucleosome spacing and varies from -1.4 to -0.9. For the predominant {10n + 5} class of nucleosome repeats found in native chromatin, our results are consistent with the DNA topology observed earlier. Thus, we reconcile the topological properties of nucleosome arrays with nucleosome core structure and provide a simple explanation for the DNA topology in native chromatin with variable DNA linker length. Topological polymorphism of the chromatin fibers described here may reflect a more general tendency of chromosomal domains containing active or repressed genes to acquire different nucleosome spacing to retain topologically distinct higher-order structures.

Regulation of chromatin folding by conformational variations of nucleosome linker DNA.

Linker DNA conformational variability has been proposed to direct nucleosome array folding into more or less compact chromatin fibers but direct experimental evidence for such models are lacking. Here, we tested this hypothesis by designing nucleosome arrays with A-tracts at specific locations in the nucleosome linkers to induce inward (AT-IN) and outward (AT-OUT) bending of the linker DNA. Using electron microscopy and analytical centrifugation techniques, we observed spontaneous folding of AT-IN nucleosome arrays into highly compact structures, comparable to those induced by linker histone H1. In contrast, AT-OUT nucleosome arrays formed less compact structures with decreased nucleosome interactions similar to wild-type nucleosome arrays. Adding linker histone H1 further increased compaction of the A-tract arrays while maintaining structural differences between them. Furthermore, restriction nuclease digestion revealed a strongly reduced accessibility of nucleosome linkers in the compact AT-IN arrays. Electron microscopy analysis and 3D computational Monte Carlo simulations are consistent with a profound zigzag linker DNA configuration and closer nucleosome proximity in the AT-IN arrays due to inward linker DNA bending. We propose that the evolutionary preferred positioning of A-tracts in DNA linkers may control chromatin higher-order folding and thus influence cellular processes such as gene expression, transcription and DNA repair.

Genome-wide mapping of histone H3K9me2 in acute myeloid leukemia reveals large chromosomal domains associated with massive gene silencing and sites of genome instability.

A facultative heterochromatin mark, histone H3 lysine 9 dimethylation (H3K9me2), which is mediated by histone methyltransferases G9a/GLP (EHMT2/1), undergoes dramatic rearrangements during myeloid cell differentiation as observed by chromatin imaging. To determine whether these structural transitions also involve genomic repositioning of H3K9me2, we used ChIP-sequencing to map genome-wide topography of H3K9me2 in normal human granulocytes, normal CD34+ hematopoietic progenitors, primary myeloblasts from acute myeloid leukemia (AML) patients, and a model leukemia cell line K562. We observe that H3K9me2 naturally repositions from the previously designated "repressed" chromatin state in hematopoietic progenitors to predominant association with heterochromatin regions in granulocytes. In contrast, AML cells accumulate H3K9me2 on previously undefined large (> 100 Kb) genomic blocks that are enriched with AML-specific single nucleotide variants, sites of chromosomal translocations, and genes downregulated in AML. Specifically, the AML-specific H3K9me2 blocks are enriched with genes regulated by the proto-oncogene ERG that promotes stem cell characteristics. The AML-enriched H3K9me2 blocks (in contrast to the heterochromatin-associated H3K9me2 blocks enriched in granulocytes) are reduced by pharmacological inhibition of the histone methyltransferase G9a/GLP in K562 cells concomitantly with transcriptional activation of ERG and ETS1 oncogenes. Our data suggest that G9a/GLP mediate formation of transient H3K9me2 blocks that are preserved in AML myeloblasts and may lead to an increased rate of AML-specific mutagenesis and chromosomal translocations.

Nucleosome-like, Single-stranded DNA (ssDNA)-Histone Octamer Complexes and the Implication for DNA Double Strand Break Repair.

Repair of DNA double strand breaks (DSBs) is key for maintenance of genome integrity. When DSBs are repaired by homologous recombination, DNA ends can undergo extensive processing, producing long stretches of single-stranded DNA (ssDNA). In vivo, DSB processing occurs in the context of chromatin, and studies indicate that histones may remain associated with processed DSBs. Here we demonstrate that histones are not evicted from ssDNA after in vitro chromatin resection. In addition, we reconstitute histone-ssDNA complexes (termed ssNucs) with ssDNA and recombinant histones and analyze these particles by a combination of native gel electrophoresis, sedimentation velocity, electron microscopy, and a recently developed electrostatic force microscopy technique, DREEM (dual-resonance frequency-enhanced electrostatic force microscopy). The reconstituted ssNucs are homogenous and relatively stable, and DREEM reveals ssDNA wrapping around histones. We also find that histone octamers are easily transferred in trans from ssNucs to either double-stranded DNA or ssDNA. Furthermore, the Fun30 remodeling enzyme, which has been implicated in DNA repair, binds ssNucs preferentially over nucleosomes, and ssNucs are effective at activating Fun30 ATPase activity. Our results indicate that ssNucs may be a hallmark of processes that generate ssDNA, and that posttranslational modification of ssNucs may generate novel signaling platforms involved in genome stability.

Hierarchical looping of zigzag nucleosome chains in metaphase chromosomes.

The architecture of higher-order chromatin in eukaryotic cell nuclei is largely unknown. Here, we use electron microscopy-assisted nucleosome interaction capture (EMANIC) cross-linking experiments in combination with mesoscale chromatin modeling of 96-nucleosome arrays to investigate the internal organization of condensed chromatin in interphase cell nuclei and metaphase chromosomes at nucleosomal resolution. The combined data suggest a novel hierarchical looping model for chromatin higher-order folding, similar to rope flaking used in mountain climbing and rappelling. Not only does such packing help to avoid tangling and self-crossing, it also facilitates rope unraveling. Hierarchical looping is characterized by an increased frequency of higher-order internucleosome contacts for metaphase chromosomes compared with chromatin fibers in vitro and interphase chromatin, with preservation of a dominant two-start zigzag organization associated with the 30-nm fiber. Moreover, the strong dependence of looping on linker histone concentration suggests a hierarchical self-association mechanism of relaxed nucleosome zigzag chains rather than longitudinal compaction as seen in 30-nm fibers. Specifically, concentrations lower than one linker histone per nucleosome promote self-associations and formation of these looped networks of zigzag fibers. The combined experimental and modeling evidence for condensed metaphase chromatin as hierarchical loops and bundles of relaxed zigzag nucleosomal chains rather than randomly coiled threads or straight and stiff helical fibers reconciles aspects of other models for higher-order chromatin structure; it constitutes not only an efficient storage form for the genomic material, consistent with other genome-wide chromosome conformation studies that emphasize looping, but also a convenient organization for local DNA unraveling and genome access.

A role for stefin B (cystatin B) in inflammation and endotoxemia.

Stefin B (cystatin B) is an endogenous cysteine cathepsin inhibitor, and the loss-of-function mutations in the stefin B gene were reported in patients with Unverricht-Lundborg disease (EPM1). In this study we demonstrated that stefin B-deficient (StB KO) mice were significantly more sensitive to the lethal LPS-induced sepsis and secreted higher amounts of pro-inflammatory cytokines IL-1ß and IL-18 in the serum. We further showed that increased caspase-11 gene expression and better pro-inflammatory caspase-1 and -11 activation determined in StB KO bone marrow-derived macrophages resulted in enhanced IL-1ß processing. Pretreatment of macrophages with the cathepsin inhibitor E-64d did not affect secretion of IL-1ß, suggesting that the increased cathepsin activity determined in StB KO bone marrow-derived macrophages is not essential for inflammasome activation. Upon LPS stimulation, stefin B was targeted into the mitochondria, and the lack of stefin B resulted in the increased destabilization of mitochondrial membrane potential and mitochondrial superoxide generation. Collectively, our study demonstrates that the LPS-induced sepsis in StB KO mice is dependent on caspase-11 and mitochondrial reactive oxygen species but is not associated with the lysosomal destabilization and increased cathepsin activity in the cytosol.

Nucleosome-positioning sequence repeats impact chromatin silencing in yeast minichromosomes.

Eukaryotic gene expression occurs in the context of structurally distinct chromosomal domains such as the relatively open, gene-rich, and transcriptionally active euchromatin and the condensed and gene-poor heterochromatin where its specific chromatin environment inhibits transcription. To study gene silencing by heterochromatin, we created a minichromosome reporter system where the gene silencer elements were used to repress the URA3 reporter gene. The minichromosome reporters were propagated in yeast Saccharomyces cerevisiae at a stable copy number. Conduction of gene silencing through nucleosome arrays was studied by placing various repeats of clone-601 DNA with high affinity for histones between the silencer and reporter in the yeast minichromosomes. High-resolution chromatin mapping with micrococcal nuclease showed that the clone-601 nucleosome positioning downstream of the HML-E gene silencing element was not significantly altered by chromatin silencing. Using URA3 reporter assays, we observed that gene silencing was conducted through arrays of up to eight nucleosomes. We showed that the shorter nucleosome repeat lengths, typical of yeast (167 and 172 bp), were more efficient in conducting silencing in vivo compared to the longer repeats (207 bp) typical of higher eukaryotes. Both the longer and the shorter repeat lengths were able to conduct silencing in minichromosomes independently of clone-601 nucleosome positioning orientations vs. the silencer element. We suggest that the shorter nucleosome linkers are more suitable for conducting gene silencing than the long repeats in yeast due to their higher propensity to support native-like chromatin higher-order folding.

Developmentally regulated linker histone H1c promotes heterochromatin condensation and mediates structural integrity of rod photoreceptors in mouse retina.

Mature rod photoreceptor cells contain very small nuclei with tightly condensed heterochromatin. We observed that during mouse rod maturation, the nucleosomal repeat length increases from 190 bp at postnatal day 1 to 206 bp in the adult retina. At the same time, the total level of linker histone H1 increased reaching the ratio of 1.3 molecules of total H1 per nucleosome, mostly via a dramatic increase in H1c. Genetic elimination of the histone H1c gene is functionally compensated by other histone variants. However, retinas in H1c/H1e/H1(0) triple knock-outs have photoreceptors with bigger nuclei, decreased heterochromatin area, and notable morphological changes suggesting that the process of chromatin condensation and rod cell structural integrity are partly impaired. In triple knock-outs, nuclear chromatin exposed several epigenetic histone modification marks masked in the wild type chromatin. Dramatic changes in exposure of a repressive chromatin mark, H3K9me2, indicate that during development linker histone plays a role in establishing the facultative heterochromatin territory and architecture in the nucleus. During retina development, the H1c gene and its promoter acquired epigenetic patterns typical of rod-specific genes. Our data suggest that histone H1c gene expression is developmentally up-regulated to promote facultative heterochromatin in mature rod photoreceptors.

Combined micrococcal nuclease and exonuclease III digestion reveals precise positions of the nucleosome core/linker junctions: implications for high-resolution nucleosome mapping.

Micrococcal nuclease (MNase) is extensively used in genome-wide mapping of nucleosomes but its preference for AT-rich DNA leads to errors in establishing precise positions of nucleosomes. Here, we show that the MNase digestion of nucleosomes assembled on a strong nucleosome positioning sequence, Widom's clone 601, releases nucleosome cores whose sizes are strongly affected by the linker DNA sequence. Our experiments produced nucleosomal DNA sizes varying between 147 and 155 bp, with positions of the MNase cuts reflecting positions of the A⋅T pairs rather than the nucleosome core/linker junctions determined by X-ray crystallography. Extent of chromatosomal DNA protection by linker histone H1 also depends on the linker DNA sequence. Remarkably, we found that a combined treatment with MNase and exonuclease III (exoIII) overcomes MNase sequence preference producing nucleosomal DNA trimmed symmetrically and precisely at the core/linker junctions regardless of the underlying DNA sequence. We propose that combined MNase/exoIII digestion can be applied to in situ chromatin for unbiased genome-wide mapping of nucleosome positions that is not influenced by DNA sequences at the core/linker junctions. The same approach can be also used for the precise mapping of the extent of linker DNA protection by H1 and other protein factors associated with nucleosome linkers.

Nucleosome spacing and chromatin higher-order folding.

Packing of about two meters of the human genome DNA into chromatin occupying a several micron-sized cell nucleus requires a high degree of compaction in a manner that allows the information encoded on DNA to remain easily accessible. This packing is mediated by repeated coiling of DNA double helix around histones to form nucleosome arrays that are further folded into higher-order structures. Relatively straight DNA linkers separate the nucleosomes and the spacing between consecutive nucleosome varies between different cells and between different chromosomal loci. In a recent work ( 1) our group used a biochemically defined in vitro reconstituted system to explore how do various DNA linkers mediate nucleosome array packing into higher-order chromatin structures. For long nucleosome linkers (about 60 bp) we observed a more open chromatin structure and no effect of small linker length alterations (±2-4 bp) on chromatin folding. In striking contrast, for shorter linkers (20-32 bp) we found more compact packing with strong periodical dependence upon the linker DNA lengths. Our data together with high-resolution nucleosome position mapping provide evidence for the natural nucleosome repeats to support a chromatin architecture that, by default, restricts spontaneous folding of nucleosome arrays into compact chromatin fibers. We suggest that incomplete folding of the nucleosome arrays may promote global inter-array interactions that lead to chromatin condensation in metaphase chromosomes and heterochromatin.

Short nucleosome repeats impose rotational modulations on chromatin fibre folding.

In eukaryotic cells, DNA is organized into arrays of repeated nucleosomes where the shorter nucleosome repeat length (NRL) types are associated with transcriptionally active chromatin. Here, we tested a hypothesis that systematic variations in the NRL influence nucleosome array folding into higher-order structures. For NRLs with fixed rotational settings, we observed a negative correlation between NRL and chromatin folding. Rotational variations within a range of longer NRLs (188 bp and above) typical of repressed chromatin in differentiated cells did not reveal any changes in chromatin folding. In sharp contrast, for the shorter NRL range of 165-177 bp, we observed a strong periodic dependence of chromatin folding upon the changes in linker DNA lengths, with the 172 bp repeat found in highly transcribed yeast chromatin imposing an unfolded state of the chromatin fibre that could be reversed by linker histone. Our results suggest that the NRL may direct chromatin higher-order structure into either a nucleosome position-dependent folding for short NRLs typical of transcribed genes or an architectural factor-dependent folding typical of longer NRLs prevailing in eukaryotic heterochromatin.

Chromatin organization - the 30 nm fiber.

Despite over 30 years of work, the fundamental structure of eukaryotic chromatin remains controversial. Here, we review the roots of this controversy in disparities between results derived from studies of chromatin in nuclei, chromatin isolated from nuclei, and chromatin reconstituted from defined components. Thanks to recent advances in imaging, modeling, and other approaches, it is now possible to recognize some unifying principles driving chromatin architecture at the level of the ubiquitous '30 nm' chromatin fiber. These suggest that fiber architecture involves both zigzag and bent linker motifs, and that such heteromorphic structures facilitate the observed high packing ratios. Interactions between neighboring fibers in highly compact chromatin lead to extensive interdigitation of nucleosomes and the inability to resolve individual fibers in compact chromatin in situ.

A single heterochromatin boundary element imposes position-independent antisilencing activity in Saccharomyces cerevisiae minichromosomes.

Chromatin boundary elements serve as cis-acting regulatory DNA signals required to protect genes from the effects of the neighboring heterochromatin. In the yeast genome, boundary elements act by establishing barriers for heterochromatin spreading and are sufficient to protect a reporter gene from transcriptional silencing when inserted between the silencer and the reporter gene. Here we dissected functional topography of silencers and boundary elements within circular minichromosomes in Saccharomyces cerevisiae. We found that both HML-E and HML-I silencers can efficiently repress the URA3 reporter on a multi-copy yeast minichromosome and we further showed that two distinct heterochromatin boundary elements STAR and TEF2-UASrpg are able to limit the heterochromatin spreading in circular minichromosomes. In surprising contrast to what had been observed in the yeast genome, we found that in minichromosomes the heterochromatin boundary elements inhibit silencing of the reporter gene even when just one boundary element is positioned at the distal end of the URA3 reporter or upstream of the silencer elements. Thus the STAR and TEF2-UASrpg boundary elements inhibit chromatin silencing through an antisilencing activity independently of their position or orientation in S. cerevisiae minichromosomes rather than by creating a position-specific barrier as seen in the genome. We propose that the circular DNA topology facilitates interactions between the boundary and silencing elements in the minichromosomes.

A structural perspective on the where, how, why, and what of nucleosome positioning.

The DNA in eukaryotic chromatin is packed by histones into arrays of repeating units called nucleosomes. Each nucleosome contains a nucleosome core, where the DNA is wrapped around a histone octamer, and a stretch of relatively unconstrained DNA called the linker DNA. Since nucleosome cores occlude the DNA from many DNA-binding factors, their positions provide important clues for understanding chromatin packing and gene regulation. Here we review the recent advances in the genome-wide mapping of nucleosome positions, the molecular and structural determinants of nucleosome positioning, and the importance of nucleosome positioning in chromatin higher order folding and transcriptional regulation.

Rearrangement of upstream sequences of the hTERT gene during cellular immortalization.

Telomerase expression, resulting from transcriptional activation of the hTERT gene, allows cells to acquire indefinite proliferative potential during cellular immortalization and tumorigenesis. However, mechanisms of hTERT gene activation in many immortal cell lines and cancer cells are poorly understood. Here, we report our studies on hTERT activation using genetically related pairs of telomerase-negative (Tel(-)) and -positive (Tel(+)) fibroblast lines. First, whereas transiently transfected plasmid reporters did not recapitulate the endogenous hTERT promoter, the promoter in chromosomally integrated bacterial artificial chromosome (BAC) reporters was activated in a subset of Tel(+) cells, indicating that activation of the hTERT promoter required native chromatin context and/or distal regulatory elements. Second, the hTERT gene, located near the telomere of chromosome 5p, was translocated in all three Tel(+) cell lines but not in their parental precrisis cells and Tel(-) immortal siblings. The breakage points were mapped to regions upstream of the hTERT promoter, indicating that the hTERT gene was the target of these chromosomal rearrangements. In two Tel(+) cell lines, translocation of the endogenous hTERT gene appeared to be the major mechanism of its activation as the activity of hTERT promoter in many chromosomally integrated BAC reporters, with intact upstream and downstream neighboring loci, remained relatively low. Therefore, our results suggest that rearrangement of upstream sequences is an important new mechanism of hTERT promoter activation during cellular immortalization. The chromosomal rearrangements likely occurred during cellular crisis and facilitated by telomere dysfunction. Such translocations allowed the hTERT promoter to escape from the native condensed chromatin environment.

Evidence for heteromorphic chromatin fibers from analysis of nucleosome interactions.

The architecture of the chromatin fiber, which determines DNA accessibility for transcription and other template-directed biological processes, remains unknown. Here we investigate the internal organization of the 30-nm chromatin fiber, combining Monte Carlo simulations of nucleosome chain folding with EM-assisted nucleosome interaction capture (EMANIC). We show that at physiological concentrations of monovalent ions, linker histones lead to a tight 2-start zigzag dominated by interactions between alternate nucleosomes (i +/- 2) and sealed by histone N-tails. Divalent ions further compact the fiber by promoting bending in some linker DNAs and hence raising sequential nucleosome interactions (i +/- 1). Remarkably, both straight and bent linker DNA conformations are retained in the fully compact chromatin fiber as inferred from both EMANIC and modeling. This conformational variability is energetically favorable as it helps accommodate DNA crossings within the fiber axis. Our results thus show that the 2-start zigzag topology and the type of linker DNA bending that defines solenoid models may be simultaneously present in a structurally heteromorphic chromatin fiber with uniform 30 nm diameter. Our data also suggest that dynamic linker DNA bending by linker histones and divalent cations in vivo may mediate the transition between tight nucleosome packing within discrete 30-nm fibers and self-associated higher-order chromosomal forms.