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.

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 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.

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 Mg-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.

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.

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.

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.

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). , DSB processing occurs in the context of chromatin, and studies indicate that histones may remain associated with processed DSBs.

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.

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-1β 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-1β processing.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Conformational change in the chromatin remodelling protein MENT.

Chromatin condensation to heterochromatin is a mechanism essential for widespread suppression of gene transcription, and the means by which a chromatin-associated protein, MENT, induces a terminally differentiated state in cells. MENT, a protease inhibitor of the serpin superfamily, is able to undergo conformational change in order to effect enzyme inhibition. Here, we sought to investigate whether conformational change in MENT is 'fine-tuned' in the presence of a bound ligand in an analogous manner to other serpins, such as antithrombin where such movements are reflected by a change in intrinsic tryptophan fluorescence.

Chromatin condensation in terminally differentiating mouse erythroblasts does not involve special architectural proteins but depends on histone deacetylation.

Terminal erythroid differentiation in vertebrates is characterized by progressive heterochromatin formation and chromatin condensation and, in mammals, culminates in nuclear extrusion. To date, although mechanisms regulating avian erythroid chromatin condensation have been identified, little is known regarding this process during mammalian erythropoiesis. To elucidate the molecular basis for mammalian erythroblast chromatin condensation, we used Friend virus-infected murine spleen erythroblasts that undergo terminal differentiation in vitro.

Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation.

Peripheral blood neutrophils form highly decondensed chromatin structures, termed neutrophil extracellular traps (NETs), that have been implicated in innate immune response to bacterial infection. Neutrophils express high levels of peptidylarginine deiminase 4 (PAD4), which catalyzes histone citrullination. However, whether PAD4 or histone citrullination plays a role in chromatin structure in neutrophils is unclear.

DNA accelerates the inhibition of human cathepsin V by serpins.

A balance between proteolytic activity and protease inhibition is crucial to the appropriate function of many biological processes. There is mounting evidence for the presence of both papain-like cysteine proteases and serpins with a corresponding inhibitory activity in the nucleus. Well characterized examples of cofactors fine tuning serpin activity in the extracellular milieu are known, but such modulation has not been studied for protease-serpin interactions within the cell.

MeCP2-chromatin interactions include the formation of chromatosome-like structures and are altered in mutations causing Rett syndrome.

hMeCP2 (human methylated DNA-binding protein 2), mutations of which cause most cases of Rett syndrome (RTT), is involved in the transmission of repressive epigenetic signals encoded by DNA methylation. The present work focuses on the modifications of chromatin architecture induced by MeCP2 and the effects of RTT-causing mutants. hMeCP2 binds to nucleosomes close to the linker DNA entry-exit site and protects approximately 11 bp of linker DNA from micrococcal nuclease.