Sergei A. Grigoryev

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. In this study, we show that the hypercitrullination of histones by PAD4 mediates chromatin decondensation. Histone hypercitrullination is detected on highly decondensed chromatin in HL-60 granulocytes and blood neutrophils. The inhibition of PAD4 decreases histone hypercitrullination and the formation of NET-like structures, whereas PAD4 treatment of HL-60 cells facilitates these processes. The loss of heterochromatin and multilobular nuclear structures is detected in HL-60 granulocytes after PAD4 activation. Importantly, citrullination of biochemically defined avian nucleosome arrays inhibits their compaction by the linker histone H5 to form higher order chromatin structures. Together, these results suggest that histone hypercitrullination has important functions in chromatin decondensation in granulocytes/neutrophils.

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.

MENT, a Heterochromatin Protein That Mediates Higher Order Chromatin Folding, Is a New Serpin Family Member

Terminal cell differentiation is correlated with the extensive sequestering of previously active genes into compact transcriptionally inert heterochromatin. In vertebrate blood cells, these changes can be traced to the accumulation of a developmentally regulated heterochromatin protein, MENT. Cryoelectron microscopy of chicken granulocyte chromatin, which is highly enriched with MENT, reveals exceptionally compact polynucleosomes, which maintain a level of higher order folding above that imposed by linker histones. The amino acid sequence of MENT reveals a close structural relationship with serpins, a large family of proteins known for their ability to undergo dramatic conformational transitions. Conservation of the “hinge region” consensus in MENT indicates that this ability is retained by the protein. MENT is distinguished from the other serpins by being a basic protein, containing several positively charged surface clusters, which are likely to be involved in ionic interactions with DNA. One of the positively charged domains bears a significant similarity to the chromatin binding region of nuclear lamina proteins and with the A·T-rich DNA-binding motif, which may account for the targeting of MENT to peripheral heterochromatin. MENT ectopically expressed in a mammalian cell line is transported into nuclei and is associated with intranuclear foci of condensed chromatin.

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 ∼11 bp of linker DNA from micrococcal nuclease. MeCP2 mutants differ in this property; the R106W mutant gives very little extra protection beyond the ∼146-bp nucleosome core, whereas the large C-terminal truncation R294X reveals wild type behavior. Gel mobility assays show that linker DNA is essential for proper MeCP2 binding to nucleosomes, and electron microscopy visualization shows that the protein induces distinct conformational changes in the linker DNA. When bound to nucleosomes, MeCP2 is in close proximity to histone H3, which exits the nucleosome core close to the proposed MeCP2-binding site. These findings firmly establish nucleosomal linker DNA as a crucial binding partner of MeCP2 and show that different RTT-causing mutations of MeCP2 are correspondingly defective in different aspects of the interactions that alter chromatin architecture.

Inhibitory activity of a heterochromatin-associated serpin (MENT) against papain-like cysteine proteinases affects chromatin structure and blocks cell proliferation.

MENT (Myeloid andErythroid Nuclear Termination stage-specific protein) is a developmentally regulated chromosomal serpin that condenses chromatin in terminally differentiated avian blood cells. We show that MENT is an effective inhibitor of the papain-like cysteine proteinases cathepsins L and V. In addition, ectopic expression of MENT in mammalian cells is apparently sufficient to inhibit a nuclear papain-like cysteine proteinase and prevent degradation of the retinoblastoma protein, a major regulator of cell proliferation. MENT also accumulates in the nucleus, causes a strong block in proliferation, and promotes condensation of chromatin. Variants of MENT with mutations or deletions within the M-loop, which contains a nuclear localization signal and an AT-hook motif, reveal that this region mediates nuclear transport and morphological changes associated with chromatin condensation. Non-inhibitory mutants of MENT were constructed to determine whether its inhibitory activity has a role in blocking proliferation. These mutations changed the mode of association with chromatin and relieved the block in proliferation, without preventing transport to the nucleus. We conclude that the repressive effect of MENT on chromatin is mediated by its direct interaction with a nuclear protein that has a papain-like cysteine proteinase active site.

The interaction of NSBP1/HMGN5 with nucleosomes in euchromatin counteracts linker histone-mediated chromatin compaction and modulates transcription.

Structural changes in specific chromatin domains are essential to the orderly progression of numerous nuclear processes, including transcription. We report that the nuclear protein NSBP1 (HMGN5), a recently discovered member of the HMGN nucleosome-binding protein family, is specifically targeted by its C-terminal domain to nucleosomes in euchromatin. We find that the interaction of NSBP1 with nucleosomes alters the compaction of cellular chromatin and that in living cells, NSBP1 interacts with linker histones. We demonstrate that the negatively charged C-terminal domain of NSBP1 interacts with the positively charged C-terminal domain of H5 and that NSBP1 counteracts the linker histone-mediated compaction of a nucleosomal array. Dysregulation of the cellular levels of NSBP1 alters the transcription level of numerous genes. We suggest that mouse NSBP1 is an architectural protein that binds preferentially to euchromatin and modulates the fidelity of the cellular transcription profile by counteracting the chromatin-condensing activity of linker histones.

The Nature of the Nucleosomal Barrier to Transcription: Direct Observation of Paused Intermediates by Electron Cryomicroscopy

Transcribing SP6 RNA polymerase was arrested at unique positions in the nucleosome core, and the complexes were analyzed using biochemical methods and electron cryomicroscopy. As the polymerase enters the nucleosome, it disrupts DNA-histone interactions behind and up to approximately 20 bp ahead of the elongation complex. After the polymerase proceeds 30-40 bp into the nucleosome, two intermediates are observed. In one, only the DNA ahead of the polymerase reassociates with the octamer. In the other, DNA both ahead of and behind the enzyme reassociates. These intermediates present a barrier to elongation. When the polymerase approaches the nucleosome dyad, it displaces the octamer, which is transferred to promoter-proximal DNA.

Dynamic relocation of epigenetic chromatin markers reveals an active role of constitutive heterochromatin in the transition from proliferation to quiescence

Quiescent lymphocytes have small nuclei, filled with masses of facultative heterochromatin. Upon receiving mitogenic signals, these cells undergo nuclear enlargement, chromatin decondensation, the reactivation of cell proliferation, and changes in the intranuclear positioning of key genes. We examined the levels and intranuclear localization of major histone modifications and non-histone heterochromatin proteins in quiescent and reactivated mouse spleen lymphocytes. Dramatic and selective changes in localization of two heterochromatin-associated proteins, the histone variant macroH2A and HP1α occurred during lymphocyte reactivation. Reciprocal changes in the locations of these two proteins were observed in activated lymphocytes and cultured mouse fibroblasts induced into quiescence. We also describe a new apocentric nuclear compartment with a unique set of histone modifications that occurs as a zone of chromatin surrounding centromeric heterochromatin in differentiated lymphocytes. It is within this zone that the most significant changes occur in the transition from proliferation to quiescence. Our results suggest that constitutive centromeric heterochromatin plays an active role in cell differentiation and reactivation.

Toward Convergence of Experimental Studies and Theoretical Modeling of the Chromatin Fiber

Understanding the structural organization of eukaryotic chromatin and its control of gene expression represents one of the most fundamental and open challenges in modern biology. Recent experimental advances have revealed important characteristics of chromatin in response to changes in external conditions and histone composition, such as the conformational complexity of linker DNA and histone tail domains upon compact folding of the fiber. In addition, modeling studies based on high-resolution nucleosome models have helped explain the conformational features of chromatin structural elements and their interactions in terms of chromatin fiber models. This minireview discusses recent progress and evidence supporting structural heterogeneity in chromatin fibers, reconciling apparently contradictory fiber models.

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

Abstract 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.