Micrococcal Nuclease Digestion Research Articles

Open chromatin reveals the functional maize genome

Cellular processes mediated through nuclear DNA must contend with chromatin. Chromatin structural assays can efficiently integrate information across diverse regulatory elements, revealing the functional noncoding genome. In this study, we use a differential nuclease sensitivity assay based on micrococcal nuclease (MNase) digestion to discover open chromatin regions in the maize genome. We find that maize MNase-hypersensitive (MNase HS) regions localize around active genes and within recombination hotspots, focusing biased gene conversion at their flanks. Although MNase HS regions map to less than 1% of the genome, they consistently explain a remarkably large amount (∼40%) of heritable phenotypic variance in diverse complex traits. MNase HS regions are therefore on par with coding sequences as annotations that demarcate the functional parts of the maize genome. These results imply that less than 3% of the maize genome (coding and MNase HS regions) may give rise to the overwhelming majority of phenotypic variation, greatly narrowing the scope of the functional genome.

MNase titration reveals differences between nucleosome occupancy and chromatin accessibility.

Chromatin accessibility plays a fundamental role in gene regulation. Nucleosome placement, usually measured by quantifying protection of DNA from enzymatic digestion, can regulate accessibility. We introduce a metric that uses micrococcal nuclease (MNase) digestion in a novel manner to measure chromatin accessibility by combining information from several digests of increasing depths. This metric, MACC (MNase accessibility), quantifies the inherent heterogeneity of nucleosome accessibility in which some nucleosomes are seen preferentially at high MNase and some at low MNase. MACC interrogates each genomic locus, measuring both nucleosome location and accessibility in the same assay. MACC can be performed either with or without a histone immunoprecipitation step, and thereby compares histone and non-histone protection. We find that changes in accessibility at enhancers, promoters and other regulatory regions do not correlate with changes in nucleosome occupancy. Moreover, high nucleosome occupancy does not necessarily preclude high accessibility, which reveals novel principles of chromatin regulation.

The Pioneer Transcription Factor FoxA Maintains an Accessible Nucleosome Configuration at Enhancers for Tissue-Specific Gene Activation

Nuclear DNA wraps around core histones to form nucleosomes, which restricts the binding of transcription factors to gene regulatory sequences. Pioneer transcription factors can bind DNA sites on nucleosomes and initiate gene regulatory events, often leading to the local opening of chromatin. However, the nucleosomal configuration of open chromatin and the basis for its regulation is unclear. We combined low and high levels of micrococcal nuclease (MNase) digestion along with core histone mapping to assess the nucleosomal configuration at enhancers and promoters in mouse liver. We find that MNase-accessible nucleosomes, bound by transcription factors, are retained more at liver-specific enhancers than at promoters and ubiquitous enhancers. The pioneer factor FoxA displaces linker histone H1, thereby keeping enhancer nucleosomes accessible in chromatin and allowing other liver-specific transcription factors to bind and stimulate transcription. Thus, nucleosomes are not exclusively repressive to gene regulation when they are retained with, and exposed by, pioneer factors.

Analysis of nuclease hypersensitive‐equivalent sites in SV40 by Next Generation Sequencing

Chromatin fragments obtained by sonication or Micrococcal nuclease digestion of SV40 minichromosomes or disrupted virions were analyzed by Next Generation Sequencing (NGS) to determine the location of regions of the SV40 genome differentially sensitive to either treatment. Both treatments yielded samples which upon analysis allowed us to identify regions of hypersensitivity and moderate sensitivity. With sonication all of the samples contained regions of hypersensitivity centered on nucleotides 141 and 221 and regions of moderate sensitivity surrounding these sites which included the 21 base pair repeat regions and enhancers. Following Micrococcal nuclease treatment Hypersensitive sites were found in minichromosomes centered on nucleotides 30 and 92 and in chromatin from disrupted virions centered at nucleotide 106. Micrococcal nuclease also generated a pattern of peaks and valleys based upon the number of reads at each nucleotide consistent with the location of nucleosomes. While the pattern was very similar throughout most of the SV40 genome in minichromosomes and chromatin from disrupted virions, the pattern differed significantly in the regulatory region. In minichromosomes we observed a nucleosome‐free region extending from approximately nucleotide 5000 to nucleotide 250 encompassing most of the early and late transcription regulatory sequences and the origin of replication. In chromatin from disrupted virions we observed a very small open region which at most included only the enhancers.Support or Funding InformationThis work was supported by grants from NIH AI094441 (to BM) and NIH GM098328 (to LB)

Practical Guidelines for High-Resolution Epigenomic Profiling of Nucleosomal Histones in Postmortem Human Brain Tissue

BackgroundThe nervous system may include more than 100 residue-specific posttranslational modifications of histones forming the nucleosome core that are often regulated in cell-type-specific manner. On a genome-wide scale, some of the histone posttranslational modification landscapes show significant overlap with the genetic risk architecture for several psychiatric disorders, fueling PsychENCODE and other large-scale efforts to comprehensively map neuronal and nonneuronal epigenomes in hundreds of specimens. However, practical guidelines for efficient generation of histone chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) datasets from postmortem brains are needed. MethodsProtocols and quality controls are given for the following: 1) extraction, purification, and NeuN neuronal marker immunotagging of nuclei from adult human cerebral cortex; 2) fluorescence-activated nuclei sorting; 3) preparation of chromatin by micrococcal nuclease digest; 4) ChIP for open chromatin-associated histone methylation and acetylation; and 5) generation and sequencing of ChIP-seq libraries. ResultsWe present a ChIP-seq pipeline for epigenome mapping in the neuronal and nonneuronal nuclei from the postmortem brain. This includes a stepwise system of quality controls and user-friendly data presentation platforms. ConclusionsOur practical guidelines will be useful for projects aimed at histone posttranslational modification mapping in chromatin extracted from hundreds of postmortem brain samples in cell-type-specific manner.

Mammalian NET-seq analysis defines nascent RNA profiles and associated RNA processing genome-wide.

The transcription cycle of RNA polymerase II (Pol II) correlates with changes to the phosphorylation state of its large subunit C-terminal domain (CTD). We recently developed Native Elongation Transcript sequencing using mammalian cells (mNET-seq), which generates single-nucleotide-resolution genome-wide profiles of nascent RNA and co-transcriptional RNA processing that are associated with different CTD phosphorylation states. Here we provide a detailed protocol for mNET-seq. First, Pol II elongation complexes are isolated with specific phospho-CTD antibodies from chromatin solubilized by micrococcal nuclease digestion. Next, RNA derived from within the Pol II complex is size fractionated and Illumina sequenced. Using mNET-seq, we have previously shown that Pol II pauses at both ends of protein-coding genes but with different CTD phosphorylation patterns, and we have also detected phosphorylation at serine 5 (Ser5-P) CTD-specific splicing intermediates and Pol II accumulation over co-transcriptionally spliced exons. With moderate biochemical and bioinformatic skills, mNET-seq can be completed in ∼6 d, not including sequencing and data analysis.

Lamin ChIP from Chromatin Prepared by Micrococcal Nuclease Digestion.

It is now clearly demonstrated that nuclear lamins interact with the genomic DNA and largely contribute to its three-dimensional organization and transcriptional regulation. Emergence of genome-wide mapping techniques such as DamID technology or chromatin immunoprecipitation (ChIP) followed by array hybridization or high-throughput sequencing has allowed the mapping of large lamin-interacting genomic areas called lamina-associated domains. These cover up to 40 % of the genome and are preferentially located in transcriptionally silent heterochromatin at the nuclear periphery. We recently showed that the use of enzymatic rather than physical fragmentation of chromatin in ChIP experiments uncovers new chromatin compartments with features of euchromatin that interacts with A-type lamins. We describe here a detailed ChIP procedure to covalently cross-link protein-DNA, fragment the chromatin fibers by micrococcal nuclease digestion, and solubilize the lamin network with a short sonication pulse prior to immunoprecipitating the lamin-DNA complexes using specific antibodies. Enriched DNA fragments from the lamin-binding sites are then purified as suitable samples for qPCR analysis or high-throughput sequencing.

The State of Chromatin Condensation, the Expression of Genes Involved in DNA Damage Response and the DNA Repair Capacity Affect the Drug Sensitivity of PBMCs of Myeloma Patients Treated with Melphalan

Melphalan is an interstrand cross-link (ICL)-inducing agent and one of the most active chemotherapeuticdrugs in the treatment of multiple myeloma (MM). However, the molecular mechanisms contributing to differential response of MM patients to melphalan are poorly understood. Herein, we investigated the underlying mechanisms in processing and repair of melphalan-induced DNA lesions and their potent contribution to the outcome of anti-myeloma therapy.We studied peripheral blood mononuclear cells (PBMCs) from 15 newly diagnosed multiple myeloma (MM) patients (8M/7F; median age 61 years) responders (≥PR, n=9) and non-responders (< PR, n=6) to subsequent melphalan therapy. PBMCs from 25 healthy controls were also included in this study (HC; 14M/11F, median age 58 years). PBMCs were ex vivo treated with melphalan and the extent of the DNA damage formation/repair (monoadducts by using Southern blot; ICLs by using a quantitative PCR-based assay; double strand breaks (DSBs) using immunofluorescence quantification of γH2AX foci), the induction of the apoptotic pathway by using a photometric enzyme-immunoassay and the local chromatin condensation by using micrococcal nuclease digestion were examined. Finally, the expression of a focused panel of 84 genes involved in DNA damage response (DDR) pathways (ATM/ATR signaling, DNA repair pathways, cell cycle regulation, apoptosis) was also evaluated.Following ex vivo treatment of PBMCs with melphalan, in all individuals examined biphasic DNA repair kinetics were observed, including a fast first-phase of repair and a much slower second phase. Interestingly, the accumulation of monoadducts was inversely correlated with the first-phase repair capacity of the PBMCs, being significantly higher in HC than in responders and lowest in non-responders (all P<0.001). The second phase repair capacity showed no differences in all individuals analyzed. Also, although ICLs "unhooking" rates were similar in all individuals, accumulation of ICLs was significantly higher in HC compared to responders' PBMCs (P<0.01), due to higher levels of monoadducts that are precursors of ICLs left unrepaired in these cells, resulting in higher formation of ICLs. Minimal amounts of ICLs were observed in non-responders. Moreover, DSBs burden was significantly higher in HC than in responders, due to higher accumulation of ICL, which are precursors of DSBs and lower rates of DSB repair in these cells (P<0.01). Again, minimal amounts of DSBs were observed in non-responders.Interestingly, apoptosis rate of PBMCs was inversely correlated with the repair efficiency of both the first-phase monoadducts and the DSBs, with the apoptosis being significantly higher in HC compared to responders and lowest in non-responders (all P<0.05). In untreated PBMCs, we found an inverse correlation between the local chromatin condensation and the repair capacity. Therefore, we observed a progressive, significant increase in the looseness of the local chromatin structure, from HC to MM patients, with responders showing more condensed chromatin structure compared to non-responders (all P<0.05). Interestingly, by using a-amanitin, an inducer of chromatin condensation, PBMCs from non-responders showed the DNA repair capacity and the melphalan sensitivity similar to PBMCs from responders, suggesting that the state of the chromatin structure contributes to the response to melphalan therapy.Finally, microarray analyses of untreated PBMCs consistently point to an altered expression of several DNA damage response-related genes in MM patients compared to HC. Particularly, responders' PBMCs showed upregulation of 4 genes (ATR, CHEK2, XPA, XRCC1) and downregulation of 5 genes (ATM, MPG, UNG, CDKN1A, CDC25C) compared to non-responders (in all cases, fold changes between groups were >2, P<0.001), suggesting that changes in the molecular components of the DDR pathways correlate with the outcome of melphalan therapy.We conclude that the state of chromatin condensation, the expression of genes involved in DDR pathways and the repair capacity of monoadducts and DSBs affect the drug sensitivity of PBMCs and maybe used for the prediction of response of myeloma patients to melphalan therapy. DisclosuresTerpos:Celgene: Honoraria, Other: travel expenses; Novartis: Honoraria; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel expenses. Munshi:celgene: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; novartis: Membership on an entity's Board of Directors or advisory committees. Dimopoulos:Janssen-Cilag: Honoraria; Novartis: Honoraria; Genesis: Honoraria; Amgen: Honoraria; Onyx: Honoraria; Celgene: Honoraria; Janssen: Honoraria.

Nucleosome Stability Distinguishes Two Different Promoter Types at All Protein-Coding Genes in Yeast

Previous studies indicate that eukaryotic promoters display a stereotypical chromatin landscape characterized by a well-positioned +1 nucleosome near the transcription start site and an upstream -1 nucleosome that together demarcate a nucleosome-free (or -depleted) region. Here we present evidence that there are two distinct types of promoters distinguished by the resistance of the -1 nucleosome to micrococcal nuclease digestion. These different architectures are characterized by two sequence motifs that are broadly deployed at one set of promoters where a nuclease-sensitive ("fragile") nucleosome forms, but concentrated in a narrower, nucleosome-free region at all other promoters. The RSC nucleosome remodeler acts through the motifs to establish stable +1 and -1 nucleosome positions, while binding of a small set of general regulatory (pioneer) factors at fragile nucleosome promoters plays a key role in their destabilization. We propose that the fragile nucleosome promoter architecture is adapted for regulation of highly expressed, growth-related genes.

DNA sequence templates adjacent nucleosome and ORC sites at gene amplification origins in Drosophila.

Eukaryotic origins of DNA replication are bound by the origin recognition complex (ORC), which scaffolds assembly of a pre-replicative complex (pre-RC) that is then activated to initiate replication. Both pre-RC assembly and activation are strongly influenced by developmental changes to the epigenome, but molecular mechanisms remain incompletely defined. We have been examining the activation of origins responsible for developmental gene amplification in Drosophila. At a specific time in oogenesis, somatic follicle cells transition from genomic replication to a locus-specific replication from six amplicon origins. Previous evidence indicated that these amplicon origins are activated by nucleosome acetylation, but how this affects origin chromatin is unknown. Here, we examine nucleosome position in follicle cells using micrococcal nuclease digestion with Ilumina sequencing. The results indicate that ORC binding sites and other essential origin sequences are nucleosome-depleted regions (NDRs). Nucleosome position at the amplicons was highly similar among developmental stages during which ORC is or is not bound, indicating that being an NDR is not sufficient to specify ORC binding. Importantly, the data suggest that nucleosomes and ORC have opposite preferences for DNA sequence and structure. We propose that nucleosome hyperacetylation promotes pre-RC assembly onto adjacent DNA sequences that are disfavored by nucleosomes but favored by ORC.

Abstract 134: Reciprocal Cardiac Chromatin Regulation by Ctcf and Hmgb2

Chromatin remodeling plays an essential role in cardiac gene reprogramming under pathological conditions. However, we currently lack a detailed understanding of how this process is structurally coordinated across the genome. Previous RNA and proteomic analyses of an in vivo model of heart failure performed by our group identified the chromatin structural proteins CTCF and high mobility group protein B2 (HMGB2) as regulators of heart disease. CTCF is an essential factor in the maintenance of global chromatin organization through the facilitation of long-range interactions and chromatin looping. HMGB2 is a non-nucleosomal structural protein that packages nucleosomes into higher order structures. An unbiased genomic analysis of ~100 strains of mice treated with isoproterenol (ISO) followed by transcriptome and phenotype analyses revealed a general down-regulation of CTCF at the mRNA level across the different strains after ISO treatment, whereas the HMGB2 response was strongly influenced by common genetic variation (i.e. between strains). ChIP-seq data, ChIP-reChIP-PCR experiments (detection of concomitant presence of two proteins at a given locus) and immunofluorescence showed CTCF and HMGB2 bind the same regions of the genome, yet they do not physically co-localize in myocyte nuclei. Loss of HMGB2, which induces hypertrophy in myocytes, causes de-condensation of chromatin, as directly measured by micrococcal nuclease digestion; CTCF knockdown has no effect on global chromatin accessibility by this assay. Examination of other chromatin structural proteins like histone H1 suggests this interaction between CTCF and HMGB2 is specific. Lastly, we showed that CTCF and HMGB2 regulate each other’s expression as well as the transcription of cardiac hypertrophy genes and ribosomal RNA. Our data show that basal HMGB2 (but not CTCF) expression correlates with cardiac mass. The response of HMGB2 levels to heart failure stimuli is highly influenced by genetics, whereas the decrease in CTCF following (ISO) is unaffected by genetic variation. These findings reveal molecular mechanisms of chromatin compaction in cardiac cells, linking them with genetic variability, and revealing a reciprocal relationship between HMGB2 and CTCF in cardiac hypertrophy.

MARCC (Matrix-Assisted Reader Chromatin Capture): An Antibody-Free Method to Enrich and Analyze Combinatorial Nucleosome Modifications.

Combinatorial patterns of histone modifications are key indicators of different chromatin states. Most of the current approaches rely on the usage of antibodies to analyze combinatorial histone modifications. Here we detail an antibody-free method named MARCC (Matrix-Assisted Reader Chromatin Capture) to enrich combinatorial histone modifications. The combinatorial patterns are enriched on native nucleosomes extracted from cultured mammalian cells and prepared by micrococcal nuclease digestion. Such enrichment is achieved by recombinant chromatin-interacting protein modules, or so-called reader domains, which can bind in a combinatorial modification-dependent manner. The enriched chromatin can be quantified by immunoblotting or mass spectrometry for the co-existence of histone modifications, while the associated DNA content can be analyzed by qPCR or next-generation sequencing. Altogether, MARCC provides a reproducible, efficient and customizable solution to enrich and analyze combinatorial histone modifications.

Mapping regulatory factors by immunoprecipitation from native chromatin.

Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin (ORGANIC) is a high-resolution method that can be used to quantitatively map protein-DNA interactions with high specificity and sensitivity. This method uses micrococcal nuclease (MNase) digestion of chromatin and low-salt solubilization to preserve protein-DNA complexes, followed by immunoprecipitation and paired-end sequencing for genome-wide mapping of binding sites. In this unit, we describe methods for isolation of nuclei and MNase digestion of unfixed chromatin, immunoprecipitation of protein-DNA complexes, and high-throughput sequencing to map sites of bound factors.

Differential association of chromatin proteins identifies BAF60a/SMARCD1 as a regulator of embryonic stem cell differentiation.

Embryonic stem cells (ESCs) possess a distinct chromatin conformation maintained by specialized chromatin proteins. To identify chromatin regulators in ESCs, we developed a simple biochemical assay named D-CAP (differential chromatin-associated proteins), using brief micrococcal nuclease digestion of chromatin, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Using D-CAP, we identified several differentially chromatin-associated proteins between undifferentiated and differentiated ESCs, including the chromatin remodeling protein SMARCD1. SMARCD1 depletion in ESCs led to altered chromatin and enhanced endodermal differentiation. Gene expression and chromatin immunoprecipitation sequencing (ChIP-seq) analyses suggested that SMARCD1 is both an activator and arepressor and is enriched at developmental regulators and that its chromatin binding coincides withH3K27me3. SMARCD1 knockdown caused H3K27me3 redistribution and increased H3K4me3 around the transcription start site (TSS). One of the identified SMARCD1 targets was Klf4. In SMARCD1-knockdown clones, KLF4, as well as H3K4me3 at the Klf4 locus, remained high and H3K27me3 was abolished. These results propose a role for SMARCD1 in restricting pluripotency and activating lineage pathways by regulating H3K27 methylation.

Genome wide nucleosome mapping for HSV-1 shows nucleosomes are deposited at preferred positions during lytic infection.

HSV is a large double stranded DNA virus, capable of causing a variety of diseases from the common cold sore to devastating encephalitis. Although DNA within the HSV virion does not contain any histone protein, within 1 h of infecting a cell and entering its nucleus the viral genome acquires some histone protein (nucleosomes). During lytic infection, partial micrococcal nuclease (MNase) digestion does not give the classic ladder band pattern, seen on digestion of cell DNA or latent viral DNA. However, complete digestion does give a mono-nucleosome band, strongly suggesting that there are some nucleosomes present on the viral genome during the lytic infection, but that they are not evenly positioned, with a 200bp repeat pattern, like cell DNA. Where then are the nucleosomes positioned? Here we perform HSV-1 genome wide nucleosome mapping, at a time when viral replication is in full swing (6hr PI), using a microarray consisting of 50mer oligonucleotides, covering the whole viral genome (152kb). Arrays were probed with MNase-protected fragments of DNA from infected cells. Cells were not treated with crosslinking agents, thus we are only mapping tightly bound nucleosomes. The data show that nucleosome deposition is not random. The distribution of signal on the arrays suggest that nucleosomes are located at preferred positions on the genome, and that there are some positions that are not occupied (nucleosome free regions -NFR or Nucleosome depleted regions -NDR), or occupied at frequency below our limit of detection in the population of genomes. Occupancy of only a fraction of the possible sites may explain the lack of a typical MNase partial digestion band ladder pattern for HSV DNA during lytic infection. On average, DNA encoding Immediate Early (IE), Early (E) and Late (L) genes appear to have a similar density of nucleosomes.

Glycolytic metabolism influences global chromatin structure.

Metabolic rewiring, specifically elevated glycolytic metabolism is a hallmark of cancer. Global chromatin structure regulates gene expression, DNA repair, and also affects cancer progression. But the interrelationship between tumor metabolism and chromatin architecture remain unclear. Here we show that increased glycolysis in cancer cells promotes an open chromatin configuration. Using complementary methods including Micrococcal nuclease (MNase) digestion assay, electron microscope and immunofluorescence staining, we demonstrate that glycolysis inhibition by pharmacological and genetic approaches was associated with induction of compacted chromatin structure. This condensed chromatin status appeared to result chiefly from histone hypoacetylation as restoration of histone acetylation with an HDAC inhibitor reversed the compacted chromatin state. Interestingly, glycolysis inhibition-induced chromatin condensation impeded DNA repair efficiency leading to increased sensitivity of cancer cells to DNA damage drugs, which may represent a novel molecular mechanism that can be exploited for cancer therapy.

Histone Phosphorylation Combined with Acetylation Dramatically Increase Nucleosome Accessibility

Histone post translational modifications (PTMs) regulate DNA transcription, replication and repair. A number of histone PTMs have been identified within the DNA entry-exit region of the nucleosome including phosphorylation of H3 at tyrosine 41 (H3Y41ph) and threonine 45 (H3T45ph). Each of these modifications are implicated to occur with H3K56ac, which is known to increase DNA unwrapping and accessibility. However, the influence of H3Y41ph and H3T45ph on nucleosome dynamics and stability with and without H3K56ac remains undetermined. Micrococcal nuclease digestion, small angle x-ray scattering and fluorescence resonance energy transfer (FRET) measurements reveal that each phosphorylation mimics H3Y41E and H3T45E, and the exact modification, H3Y41ph that was prepared by sequential native ligation, increase nucleosome unwrapping and enhance DNA accessibility to protein binding by three fold. Combinations of either phosphorylation site with H3K56ac increased DNA accessibility multiplicatively, while the combination of the phosphorylation sites did not increase accessibility beyond single phosphorylations. These results show that H3Y41 and H3T45 phosphorylation increase nucleosome unwrapping and accessibility similarly to H3K56 acetylation, and that combinations of PTMs can function multiplicatively to increase DNA accessibility by more than an order of magnitude.

Columnar structure of SV40 minichromosome

Like the sequence of the strongest 601 clone nucleosome of Lowary and Widom, the SV40 genome sequence contains tracks of YR dinucleotides separated by small integers of the 10.4n base series (10, 11, 21 and 30 bases). The tracks, however, substantially exceed the nucleosome DNA size and, thus, correspond to more extended structure - columnar chromatin. The micrococcal nuclease digests of the SV40 chromatin do not show uniquely positioned individual nucleosomes. This confirms the columnar structure of the minichromosome, as well as earlier electron microscopy studies.

Metaphase Chromatin Plates Explain the Structure and Physical Properties of Condensed Chromosomes

Previous studies showed that during mitosis chromatin filaments are folded into multilayer plates (1). These structures can be self-assembled from chromatin fragments obtained by micrococcal nuclease digestion of metaphase chromosomes (2). Chromosomes of different animal and plant species show great differences in size (which are dependent on the amount of DNA that they contain), but in all cases chromosomes are elongated cylinders that have relatively similar shape proportions (the length to diameter ratio is approximately 13). It is possible to explain this morphology by considering that chromosomes are self-organizing supramolecular structures formed by stacked layers of planar chromatin having different nucleosome-nucleosome interaction energies in different regions (3). The nucleosomes in the periphery of the chromosome are less stabilized by the attractive interactions with other nucleosomes and this generates a surface potential that destabilizes the structure. Chromosomes are smooth cylinders because this morphology has a lower surface energy than structures having irregular surfaces. The symmetry breaking produced by the different values of the surface energies in the telomeres and in the lateral surface explains the elongated structure of the chromosomes. The results obtained by other authors in nanomechanical studies of chromatin and chromosome stretching have been used to test the proposed supramolecular structure. It is demonstrated quantitatively that internucleosome interactions between chromatin layers can justify the work required for elastic chromosome stretching. Chromosomes can be considered as hydrogels with a lamellar liquid crystal organization. The good mechanical properties of this structure may be useful for the maintenance of chromosome integrity during mitosis. Furthermore this chromatin organization avoids random entanglement of the extremely long genomic DNA molecules in chromosomes.(1) Daban (2011) Micron 42:733-750.(2) Milla and Daban (2012) Biophys J 103:567-575.(3) Daban (2014) J. R. Soc. Interface 11:20131043.

Nuclear Condensation during Mouse Erythropoiesis Requires Caspase-3 Mediated Nuclear Opening Formation

Mammalian erythropoiesis is a dynamic process in which erythroid progenitors proliferate and differentiate into mature enucleated red blood cells. In the late stage of terminal erythropoiesis, erythroblasts undergo cell-cycle exit, chromatin condensation, and extrusion of the pycnotic nucleus via an asymmetric cell division. Recent genetic and biochemical studies illustrated that various signaling pathways, including histone deacetylation and other chromatin modifications, are involved in chromatin condensation and enucleation. However, it is unclear the global dynamic changes of the nucleosome and how different histones are regulated during chromatin condensation. We proposed to directly characterize the expression levels and localization of different histones and their variants during erythropoiesis. Using a mouse fetal liver erythroblast in vitro culture system and immunofluorescence analysis, we found an unexpected, gradually enlarged nuclear opening through which most histones, except H2AZ, were released out of the nucleus. The same phenotype was observed in freshly purified fetal liver and bone marrow erythroblasts (Figure 1). These openings lack nuclear lamina, nuclear pore complexes, and nuclear membrane but are distinct from nuclear envelope changes during apoptosis and mitosis. Western blot analysis also demonstrated the nuclear release of a fraction of the major histones, however, many well-known nuclear proteins remained in the nucleus in this process. We also demonstrated that the histone release was cell cycle regulated and independent of nuclear exportin.Using micrococcal nuclease digestion of chromatin followed by next generation sequencing (MNase-seq) technique, we demonstrated that histone release from the nuclear opening is associated with dynamic nucleosomal changes during mouse terminal erythropoiesis. Mechanistically, caspase-3 is involved in the regulation of nuclear openings during erythropoiesis. Inhibition or knockdown of caspase-3 completely blocked nuclear opening formation and histone release, which led to inhibition of chromatin condensation, cell differentiation, and ultimate cell death.In summary, our study revealed a caspase-3 mediated nuclear opening formation with histone release in mouse erythroblasts that is unique in mammalian cells. The dynamic nuclear opening formation is required for fast release of major histones into cytoplasm to facilitate chromatin condensation throughout erythropoiesis. This “prokaryotic phase” of erythroblast is also associated with genome wide nucleosome localization changes that correlate with the size of the nuclear opening and histone release, which may provide clues for the pathogenesis of erythroid related diseases with unknown etiology. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.