Histone Fractions Research Articles

Histidine Nτ-methylation identified as a new posttranslational modification in histone H2A at His-82 and H3 at His-39

Histone posttranslational modifications play critical roles in a variety of eukaryotic cellular processes. In particular, methylation at lysine and arginine residues is an epigenetic mark that determines the chromatin state. In addition, histone "histidine" methylation was initially reported over 50years ago; however, further studies in this area were not conducted, leaving a gap in our understanding. Here, we aimed to investigate the occurrence of histidine methylation in histone proteins using highly sensitive mass spectrometry. We found that acid hydrolysates of whole histone fraction from calf thymus contained Nτ-methylhistidine, but not Nπ-methylhistidine. Both core and linkerhistones carried a Nτ-methylhistidine modification, and methylation levels were relatively high in histone H3. Furthermore, through MALDI-TOF MS, we identified two histidine methylation sites at His-82 in the structured globular domain of histone H2A and His-39 in the N-terminal tail of histones H3. Importantly, these histidine methylation signals were also detected in histones purified from a human cell line HEK293T. Moreover, we revealed the overall methylation status of histone H3, suggesting that methylation is enriched primarily at lysine residues and to a lesser extent at arginine and histidine residues. Thus, our findings established histidine Nτ-methylation as a new histone modification, which may serve as a chemical flag that mediates the epigenetic mark of adjacent residues of the N-terminal tail and the conformational properties of the globular domain.

Direct and cost-effective method for histone isolation from cultured mammalian cells

Histones are an essential part of nucleosomes that regulate chromatin structure and function. Histone exchanges and modifications represent a scaffold for DNA transcription, repair, and replication. Studying histones and histone code is an important and fast-developing branch of epigenetic science. Here we propose a fast, efficient, and versatile assay for nucleosomal histone isolation from mammalian cells, without the use of acids or high salt solutions which are common for other histone isolation techniques. All components used in the protocol are common and inexpensive laboratory chemicals. The protocol has been evaluated on six commonly used cell lines and two animal tissue samples. The mild extraction conditions preserve delicate histone epigenetic changes, allowing its downstream analyses. We have demonstrated the assays’ successful application during changes in the transcriptional activity of histone genes, cell cycle transitions, and DNA-damaging conditions. Histone fractions, obtained by the protocol, can be used for further applications, such as electrophoresis, immunoblot, and mass spectrometry. Therefore, the new proposed nucleosomal histone isolation method is sensitive, specific, and suitable for downstream applications of various kinds.

Expeditious Extraction of Histones from Limited Cells or Tissue Samples and Quantitative Top-Down Proteomic Analysis.

Histones are the primary protein component of chromatin and are involved in virtually all DNA-templated processes. Histones are abundantly post-translationally modified by a variety of chromatin-modifying machinery. These post-translational modifications (PTMs) are recognized by a range of "reader" proteins, which recruit additional proteins to specific locations on chromatin and impart precise and powerful effects on gene regulation. Each PTM typically exerts a positive or negative effect on transcription, and recent studies have shown that histone PTMs function in a combinatorial histone code: that is, histone PTMs function in combination to exert precise DNA-templated regulation. Thus, there is a need to identify and understand proteoforms, or unambiguously defined single protein molecules with all combinations of modifications. Top-down proteomics is currently the only viable approach for identifying and quantitating histone proteoforms, and mass spectrometry instruments have become sufficiently powerful to perform these quantitative analyses in a robust and high-throughput fashion. These recent innovations have enabled new experimental directions in chromatin research but have also introduced temporal and other constraints. This has led us to develop the protocols described here, which increase throughput, reduce sample requirements, and maintain robust quantitation. Although originally designed for high-throughput quantitative top-down proteomics, the protocols described here are useful for a wide range of chromatin biology applications. Starting with small amounts of cells or tissue, we describe two basic protocols for exceptionally rapid and efficient nuclei isolation, acid extraction of histones, and high-performance liquid chromatography fractionation of histones into histone families. We additionally describe the quantitative top-down proteomic analysis of histone H4 proteoforms. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Nuclei isolation and acid extraction of histones from mammalian cells in culture/tissues Basic Protocol 2: HPLC fractionation of histones and histone H4 HPLC-MS/MS Support Protocol: Preparation of intact H3 histone tails by Glu-C digestion.

Epigenetic homogeneity in histone methylation underlies sperm programming for embryonic transcription

Sperm contributes genetic and epigenetic information to the embryo to efficiently support development. However, the mechanism underlying such developmental competence remains elusive. Here, we investigated whether all sperm cells have a common epigenetic configuration that primes transcriptional program for embryonic development. Using calibrated ChIP-seq, we show that remodelling of histones during spermiogenesis results in the retention of methylated histone H3 at the same genomic location in most sperm cell. This homogeneously methylated fraction of histone H3 in the sperm genome is maintained during early embryonic replication. Such methylated histone fraction resisting post-fertilisation reprogramming marks developmental genes whose expression is perturbed upon experimental reduction of histone methylation. A similar homogeneously methylated histone H3 fraction is detected in human sperm. Altogether, we uncover a conserved mechanism of paternal epigenetic information transmission to the embryo through the homogeneous retention of methylated histone in a sperm cells population.

The Impact of Iron–Molybdenum Polyoxometalates and a Mixture of Nanoparticle Components on the Content of Nucleic Acids and Histone Proteins in Rat Blood Lymphocytes

Nanomaterials can exhibit a more pronounced genotoxic effect compared to the individual components contained in them, changing the structure of DNA and histone proteins, as well as the amount of these substances. We analyzed the impact of the Keplerate-type nanocluster iron–molybdenum polyoxometalate (POM) and POM degradation products in solutions on the content of nucleic acids, free nucleotides, and histone proteins in blood lymphocytes of Wistar rats. We revealed a reversible increase in the amount of DNA after a single injection of POM with a dose of 0.15 mg/100 g and no decrease in the histone content after 1, 7, and 30 injections. On the contrary, the products of degradation of the same POM dose changed the level of DNA, RNA, free nucleotides, total histone number, and the histone fractions. Preservation of the total number of lymphocytes during thirty-fold exposure to POM, in contrast to the effects of degradation products, confirms the less pronounced toxic effect of POM compared to the action of the POM component mixture.

Demonstration of exogenous nuclear histone H3 binding to mitochondria and subsequent cytochrome c release in cauliflower

The life cycle of a cell is partly regulated by the programmed cell death (PCD) process. From development to demise, a cell's PCD process must respond to external signals and internal factors mediated by mitochondria. Previous studies show that the release of histones into the cytosol caused by DNA damage or loss of nuclear integrity is correlated with apoptosis in mammalian cells. These released histones bind to mitochondria and permeabilize its inner and outer membranes, which causes the release of cytochrome c into the cytosol that leads to caspase activation and the demise of the cell. Owing to the high conservation of histones, we hypothesize that histone-mediated cytochrome c release from mitochondria may be conserved across a wide range of eukaryotes. We investigated this histone-mitochondrial interaction in cauliflower using density-gradient purified mitochondria and exogenous histones from a crude histone fraction, then added the exogenous histone fractions to the purified cauliflower mitochondria and analyzed the mitochondrial pellets and supernatants by immunoblotting against cytochrome c and H3. Our data clearly shows that histone-enriched fractions elicited cytochrome c release from mitochondria, and that mitochondria bind exogenous histone H3.

Physiological and biochemical aspects of the functioning of Arg-X proteolytic system in the cell nuclei of plants

Plants are structural and biochemical systems in which at each moment of ontogenesis are established inherent only to this plant, the ratio of growth, biorhythmic and morphogenetic processes. The conjugacy of circadian rhythm and the activity of chromatin-modifying enzymes is becoming increasingly important. In this aspect, histones play an important role, since they are able to fine-tune transcriptional programs through a series of accurately organized post-translational modifications that combinatorially regulate chromatin function. It is proved that almost all histones are proteolytically modified. It was shown that differences in the exhibition of activity Arg-X proteolysis between spring and winter seedlings are observed mainly in the fractions of core histones at the level of chromatin tightly bound. The preferential localization of Arg-X protease activity was found in the histone (H3 + H4) block as spring and winter varieties. In addition, activity Arg-X proteolysis at the level of nuclear matrix correlated with the growth characteristics of spring and winter seedlings in coleoptiles and roots growing by cell stretching. Results can be viewed as an illustration of a multi-layered regulatory proteinase network involved in proteolytic processing of nuclear proteins, taking part in circadian spatial and temporal structural changes of chromatin states.

Quantifying Ubiquitination Signaling with a Chemical Proteomics Strategy

Stoichiometric analysis of posttranslational modifications is an emerging strategy for absolute quantification of the modification's fractional abundance. In this study, we present a quantitative chemical proteomic workflow for stoichiometric analysis of ubiquitination, named Isotope‐BAlanced Quantification of Ubiquitination (IBAQ‐Ub). The strategy utilizes a new amine‐reactive chemical tag (AcGG‐NHS) for highly efficient labeling of amine residues. The new chemical tag is structurally homologue to the GG remnant of ubiquitin on modified lysine following trypsin cleavage and therefore enables the generation of structurally identical peptides from ubiquitinated and unmodified lysine residues following trypsin digestion and the secondary stable isotopic labeling. Quantitative analysis of labeled peptides with HPLC and high resolution mass spectrometry allows unbiased measurements of absolute site‐specific ubiquitination stoichiometry. Using recombinant protein standards and isotopic serial dilution, we showed that the strategy is highly robust, sensitive and accurate. We further designed a SILAC‐based experiment to demonstrate the efficient labeling of lysine with the new AcGG tag in complex cell lysates. Application of the IBAQ‐Ub workflow for the analysis of enriched histone fractions allowed us to measure the stoichiometry of histone H2B C‐terminal ubiquitination. Comparative analysis of stoichiometric dynamics upon proteasome inhibitor treatments showed a dramatic decrease of histone H2B ubiquitination levels and a significant increase of polyubiquitination K48 linkage. Thus, this work provides an efficient chemical proteomics tool for quantitative stoichiometric analysis of ubiquitination signaling pathways in cells and diseases.Support or Funding InformationUniversity of Minnesota start‐up fund and National Science Foundation (CHE‐1753154)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impairment of production and posttranslational changes of placental nuclear and membrane proteins with complicated pregnancy

The content of nuclear and membrane proteins of the placenta, as well as posttranslational modification of these proteins in physiological pregnancy and placental insufficiency (PI) were studied. Differential centrifugation, electrophoresis in polyacrylamide gel, spectrophotometric methods were used. It was found that with PN there is a decrease in the degree of production of the studied proteins of varying degrees relative to control parameters. For chromatin proteins, a more pronounced decrease in the content of non-histone proteins was found in comparison with histones. Among histone fractions, the maximum decrease was detected in the H2A fraction. The degree of change in the amount of membrane proteins depends on the detergent used. Changes in posttranslational protein modifications disorders are characterized by a decrease in the content of amine and amide (especially difficult to hydrolyze) groups and an increase in carbonyl derivatives of proteins. The revealed changes in the composition and structure of the nuclear and membrane proteins of the placenta, performing numerous regulatory functions, can be triggering links in the chain of molecular damage in the placenta at PI.

In vitro interactions of histones and α-crystallin

The aggregation of crystallins in lenses is associated with cataract formation. We previously reported that mutant crystallins are associated with an increased abundance of histones in knock-in and knockout mouse models. However, very little is known about the specific interactions between lens crystallins and histones. Here, we performed in vitro analyses to determine whether α-crystallin interacts with histones directly. Isothermal titration calorimetry revealed a strong histone–α-crystallin binding with a Kd of 4 × 10−7 M, and the thermodynamic parameters suggested that the interaction was both entropy and enthalpy driven. Size-exclusion chromatography further showed that histone–α-crystallin complexes are water soluble but become water insoluble as the concentration of histones is increased. Right-angle light scattering measurements of the water-soluble fractions of histone–α-crystallin mixtures showed a decrease in the oligomeric molecular weight of α-crystallin, indicating that histones alter the oligomerization of α-crystallin. Taken together, these findings reveal for the first time that histones interact with and affect the solubility and aggregation of α-crystallin, indicating that the interaction between α-crystallin and histones in the lens is functionally important.

Changes of Arg-X proteolysis localization under conditions of deacetylation inhibition of nuclear proteins in spring and winter wheat seedlings

The investigation of the nuclear proteins has always been relevant, because they are involved in the expression, replication, storage, reparation and transmission of genetic information. It is known that sodium butyrate, as an inhibitor of protein deacetylation, leads to acetylation of histones and activates the transcriptional function of chromatin, and also it can effectively inhibit cell proliferation at low concentrations. However, it has been shown that the supplement or deletion of acetyl groups on lysine residues near the cleavage sites can regulate the sensibility of histones to proteolysis in chromatin. In the present work, the differences between 24-h seedlings of spring wheat and winter wheat in allocation of Arg-X proteolytic activity in trypsin-like complexes extracted from the fractions of non-histones and histones isolated from the nuclear structures (nucleoplasm, chromatin loosely and tightly bound and nuclear matrix) were shown. It is possible that the appearance of additional Arg-X proteolytic activity which we found in the non-histone and histone fractions that we were isolated primarily from the chromatin loosely bound and from the nuclear matrix are linked to increased histone acetylation under conditions of deacetylation inhibition of nuclear proteins. We suggest the revealed changes of Arg-X proteolysis may be associated with assembly of nucleosomes and possibly nucleosome control of gene expression.

Nucleosome stability measured in situ by automated quantitative imaging

Current approaches have limitations in providing insight into the functional properties of particular nucleosomes in their native molecular environment. Here we describe a simple and powerful method involving elution of histones using intercalators or salt, to assess stability features dependent on DNA superhelicity and relying mainly on electrostatic interactions, respectively, and measurement of the fraction of histones remaining chromatin-bound in the individual nuclei using histone type- or posttranslational modification- (PTM-) specific antibodies and automated, quantitative imaging. The method has been validated in H3K4me3 ChIP-seq experiments, by the quantitative assessment of chromatin loop relaxation required for nucleosomal destabilization, and by comparative analyses of the intercalator and salt induced release from the nucleosomes of different histones. The accuracy of the assay allowed us to observe examples of strict association between nucleosome stability and PTMs across cell types, differentiation state and throughout the cell-cycle in close to native chromatin context, and resolve ambiguities regarding the destabilizing effect of H2A.X phosphorylation. The advantages of the in situ measuring scenario are demonstrated via the marked effect of DNA nicking on histone eviction that underscores the powerful potential of topological relaxation in the epigenetic regulation of DNA accessibility.

A novel hypothesis for histone-to-protamine transition in Bos taurus spermatozoa.

DNA compaction with protamines in sperm is essential for successful fertilization. However, a portion of sperm chromatin remains less tightly packed with histones, which genomic location and function remain unclear. We extracted and sequenced histone-associated DNA from sperm of nine ejaculates from three bulls. We found that the fraction of retained histones varied between samples, but the variance was similar between samples from the same and different individuals. The most conserved regions showed similar abundance across all samples, whereas in other regions, their presence correlated with the size of histone fraction. This may refer to gradual histone–protamine transition, where easily accessible genomic regions, followed by the less accessible regions are first substituted by protamines. Our results confirm those from previous studies that histones remain in repetitive genome elements, such as centromeres, and added new findings of histones in rRNA and SRP RNA gene clusters and indicated histone enrichment in some spermatogenesis-associated genes, but not in genes of early embryonic development. Our functional analysis revealed significant overrepresentation of cGMP-dependent protein kinase G (cGMP-PKG) pathway genes among histone-enriched genes. This pathway is known for its importance in pre-fertilization sperm events. In summary, a novel hypothesis for gradual histone-to-protamine transition in sperm maturation was proposed. We believe that histones may contribute structural information into early embryo by epigenetically modifying centromeric chromatin and other types of repetitive DNA. We also suggest that sperm histones are retained in genes needed for sperm development, maturation and fertilization, as these genes are transcriptionally active shortly prior to histone-to-protamine transition.

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

Mammalian erythropoiesis involves chromatin condensation that is initiated in the early stage of terminal differentiation. The mechanisms of chromatin condensation during erythropoiesis are unclear. Here, we show that the mouse erythroblast forms large, transient, and recurrent nuclear openings that coincide with the condensation process. The opening lacks nuclear lamina, nuclear pore complexes, and nuclear membrane, but it is distinct from nuclear envelope changes that occur during apoptosis and mitosis. A fraction of the major histones are released from the nuclear opening and degraded in the cytoplasm. We demonstrate that caspase-3 is required for the nuclear opening formation throughout terminal erythropoiesis. Loss of caspase-3 or ectopic expression of a caspase-3 non-cleavable lamin B mutant blocks nuclear opening formation, histone release, chromatin condensation, and terminal erythroid differentiation. We conclude that caspase-3-mediated nuclear opening formation accompanied by histone release from the opening is a critical step toward chromatin condensation during erythropoiesis in mice.

Analyses of Histone Proteoforms Using Front-end Electron Transfer Dissociation-enabled Orbitrap Instruments

Histones represent a class of proteins ideally suited to analyses by top-down mass spectrometry due to their relatively small size, the high electron transfer dissociation-compatible charge states they exhibit, and the potential to gain valuable information concerning combinatorial post-translational modifications and variants. We recently described new methods in mass spectrometry for the acquisition of high-quality MS/MS spectra of intact proteins (Anderson, L. C., English, A. M., Wang, W., Bai, D. L., Shabanowitz, J., and Hunt, D. F. (2015) Int. J. Mass Spectrom. 377, 617-624). Here, we report an extension of these techniques. Sequential ion/ion reactions carried out in a modified Orbitrap Velos Pro/Elite(TM) capable of multiple fragment ion fills of the C-trap, in combination with data-dependent and targeted HPLC-MS experiments, were used to obtain high resolution MS/MS spectra of histones from butyrate-treated HeLa cells. These spectra were used to identify several unique intact histone proteoforms with up to 81% sequence coverage. We also demonstrate that parallel ion parking during ion/ion proton transfer reactions can be used to separate species of overlapping m/z that are not separated chromatographically, revealing previously indiscernible signals. Finally, we characterized several truncated forms of H2A and H2B found within the histone fractions analyzed, achieving up to 93% sequence coverage by electron transfer dissociation MS/MS. Results of follow-up in vitro experiments suggest that some of the truncated histone H2A proteoforms we observed can be generated by cathepsin L, an enzyme known to also catalyze clipping of histone H3.

The histone proteins of blood leukocytes under miscarriage.

To analyze fractions of histones in lymphocytes of peripheral blood of women under physiological and complicated course of pregnancy. Three groups of female patients were examined (85 persons in all). The main group (I) included 30 women with physiological course of pregnancy; the main group (II) included women with threat of miscarriage (35 persons); control group (III) consisted of 20 healthy and without pregnancy women. The study established significant increasing of content of histone proteins under physiological development of pregnancy, especially fractions (H2A, H3, H4) and H2B as compared with control. In female patients from group II content of total histone fraction (H2A, H3, H4) remained at the level of women without pregnancy and reliably differed from similar indices of group I. The established alterations possibly are necessary for decreasing of activity of lymphocytes and development of tolerance to tissues of fetus.

The Study of Cellular and Molecular Physiological Characteristics of Sperm in Men Living in the Aral Sea Region.

BACKGROUND:Extreme environmental situation in the Aral crisis has caused a massive chemical pollution of the territory for decades with high doses of pesticides, herbicides. Discharge of industrial waste into the rivers that feed the Aral Sea has lead to the development of various pathological processes in the human body, as well as disruption of reproductive function in young men.AIM:To evaluate the performance of molecular cellular changes in the sperm of men under the influence of dust and salt aerosols in Aral Sea region.MATERIAL AND METHODS:Clinical and laboratory studies were conducted in men 5 settlements (Aralsk-city, v. Aiteke-Bi, v. Zhalagash, v. Zhusaly, v. Shieli). We have studied male ejaculate obtained after 4-5 days of abstinence, and placed it in a warm tube with a glass stopper. On the investigation proceeded ejaculate within 20-30 minutes after its preparation, during which time he was subjected to liquefaction. Isolation and quantification of ASF, RNA, DNA, and determining the fraction of histones in sperm was performed by the method of Markusheva and Savina.RESULTS:It was found that the value of ASF in the semen of men living in the zone of ecological disaster higher compared with the values of parameters in men living in the area of environmental crisis, and this trend is observed in all age groups. The study of circulating extracellular DNA and RNA in the sperm of men registered their decline with a corresponding increase of acid precursors that can be attributed to the degradation of nucleic acids under the influence of negative factors in the complex area of ecological trouble. Also, according to a study in men residing in the areas of environmental catastrophe at the age of 18-29 years, found an increased content of the H1 histone H2A lower total fraction, H3, H4 - and a sharp increase in histone H2B content - histones.CONCLUSIONS:Men living in environmentally disadvantaged areas of Kyzylorda region under the influence of dust and salt aerosols and other toxicants leads to disruption of the reproductive function in men.

Middle-down electron capture dissociation and electron transfer dissociation for histone analysis

Abstract The post-translational modifications (PTMs) of histones play a major role in activating or silencing gene transcription. To gain better understanding of the interplay between the PTMs that occur on histones, they are extensively studied using mass spectrometry techniques. Due to the abundance of lysines and arginines, the typical trypsin digestion has been found less favorable and GluC-digests have been explored as an alternative to yield larger peptides amenable to middle-down approaches. In addition, the use of weak cation exchange hydrophilic interaction liquid chromatography (WCX-HILIC) and the use of electron-based fragmentation techniques were found to be advantageous for the in-depth characterization of histone variants containing multiple PTMs. As a test model, we used histones from MEL (murine erythroleukemia) cells treated with butyric acid or DMSO. After acid extraction, histone pellets were dried and fractionated using a reversed-phase C3 column. For middle-down analysis, selected histone fractions were digested using GluC. The digested samples were separated on a WCX-HILIC capillary column packed in-house with PolyCAT A resin, coupled to a linear trap quadrupole Fourier transformation ion cyclotron resonance (LTQFT-ICR) instrument. Raw data was acquired on the LTQFT-ICR using electron capture dissociation (ECD). After deconvolution of the raw data, we generated heatmaps to illustrate differential maps between differentially treated histone samples. We also explored the innovative use of Skyline to quantify histone tails. In addition, we report some preliminary data using a synthetic histone peptide acquired on an Orbitrap Fusion using electron transfer dissociation (ETD). Both, ECD and ETD methods are capable of comprehensively analyzing complex histone variations not accessible with conventional techniques.

Spermatozoa of Sminthopsis murina (Mammalia: Metatheria) exhibit an unusually high degree of chromatin stability in the absence of disulphide bonding in protamine 1.

Although all but a single genus (Planigale) of the metatheria so far examined contain no cysteine residues in protamine 1, we report a remarkable level of chromatin stability in the spermatozoa of the common dunnart, Sminthopsis murina. S. murina cauda epididymal spermatozoa and somatic epithelial cells were exposed to a combination of graded treatments to lyse sperm protein and induce sperm DNA damage via standard freeze-thaw protocols and post-thaw incubation at 37°C for 48h, exposure to sodium nitroprusside (SNP) and the enzyme AluI restriction endonuclease. Sperm DNA fragmentation was assessed using the comet assay and sperm chromatin dispersal test. Although S. murina somatic cells showed DNA fragmentation following protein lysis and after treatment with all the protocols specifically designed to induce chromatin damage, sperm DNA fragmentation was only observed following moderate to severe proteolytic exposure and treatment with the restriction endonuclease; there was also an increase in the baseline halo of spermatozoa treated with an aggressive reducing agent, but no corresponding evidence of fragmented DNA, suggesting that cysteine residues may be functioning to conform tertiary and/or quaternary chromatin structure. Given that the protamine 1 of S. murina contains no cysteine, we suggest that the source of these residues is possibly the histone fraction of the chromatin and that the high level of stability is potentially related to prolonged sperm survival in the female's reproductive tract.

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.