Chromatin loading of MCM hexamers is associated with di-/tri-methylation of histone H4K20 toward S phase entry.

DNA replication is a key step in initiating cell proliferation. Loading hexameric complexes of minichromosome maintenance (MCM) helicase onto DNA replication origins during the G1 phase is essential for initiating DNA replication. Here, we examined MCM hexamer states during the cell cycle in human hTERT-RPE1 cells using multicolor immunofluorescence-based, single-cell plot analysis, and biochemical size fractionation. Experiments involving cell-cycle arrest at the G1 phase and release from the arrest revealed that a double MCM hexamer was formed via a single hexamer during G1 progression. A single MCM hexamer was recruited to chromatin in the early G1 phase. Another single hexamer was recruited to form a double hexamer in the late G1 phase. We further examined relationship between the MCM hexamer states and the methylation levels at lysine 20 of histone H4 (H4K20) and found that the double MCM hexamer state was correlated with di/trimethyl-H4K20 (H4K20me2/3). Inhibiting the conversion from monomethyl-H4K20 (H4K20me1) to H4K20me2/3 retained the cells in the single MCM hexamer state. Non-proliferative cells, including confluent cells or Cdk4/6 inhibitor-treated cells, also remained halted in the single MCM hexamer state. We propose that the single MCM hexamer state is a halting step in the determination of cell cycle progression.

Histone modification dynamics as revealed by multicolor immunofluorescence-based single-cell analysis.

Post-translational modifications on histones can be stable epigenetic marks or transient signals that can occur in response to internal and external stimuli. Levels of histone modifications fluctuate during the cell cycle and vary among different cell types. Here, we describe a simple system to monitor the levels of multiple histone modifications in single cells by multicolor immunofluorescence using directly labeled modification-specific antibodies. We analyzed histone H3 and H4 modifications during the cell cycle. Levels of active marks, such as acetylation and H3K4 methylation, were increased during the S phase, in association with chromatin duplication. By contrast, levels of some repressive modifications gradually increased during G2 and the next G1 phases. We applied this method to validate the target modifications of various histone demethylases in cells using a transient overexpression system. In extracts of marine organisms, we also screened chemical compounds that affect histone modifications and identified psammaplin A, which was previously reported to inhibit histone deacetylases. Thus, the method presented here is a powerful and convenient tool for analyzing the changes in histone modifications.

Human Ebp1 rescues the synthetic lethal growth of fission yeast cells lacking Cdb4 and Nup184.

Cdb4 is a protein with unknown functions that binds to curved DNA in vitro in the fission yeast Schizosaccharomyces pombe. Homologues of Cdb4 were identified in a wide range of eukaryotes, including human Ebp1. Both S. pombe Cdb4 and human Ebp1 are nonpeptidase members of the methionine aminopeptidase family. It has been reported that Ebp1 homologues are involved in cell growth regulation and differentiation. However, opposing functions have also been considered and debated upon, and the precise biological functions of this conserved protein are largely unknown. S. pombe cdb4 is a nonessential gene, and no obvious phenotypes have been detected in cells with cdb4 gene deletion. In this study, we identified nup184, encoding a component of the nuclear pore complex, as a gene responsible for the synthetic lethal phenotype associated with cdb4. Furthermore, the synthetic lethal phenotype of Cdb4 was suppressed by over-expression of human Ebp1, suggesting that it has conserved crucial functions in S. pombe Cdb4 and human Ebp1. This synthetic lethal phenotype associated with Cdb4 and Nup184 provides a molecular genetics tool to study the functions of S. pombe Cdb4 and its conserved members of proteins, including human Ebp1.

Comprehensive epigenome characterization reveals diverse transcriptional regulation across human vascular endothelial cells.

BACKGROUND: Endothelial cells (ECs) make up the innermost layer throughout the entire vasculature. Their phenotypes and physiological functions are initially regulated by developmental signals and extracellular stimuli. The underlying molecular mechanisms responsible for the diverse phenotypes of ECs from different organs are not well understood. RESULTS: To characterize the transcriptomic and epigenomic landscape in the vascular system, we cataloged gene expression and active histone marks in nine types of human ECs (generating 148 genome-wide datasets) and carried out a comprehensive analysis with chromatin interaction data. We developed a robust procedure for comparative epigenome analysis that circumvents variations at the level of the individual and technical noise derived from sample preparation under various conditions. Through this approach, we identified 3765 EC-specific enhancers, some of which were associated with disease-associated genetic variations. We also identified various candidate marker genes for each EC type. We found that the nine EC types can be divided into two subgroups, corresponding to those with upper-body origins and lower-body origins, based on their epigenomic landscape. Epigenomic variations were highly correlated with gene expression patterns, but also provided unique information. Most of the deferentially expressed genes and enhancers were cooperatively enriched in more than one EC type, suggesting that the distinct combinations of multiple genes play key roles in the diverse phenotypes across EC types. Notably, many homeobox genes were differentially expressed across EC types, and their expression was correlated with the relative position of each organ in the body. This reflects the developmental origins of ECs and their roles in angiogenesis, vasculogenesis and wound healing. CONCLUSIONS: This comprehensive analysis of epigenome characterization of EC types reveals diverse transcriptional regulation across human vascular systems. These datasets provide a valuable resource for understanding the vascular system and associated diseases.

Effect of mycalolides isolated from a marine sponge Mycale aff. nullarosette on actin in living cells.

Discovery of novel bioactive compounds is important not only for therapeutic purposes but also for understanding the mechanisms of biological processes. To screen bioactive compounds that affect nuclear morphology in marine organism extracts, we employed a microscopy-based assay using DNA staining of human cancer cells. A crude extract from a marine sponge Mycale aff. nullarosette, collected from the east coast of Japan, induced cellular binucleation. Fractionation of the extract led to the isolation of mycalolides A and B, and 38-hydroxymycalolide B as the active components. Mycalolides have been identified as marine toxins that induce depolymerization of the actin filament. Live cell imaging revealed that low concentrations of mycalolide A produce binucleated cells by inhibiting the completion of cytokinesis. At higher concentrations, however, mycalolide A causes immediate disruption of actin filaments and changes in cell morphology, yielding rounded cells. These results suggest that the completion of cytokinesis is a process requiring high actin polymerization activity. Furthermore, luciferase reporter assays with mycalolide A treatments support the view that the level of globular actin can affect transcription of a serum response gene.

A chromatin integration labelling method enables epigenomic profiling with lower input.

Chromatin plays a crucial role in gene regulation, and chromatin immunoprecipitation followed by sequencing (ChIP-seq) has been the standard technique for examining protein-DNA interactions across the whole genome. However, it is difficult to obtain epigenomic information from limited numbers of cells by ChIP-seq because of sample loss during chromatin preparation and inefficient immunoprecipitation. In this study, we established an immunoprecipitation-free epigenomic profiling method named chromatin integration labelling (ChIL), which enables the amplification of genomic sequences closely associated with the target molecules before cell lysis. Using ChIL followed by sequencing (ChIL-seq), we reliably detected the distributions of histone modifications and DNA-binding factors in 100-1,000 cells. In addition, ChIL-seq successfully detected genomic regions associated with histone marks at the single-cell level. Thus, ChIL-seq offers an alternative method to ChIP-seq for epigenomic profiling using small numbers of cells, in particular, those attached to culture plates and after immunofluorescence.

Histone H4 lysine 20 acetylation is associated with gene repression in human cells.

Histone acetylation is generally associated with gene activation and chromatin decondensation. Recent mass spectrometry analysis has revealed that histone H4 lysine 20, a major methylation site, can also be acetylated. To understand the function of H4 lysine 20 acetylation (H4K20ac), we have developed a specific monoclonal antibody and performed ChIP-seq analysis using HeLa-S3 cells. H4K20ac was enriched around the transcription start sites (TSSs) of minimally expressed genes and in the gene body of expressed genes, in contrast to most histone acetylation being enriched around the TSSs of expressed genes. The distribution of H4K20ac showed little correlation with known histone modifications, including histone H3 methylations. A motif search in H4K20ac-enriched sequences, together with transcription factor binding profiles based on ENCODE ChIP-seq data, revealed that most transcription activators are excluded from H4K20ac-enriched genes and a transcription repressor NRSF/REST co-localized with H4K20ac. These results suggest that H4K20ac is a unique acetylation mark associated with gene repression.

Distribution of histone H4 modifications as revealed by a panel of specific monoclonal antibodies.

Post-translational histone modifications play a critical role in genome functions such as epigenetic gene regulation and genome maintenance. The tail of the histone H4 N-terminus contains several amino acids that can be acetylated and methylated. Some of these modifications are known to undergo drastic changes during the cell cycle. In this study, we generated a panel of mouse monoclonal antibodies against histone H4 modifications, including acetylation at K5, K8, K12, and K16, and different levels of methylation at K20. Their specificity was evaluated by ELISA and immunoblotting using synthetic peptide and recombinant proteins that harbor specific modifications or amino acid substitutions. Immunofluorescence confirmed the characteristic distributions of target modifications. An H4K5 acetylation (H4K5ac)-specific antibody CMA405 reacted with K5ac only when the neighboring K8 was unacetylated. This unique feature allowed us to detect newly assembled H4, which is diacetylated at K5 and K12, and distinguish it from hyperacetylated H4, where K5 and K8 are both acetylated. Chromatin immunoprecipiation combined with deep sequencing (ChIP-seq) revealed that acetylation of both H4K8 and H4K16 were enriched around transcription start sites. These extensively characterized and highly specific antibodies will be useful for future epigenetics and epigenome studies.

Visualizing posttranslational and epigenetic modifications of endogenous proteins in vivo.

Protein localization and dynamics can now be visualized in living cells using the fluorescent protein fusion technique, but it is still difficult to selectively detect molecules with a specific function. As a posttranslational protein modification is often associated with a specific function, marking specifically modified protein molecules in living cells is a way to track an important fraction of protein. In the nucleus, histones are subjected to a variety of modifications such as acetylation and methylation that are associated with epigenetic gene regulation. RNA polymerase II, an enzyme that transcribes genes, is also differentially phosphorylated during the initiation and elongation of transcription. To understand the mechanism of gene regulation in vivo, we have developed methods to track histone and RNA polymerase II modifications using probes derived from modification-specific monoclonal antibodies. In Fab-based live endogenous modification labeling (FabLEM), fluorescently labeled antigen-binding fragments (Fabs) are loaded into cells. Fabs bind to target modifications in the nucleus with a binding time of a second to tens of seconds, and so the modification can be tracked without disturbing cell function. For tracking over longer periods of time or in living animals, we have also developed a genetically encoded system to express a modification-specific intracellular antibody (mintbody). Transgenic fruit fly and zebrafish that express histone H3 Lys9 acetylation-specific mintbody developed normally and remain fertile, suggesting that visualizing histone modifications in any tissue in live animals has become possible. These live cell modification tracking techniques will facilitate future studies on epigenetic regulation related to development, differentiation, and disease. Moreover, these techniques can be applied to any other protein modification, opening up new avenues in broad areas in biology and medicine.

Early development of cloned bovine embryos produced from oocytes enucleated by fluorescence metaphase II imaging using a conventional halogen-lamp microscope.

Enucleation of a recipient oocyte is one of the key processes in the procedure of somatic cell nuclear transfer (SCNT). However, especially in bovine species, lipid droplets spreading in the ooplasm hamper identification and enucleation of metaphase II (MII) chromosomes, and thereby the success rate of the cloning remains low. In this study we used a new experimental system that enables fluorescent observation of chromosomes in living oocytes without any damage. We succeeded in visualizing and removing the MII chromosome in matured bovine oocytes. This experimental system consists of injecting fluorescence-labeled antibody conjugates that bind to chromosomes and fluorescent observation using a conventional halogen-lamp microscope. The cleavage rates and blastocyst rates of bovine embryos following in vitro fertilization (IVF) decreased as the concentration of the antibody increased (p<0.05). The enucleation rate of the conventional method (blind enucleation) was 86%, whereas all oocytes injected with the antibody conjugates were enucleated successfully. Fusion rates and developmental rates of SCNT embryos produced with the enucleated oocytes were the same as those of the blind enucleation group (p>0.05). For the production of SCNT embryos, the new system can be used as a reliable predictor of the location of metaphase plates in opaque oocytes, such as those in ruminant animals.

Regulation of RNA polymerase II activation by histone acetylation in single living cells.

In eukaryotic cells, post-translational histone modifications have an important role in gene regulation. Starting with early work on histone acetylation, a variety of residue-specific modifications have now been linked to RNA polymerase II (RNAP2) activity, but it remains unclear if these markers are active regulators of transcription or just passive byproducts. This is because studies have traditionally relied on fixed cell populations, meaning temporal resolution is limited to minutes at best, and correlated factors may not actually be present in the same cell at the same time. Complementary approaches are therefore needed to probe the dynamic interplay of histone modifications and RNAP2 with higher temporal resolution in single living cells. Here we address this problem by developing a system to track residue-specific histone modifications and RNAP2 phosphorylation in living cells by fluorescence microscopy. This increases temporal resolution to the tens-of-seconds range. Our single-cell analysis reveals histone H3 lysine-27 acetylation at a gene locus can alter downstream transcription kinetics by as much as 50%, affecting two temporally separate events. First acetylation enhances the search kinetics of transcriptional activators, and later the acetylation accelerates the transition of RNAP2 from initiation to elongation. Signatures of the latter can be found genome-wide using chromatin immunoprecipitation followed by sequencing. We argue that this regulation leads to a robust and potentially tunable transcriptional response.

Evaluation of chemical fluorescent dyes as a protein conjugation partner for live cell imaging.

To optimize live cell fluorescence imaging, the choice of fluorescent substrate is a critical factor. Although genetically encoded fluorescent proteins have been used widely, chemical fluorescent dyes are still useful when conjugated to proteins or ligands. However, little information is available for the suitability of different fluorescent dyes for live imaging. We here systematically analyzed the property of a number of commercial fluorescent dyes when conjugated with antigen-binding (Fab) fragments directed against specific histone modifications, in particular, phosphorylated H3S28 (H3S28ph) and acetylated H3K9 (H3K9ac). These Fab fragments were conjugated with a fluorescent dye and loaded into living HeLa cells. H3S28ph-specific Fab fragments were expected to be enriched in condensed chromosomes, as H3S28 is phosphorylated during mitosis. However, the degree of Fab fragment enrichment on mitotic chromosomes varied depending on the conjugated dye. In general, green fluorescent dyes showed higher enrichment, compared to red and far-red fluorescent dyes, even when dye:protein conjugation ratios were similar. These differences are partly explained by an altered affinity of Fab fragment after dye-conjugation; some dyes have less effect on the affinity, while others can affect it more. Moreover, red and far-red fluorescent dyes tended to form aggregates in the cytoplasm. Similar results were observed when H3K9ac-specific Fab fragments were used, suggesting that the properties of each dye affect different Fab fragments similarly. According to our analysis, conjugation with green fluorescent dyes, like Alexa Fluor 488 and Dylight 488, has the least effect on Fab affinity and is the best for live cell imaging, although these dyes are less photostable than red fluorescent dyes. When multicolor imaging is required, we recommend the following dye combinations for optimal results: Alexa Fluor 488 (green), Cy3 (red), and Cy5 or CF640 (far-red).

Structural polymorphism in the L1 loop regions of human H2A.Z.1 and H2A.Z.2.

The histone H2A.Z variant is widely conserved among eukaryotes. Two isoforms, H2A.Z.1 and H2A.Z.2, have been identified in vertebrates and may have distinct functions in cell growth and gene expression. However, no structural differences between H2A.Z.1 and H2A.Z.2 have been reported. In the present study, the crystal structures of nucleosomes containing human H2A.Z.1 and H2A.Z.2 were determined. The structures of the L1 loop regions were found to clearly differ between H2A.Z.1 and H2A.Z.2, although their amino-acid sequences in this region are identical. This structural polymorphism may have been induced by a substitution that evolutionally occurred at the position of amino acid 38 and by the flexible nature of the L1 loops of H2A.Z.1 and H2A.Z.2. It was also found that in living cells nucleosomal H2A.Z.1 exchanges more rapidly than H2A.Z.2. A mutational analysis revealed that the amino-acid difference at position 38 is at least partially responsible for the distinctive dynamics of H2A.Z.1 and H2A.Z.2. These findings provide important new information for understanding the differences in the regulation and functions of H2A.Z.1 and H2A.Z.2 in cells.

Genetically encoded system to track histone modification in vivo.

Post-translational histone modifications play key roles in gene regulation, development, and differentiation, but their dynamics in living organisms remain almost completely unknown. To address this problem, we developed a genetically encoded system for tracking histone modifications by generating fluorescent modification-specific intracellular antibodies (mintbodies) that can be expressed in vivo. To demonstrate, an H3 lysine 9 acetylation specific mintbody (H3K9ac-mintbody) was engineered and stably expressed in human cells. In good agreement with the localization of its target acetylation, H3K9ac-mintbody was enriched in euchromatin, and its kinetics measurably changed upon treatment with a histone deacetylase inhibitor. We also generated transgenic fruit fly and zebrafish stably expressing H3K9ac-mintbody for in vivo tracking. Dramatic changes in H3K9ac-mintbody localization during Drosophila embryogenesis could highlight enhanced acetylation at the start of zygotic transcription around mitotic cycle 7. Together, this work demonstrates the broad potential of mintbody and lays the foundation for epigenetic analysis in vivo.

Independence of repressive histone marks and chromatin compaction during senescent heterochromatic layer formation.

The expansion of repressive epigenetic marks has been implicated in heterochromatin formation during embryonic development, but the general applicability of this mechanism is unclear. Here we show that nuclear rearrangement of repressive histone marks H3K9me3 and H3K27me3 into nonoverlapping structural layers characterizes senescence-associated heterochromatic foci (SAHF) formation in human fibroblasts. However, the global landscape of these repressive marks remains unchanged upon SAHF formation, suggesting that in somatic cells, heterochromatin can be formed through the spatial repositioning of pre-existing repressively marked histones. This model is reinforced by the correlation of presenescent replication timing with both the subsequent layered structure of SAHFs and the global landscape of the repressive marks, allowing us to integrate microscopic and genomic information. Furthermore, modulation of SAHF structure does not affect the occupancy of these repressive marks, nor vice versa. These experiments reveal that high-order heterochromatin formation and epigenetic remodeling of the genome can be discrete events.

Fluorescence cell imaging and manipulation using conventional halogen lamp microscopy.

Technologies for vitally labeling cells with fluorescent dyes have advanced remarkably. However, to excite fluorescent dyes currently requires powerful illumination, which can cause phototoxic damage to the cells and increases the cost of microscopy. We have developed a filter system to excite fluorescent dyes using a conventional transmission microscope equipped with a halogen lamp. This method allows us to observe previously invisible cell organelles, such as the metaphase spindle of oocytes, without causing phototoxicity. Cells remain healthy even after intensive manipulation under fluorescence observation, such as during bovine, porcine and mouse somatic cell cloning using nuclear transfer. This method does not require expensive epifluorescence equipment and so could help to reduce the science gap between developed and developing countries.

Crystal structure of the human centromeric nucleosome containing CENP-A.

In eukaryotes, accurate chromosome segregation during mitosis and meiosis is coordinated by kinetochores, which are unique chromosomal sites for microtubule attachment. Centromeres specify the kinetochore formation sites on individual chromosomes, and are epigenetically marked by the assembly of nucleosomes containing the centromere-specific histone H3 variant, CENP-A. Although the underlying mechanism is unclear, centromere inheritance is probably dictated by the architecture of the centromeric nucleosome. Here we report the crystal structure of the human centromeric nucleosome containing CENP-A and its cognate α-satellite DNA derivative (147 base pairs). In the human CENP-A nucleosome, the DNA is wrapped around the histone octamer, consisting of two each of histones H2A, H2B, H4 and CENP-A, in a left-handed orientation. However, unlike the canonical H3 nucleosome, only the central 121 base pairs of the DNA are visible. The thirteen base pairs from both ends of the DNA are invisible in the crystal structure, and the αN helix of CENP-A is shorter than that of H3, which is known to be important for the orientation of the DNA ends in the canonical H3 nucleosome. A structural comparison of the CENP-A and H3 nucleosomes revealed that CENP-A contains two extra amino acid residues (Arg 80 and Gly 81) in the loop 1 region, which is completely exposed to the solvent. Mutations of the CENP-A loop 1 residues reduced CENP-A retention at the centromeres in human cells. Therefore, the CENP-A loop 1 may function in stabilizing the centromeric chromatin containing CENP-A, possibly by providing a binding site for trans-acting factors. The structure provides the first atomic-resolution picture of the centromere-specific nucleosome.

Tracking epigenetic histone modifications in single cells using Fab-based live endogenous modification labeling.

Histone modifications play an important role in epigenetic gene regulation and genome integrity. It remains largely unknown, however, how these modifications dynamically change in individual cells. By using fluorescently labeled specific antigen binding fragments (Fabs), we have developed a general method to monitor the distribution and global level of endogenous histone H3 lysine modifications in living cells without disturbing cell growth and embryo development. Fabs produce distinct nuclear patterns that are characteristic of their target modifications. H3K27 trimethylation-specific Fabs, for example, are concentrated on inactive X chromosomes. As Fabs bind their targets transiently, the ratio of bound and free molecules depends on the target concentration, allowing us to measure changes in global modification levels. High-affinity Fabs are suitable for mouse embryo imaging, so we have used them to monitor H3K9 and H3K27 acetylation levels in mouse preimplantation embryos produced by in vitro fertilization and somatic cell nuclear transfer. The data suggest that a high level of H3K27 acetylation is important for normal embryo development. As Fab-based live endogenous modification labeling (FabLEM) is broadly useful for visualizing any modification, it should be a powerful tool for studying cell signaling and diagnosis in the future.

Structural basis of instability of the nucleosome containing a testis-specific histone variant, human H3T.

A histone H3 variant, H3T, is highly expressed in the testis, suggesting that it may play an important role in the chromatin reorganization required for meiosis and/or spermatogenesis. In the present study, we found that the nucleosome containing human H3T is significantly unstable both in vitro and in vivo, as compared to the conventional nucleosome containing H3.1. The crystal structure of the H3T nucleosome revealed structural differences in the H3T regions on both ends of the central alpha2 helix, as compared to those of H3.1. The H3T-specific residues (Met71 and Val111) are the source of the structural differences observed between H3T and H3.1. A mutational analysis revealed that these residues are responsible for the reduced stability of the H3T-containing nucleosome. These physical and structural properties of the H3T-containing nucleosome may provide the basis of chromatin reorganization during spermatogenesis.

Visualization of DNA methylation and histone modifications in living cells.

DNA methylation and histone modifications play important roles in genome function, including epigenetic gene regulation. These modifications undergo drastic changes when nuclei are reprogrammed during development and differentiation. Recent studies have enabled the detection of the dynamics of modifications in living cultured cells and mouse preimplantation embryos. DNA methylation was visualized using the methyl-CpG-binding domain of the human MBD1 protein. The level and distribution of histone modifications can be monitored by two different methods. One approach uses fluorescence/Förster resonance energy transfer (FRET)-based sensors and another uses fluorescently labeled antigen binding fragments of specific antibodies. These visualization techniques will facilitate future studies on epigenetic regulation related to development, differentiation, and disease.