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Live human myeloid leukemia (HL-60/S4) cells exposed to acute hyperosmotic stress with sucrose undergo dehydration and cell shrinkage. Interphase chromatin and mitotic chromosomes congeal, and exhibit altered phase separation (demixing) of chromatin-associated proteins. To investigate concurrent changes in the transcriptome, we exposed exponentially growing HL-60/S4 cells to acute hyperosmotic stress (∼600 milliOsmolar) for 30 and 60 minutes by addition of sucrose to the culture medium. We employed RNA-Seq of polyA mRNA to identify genes with significantly increased or decreased transcript levels relative to untreated control cells (i.e., differential gene expression). These identified genes were examined for over-representation of Gene Ontology (GO) terms. In hyperosmotically-stressed cells, multiple GO terms associated with transcription, translation, mitochondrial function and proteosome activity, as well as the gene set “replication-dependent histones”, were over-represented among genes with increased transcript levels; whereas, genes with decreased transcript levels were over-represented in various GO terms for transcription repressors. The overall transcriptome profiles of these stressed cells suggest a rapid acquisition of cellular rebuilding, a futile homeostatic response, as these cells are ultimately doomed to a dehydrated death.“Do not go gentle into that good nightRage, rage against the dying of the light”Dylan Thomas (1914-1953)

"Interphase epichromatin" describes the surface of chromatin located adjacent to the interphase nuclear envelope. It was discovered in 2011 using a bivalent anti-nucleosome antibody (mAb PL2-6), now known to be directed against the nucleosome acidic patch. The molecular structure of interphase epichromatin is unknown, but is thought to be heterochromatic with a high density of "exposed" acidic patches. In the 1960s, transmission electron microscopy of fixed, dehydrated, sectioned, and stained inactive chromatin revealed "unit threads," frequently organized into parallel arrays at the nuclear envelope, which were interpreted as regular helices with similar to 30-nm center-to-center distance. Also observed in certain cell types, the nuclear envelope forms a "sandwich" around a layer of closely packed unit threads (ELCS, envelope-limited chromatin sheets). Discovery of the nucleosome in 1974 led to revised helical models of chromatin. But these models became very controversial and the existence of in situ 30-nm chromatin fibers has been challenged. Development of cryo-electron microscopy (Cryo-EM) gave hope that in situ chromatin fibers, devoid of artifacts, could be structurally defined. Combining a contrast-enhancing phase plate and cryo-electron tomography (Cryo-ET), it is now possible to visualize chromatin in a "close-to-native" situation. ELCS are particularly interesting to study by Cryo-ET. The chromatin sheet appears to have two layers of similar to 30-nm chromatin fibers arranged in a criss-crossed pattern. The chromatin in ELCS is continuous with adjacent interphase epichromatin. It appears that hydrated similar to 30-nm chromatin fibers are quite rare in most cells, possibly confined to interphase epichromatin at the nuclear envelope.

Cancer cell lines often have large structural variants (SVs) that evolve over time. There are many reported differences in large scale SVs between HL-60 and HL-60/S4, cell lines derived from the same acute promyelocytic leukemia sample. However, the stability and variability of inter- and intra-chromosomal SVs between different sources is unknown. Here, we used Hi-C and RNA-seq to identify and compare large SVs in HL-60 and HL-60/S4 cell lines. Comparisons with previously reported karyotypes identified two non-canonical SVs in HL-60. Ten previously unreported SVs were identified in HL-60/S4. The unreported SVs were generally small enough to be plausibly undetected with traditional karyotyping methods. An expansion centered on MYC was found in a novel genomic location in HL-60. The HL-60 cell line has more within-line structural variation than the HL-60/S4 derivative cell line. HL-60 and HL-60/S4 karyotypes are generally consistent with the current literature, with some exceptions. Hic_breakfinder is an effective tool for identifying all SVs, but intra-chromosomal SVs are less reliably detected across different samples. The orientation of SV patterns, and strandedness of gene fusions, allowed us to differentiate inversions from other forms of intra-chromosomal SV. Visual inspection of Hi-C heatmap patterns allow further characterization of SVs without additional methods, although orthogonal information such as gene fusions contribute to the characterization.

Dehydration of cells by acute hyperosmotic stress has profound effects upon cell structure and function. Interphase chromatin and mitotic chromosomes collapse (“congelation”). HL-60/S4 cells remain ~100% viable for, at least, 1 hour, exhibiting shrinkage to ~2/3 their original volume, when placed in 300mM sucrose in tissue culture medium. Fixed cells were imaged by immunostaining confocal and STED microscopy. At a “global” structural level (μm), mitotic chromosomes congeal into a residual gel with apparent (phase) separations of Ki67, CTCF, SMC2, RAD21, H1 histones and HMG proteins. At an “intermediate” level (sub-μm), radial distribution analysis of STED images revealed a most probable peak DNA density separation of ~0.16 μm, essentially unchanged by hyperosmotic stress. At a “local” structural level (~1-2 nm), in vivo crosslinking revealed essentially unchanged crosslinked products between H1, HMG and inner histones. Hyperosmotic cellular stress is discussed in terms of concepts of mitotic chromosome structure and liquid-liquid phase separation.

Epigenomic regulation plays a vital role in cell differentiation. The leukemic HL-60/S4 [human myeloid leukemic cell line HL-60/S4 (ATCC CRL-3306)] promyelocytic cell can be easily differentiated from its undifferentiated promyelocyte state into neutrophil- and macrophage-like cell states. In this study, we present the underlying genome and epigenome architecture of HL-60/S4 through its differentiation. We performed whole-genome bisulphite sequencing of HL-60/S4 cells and their differentiated counterparts. With the support of karyotyping, we show that HL-60/S4 maintains a stable genome throughout differentiation. Analysis of differential Cytosine-phosphate-Guanine dinucleotide methylation reveals that most methylation changes occur in the macrophage-like state. Differential methylation of promoters was associated with immune-related terms. Key immune genes, CEBPA, GFI1, MAFB and GATA1 showed differential expression and methylation. However, we observed the strongest enrichment of methylation changes in enhancers and CTCF binding sites, implying that methylation plays a major role in large-scale transcriptional reprogramming and chromatin reorganisation during differentiation. Correlation of differential expression and distal methylation with support from chromatin capture experiments allowed us to identify putative proximal and long-range enhancers for a number of immune cell differentiation genes, including CEBPA and CCNF. Integrating expression data, we present a model of HL-60/S4 differentiation in relation to the wider scope of myeloid differentiation., Summary: Epigenomics play a role in cell identity and differentiation. We present the DNA methylation landscape of leukemic cells during in vitro differentiation, adding another omics layer to better understand differentiation mechanisms.

Epichromatin is identified by immunostaining fixed and permeabilized cells with particular bivalent anti-nucleosome antibodies (mAbs PL2-6 and 1H6). During interphase, epichromatin resides adjacent to the inner nuclear membrane; during mitosis, at the outer surface of mitotic chromosomes. By STED (stimulated emission depletion) microscopy, PL2-6 stained interphase epichromatin is ∼76 nm thick and quite uniform; mitotic epichromatin is more variable in thickness, exhibiting a "wrinkled" surface with an average thickness of ∼78 nm. Co-immunostaining with anti-Ki-67 demonstrates Ki-67 deposition between the PL2-6 "ridges" of mitotic epichromatin. Monovalent papain-derived Fab fragments of PL2-6 yield a strikingly different punctate "chromomeric" immunostaining pattern throughout interphase nuclei and along mitotic chromosome arms. Evidence from electrophoretic mobility shift assay (EMSA) and from analytical ultracentrifugation characterize the Fab/mononucleosome complex, supporting the concept that there are two binding sites per nucleosome. The peptide sequence of the Hv3 region (heavy chain variable region 3) of the PL2-6 antibody binding site strongly resembles other nucleosome acidic patch binding proteins (especially, LANA and CENPC), supporting that the nucleosome acidic patch is included within the epichromatin epitope. It is speculated that the interphase epichromatin epitope is "exposed" with favorable geometric arrangements for binding bivalent PL2-6 at the surface chromatin; whereas, the epitope is "hidden" within internal chromatin. Furthermore, it is suggested that the "exposed" nucleosome surface of mitotic epichromatin may play a role in post-mitotic nuclear envelope reformation.

To understand the chromatin changes underlying differential gene expression during induced differentiation of human leukemic HL-60/S4 cells, we conducted RNA-Seq analysis on quadruplicate cultures of undifferentiated, granulocytic- and macrophage-differentiated cell forms. More than half of mapped genes exhibited altered transcript levels in the differentiated cell forms. In general, more genes showed increased mRNA levels in the granulocytic form and in the macrophage form, than showed decreased levels. The majority of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were significantly enriched in genes that exhibited differential transcript levels after either RA or TPA treatment. Changes in transcript levels for groups of genes with characteristic protein phenotypes, such as genes encoding cytoplasmic granular proteins, nuclear envelope and cytoskeletal proteins, cell adhesion proteins, and proteins involved in the cell cycle and apoptosis illustrate the profound differences among the various cell states. In addition to the transcriptome analyses, companion karyotyping by M-FISH of undifferentiated HL-60/S4 cells revealed a plethora of chromosome alterations, compared with normal human cells. The present mRNA profiling provides important information related to nuclear shape changes (e.g., granulocyte lobulation), deformability of the nuclear envelope and linkage between the nuclear envelope and cytoskeleton during induced myeloid chromatin differentiation.

Interactions of chromatin with bivalent immunoglobin nucleosome-binding antibodies and their monovalent (papain-derived) antigen-binding fragment analogs are useful probes for examining chromatin conformational states. To help interpret antibody-chromatin interactions and explore how antibodies might compete for interactions with chromatin components, we incorporate coarse-grained PL2-6 antibody modeling into our mesoscale chromatin model. We analyze interactions and fiber structures for the antibody-chromatin complexes in open and condensed chromatin, with and without H1 linker histone (LH). Despite minimal and transient interactions at physiological salt, we capture significant differences in antibody-chromatin complex configurations in open fibers, with more intense interactions between the bivalent antibody and chromatin compared to monovalent antigen-binding fragments. For these open chromatin fiber morphologies, antibody binding to histone tails is increased and compaction is greater for bivalent compared to monovalent and antibody-free systems. Differences between monovalent and bivalent binding result from antibody competition with internal chromatin fiber components (nucleosome core and linker DNA) for histone tail (H3, H4, H2A, H2B) interactions. This antibody competition for tail contacts reduces tail-core and tail-linker interactions and increases tail-antibody interactions. Such internal structural changes in open fibers resemble mechanisms of LH condensation, driven by charge screening and entropy changes. For condensed fibers at physiological salt, the three systems are much more similar overall, but some subtle tail interaction differences can be noted. Adding LH results in less-dramatic changes for all systems, except that the bivalent complex at physiological salt shows cooperative effects between LH and the antibodies in condensing chromatin fibers. Such dynamic interactions that depend on the internal structure and complex-stabilizing interactions within the chromatin fiber have implications for gene regulation and other chromatin complexes such as with LH, remodeling proteins, and small molecular chaperones that bind and modulate chromatin structure.

Visualizing chromatin adjacent to the nuclear envelope (denoted “epichromatin”) by in vitro immunostaining with a bivalent nucleosome-binding antibody (termed monoclonal antibody PL2-6) has suggested a distinct and conserved chromatin structure. Moreover, different staining patterns for chromatin complexed with the monovalent “Fab” fragment of PL2-6, compared to the bivalent form, point to distinct binding interactions. To help interpret antibody/chromatin interactions and these differential binding modes, we incorporate coarse-grained PL2-6 antibody modeling into our mesoscale chromatin model and analyze interactions and fiber structures for the antibody/chromatin complexes in open and condensed chromatin, with and without linker histone H1 (LH). Despite minimal and transient interactions at physiological salt, we capture differential binding for monomer and dimer antibody forms to open fibers, with much more intense interactions in the bivalent antibody/chromatin complex. For these open “zigzag” fiber morphologies, differences result from antibody competition for peptide tail contacts with internal chromatin fiber components (nucleosome core and linker DNA). Antibody competition results in dramatic conformational and energetic differences among monovalent, bivalent, and free chromatin systems in the parental linker DNA / tail interactions. These differences in binding modes and changes in internal fiber structure, driven by conformational entropy gains, help interpret the differential staining patterns for the monovalent versus bivalent antibody/chromatin complexes. More generally, such dynamic interactions which depend on the complex internal structure and self-interactions of the chromatin fiber have broader implications to other systems that bind to chromatin, such as linker histones and remodeling proteins.STATEMENT OF SIGNIFICANCEUsing mesoscale modeling, we help interpret differential binding modes for antibody/chromatin interactions to elucidate the structural details of “epichromatin” (chromatin adjacent to the nuclear envelope), which had been visualized to produce different staining patterns for monovalent and bivalent forms of the PL2-6 antibody. To our knowledge, this is the first application of such a coarse-grained computational antibody model to probe chromatin structure and mechanisms of antibody/chromatin binding. Our work emphasizes how antibody units compete with native internal chromatin fiber units (histone tails, nucleosome core, and linker DNA) for fiber-stabilizing interactions and thereby drive differential antibody binding for open zigzag chromatin fibers. Such competition, which dynamically alters internal chromatin structure upon binding, could be relevant to other chromatin binding mechanisms such as those involving linker histones or chromatin remodeling proteins.

Tumor necrosis factor alpha (TNF-α) is a potent cytokine involved in systemic inflammation and immune modulation. Signaling responses that involve TNF-α are context dependent and capable of stimulating pathways promoting both cell death and survival. TNF-α treatment has been investigated as part of a combined therapy for acute myeloid leukemia due to its modifying effects on all-trans retinoic acid (ATRA) mediated differentiation into granulocytes.To investigate the interaction between cellular differentiation and TNF-α, we performed RNA-sequencing on two forms of the human HL-60/S4 promyelocytic leukemia cell line treated with TNF-α. The ATRA-differentiated granulocytic form of HL-60/S4 cells had an enhanced transcriptional response to TNF-α treatment compared to the undifferentiated promyelocytes. The observed TNF-α responses included differential expression of cell cycle gene sets, which were generally upregulated in TNF-α treated promyelocytes, and downregulated in TNF-α treated granulocytes. This is consistent with TNF-α induced cell cycle repression in granulocytes and cell cycle progression in promyelocytes. Moreover, comparisons with gene expression changes associated with differentiation indicated that TNF-α treatment of granulocytes shifts the transcriptome towards that of a macrophage.We conclude that TNF-α treatment promotes a divergent transcriptional program in promyelocytes and granulocytes. TNF-α promotes cell cycle associated gene expression in promyelocytes. In contrast, TNF-α stimulated granulocytes have reduced cell cycle gene expression, and a macrophage-like transcriptional program.

Background Mammalian cells are flexible and can rapidly change shape when they contract, adhere, or migrate. The nucleus must be stiff enough to withstand cytoskeletal forces, but flexible enough to remodel as the cell changes shape. This is particularly important for cells migrating through confined spaces, where the nuclear shape must change in order to fit through a constriction. This occurs many times in the life cycle of a neutrophil, which must protect its chromatin from damage and disruption associated with migration. Here we characterized the effects of constricted migration in neutrophil-like cells. Results Total RNA sequencing identified that migration of neutrophil-like cells through 5- or 14-μm pores was associated with changes in the transcript levels of inflammation and chemotaxis-related genes when compared to unmigrated cells. Differentially expressed transcripts specific to migration with constriction were enriched for groups of genes associated with cytoskeletal remodeling. Hi-C was used to capture the genome organization in control and migrated cells. Limited switching was observed between the active (A) and inactive (B) compartments after migration. However, global depletion of short-range contacts was observed following migration with constriction compared to migration without constriction. Regions with disrupted contacts, TADs, and compartments were enriched for inactive chromatin. Conclusion Short-range genome organization is preferentially altered in inactive chromatin, possibly protecting transcriptionally active contacts from the disruptive effects of migration with constriction. This is consistent with current hypotheses implicating heterochromatin as the mechanoresponsive form of chromatin. Further investigation concerning the contribution of heterochromatin to stiffness, flexibility, and protection of nuclear function will be important for understanding cell migration in relation to human health and disease. Electronic supplementary material The online version of this article (10.1186/s12915-018-0608-2) contains supplementary material, which is available to authorized users.

Background With the passing of Jörg Langowski 6 May 2017 in a sailplane accident, the scientific community was deprived of a strident and effective voice for DNA and chromatin molecular and computational biophysics, for open access publishing and for the creation of effective scientific research networks. Methods Here, after reviewing some of Jörg’s key research contributions and ideas, we offer through the personal remembrance of his closest collaborators, a deep analysis of the major results of his research and the future directions they have engendered. Conclusions The legacy of Jörg Langowski has been to propel a way of viewing biological function that considers living systems as dynamic and in three dimensions. This physical view of biology that he pioneered is now, finally, becoming established also because of his great effort.

‘Epichromatin’, the surface of chromatin beneath the interphase nuclear envelope (NE) or at the surface of mitotic chromosomes, was discovered by immunostaining with a specific bivalent mouse monoclonal anti-nucleosome antibody (mAb PL2-6). ‘Chromomeres’, punctate chromatin particles approximately 200–300 nm in diameter, identified throughout the interphase chromatin and along mitotic chromosomes, were observed by immunostaining with the monovalent papain-derived Fab fragments of bivalent PL2-6. The specific target for PL2-6 appears to include the nucleosome acidic patch. Thus, within the epichromatin and chromomeric regions, this epitope is ‘exposed’. Considering that histones possess unstructured ‘tails’ (i.e. intrinsically disordered peptide regions, IDPR), our perception of these chromatin regions becomes more ‘fuzzy’ (less defined). We suggest that epichromatin cationic tails facilitate interactions with anionic components of NE membranes. We also suggest that the unstructured histone tails (especially, histone H1 tails), with their presumed promiscuous binding, establish multivalent binding that stabilizes each chromomere as a unit of chromatin higher order structure. We propose an ‘unstructured stability’ hypothesis, which postulates that the stability of epichromatin and chromomeres (as well as other nuclear chromatin structures) is a consequence of the collective contributions of numerous weak histone IDPR binding interactions arising from the multivalent nucleosome, analogous to antibody avidity.

Cell differentiation is associated with changes in chromatin organization and gene expression. In this study, we examine chromatin structure following differentiation of the human myeloid leukemia cell line (HL-60/S4) into granulocytes with retinoic acid (RA) or into macrophage with phorbol ester (TPA). We performed ChIP-seq of histone H3 and its modifications, analyzing changes in nucleosome occupancy, nucleosome repeat length, eu-/heterochromatin redistribution and properties of epichromatin (surface chromatin adjacent to the nuclear envelope). Nucleosome positions changed genome-wide, exhibiting a specific class of alterations involving nucleosome loss in extended (∼1kb) regions, pronounced in enhancers and promoters. Genes that lost nucleosomes at their promoters showed a tendency to be upregulated. On the other hand, nucleosome gain did not show simple effects on transcript levels. The average genome-wide nucleosome repeat length (NRL) did not change significantly with differentiation. However, we detected an approximate 10 bp NRL decrease around the haematopoietic transcription factor (TF) PU.1 and the architectural protein CTCF, suggesting an effect on NRL proximal to TF binding sites. Nucleosome occupancy changed in regions associated with active promoters in differentiated cells, compared with untreated HL-60/S4 cells. Epichromatin regions revealed an increased GC content and high nucleosome density compared with surrounding chromatin. Epichromatin showed depletion of major histone modifications and revealed enrichment with PML body-associated genes. In general, chromatin changes during HL-60/S4 differentiation appeared to be more localized to regulatory regions, compared with genome-wide changes among diverse cell types studied elsewhere.

Cycling eukaryotic cells rapidly re-establish the nuclear envelope and internal architecture following mitosis. Studies with a specific anti-nucleosome antibody recently demonstrated that the surface ("epichromatin") of interphase and mitotic chromatin possesses a unique and conserved conformation, suggesting a role in postmitotic nuclear reformation. Here we present evidence showing that the anionic glycerophospholipid phosphatidylserine is specifically located in epichromatin throughout the cell cycle and is associated with nucleosome core histones. This suggests that chromatin bound phosphatidylserine may function as a nucleation site for the binding of ER and re-establishment of the nuclear envelope.

The interphase nucleus and nuclear envelope can acquire a myriad of shapes in normal or pathological cell states. There exist a wide variety of indentations and invaginations, of protrusions and evaginations. It has been difficult to classify and name all of these nuclear shapes and, consequently, a barrier to understanding the biochemical and biophysical causes. This review focuses upon one type of nuclear envelope shape change, named "nuclear envelope-limited chromatin sheets" (ELCS), which appears to involve exaggerated nuclear envelope growth, carrying with it one or more layers of approximately 30 nm diameter heterochromatin. A hypothesis on the formation of ELCS is proposed, relating higher order heterochromatin structure in an interphase nucleus, nuclear envelope growth, and nuclear envelope-heterochromatin interactions.

Objective Lamin B receptor (LBR) is an integral protein of the inner nuclear membrane. Recent studies have demonstrated that genetic deficiency of LBR during granulopoiesis results in hypolobulation of the mature neutrophil nucleus, as observed in human Pelger-Huet anomaly and mouse ichthyosis ( ic ). In this study, we utilized differentiated early promyelocytes (EPRO cells) that were derived from the bone marrow of homozygous and heterozygous ichthyosis mice to examine changes to the expression of nuclear envelope proteins and heterochromatin structure that result from deficient LBR expression. Materials and Methods Wild-type (+/+), heterozygous (+/ ic ), and homozygous ( ic / ic ) granulocytic forms of EPRO cells were analyzed for the expression of multiple lamins and inner nuclear envelope proteins by immunostaining and immunoblotting techniques. The heterochromatin architecture was also examined by immunostaining for histone lysine methylation. Results Wild-type (+/+) and heterozygous (+/ ic ) granulocytic forms revealed ring-shaped nuclei and contained LBR within the nuclear envelope; ic / ic granulocytes exhibited smaller ovoid nuclei devoid of LBR. The pericentric heterochromatin of undifferentiated and granulocytic ic / ic cells was condensed into larger spots and shifted away from the nuclear envelope, compared to +/+ and +/ ic cell forms. Lamin A/C, which is normally not present in mature granulocytes, was significantly elevated in LBR-deficient EPRO cells. Conclusions Our observations suggest roles for LBR during granulopoiesis, which can involve augmenting nuclear membrane growth, facilitating compartmentalization of heterochromatin, and promoting downregulation of lamin A/C expression.

The major human blood granulocyte, the neutrophil, is an essential component of the innate immunity system, emigrating from blood vessels and migrating through tight tissue spaces to the site of bacterial or fungal infection where they kill and phagocytose invading microbes. Since the late nineteenth century, it has been recognized that the human neutrophil nucleus is distinctly not ovoid as in other cell types, but possesses a lobulated (segmented) shape. This deformable nucleus enhances rapid migration. Recent studies have demonstrated that lamin B receptor (LBR) is necessary for the non-ovoid shape. LBR is an integral membrane protein of the nuclear envelope. A single dominant mutation in humans leads to neutrophils with hypolobulated nuclei (Pelger-Huet anomaly); homozygosity leads to ovoid granulocyte nuclei. Interestingly, LBR is also an enzyme involved in cholesterol metabolism. Homozygosity for null mutations is frequently lethal and associated with severe skeletal deformities. In addition to the necessity for LBR, formation of the mature granulocyte nucleus also depends upon lamin composition and microtubule integrity. These observations are part of a larger question on the relationships between nuclear shape and cellular function.

Mature blood neutrophils (polymorphonuclear granulocytes) have characteristically complex nuclear shapes. The human neutrophil nucleus generally possesses 3-4 lobes; the mouse neutrophil nucleus frequently resembles a twisted toroid with a central hole. Myeloid tissue culture systems (e.g., human HL-60 and murine MPRO) can be induced to differentiate in vitro towards neutrophils by addition of retinoic acid, exhibiting the characteristic nuclear shape changes. Confocal immunostaining and thin-section transmission electron microscopic image data from differentiated HL-60 and MPRO cells clearly demonstrate proximity of the centrosomal region (containing dynein, gamma-tubulin and C-Nap1) to regions of granulocytic nuclear indentations. In addition, the centrosomal region, flanked by the Golgi apparatus, is shown to be present within the central hole of the toroidal mouse granulocyte nucleus. A role for the centrosomal region and associated microtubules in molding granulocytic nuclear shape is suggested.

Thirty years ago, our conception of chromatin structure underwent a total metamorphosis as the nucleosome era began. In Kurosawa's classic movie 'Rashomon' (1951), each participant had a different perspective of the same pivotal event. This review outlines our perception of history.

Epichromatin, the surface of chromatin facing the nuclear envelope in an interphase nucleus, reveals a “rim” staining pattern with specific mouse monoclonal antibodies against histone H2A/H2B/DNA and phosphatidylserine epitopes. Employing a modified ChIP-Seq procedure on undifferentiated and differentiated human leukemic (HL-60/S4) cells, >95% of assembled epichromatin regions overlapped with Alu retrotransposons. They also exhibited enrichment of the AluS subfamily and of Alu oligomers. Furthermore, mapping epichromatin regions to the human chromosomes revealed highly similar localization patterns in the various cell states and with the different antibodies. Comparisons with available epigenetic databases suggested that epichromatin is neither “classical” heterochromatin nor highly expressing genes, implying another function at the surface of interphase chromatin. A modified chromatin immunoprecipitation procedure (xxChIP) was developed because the studied antibodies react generally with mononucleosomes and lysed chromatin. A second fixation is necessary to securely attach the antibodies to the epichromatin epitopes of the intact nucleus.

Ultrastructural studies conducted in situ using conventional transmission electron microscopy have had relatively little impact on defining the structural organization of chromatin. This is due to the fact that in routine transmission electron microscopy, together with the deoxyribonucleoprotein, many different intermingled substances are contrasted, masking the ultrastructure of chromatin. By selective staining of DNA in thin sections, using the Feulgen-like osmium-ammine reaction, these drawbacks have been overcome and worthwhile data have been obtained both on the gross morphology and the ultrastructural-functional organization of chromatin in situ. In the present study these results are reviewed and discussed in light of recent achievements in both interphase nuclear chromatin compartmentalization in interphase nuclei and in the structural organization of chromatin fibers in transcriptionally active and inactive chromatin.

Retinoic acid (RA) treatment of HL-60 cells in vitro induces granulocytic differentiation, involving reorganization of the nucleus and cytoplasm, development of chemoattractant-directed migration, and eventual apoptosis. The present studies with HL-60/S4 cells document that major elements of the cytoskeleton are changed: actin increases by 50%; vimentin decreases by more than 95%. The cellular content of α-tubulin does not significantly change; but the centrosomal–microtubule (MT) array moves away from the lobulating nucleus. Cytoskeletal-modifying chemicals modulate this polarized reorganization: Taxol and cytochalasin D enhance centrosome movement; nocodazole reverses it. Cytoskeletal-modifying chemicals do not appear to affect nuclear lobulation or the integrity of envelope-limited chromatin sheets (ELCS). Employing bcl-2-overexpressing HL-60 cells permitted demonstration of nuclear lobulation, ELCS formation, and centrosome–MT movement concomitantly during RA-induced differentiation, implying independence between the cellular reorganization and apoptotic programs. RA appears to promote an inherent potential in HL-60 cells for cytoskeletal polarization, likely to be important for chemoattractant-directed cell migration, an established characteristic of mature granulocytes.

Molecular features of imprinted genes include differences in expression, methylation, and the timing of DNA replication between parental alleles. Whereas methylation differences always seem to be associated with differences in expression, differences in the timing of replication between parental homologs are not always seen at imprinted loci. These observations raise the possibility that differences in replication timing may not be an essential feature underlying genomic imprinting. In this study, we examined the timing of replication of the two alleles of the imprinted RSVIgmyc transgene in individual embryonic cells using fluorescence in situ hybridization (FISH). The cis-acting signals for RSVIgmyc imprinting are within RSVIgmyc itself. Thus, allele-specific differences in replication, if they indeed govern RSVIgmyc imprinting, should be found in RSVIgmyc sequences. We found that the parental alleles of RSVIgmyc, which exhibit differences in methylation, replicated at the same time. Synchronous replication was also seen in embryonic cells containing a modified version of RSVIgmyc that exhibited parental allele differences in both methylation and expression. These findings indicate that maintenance of expression and methylation differences between alleles does not require a difference in replication timing. The differences in replication timing of endogenous imprinted alleles detected by FISH might therefore reflect structural differences between the two alleles that could be a consequence of imprinting or, alternatively, could be unrelated to imprinting.

Neutrophils are characterized by their distinct nuclear shape, which is thought to facilitate the transit of these cells through pore spaces less than one-fifth of their diameter. We used human promyelocytic leukemia (HL-60) cells as a model system to investigate the effect of nuclear shape in whole cell deformability. We probed neutrophil-differentiated HL-60 cells lacking expression of lamin B receptor, which fail to develop lobulated nuclei during granulopoiesis and present an in vitro model for Pelger-Huet anomaly; despite the circular morphology of their nuclei, the cells passed through micron-scale constrictions on similar timescales as scrambled controls. We then investigated the unique nuclear envelope composition of neutrophil-differentiated HL-60 cells, which may also impact their deformability; although lamin A is typically down-regulated during granulopoiesis, we genetically modified HL-60 cells to generate a subpopulation of cells with well defined levels of ectopic lamin A. The lamin A-overexpressing neutrophil-type cells showed similar functional characteristics as the mock controls, but they had an impaired ability to pass through micron-scale constrictions. Our results suggest that levels of lamin A have a marked effect on the ability of neutrophils to passage through micron-scale constrictions, whereas the unusual multilobed shape of the neutrophil nucleus is less essential.

Ultra-thin sections of Chironomus salivary glands were stained in a non-Feulgen procedure with osmium ammine-B and imaged at several electron energy-loss windows. For two types of RNP-containing structures (ie Balbiani ring granules and endoplasmic reticulum), a significant spatial correlation was observed between stain distribution and net phosphorus distribution. Non-Feulgen osmium ammine-B staining does not require the use of ultra-thin sections and can approximate the distribution of nucleic acid phosphorus.

Mature Balbiani Ring (BR) granules in situ were stained with the nucleic acid specific stain, osmium ammine-B, recorded by electron spectroscopic imaging and reconstructed by electron microscope tomography to examine the three-dimensional (3-D) distribution of BR heterogeneous nuclear RNA (hnRNA). The BR2 granules contain ca. 37 kb of mRNA. Reconstructed BR granules were selected to emphasize one of the prevalent conformations seen in the sectioned salivary glands, the en face or "pin-wheel" conformation. A variety of image processing and volume-rendering operations were applied to the set of reconstructed BR granules. Some of the conclusions of this study are the following: (1) RNA distribution is not uniform throughout the granule; (2) RNA is condensed into about ten particles per granule, which all appear to possess approximately the same RNA stain density; (3) heterogeneity exists in the positions and sizes of particles within the various BR granules. These data argue for the folding of a beaded ribbon, consisting of connected particulate condensations of BR mRNA, possessing considerable 3-D flexibility, even in the packaged state. A comparison of this beaded-ribbon model and a prior folded hnRNP fiber model is also presented.

The telomere binding protein (TP) from the macronucleus of the ciliate Euplotes eurystomus was purified by removal of tenaciously bound DNA with hydroxylapatite, and the purified TP partially sequenced. Rabbit antiserum was generated against a synthetic peptide of 14 amino acids at the amino-terminus of the TP. This antiserum was employed to examine the accessibility of TP antigenic determinants in nuclei and chromatin. Immunofluorescent staining of isolated macronuclei revealed only weak reactivity with specific antiserum. Reactivity within replication bands was demonstrated, and could be augmented by preparation of nuclear scaffolds. Employing a dot immunoblot analysis, the amino-terminal antigenic determinants of TP were revealed after extraction of histone H1 (and some nonhistones). A different aspect of TP inaccessibility was demonstrated by immunoblot analysis of trypsin-treated macronuclei and chromatin; TP was considerably less susceptible to digestion by trypsin than were histones H1 and H3. The relative inaccessibility of TP was not a consequence of chromatin higher-order structure, since soluble macronuclear chromatin in low salt exhibited the same burying of antigenic determinants by dot blot analysis, and the same decreased susceptibility to trypsin, as did isolated nuclei. Electron microscopy of soluble macronuclear chromatin spread in low salt revealed that most telomeres appear unfolded, without stable higher-order structure. The mechanisms for the relative inaccessibility of TP are not yet known, but probably arise as a consequence of the strong interactions of TP with the telomere nucleotide sequence and additional interactions of TP with various chromatin proteins, perhaps including histone H1.

The replication band is a large, migrating, macronuclear domain that is the site of DNA synthesis in hypotrichous ciliated protozoa. A number of agents that produce inactivation of this structure and its replicational activity are described here. These agents include heat shock, aphidicolin, cell crowding, various cAMP phosphodiesterase inhibitors and a calmodulin inhibitor. With the exception of aphidicolin, which has a direct inhibitory effect upon DNA polymerases, the mechanisms of inactivation are presently unknown. The inactivating properties of cAMP phosphodiesterase inhibitors suggest that intracellular cAMP levels may influence replication band structure and function. Supplementary key words. Autoradiography, electron microscopy, in situ hybridization. HE sole site of DNA synthesis in the macronuclei (MAC) T of hypotrichous ciliated protozoa is the replication band (RB) 115, 20, 39, 401. It has been most studied in the MAC of the fresh water ciliate Euplotes eurystomus. The single MAC is elongate and C-shaped; i.e. ca. 100-150 pm long by 10-15 pm wide. The single MIC is the "germline" micronucleus, ca. 3 pm diameter, with an independent S phase immediately following cytokinesis. During MAC S phase, two RB appear simulta- neously at the tips of a MAC. Both RB migrate to the midpoint of the elongate MAC as S phase progresses for about 10-12 hr. The two RB fuse at the termination of S, the MAC shortens and becomes pinched in half during cytokinesis. During the entire S phase the RB maintain a unique ultrastructural strati- fication (30, 3 1, 401. The migrating RE is ca. 5 pm in length and composed of two structural zones: a forward zone (FZ) consisting of a mesh of 40-50-nm chromatin fibers; and a pale- staining rear zone (RZ) containing fine ca. 10-nm fibers. In front of and behind the RB, chromatin is organized into condensed granules. Autoradiographic EM studies have demonstrated that the site of DNA synthesis is the junction between FZ and RZ (25) (Stevens, A. R. 1963, Electron microscope autoradiog- raphy of DNA and RNA synthesis in Euplotes eurystomus. J. Cell Biol., 19:67a). Replication bands exhibit distinctive cyto- chemical and immunochemical properties ( 1-3, 30, 33, 34, 361. A clue towards understanding the structural integrity of RB may be derived from the recent observation of non-chromatin 10- nm filaments within Euplotes MAC in condensed chromatin granules (CG) and in the FZ (29). Conceivably these 10-nm filaments might function to organize the chromatin molecules into the various nuclear compartments and functional states. Organization of MAC chromatin is an obvious necessity when it is recalled that each MAC contains ca. lo8 gene-size linear DNA fragments of average size ca. 2 kb (21, 221. Each type of MAC DNA fragment appears to possess a single coding region flanked by non-transcribed regions and terminated by identical telomeric nucleotide sequences. Otherwise, the MAC DNA mol- ecules exist as reasonably normal chromatin molecules with inner histones, histone H1 and nucleosomes (8, 9, 171.

Nuclear envelope-limited chromatin sheets (ELCS) form during excessive interphase nuclear envelope growth in a variety of cells. ELCS appear as extended sheets within the cytoplasm connecting distant nuclear lobes. Cross-section stained images of ELCS, viewed by transmission electron microscopy, resemble a sandwich of apposed nuclear envelopes separated by ∼30 nm, containing a layer of parallel chromatin fibers. In this study, the ultrastructure of ELCS was compared by three different methods: (1) aldehyde fixation/dehydration/plastic embedding/sectioning and staining, (2) high-pressure freezing/freeze substitution into plastic/sectioning and staining, and (3) high-pressure freezing/cryo-sectioning/cryo-electron microscopy. ELCS could be clearly visualized by all three methods and, consequently, must exist in vivo and are not fixation artifacts. The ∼30-nm chromatin fibers could only be observed following aldehyde fixation; none were seen in cryo-sections. Electron microscopic tomography tangential views of aldehyde-fixed ELCS suggested an ordering of the separate chromatin fibers adjacent to the nuclear envelope. Possible mechanisms of this chromatin ordering are discussed.

The principal human blood granulocyte (neutrophil) possesses a lobulated and deformable nucleus, important to facilitate rapid egress from blood vessels as these cells migrate to sites of bacterial or fungal infection. This unusual nuclear shape is a product of elevated levels of an integral membrane protein of the nuclear envelope lamin B receptor (LBR) and of decreased amounts of lamin A/C. In humans, a genetic deficiency of LBR produces Pelger-Huet anomaly, resulting in blood neutrophils that exhibit hypolobulated nuclei with redistributed heterochromatin. Structural changes in nuclear architecture occur during granulopoiesis within bone marrow. The exact mechanisms of this nuclear shape change and of heterochromatin redistribution remain largely unknown. As a tool to facilitate analysis of these mechanisms, a stable LBR knockdown subline of HL-60 cells was established. During in vitro granulopoiesis induced with retinoic acid, the LBR knockdown cells retain an ovoid shaped nucleus with reduced levels of lamin A/C; while, the parent cells develop highly lobulated nuclei. In contrast, macrophage forms induced in LBR knockdown cells by in vitro treatment with phorbol ester were indistinguishable from the parent cells, judged by both nuclear shape and attached cell morphology. The capability of differentiation of LBR knockdown HL-60 cells should facilitate a detailed analysis of the molecular relationship between LBR levels, granulocyte nuclear shape and heterochromatin distribution.

SUMMARY An optimized distribution of tilt angles for tomography of specimens of non-circular cross-section is derived and tested with reconstructions of a phantom model.

The Balbiani Rings (BR) in the polytene chromosomes of Chironomus salivary glands are intense sites of transcription. The nascent RNPs fold during transcription into 40-50-nm granules, containing in the mature transcript approximately 37-kb RNA. Using a new nucleic acid specific stain, osmium ammine B on Lowicryl sections, in combination with electron energy filtered imaging of sections containing BR granules, we demonstrate a RNA-rich particulate substructure (10-nm particle diameter; 10-12 particles per BR granule). Elemental imaging supports that these particles are enriched in phosphorus. The possible relationship of these RNA-rich particles to ribonucleosomes is discussed, as well as models for their arrangement in the mature BR granules.

Lamin B receptor (LBR) is an integral membrane protein of the interphase nuclear envelope (NE). The N-terminal end resides in the nucleoplasm, binding to lamin B and heterochromatin, with the interactions disrupted during mitosis. The C-terminal end resides within the inner nuclear membrane, retreating with the ER away from condensing chromosomes during mitotic NE breakdown. Some of these properties are interpretable in terms of our current structural knowledge of LBR, but many of the structural features remain unknown. LBR apparently has an evolutionary history which brought together at least two ancient conserved structural domains (i.e., Tudor and sterol reductase). This convergence may have occurred with the emergence of the chordates and echinoderms. It is not clear what survival values have maintained LBR structure during evolution. But it seems likely that roles in post-mitotic nuclear reformation, interphase NE growth and compartmentalization of nuclear architecture might have provided some evolutionary advantage to preservation of the LBR gene. REVIEW

Antisera specific to acetylated and unacetylated N-terminal domains of histone H4 were employed to map the spatial distribution of exposed antigenic determinants in the macronucleus of Euplotes eurystomus. Sites of putative transcription-related acetylation appear to reside in the regions between condensed chromatin granules and disappear during chromatin restructuring in the forward zone of replication bands. Coincident with synthesis of newly replicated chromatin, the specific antisera reveal a resumption of exposed sites of H4 acetylation in the rear zone of replication bands.

The major blood granulocyte (neutrophil) is rapidly recruited to sites of bacterial and fungal infections. It is a highly malleable cell, allowing it to squeeze out of blood vessels and migrate through tight tissue spaces. The human granulocyte nucleus is lobulated and exhibits a paucity of nuclear lamins, increasing its capability for deformation. The present study examined the existence of protein connections between the nuclear envelope and cytoskeletal elements (the LINC complex) in differentiated cell states (i.e. granulocytic, monocytic and macrophage) of the human leukemic cell line HL-60, as well as in human blood leukocytes. HL-60 granulocytes exhibited a deficiency of several LINC complex proteins (i.e. nesprin 1 giant, nesprin 2 giant, SUN1, plectin and vimentin); whereas, the macrophage state revealed nesprin 1 giant, plectin and vimentin. Both states possessed SUN2 in the nuclear envelope. Parallel differences were observed with some of the LINC complex proteins in isolated human blood leukocytes, including macrophage cells derived from blood monocytes. The present study documenting the paucity of LINC complex proteins in granulocytic forms, in combination with previous data on granulocyte nuclear shape and nuclear envelope composition, suggest the hypothesis that these adaptations evolved to facilitate granulocyte cellular malleability.

Objective. The capacity of neutrophils to eradicate bacterial infections is dependent on normal development and activation of functional responses, which include chemotaxis and generation of oxygen radicals during the respiratory burst. A unique feature of the neutrophil is its highly lobulated nucleus, which is thought to facilitate chemotaxis, but may also play a role in other critical neutrophil functions. Nuclear lobulation is dependent on expression of the inner nuclear envelope protein, the lamin B receptor (LBR), mutations of which cause hypolobulated neutrophil nuclei in human Pelger-Huet anomaly and the ''ichthyosis'' (ic) phe- notype in mice. In this study, we have investigated roles for LBR in mediating neutrophil development and activation of multiple neutrophil functions, including chemotaxis and the respiratory burst. Materials and Methods. A progenitor EML cell line was generated from an ic/ic mouse, and derived cells that lacked LBR expression were induced to mature neutrophils and then exam- ined for abnormal morphology and functional responses. Results. Neutrophils derived from EML-ic/ic cells exhibited nuclear hypolobulation identical to that observed in ichthyosis mice. The ic/ic neutrophils also displayed abnormal chemotaxis, supporting the notion that nuclear segmentation augments neutrophil extravasation. Further- more, promyelocytic forms of ic/ic cells displayed decreased proliferative responses and pro- duced a deficient respiratory burst upon terminal maturation. Conclusions. Our studies of promyelocytes that lack LBR expression have identified roles for LBR in regulating not only the morphologic maturation of the neutrophil nucleus, but also proliferative and functional responses that are critical to innate immunity. 2008 ISEH

The human blood granulocyte (neutrophil) is adapted to find and destroy infectious agents. The nucleus of the human neutrophil has a segmented appearance, consisting of a linear or branched array of three or four lobes. Adequate levels of lamin B receptor (LBR) are necessary for differentiation of the lobulated nucleus. The levels of other components of the nuclear envelope may also be important for nuclear shape determination. In the present study, immunostaining and immunoblotting procedures explored the levels of various components of the nuclear envelope and heterochromatin, comparing freshly isolated human neutrophils with granulocytic forms of HL-60 cells, a tissue culture model system. In comparison to granulocytic HL-60 cells, blood neutrophil nuclear envelopes contain low-to-negligible amounts of LBR, lamins A/C, B1 and B2, LAP2beta and emerin. Surprisingly, a "mitotic" chromosome marker, H3(S10)phos, is elevated in neutrophil nuclei, compared to granulocytic HL-60 cells. Furthermore, neutrophil nuclei appear to be more fragile to methanol fixation, than observed with granulocytic HL-60 cells. Thus, the human neutrophil nucleus appears to be highly specialized, possessing a paucity of nuclear envelope-stabilizing proteins. In consequence, the neutrophil nucleus appears to be very malleable, supporting rapid migration through tight tissue spaces.

Distinct in situ 10 nm non-chromatin fibers exist within the macronucleus of the ciliated protozoanEuplotes eurystomus. Their presence is detected after permeabilizing cells in a cytoskeleton-stabilizing buffer and then fixation with glutaraldehyde-tannic acid, followed by OsO4. The 10 nm fibers are primarily localized within condensed chromatin and within the forward zone of the replication band. Although their functional role is unclear, it is suggested that they may constitute a structural framework for organization of the very large number (ca. 108) of macronuclear minichromosomes.

The nature of the wild-type gene product at the mouse ichthyosis (ic) locus has been of great interest because mutations at this locus cause marked abnormalities in nuclear heterochromatin, similar to those observed in Pelger-Huet anomaly (PHA). We recently found that human PHA is caused by mutations in the gene (LBR) encoding lamin B receptor, an evolutionarily conserved inner nuclear membrane protein involved in nuclear assembly and chromatin binding. Mice homozygous for deleterious alleles at the ichthyosis (ic) locus present with a blood phenotype similar to PHA, and develop other phenotypic abnormalities, including alopecia, variable expression of syndactyly and hydrocephalus. The ic locus on mouse chromosome 1 shares conserved synteny with the chromosomal location of the human LBR locus on human chromosome 1. In this study, we identified one nonsense (815ins) and two frameshift mutations (1088insCC and 1884insGGAA) within the Lbr gene of mice homozygous for either of three independent mutations (ic, ic J and ic 4J , respectively) at the ichthyosis locus. These allelic mutations are predicted to result in truncated or severely impaired LBR protein. Our studies of mice homozygous for the ic J mutation revealed a complete loss of LBR protein as shown by immunofluorescence microscopy and immunoblotting. The findings provide the molecular basis for the heterochromatin clumping and other distinct phenotypes caused by ic mutations. These spontaneous Lbr mutations confirm the molecular basis of human PHA and provide a small animal model for determination of the precise function of LBR in normal and pathological states.

Pelger-Huet anomaly (PHA; OMIM *169400) is an autosomal dominant disorder characterized by abnormal nuclear shape and chromatin organization in blood granulocytes. Affected individuals show hypolobulated neutrophil nuclei with coarse chromatin. Presumed homozygous individuals have ovoid neutrophil nuclei, as well as varying degrees of developmental delay, epilepsy and skeletal abnormalities. Homozygous offspring in an extinct rabbit lineage showed severe chondrodystrophy, developmental anomalies and increased pre- and postnatal mortality. Here we show, by carrying out a genome-wide linkage scan, that PHA is linked to chromosome 1q41-43. We identified four splice-site, two frameshift and two nonsense mutations in LBR, encoding the lamin B receptor. The lamin B receptor (LBR), a member of the sterol reductase family, is evolutionarily conserved and integral to the inner nuclear membrane; it targets heterochromatin and lamins to the nuclear membrane. Lymphoblastoid cells from heterozygous individuals affected with PHA show reduced expression of the lamin B receptor, and cells homozygous with respect to PHA contain only trace amounts of it. We found that expression of the lamin B receptor affects neutrophil nuclear shape and chromatin distribution in a dose-dependent manner. Our findings have implications for understanding nuclear envelope-heterochromatin interactions, the pathogenesis of Pelger-like conditions in leukemia, infection and toxic drug reactions, and the evolution of neutrophil nuclear shape.

The human leukemic cell line (HL-60) can be induced to differentiate in vitro to granulocytic form with retinoic acid (RA), or to monocytic/macrophage form with phorbol ester (TPA). The granulocytic form acquires nuclear lobulation, nuclear envelope-limited chromatin sheets (ELCS), and cytoskeletal polarization, none of which are acquired following treatment with TPA. Immunoblotting analyses and capillary zone electrophoresis demonstrated that following RA treatment: lamins A/C and B1, and vimentin decreased to negligible amounts; LAP2β, lamin B2 and emerin remained essentially unchanged; lamin B receptor (LBR) increased markedly; histone subtypes H1.4 and 1.5 exhibited dephosphorylation. Following TPA treatment: lamins A/C and B1, B2 and vimentin increased in amount; LAP2β and emerin remained essentially unchanged; LBR increased markedly; histone subtypes H1.4 and 1.5 exhibited dephosphorylation. Emerin, which was cytoplasmic in undifferentiated or granulocytic cells, localized into the nuclear envelope following TPA. Normal human granulocytes revealed compositional differences compared to granulocytic forms of HL-60, namely increased vimentin and appearance of histone subtype H1.3. A working hypothesis for nuclear lobulation postulates a combination of: increased nuclear envelope deformability due to lamins A/C and B1 deficiency; an increase in nuclear surface area/volume; an increase in chromatin–nuclear envelope interactions.

The tightly centromere-linked gene SPO15 is essential for meiotic cell division in the yeast Saccharomyces cerevisiae. Diploid cells without the intact SPO15 gene product are able to complete premeiotic DNA synthesis and genetic recombination, but are unable to traverse the division cycles. Electron microscopy of blocked cells reveals a duplicated but unseparated spindle-pole body. Thus cells are unable to form a bipolar spindle. Sequence analysis of SPO15 DNA reveals an open reading frame that predicts a protein of 704 amino acids. This protein is identical to VPS1, a gene involved in vacuolar protein sorting in yeast which has significant sequence homology (45% overall, 66% over 300 amino acids) to the microtubule bundling-protein, dynamin. The SPO15 gene product expressed in Escherichia coli can be affinity-purified with microtubules. SPO15 encodes a protein that is likely to be involved in a microtubule-dependent process required for the timely separation of spindle-pole bodies in meiosis.

Exposure of the human leukemic cell line (HL-60) to 1 microM retinoic acid (RA) induces in vitro granulopoiesis, including the development of lobulated nuclei. Ultrastructural studies, presented here, demonstrate the formation of extensive quantities of nuclear envelope-limited chromatin sheets (ELCS), in addition to nuclear lobulation, following treatment with RA. ELCS contain DNA, as shown by the Feulgen-like electron microscope stain osmium ammine-B. Lamin B was demonstrated in ELCS by immunoelectron microscopy with colloidal gold-labeled antibody. Formation of ELCS occurred in Bcl2-overexpressing HL-60 cell sublines with suppressed apoptotic cell death, indicating separable mechanisms for ELCS formation and apoptosis. Immunofluorescent and immunoblotting procedures demonstrated modulations in the amounts and distribution of nuclear envelope-associated components. Total amounts of lamins A/C and cytoplasmic vimentin were reduced by RA treatment. The amounts of lamin B, lamin B receptor (LBR), and lamina-associated polypeptide 2 (LAP2) did not exhibit significant quantitative changes, but acquired heterogeneous staining patterns on the nuclear envelope. RA induced the appearance of low-molecular-weight LBR-related proteins. This study demonstrated the parallel induction of lobulated nuclei and of ELCS and the modulation of nuclear envelope components following exposure of HL-60 to retinoic acid.