Interphase epichromatin: last refuge for the 30-nm chromatin fiber?

"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 ~ 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 ~ 30-nm chromatin fibers arranged in a criss-crossed pattern. The chromatin in ELCS is continuous with adjacent interphase epichromatin. It appears that hydrated ~ 30-nm chromatin fibers are quite rare in most cells, possibly confined to interphase epichromatin at the nuclear envelope.

DNA Replication: An in vivo Space-Time Continuum in the Ciliate Replication Band.

The replication band in the macronucleus of ciliated protozoa has fascinated microscopists since the 19th Century. It migrates through the nucleus, corresponding to a region of DNA replication and nascent chromatin assembly. A new study shows that calcium and actin filaments may participate in the formation and migration of the replication band.

Linker histone epitopes are hidden by in situ higher-order chromatin structure.

BACKGROUND: Histone H1 is the most mobile histone in the cell nucleus. Defining the positions of H1 on chromatin in situ, therefore, represents a challenge. Immunoprecipitation of formaldehyde-fixed and sonicated chromatin, followed by DNA sequencing (xChIP-seq), is traditionally the method for mapping histones onto DNA elements. But since sonication fragmentation precedes ChIP, there is a consequent loss of information about chromatin higher-order structure. Here, we present a new method, xxChIP-seq, employing antibody binding to fixed intact in situ chromatin, followed by extensive washing, a second fixation, sonication and immunoprecipitation. The second fixation is intended to prevent the loss of specifically bound antibody during washing and subsequent sonication and to prevent antibody shifting to epitopes revealed by the sonication process. In many respects, xxChIP-seq is comparable to immunostaining microscopy, which also involves interaction of the primary antibody with fixed and permeabilized intact cells. The only epitopes displayed after immunostaining are the "exposed" epitopes, not "hidden" by the fixation of chromatin higher-order structure. Comparison of immunoprecipitated fragments between xChIP-seq versus xxChIP-seq should indicate which epitopes become inaccessible with fixation and identify their associated DNA elements. RESULTS: We determined the genomic distribution of histone variants H1.2 and H1.5 in human myeloid leukemia cells HL-60/S4 and compared their epitope exposure by both xChIP-seq and xxChIP-seq, as well as high-resolution microscopy, illustrating the influences of preserved chromatin higher-order structure in situ. We found that xChIP and xxChIP H1 signals are in general negatively correlated, with differences being more pronounced near active regulatory regions. Among the intriguing observations, we find that transcription-related regions and histone PTMs (i.e., enhancers, promoters, CpG islands, H3K4me1, H3K4me3, H3K9ac, H3K27ac and H3K36me3) exhibit significant deficiencies (depletions) in H1.2 and H1.5 xxChIP-seq reads, compared to xChIP-seq. These observations suggest the existence of in situ transcription-related chromatin higher-order structures stabilized by formaldehyde. CONCLUSION: Comparison of H1 xxChIP-seq to H1 xChIP-seq allows the development of hypotheses on the chromosomal localization of (stabilized) higher-order structure, indicated by the generation of "hidden" H1 epitopes following formaldehyde crosslinking. Changes in H1 epitope exposure surrounding averaged chromosomal binding sites or epigenetic modifications can also indicate whether these sites have chromatin higher-order structure. For example, comparison between averaged active or inactive promoter regions suggests that both regions can acquire stabilized higher-order structure with hidden H1 epitopes. However, the H1 xChIP-seq comparison cannot define their differences. Application of the xxChIP-seq versus H1 xChIP-seq method is particularly relevant to chromatin-associated proteins, such as linker histones, that play dynamic roles in establishing chromatin higher-order structure.

Hyperosmotic stress: in situ chromatin phase separation.

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.

Hi-C detects novel structural variants in HL-60 and HL-60/S4 cell lines.

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, two cell lines derived from the same acute myeloid leukemia sample. However, the stability and variability of inter- and intra-chromosomal structural variants between different sources of the same cell line 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 published karyotypes identified novel SVs in both cell lines. Hi-C was used to characterize the known expansion centered on the MYC locus. The MYC expansion was integrated into known locations in HL-60/S4, and a novel location (chr4) in HL-60. The HL-60 cell line has more within-line structural variation than the HL-60/S4 derivative cell line. Collectively we demonstrate the usefulness of Hi-C and with RNA-seq data for the identification and characterization of SVs.

Mesoscale Modeling of Nucleosome-Binding Antibody PL2-6: Mono- versus Bivalent Chromatin Complexes.

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.

TNF-α Differentially Regulates Cell Cycle Genes in Promyelocytic and Granulocytic HL-60/S4 Cells.

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, we found evidence that TNF-α treatment of granulocytes shifts the transcriptome toward 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.

Atomic resolution cryo-EM structure of a native-like CENP-A nucleosome aided by an antibody fragment.

Genomic DNA in eukaryotes is organized into chromatin through association with core histones to form nucleosomes, each distinguished by their DNA sequences and histone variants. Here, we used a single-chain antibody fragment (scFv) derived from the anti-nucleosome antibody mAb PL2-6 to stabilize human CENP-A nucleosome containing a native α-satellite DNA and solved its structure by the cryo-electron microscopy (cryo-EM) to 2.6 Å resolution. In comparison, the corresponding cryo-EM structure of the free CENP-A nucleosome could only reach 3.4 Å resolution. We find that scFv binds to a conserved acidic patch on the histone H2A-H2B dimer without perturbing the nucleosome structure. Our results provide an atomic resolution cryo-EM structure of a nucleosome and insight into the structure and function of the CENP-A nucleosome. The scFv approach is applicable to the structural determination of other native-like nucleosomes with distinct DNA sequences.

Migration through a small pore disrupts inactive chromatin organization in neutrophil-like cells.

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.

Jörg Langowski: his scientific legacy and the future it promises.

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 and chromomeres: a 'fuzzy' perspective.

'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.

Defining the epichromatin epitope.

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.

Nucleosome repositioning during differentiation of a human myeloid leukemia cell line.

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.

Transcriptomes reflect the phenotypes of undifferentiated, granulocyte and macrophage forms of HL-60/S4 cells.

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.

Retrotransposon Alu is enriched in the epichromatin of HL-60 cells.

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.

ELCS in ice: cryo-electron microscopy of nuclear envelope-limited chromatin sheets.

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.

Nuclear envelope composition determines the ability of neutrophil-type cells to passage through micron-scale constrictions.

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-Huët 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.

Phosphatidylserine colocalizes with epichromatin in interphase nuclei and mitotic chromosomes.

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.

An epichromatin epitope: persistence in the cell cycle and conservation in evolution.

Interphase nuclear architecture is disrupted and rapidly reformed with each cell division cycle. Successive cell generations exhibit a "memory" of this nuclear architecture, as well as for gene expression. Furthermore, many features of nuclear and mitotic chromosome structure are recognizably species and tissue specific. We wish to know what properties of the underlying chromatin structure may determine these conserved features of nuclear architecture. Employing a particular mouse autoimmune anti-nucleosome monoclonal antibody (PL2-6), combined with deconvolution immunofluorescence microscopy, we present evidence for a unique epitope (involving a ternary complex of histones H2A and H2B and DNA) which is localized only at the exterior chromatin surface of interphase nuclei and mitotic chromosomes in mammalian, invertebrate and plant systems. As only the surface chromatin region is identified with antibody PL2-6, we have assigned it the name "epichromatin". We describe an "epichromatin hypothesis", suggesting that epichromatin may have a unique evolutionary conserved conformation which facilitates interaction with the reforming post-mitotic nuclear envelope and a rapid return of interphase nuclear architecture.

An in vitro model for Pelger-Huët anomaly: stable knockdown of lamin B receptor in HL-60 cells.

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-Huët 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.