Biology subjects

Olins, D. E.

Publications and source records attributed to Olins, D. E..

10 recordsLinked to original sources

bioRxiv2026

Envelope-Limited Chromatin Sheets (ELCS) Formation in The Nuclear Envelope of HL-60/S4 Cells

Envelope-Limited Chromatin Sheets (ELCS) can be induced in human promyelocytic HL-60/S4 cells by treatment with retinoic acid (RA). After 4 days, the differentiated granulocytes exhibit multilobed nuclei with outgrowths of the nuclear envelope (NE) and associated heterochromatin extending into the surrounding cytoplasm (ELCS). These fascinating structures reveal a periodic meshwork of 30 nm chromatin fibers, when viewed by Cryo-electron microscopy. Genetic and biochemical evidence indicates that RA increases the synthesis of Lamin B Receptor (LBR), which is a key enzyme for Cholesterol biosynthesis and is an essential bridge between the NE and peripheral heterochromatin. This article is in part a review of our microscopic data on the structure of ELCS, and in part a description of related transcription changes that result in the formation of ELCS. In addition, this article contains a structural and biochemical comparison of RA-induced granulocytes with phorbol ester (TPA) induced HL-60/S4 macrophages, which lack nuclear lobulation, do not form ELCS, and exhibit a reduction in LBR and Cholesterol biosynthesis. From our perspective, ELCS can be viewed as "fabric" outgrowths of the nuclear envelope, frequently connecting nuclear lobes and capable of sustaining the twisting and squeezing distortions imposed upon nuclear shape, as the granulocytes traverse narrow tissue channels.

cell biology↗

bioRxiv2026

Nuclear Pore Complexes in Various States of HL-60/S4 Cells

This study is focused upon how the structure and function of interphase nuclear pore complexes (NPCs) respond to various cellular stresses (i.e., cell differentiation, knockdown of Lamin B Receptor [LBR], and cellular dehydration) in a myeloid cell line (HL-60/S4). Each cellular stress was examined by GSEA (Gene Set Enrichment Analysis) to determine how the structure and function of the NPCs were affected. Cell differentiation into granulocytes and into macrophages resulted in widespread decreases in nucleoporin (NUP) transcription levels, affecting NPC structure and NPC transport capability. LBR knockdown (HL-60/sh1 cells) in undifferentiated cells exhibited major increases in NUP transcription, combined with improved NPC structural quality and transport capability, implying that nuclear pore function is not adversely affected by the loss of LBR. In contrast, cell dehydration of the undifferentiated HL-60/S4 cells in hyperosmotic culture medium resulted in disorganized nucleoporin transcription, with evidence of abnormal NPC structure and transport capability. The structural integrity and transport function of nuclear pores are clearly responsive to the various cellular stresses. Future investigations should examine the reversibility and resilience of these cells to such stresses as those described in this study.

cell biology↗

bioRxiv2025

Hyperosmotic Stress of HL-60/S4 Cells: Gene Set Enrichment Analysis and Microscopy Changes

Cellular homeostasis requires that internal cell conditions (e.g., pH, ionic and non-ionic solute concentrations and hydration levels) maintain a dynamic stability. To better understand the mechanisms enforcing this homeostasis, we examined the transcript abundance and cellular morphology of undifferentiated myeloid HL-60/S4 cells exposed to 30 or 60 minutes of hyperosmotic stress, resulting from 300 mM sucrose added to the tissue culture medium. We used Gene Set Expression Analyses (GSEA) to examine various cell functions (Phenotypes) of interest. Microscopy was also employed, where possible, to visualize the nuclear and chromatin physical states. We observed that hyperosmotic stress resulted in congelation of interphase and mitotic chromatin. In parallel, GSEA indicated a reduction of heterochromatin, histone lysine methyltransferase and mRNA transcription. Mitosis ceases during hyperosmotic stress, with the intact tubulin spindle remaining attached to clustered centromeres on the congealed chromosomes. GSEA indicated that ribosome biosynthesis in nucleoli and oxidative activity in mitochondria are significantly increased. In addition, the proteasome phenotype is increased, suggesting that protein synthesis and destruction are both occurring at an increased pace during hyperosmotic stress. Nuclear envelope-associated chromatin also appears to be affected by hyperosmotic stress: LBR and Heterochromatin Protein 1 alpha (CBX5) disperse into the cytoplasm. Evidence is also presented that 300 mM sucrose leads to a reduction of DNA methylation and aberrant cellular localization of MeCP2. The effects of acute hyperosmotic stress on HL-60/S4 cells are very diverse and very profound. In many respects, the stress response resembles a frantic attempt for cell survival in the face of inevitable cell death.

cell biology↗

bioRxiv2025

Phenotypic Changes in Undifferentiated and Retinoic Acid Differentiated HL-60/S4 Cells with-or-without LBR Knockdown

Many functions of the nuclear envelope protein LBR (Lamin B Receptor) have been documented. LBR influences the shape of the nuclear envelope via its connections to underlying heterochromatin and to the lamina. LBR also functions as an essential enzyme in sterol biosynthesis. This study attempts to further define the LBR gene functions in a LBR knockdown cell line (HL-60/sh1), by employing Gene Set Enrichment Analysis (GSEA) to explore: Part 1) the effects of LBR knockdown on cell differentiation to granulocytes induced by retinoic acid (RA); Part 2) the effects of LBR knockdown on phenotypic differences between undifferentiated HL-60/sh1 and undifferentiated control cell lines (HL-60/S4 and HL-60/gfp). These GSEA studies are based upon previously published mRNA transcriptome data. In Part 1, the most significant effects were the increased loss of heterochromatin and the decreased histone methyltransferase activity in RA-differentiated HL-60/sh1 granulocytes, compared to HL-60/S4 and HL-60/gfp granulocytes. In addition, HL-60/sh1 ribosome structural protein transcripts were more increased during granulocyte differentiation, than observed in HL-60/S4 and HL-60/gfp. For many predicted phenotypes (e.g., senescence, migration, chemotaxis, phagocytosis and apoptosis), no significant differences were observed among the three granulocytic cell lines. In Part 2, comparisons were mainly between undifferentiated HL-60/sh1 and HL-60/S4 cells. We noted a significant increase in ribosomal protein and ribosomal RNA synthesis in sh1 0 versus to S4 0 cells. Of further interest, we observed that the position and number of nucleoli per cell appeared to differ between these two undifferentiated cell lines. In addition, GSEA results indicated a significant gain in heterochromatin and nucleosome formation in sh1 0 versus S4 0 cells. Microscopic imaging of undifferentiated sh1 0 cells compared to S4 0 cells indicated increased DAPI stained chromatin condensates surrounding the frequently central nucleoli, possibly reflecting the increased heterochromatin and the decreased LBR. Furthermore, the sh1 0 cell nuclei appeared "rounder" than the S4 0 cell nuclei. Evidence is presented supporting that the LINC Complex ("Linker of Nucleoskeleton and Cytoskeleton") may play a role.

cell biology↗

bioRxiv2024

The Effects of Lamin B Receptor knockdown on a Myeloid Leukemia Cell

In an effort to extend our understanding of the genetic functions of the nuclear envelope protein Lamin B Receptor (LBR), we examined the effect of a stable short hairpin (sh1) RNAi knockdown of LBR on the transcriptome and immunostained morphology of the human myeloid leukemia cell line (HL-60/S4). This examination was on sh1 cells induced to granulocytic form with Retinoic Acid (RA) versus sh1 cells maintained undifferentiated (0). By comparison to control cells (i.e., not sh1), we obtained gene lists that were differentially expressed only in the LBR knockdown cell line (i.e., "only-sh1-down" and "only-sh1-up"), in RA versus 0 cells. These curated gene lists were examined by Gene Ontology (GO) analysis. Aside from chromatin related GO terms, the most surprising finding was a significant downregulation of Golgi related genes only in the sh1 cells. Possible relationships between the "Cis-Golgi-Network" and LBR are discussed. Another surprise was a significant upregulation of "Ribosome" protein transcripts only in the sh1 cells. In parallel to these findings, an immunostaining comparison of nucleoli in S4 and sh1 cells demonstrated that the number and location of nucleoli in a single nucleus differs, depending upon the presence of LBR. Speculations on the influence of LBR levels upon the liquid-liquid phase separation model of nucleolar condensation are presented.

cell biology↗

bioRxiv2024

The Multifaceted Phenotype of Senescent HL-60/S4 Macrophages

Every cell has a multifaceted phenotype. Transcriptional analysis of functionally defined groups of genes can provide insight into this phenotypic complexity. In the present study, the mRNA transcriptome of phorbol ester (TPA) differentiated HL-60/S4 macrophage cells was scrutinized using Gene Set Enrichment Analysis (GSEA), which evaluates the strengths of various cellular phenotypes by examining the enrichment of functionally different gene sets. Employing GSEA, we obtained supporting evidence that HL-60/S4 macrophages are senescent, probably a consequence of enriched TGF{beta} and NOTCH signaling transcripts. There appears to be a reduction of transcripts for heterochromatin, nucleosome formation, and chromatin remodeling phenotypes. In addition, despite upregulated oxidative stress gene transcription, we observed a reduction of DNA damage and repair transcripts. GSEA indicated that transcripts for autophagy, extracellular matrix, and inflammation/inflammasomes are enriched. We also observed that the HL-60/S4 macrophage is enriched for apoptosis gene transcripts, which may promote necrotic death by pyroptosis. The long-term goal of this research direction is to see whether this complex multifaceted phenotypic pattern is shared with other types of macrophages and to determine what mechanisms might exist to coordinate these phenotypic facets within a single cell.

cell biology↗

bioRxiv2022

The Transcriptome of Acute Dehydration in Myeloid Leukemia Cells

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 night Rage, rage against the dying of the light" Dylan Thomas (1914-1953)

cell biology↗

bioRxiv2019

Mesoscale Modeling of a Nucleosome-Binding Antibody (PL2-6): Mono- vs. Bivalent Chromatin Complexes

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.\n\nSTATEMENT 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.

biophysics↗

bioRxiv2018

TNF-α differentially regulates cell cycle genes in promyelocytic and granulocytic HL-60/S4 cells

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

genomics↗

bioRxiv2018

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

BackgroundMammalian cells are flexible and can rapidly change shape when they contract, adhere, or migrate. Their 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 constricted space, where the nuclear shape must change in order to fit through the constriction. This occurs many times in the life cycle of a neutrophil, which must protect its chromatin from damage and disruption associated with migration.\n\nResultsTotal RNA-sequencing identified that neutrophil migration through 5 or 14m 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.\n\nHi-C was used to capture the genome organization in control and migrated cells. Minimal 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.\n\nConclusionShort 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 human health and disease.

genomics↗