Decoding nucleosome positions with ATAC-seq data at single-cell level

As the basal bricks, the dynamics and arrangement of nucleosomes orchestrate the higher architecture of chromatin in a fundamental way, thereby affecting almost all nuclear biology processes. Thanks to its rather simple protocol, ATAC-seq has been rapidly adopted as a major tool for chromatin-accessible profiling at both bulk and single-cell level. However, to picture the arrangement of nucleosomes per se remains a challenge with ATAC-seq. In the present work, we introduce a novel ATAC-seq analysis toolkit, named deNOPA, to predict nucleosome positions. Assessments showed that deNOPA not only outperformed state-of-the-art tools, but it is the only tool able to predict nucleosome position precisely with ultrasparse ATAC-seq data. The remarkable performance of deNOPA was fueled by the reads from short fragments, which compose nearly half of sequenced reads and are normally discarded from nucleosome position detection. However, we found that the short fragment reads enrich information on nucleosome positions and that the linker regions were predicted by reads from both short and long fragments using Gaussian smoothing. We applied deNOPA to a single-cell ATAC-seq dataset and deciphered the intrapopulation heterogeneity of the human erythroleukemic cell line (K562). Last, using deNOPA, we showed that the dynamics of nucleosome organization may not directly couple with chromatin accessibility in the cis-regulatory regions when human cells respond to heat shock stimulation. Our deNOPA provides a powerful tool with which to analyze the dynamics of chromatin at nucleosome position level in the single-cell ATAC-seq age.

The influence of fullerenol on the cell number, cell area and colony forming unit ability in irradiated human erythroleukemic cell line

DET (dye exclusion test) cell count and cell area by computer analysis of the images were determined in cell lines of human eritroleukemia (K562), which were irradiated with X-rays in one dose of 24 Gy and pretreated with 10 nmol/mL fullerenol (Cgo(OH)24). Cell samples obtained using a citocentrifuge and May-Gr?nvald Giemsi (MGG) during, were analyzed. The cell colony formation ability was monitored using quantative CFU (colony forming unit) test. Irradiation decreases the number of K562 cells, but fullerenol significantly increases cell number on 24th and 48th hour of the experiment. Cell area is larger, and the number of formed cell colonies after irradiation is significantly smaller compared to pretreated groups during the whole experiment. Pretreatment with fullerenol maintains a smaller cell area, and the number of colony formed units was larger compared to the irradiated cells.

The influence of fullerenol on antioxidative enzyme activity in irradiated human erythroleukemic cell line (K562)

Cell culture K562 samples were treated with fullerenol (C6o(OH)24) at a concentration of 10 nmol/mL and thereafter irradiated with X-rays (24Gy). The activity of gamma-glutamyltransfrease (?-GT), total superoxide-dismutase (SOD) and glutathion-peroxidase (GSH-Px) was determined 1, 24 and 48 hours after irradiation. Irradiation induces an increase in the activity of all the investigated enzymes. Fullerenol in the applied dose decreased the ?-GT activity 24 and 48 h after irradiation. The total SOD activity is increased in both pretreated groups except in the iradiated group at the 48th hour. Treatment with fullerenol before irradiation increased GSH-Px activity in irradiated groups and decreased it in the control groups.

MicroRNAs 221 and 222 Inhibit Normal Erythropoiesis and Erythroleukemic Cell Growth Via Kit Receptor Downmodulation.

MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression primarily through translational repression. In unilineage erythropoietic (E) culture of cord blood (CB) CD34+ progenitor cells, the level of miR 221 and 222 is gradually and sharply downmodulated. Hypothetically, this decline could promote erythropoiesis by unblocking expression of key functional proteins. Our studies indicate that miR 221 and 222 target the Kit receptor: specifically, (a) the luciferase targeting assay showed that miR 221 and 222 directly interact with the 3′UTR of Kit mRNA; (b) in E culture the miR 221 and 222 level is inversely related to Kit protein expression, whereas the abundance of Kit mRNA is relatively stable. Functional studies show that treatment of CD34+ cells with miR 221 and 222, via oligonucleotide transfection or lentiviral vector infection, causes impaired proliferation and accelerated differentiation of E cells, coupled with downmodulation of Kit protein: this phenomenon, observed in E culture releasing endogenous Kit ligand (KL), is magnified in E culture supplemented with KL. Furthermore, transplantation experiments into NOD-SCID mice reveal that miR 221 or 222 treatment of CD34+ cells impairs their engraftment capacity and stem cell activity. Finally, miR 221 and 222 gene transfer impairs proliferation of the TF1 erythroleukemic cell line, expressing the Kit receptor. Altogether, our studies indicate that in human erythropoiesis the decline of miR 221 and 222 unblocks Kit protein production at translational level, thus leading to expansion of early E cells. Furthermore, overexpression of miR 221 and 222 inhibits proliferation of Kit+ erythroleukemic cells, suggesting a potential role of these microRNAs in cancer therapy.

Establishment and Characterization of a New Human Ph+ Erythroleukemic Cell Line, VES.

Continuous human malignant hematopoietic cell lines are invaluable tool for hematological research. Here we report on a new erythroleukemic cell line termed VES that was established from the bone marrow mononuclear cells (BMNC) of a 22-year-old woman with Ph+ CML during her second post-transplant period. Actually, after unsuccessful allo-transplantation, the patient received auto-transplant that also failed to engraft. At the time of initiation of BMNC into culture for the analysis of stromal cell capacity, her bone marrow was tested Ph-negative by both FISH and PCR. Instead of establishment of an adherent stromal layer in vitro, blast-like cells appeared in the supernatant on day 27. These blast cells expressed highly amplified bcr/abl locus as shown by FISH, whereas the patient’s bone marrow was still Ph-negative at the time. Following a very short period, the cultured cells started to express stable features of erythroleukemia cell line. Optimal growth was obtained by suspending the cells at concentration of 0.3x106 cells/ml in IMDM containing 10% FBS, 1% penicillin-streptomycin and 1% L-glutamine solution. After 3 days of culture, cell concentration varied between 1.3 and 1.8x106 cells/ml. VES cells were tested negative for the presence of EBV virus. For analysis of clonogenicity, VES cells at concentration of 103/ml were grown in a serum-free methylcellulose medium without cytokines (H4236; StemCell Tech., Canada). Following 7-day culture, 103 VES cells produced 251 ±42 and after 14 days 431 ±30 GF-independent colonies. Furthermore, on day 14 of the cell culture it was possible to observe reddish color of the colonies indicating the presence of hemoglobin. Cytological examination of cytospin preparations indicated homogenous population of leukemia blasts (>80%) the majority of them (>90%) being glycophorinA positive and MPO negative. Imunophenotyping and multiparameter flow cytometry revealed proerythroblastic lineage-associated profile: GlyA/CD235a+, CD11b+, CD15+, CD29+, CD33+, and CD117+. Conventional cytogenetics revealed complex karyotype with multiple numerical/structural abnormalities (MAKA), while metaphase FISH revealed the following aberrations: t(9;22), 5q31, 7q31, +8, +6. Since FISH analysis detected highly amplified bcr/abl locus, we tested VES cells’ sensitivity to imatinib (Gleevec). Treatment with imatinib for 3 days (MTS assay) inhibited proliferation of VES cells with IC50 value of 0.2mM. Following 14 days of culture in methylcellulose medium with addition of 0.2mM imatinib, the clonogenic potential of VES cells was reduced by 55% in relation to untreated control. We demonstrated that imatinib induced apoptosis in VES cells in time- and dose-dependant manner, assessed with AnnexinV and PI staining, while there were no significant changes observed in the cell cycle except for a mild increase in cells in G0/G1 phase. Although further analyses are required, we believe that VES cells represent a new Ph+ erythroleukemia cell line and a suitable model for studying Ph+ malignant hematopoiesis in vitro.

Ghrelin Is Produced by the Human Erythroleukemic HEL Cell Line and Involved in an Autocrine Pathway Leading to Cell Proliferation

Ghrelin, a ligand of the GH secretagogue receptor (GHS-R 1a), is a 28-amino acid peptide with an unusual octanoyl group on Ser3, crucial for its biological activity. For the first time, ghrelin and GHS-R 1b, a truncated variant of the receptor resulting from alternative splicing, but not GHS-R 1a, mRNAs were detected in the human erythroleukemic cell line HEL. Two antibodies, used for RIA, were directed against octanoylated and total (octanoylated and desoctanoylated) ghrelin, and the recognized epitopes were characterized. Using reverse phase HPLC analysis followed by RIA, we demonstrated that octanoylated and desoctanoylated ghrelins were present in HEL cells and their culture medium, of which more than 90% was octanoylated. The ghrelin levels were not affected after 24 h treatment with sodium butyrate, phorbol 12-myristate 13-acetate, or forskolin, but a significant 3-fold increase in desoctanoylated ghrelin was detected in the culture medium after 48 h treatment with sodium butyrate. The antighrelin SB801 and SB969 antisera inhibited HEL cell proliferation by 24% and 39%, respectively, after 72 h. Taken together, these data suggested that endogenous ghrelin stimulated HEL cell proliferation by an autocrine pathway involving an unidentified receptor, distinct from GHS-R1a, and that the HEL cell line represents a unique model to study the octanoylation of ghrelin.