Transcriptomics based on CAGE method in an anhydrobiosis research

DEVIATIIAROV R, KOZAKOVA A, DANILINA A, CORNETTE R, SHAGIMARDANOVA E, DE HOON M, KIKAWADA T, GUSEV O

INSTITUTE OF FUNDAMENTAL MEDICINE AND BIOLOGY KFU, KAZAN

Purpose: Identification of the key molecular mechanisms and regulatory elements related to desiccation tolerance in an anhydrobiotic insect Polypedilum vanderplanki.

Methods: This research based on CAGE (cap analysis of gene expression) data for isolated tissues of the larvae and Pv11 cell culture obtained from P.vanderplanki. Nervous system, fat body and gut were isolated from wet (control) and dried (case) larvae. Libraries for Pv11 cell line were collected within a time course of desiccation-rehydration experiments.

Results: Trimming and alignment of sequenced reads to the P.vanderplanki’s genome v0.9 resulted in ~40-50% mapping ratio. Further CAGE signal clustering revealed 13727 active promoter regions, and 9573 of these promoters were associated with 8062 annotated genes. Numbers of differentially expressed (DE) genes varied from 773 to 1194 for up-regulated and 600 to 1322 for down-regulated genes. Gene ontology terms enriched within these sets of DE genes represent cryptogenes-related functions, such as “glycerol ether metabolic process”, “heme binding” or “protein-L-isoaspartate (D-aspartate) O-methyltransferase activity”. Moreover, our data allow us to define universal mechanisms common for all tissues and cells. About half of annotated cryptogenes is related to this group and act without specific localization. Basic properties of the active promoter regions were characterized. Most of the desiccation-related promoters have wide CAGE signal and relatively high GC content, however a fraction of highly expressed promoters from this group are AT rich, have sharp type of the signal and enriched TATA-box motif. Among 41 common enriched motifs in the desiccation-specific promoters there are several stress related representatives like “Broad-complex core protein” binding motif related to polythene chromosome puffing process, “DNA damage-inducible protein” or “Helicase-like transcription factor” related motifs.

Discussion: Relatively low mapping ratio is mostly due to genome assembly, however, since promoter to gene association ratio is higher than 50%, we consider our data is reliable. These numbers are also comparable to Drosophila study with 12454 promoters and 8037 genes. Interestingly, activity of cryptogenes in brain and fat body was relatively higher comparing to the rest genes, suggesting that these tissues could be ready for dehydration all the time, without any additional signal. But during the desiccation experiment these genes drastically increase its activity and mostly by using “sharp” TBP-dependent promoters, while other up-regulated genes have “broad” CpG island-like promoters. Furthermore, considering relatively small genome size we expect that this data could be enough for enhancers discovery.

Conclusions: Genome-wide accurate annotation of the active promoters and its TSS positions revealed tissue-specific gene sets required for successful adaptation to complete dehydration and suggested possible signaling pathways for its regulation. Such data will be used in further wet experiments in a dry preservation system establishment.

EFFECT OF THREE WATER-SOLUBLE [C60] FULLERENCE DERIVATIVES ON DIFFERENT TYPES OF HUMAN CELLS.

V.A. SERGEEVA*, E.S. ERSHOVA, L.V. KAMENEVA, N.N VEIKO, S.V. KOSTYUK

FEDERAL STATE BUDGETARY INSTITUTION “RESEARCH CENTER FOR MEDICAL GENETICS”, MOSCOW, RUSSIA

*[e-mail]

Studies have shown that [C60]fullerenes and their derivatives can be used as anti-viral agents, nanovectors for the drug delivery, anticancer medicine or antioxidants. As the effect of such derivatives is still controversially discussed in literature, further investigation is required. In the present work we studied the effect of 3 [C60]fullerene derivatives with different moieties (F-243, F-827 and F-828) on human embryonic lung fibroblasts (HELFs), adipose-derived mesenchymal stem cells (MSCs) and breast cancer cell line MCF-7. All of the investigated derivatives were fluorescent in unfixed cells and aqueous solutions.

Concentrations of the investigated substances can be devided into three intervals. Concentrations 0,01 - 1 nM promote cell proliferation 1,15-1,2-fold (confirmed by increased levels of PCNA and Ki-67, flow cytometry). Within the range of concentrations from 4 to 15 nM (F-243), 100-150 mkM (F-827) and 10 to 19 mkM (F-828) derivatives cause DNA breaks in the nuclei (comet assay, expression of γH2AX). The activity of the transcription factors NF-kB and p53 increases. Concentrations exceeding 50 nM (F-243), 1 mM (F-827) and 25 mkM (F-828) cause necrotic cell death.

Fullerene derivatives affect ROS production. This effect was studied on HELFs incubated with F-828. During the first 24 hours of incubation F-828 localizes on the surface of the cells and drastically reduces the amount of reactive oxygen species (ROS) on the cell surface leading to the activation of NOX4 transcription. Increased level of NOX4 leads to the increment of ROS. In 3 hours after start of the incubation both NOX4 and ROS level deflates (early response). 24 hours after addition F-828 penetrates into the cytoplasm and neutralizes matrix ROS causing secondary peak in NOX4 synthesis and ROS production and thus oxidative stress (late response).

We assessed the affect of these [C60] fullerene derivatives on MSCs. After 2 weeks of exposure to F-827 in concentration 195 µM level of gene expression of Myod, Myog, Myf5 increases. Myogenic differentiation was confirmed by fluorescent microscopy and flow cytometry. F-828 and F-243 don`t affect differentiation of MSC.

As for MCF-7, F-828 and F-827 affect these type of cells in a similar way: NOX4 expression increases, level of ROS elevates leading to oxidative damage of the DNA (H2AX, 8oxodG). 3 days after start of incubation NOX4 decreases as well as ROS production and reparation starts. Expression of ATM and ATR, Rad54, survival-regulating STAT3 and antiapoptotic Bcl2 increases as a response to DNA breaks. Transcription factors NFkB and NRF2 translocate to the nucleus. After 3 days MCF-7 start overcoming the negative effect of F-827 and F-828. Derivative F-243 is more toxic for MCF-7. It causes more DNA breaks than F-828 and F-827, NOX4 expression is increased even 3 days after start of incubation and reparation system remains inactive, BRCA1, BRCA2, ATM and ATR levels drop, STAT3 is decreased.