OBJECTIVE:
Although scientists can determine the function of every gene, limited methods exist for determining how cells interpret instructions that control gene function. The instructions, (epigenetic marks), encompass chemical modifications added "on top of" DNA that do not cause a genetic mutation. Understanding how diverse patterns of epigenetic marks coordinate the regulation of gene expression is an open question because, although many epigenetic landscapes have been mapped in cell populations, limited strategies have been developed to test and/or understand how epigenetic control occurs in single cells. Epigenetic modifications respond dynamically to environmental factors, and can change within minutes. In space, organisms are exposed to powerful environmental forces (microgravity, cosmic radiation, magnetic fields) that potentially modulate the epigenome, leading to changes in gene expression that affect mammalian development. By elucidating the mechanisms that govern epigenetic programming and gene regulation in single cells, we will provide fundamental knowledge that will broadly impact the epigenetics community, basic biology, and NASA's space biology programs.
The goal of our interdisciplinary effort is to develop an integrated approach comprised of Next Generation Sequencing and single cell microscopy platforms to understand how the space environment affects DNA methylation, hydroxymethylation, and histone modifications to regulate gene expression during differentiation using an Embryonic Stem Cell model.
Through our interdisciplinary effort, we will create a framework to explain how DNA methylation (5hmeC, 5meC) and histone modifications regulate gene expression not only in response to space flight conditions, but also to environments encountered on Earth. Answers to this question, which must be tested in single cells, have heretofore remained elusive. Upon completion, our work will transform the basic biological understanding of epigenetics, propel the current space biology research program into the realm of epigenetics and impact the planning of future long-term space flight expeditions at NASA.
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APPROACH:
HL60 and TK cells were grown in the respective conditions and subjected to Next Generation Sequencing (NGS) analysis to determine the methylated regions so that we can enhance our understanding of the gene expression under microgravity conditions.
First comparison was done between the methylated regions of TK-6 cells (control: static) and under stimulated microgravity to identify the differentially methylated regions and the second comparison was done between the hydroxymethylated regions of the same to identify the differentially hydroxymethylated regions. The differentially methylated/hydroxymethylated regions identified by statistical analysis were then annotated for further biological interpretation using custom perl scripts of MeDUSA package, BEDTools and feature annotation files (GFF files from UCSC). The locality of the differentially methylated/hydroxymethylated regions and its feature type was determined using BEDTools, specifically intersectBed and windowBed. Metadata describing each differentially methylated/hydroxymethylated region (e.g., CpG density, nearest gene, genomic region in which the differentially methylated/hydroxymethylated region was found, and the read count within differentially methylated/hydroxymethylated region) and their summarized counts mapping to specified genomic features were also generated. Based on our analysis about 2000 differentially methylated genes and 100 differentially hydroxymethylated genes were identified. The next step of identifying the implicated genes, the regions within, and the pathways are in progress. Upon completion TK6 cells grown under static and under simulated microgravity conditions will be subjected to similar analysis as done for HL-60 differentiation to identify the pathways epigenetically and transcriptionally altered during microgravity conditions demonstrating proof of concept for the preliminary funding.
Upon completion we will embark on single cell approaches as Phase 2 of the study.
RESULTS:
Here, in this study we systematically examined the patterns of DNA methylation and hydroxy-methylation with its functional implications in gene regulation for the cultured TK6 lymphoblastoid cells upon exposure to micro-gravity conditions. The results reported here indicate that simulated microgravity alters methylation patterns in a limited way and subsequently the expression of genes involved in stress response. Further details of the research findings can be found by accessing the listed publication: Chowdhury B, Seetharam A, Wang Z, Liu Y et al. A Study of Alterations in DNA Epigenetic Modifications (5mC and 5hmC) and Gene Expression Influenced by Simulated Microgravity in Human Lymphoblastoid Cells. PLoS One 2016;11(1):e0147514. PMID: 26820575 (http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0147514). All processed data are available within the publication listed above and raw sequencing data is deposited at GEO, accession Number: GSE65944. (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE65944).
Chowdhury B, Seetharam A3, Wang Z, Liu Y, Lossie AC, Thimmapuram J, Irudayaraj J. A study of alterations in DNA epigenetic modifications (5mC and 5hmC) and gene expression influenced by simulated microgravity in human lymphoblastoid cells. PLoS One. 2016 Jan 28;11(1):e0147514.
[DOI]
Chowdhury B, McGovern A, Cui Y, Choudhury SR, Cho IH, Cooper B, Chevassut T, Lossie AC, Irudayaraj J. The hypomethylating agent Decitabine causes a paradoxical increase in 5-hydroxymethylcytosine in human leukemia cells. Sci Rep. 2015 Apr 22;5:9281.
