STaARS BioScience-8-Gene Control Prime-EPICON (STaARS BioScience-8-Gene Control Prime-EPICON) - 07.26.18

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STaARS BioScience-8-Gene Control Prime-EPICON examines microgravity related changes to gene expression in mammalian immune cells. The high sensitivity of immune cells to gravitational changes makes them ideal models for understanding the role Earth’s gravity plays in normal mammalian cell function. Parallel studies of cells in microgravity and on the ground help identify the primary cellular and molecular mechanisms, including regulation of gene expression, behind effects of altered gravity.
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The following content was provided by Heath Mills, Ph.D., and is maintained in a database by the ISS Program Science Office.
Experiment Details

OpNom:

Principal Investigator(s)
Oliver Ullrich, Ph.D., M.D, University of Zurich, Zurich, Switzerland

Co-Investigator(s)/Collaborator(s)
Information Pending

Developer(s)
Space Technology and Advanced Research Systems, Inc., Houston, TX, United States

Sponsoring Space Agency
National Aeronautics and Space Administration (NASA)

Sponsoring Organization
National Laboratory (NL)

Research Benefits
Information Pending

ISS Expedition Duration
February 2018 - August 2018

Expeditions Assigned
55/56

Previous Missions
Information Pending

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Experiment Description

Research Overview

  • STaARS BioScience-8-Gene Control Prime-EPICON, or Epigenetic Control of Gene Expression in Altered Gravity, tests whether epigenetic mechanisms control gene expression in response to microgravity.
  • The hypothesis for this investigation states that epigenetic mechanisms are a major factor in the regulation of gene expression by gravitational forces.
  • Mammalian immune cells,used for their sensitivity to gravitational changes, allow for better understanding of how gravity on Earth is required for normal mammalian cell function.
  • Combination of chromatin immunoprecipitation (ChIP) with genome-wide analysis using Next-Generation sequencing (ChIP-Seq) is a very well-suited approach for the genome-wide analysis of transcription factor-binding associated with epigenetic modifications (binding of acetyl-histone H3). The analysis allows for identification of every single gene, which is regulated by epigenetic mechanisms in response to microgravity.
  • Coordinated in-vitro studies with living human cells of the monocyte/macrophage system (MMS) in microgravity with ground-based controls help elucidate the primary cellular and molecular mechanisms behind the effects of altered gravity.
  • This experiment clarifies whether and to what extent gravity is involved in normal cell function, how cell function is impaired by altered gravity, and how cells adapt to the new situation.

Description

STaARS BioScience-8-Gene Control Prime-EPICON investigates the hypothesis that environmental conditions control gene expression in microgravity. The combination of chromatin immunoprecipitation (ChIP) with genome-wide analysis using Next-Generation sequencing (ChIP-Seq) is a powerful approach that may provide insights into gene regulation by identifying global binding sites for proteins of interest and which has been successfully performed in cells of the human immune system. ChIP-Seq enables transcription activity and modified histone occupancy to be mapped across the entire genome. This type of analysis may be achieved by integrating ChIP-Seq data with TranscriptionPath-Seq gene expression data. ChIP may be performed on different types of targets to answer different questions.
 
ChIP-Seq is performed on ribonucleic acid polymerase II (RNA Pol II) in order to discover, identify, and quantitate actively transcribed genes at the deoxyribonucleic acid (DNA) level. This enables the detection of genome-wide changes in gene expression without the limitations encountered when using messenger RNA (mRNA) analysis and without the influence of RNA half-life. In contrast to conventional mRNA array methods, results reflect transcription only and are not affected by mRNA degradation. To capture RNA polymerase on a gene as it is transcribing the gene and measuring the occupancy of the polymerase on the gene is a surrogate for measuring transcription rates.
 
ChIP-Seq is also performed with an antibody directed against a modified histone protein (e.g. acetylated histone H3) to map the histone modification and/or enzymes that regulate the histone modification across the genome. All analysis is performed from the same sample, therefore highest reliability for direct correlation of RNA Pol II binding and chromatin modifications are expected. All experiments are performed in quadruplicates (n=4), with 1 g in-flight controls, 1 g ground controls and 1 g cell culture controls.
 
The investigation hypothesizes that epigenetic mechanisms control gene expression in response to microgravity. Genome-wide detection of transcriptional activity is combined with genome-wide detection of modified DNA-bound histones and genome-wide detection of methylated DNA regions. Due to the planned analysis of the whole genome, microgravity-induced specific gene expression regulation may be identified at the level of every single gene.
 
Cell culture Differentiation of primary human macrophages (on launch site) is the first step to this investigation. This requires human primary M1 macrophages to be differentiated from human peripheral blood monocytic cells isolated from buffy coat from voluntary donors (blood banks) by M1 Macrophage Generation Media DXF (PromoCell), which is a chemically defined and serum-free medium and free of all animal-derived components and substances of human origin with human serum albumin as the only exception. All donors agreed by an informed consent according to Swiss law given to the “Blutspende Zürich”: “ I agree that my blood or certain components of it, as well as my associated medical records can be used anonymously for research purposes and given to third parties.” Precursor monocytes cells are isolated in Zurich, ultra-deep frozen and transported to Kennedy Space Center (KSC). Cells are thawed at KSC and finally differentiated into M1 macrophages. Differentiation of primary human M1 macrophages from human blood cells are performed on ultra-thin polycarbonate slides (to be integrated into the cell culture chamber of the flight hardware in several layers). After differentiation, primary human M1 macrophages are cultured for more than 4 weeks in temperatures between 23°C and 37°C. Handover of Hardware is done as late load L-24 hours to cold stowage. Cold stow at 37°C is required pre-launch and during ascent.
 
On orbit operations consists of Living cell cultures fixed with formaldehyde. The fixation results in protein/protein and protein/ deoxyribonucleic acid DNA crosslinks. At the moment of fixation, the protein/DNA interactions become covalently attached and the preservation of these interactions is what makes chromatin immunoprecipitation ChIP possible. Cell cultures and reagents are accommodated in Experiment Unique Equipment (EUE) in experiment containers (EC). A minimum of 4 ECs are required for each experiment group (microgravity and artificial gravity centrifuge in-flight, and 1g control on ground). The experiment containers are transferred and loaded into the STaARS-1 Experiment Facility (EF) for incubation onboard no later than 8 days after handover. All operations inside the STaARS-1 EF are remotely operated from the ground with no crew involvement. Samples are then incubated for between 3 and 6 days depending on the specific experiment protocol, then chemically fixed. After fixation, the ECs are stored 4°C storage until return. Samples are loaded into cold stow 4°C for descent. For ChIP-sequencing ChIP-Seq analysis, living cells are fixed in polymeric barrier system (PBS) with 1% formaldehyde at room temperature for 10 minutes (addition of fluid A from tank A). Fixation is stopped by the addition of 0.125 M glycine in PBS (addition of fluid B from tank B).
 
After landing STaARS, samples are returned to the principal investigator followed by sterile disassembly of hardware post-flight, recovery of sample slides, removal of fixative, and rinsing with PBS. If this is done at a location other than the science team’s laboratory, the samples are then prepared for sterile transport to home labs.
 
The cells are processed and chromatin is isolated and sonicated into short fragments. An antibody against RNA Pol II and specific histones is added in separate reactions and immunoprecipitations are performed to isolate the factor along with the associated DNA. The crosslinks are then reversed and the DNA is isolated. At this point, there is a population of DNA fragments representative of all of the genomic binding sites of the targeted factor. This DNA may then be analyzed genome-wide by Next-Generation Sequencing.
 
Chromatin immunoprecipitation consists of fixed cells being washed twice in PBS and once in deoxycholate sonication buffer. Lysates are sonicated with a microtip in order to shear the DNA to an average length of 300-500 bp. Lysates are cleared by centrifugation and stored at -80°C. Genomic DNA (input) is prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by phenol/chloroform extraction and ethanol precipitation. Purified DNA is quantified on a NanoDrop spectrophotometer. Extrapolation to the original chromatin volume allows quantitation of the total chromatin yield.
 
For each ChIP reaction, 15-30 ug of chromatin (depending on the antibody) is precleared with protein A agarose beads (Invitrogen). ChIP reactions are set up using precleared chromatin and the antibody and incubated overnight at 4°C. Protein A agarose beads are added and incubation at 4°C is continued for another 3 hours. Immune complexes are washed, eluted from the beads with sodium dodecyl sulphate (SDS) buffer, and subjected to RNase treatment and proteinase K treatment. Crosslinks are reversed by incubation overnight at 65°C, and ChIP DNA is purified by phenol-chloroform extraction and ethanol precipitation.
 
ChIP Sequencing (Illumina) starts with ChIP DNA being prepared for amplification by converting overhangs into phosphorylated blunt ends and the addition of an adenine to the 3’-ends. Illumina genomic adapters are ligated and the sample is size-fractionated (200-300 bp) on an agarose gel. After a final polymerase chain reaction (PCR) amplification step, the resulting DNA libraries are quantified and sequenced on the Illumina HiSeq. Sequences (50 nt reads, single end) are aligned to the corresponding reference genome using the Burrows-Wheeler Aligner (BWA) algorithm. Wig files are generated using Model-based Analysis of ChIP-Seq.

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Applications

Space Applications
The investigation analyzes the whole genome to identify microgravity-induced specific gene expression regulation at the single-gene level. Knowing how and to what extent gravity is involved in and alters normal cell function contributes to better mediation of space-related immunological effects, protecting humans on deep space missions.

Earth Applications
This investigation advances understanding of the function of mammalian immune cells, providing insight into how to address problems associated with altered immune function on Earth.

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Operations

Operational Requirements and Protocols
Precursor monocytes cells are isolated in Zurich, ultra-deep frozen and transported to KSC. Cells are thawed at KSC and finally differentiated into M1 macrophages. Handover of hardware is done as late load L-24 hours to cold stowage. Cold stow at 37°C is required pre-launch and during ascent. The experiment containers are transferred and loaded into the STaARS-1 experiment facility for incubation aboard no later than 8 days after handover. All operations inside the STaARS-1 EF are remotely operated from the ground with no crew involvement. Once experiment is completed the ECs are chemically fixed and stored at 4°C until return. Upon return samples are handed over to principal investigator.

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Decadal Survey Recommendations

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Results/More Information

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Related Websites

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Imagery