RESEARCH PRODUCTS. RESEARCH PRODUCTS OF THIS PROJECT INCLUDE STUDIES OF GENES THAT MAY BE EPIGENETICALLY SUSCEPTIBLE TO SPACE IONIZING RADIATION (IR) AND MICROGRAVITY AND HOW THESE CHANGES AFFECT ENDOCRINE REGULATION OF GENES INVOLVED IN DNA DAMAGE RESPONSE (DDR) AND CELL CYCLE CONTROL. THE TERM EPIGENETICS REFERS TO CHANGES IN GENE EXPRESSION IN THE ABSENCE OF DNA SEQUENCE MODIFICATIONS AND INCLUDES CPG METHYLATION HISTONE MODIFICATIONS (E.G. HISTONE ACETYLATION METHYLATION) AND EXPRESSION OF NON-CODING RNAS (E.G. MICRORNAS). TYPE OF INVESTIGATION. THIS IS A GROUND-BASED NEW SPACE BIOLOGY (NSB) PROPOSAL THAT INVESTIGATES ONE OF THE MAJOR RECOMMENDATIONS OF THE 2011 DECADAL SURVEY RELATED TO THE INFLUENCE OF THE SPACE ENVIRONMENT ON ANIMAL CELL AND MOLECULAR BIOLOGY. THIS WORK IS RESPONSIVE TO THE SPACE CELLULAR AND MOLECULAR BIOLOGY 2018 NASA OMNIBUS RESEARCH ANNOUNCEMENT SOLICITING RESEARCH ANSWERS AS TO HOW THE SPACE ENVIRONMENT CAUSES EPIGENETIC CHANGES. KEY ELEMENTS OF THE SPACE ENVIRONMENT INCLUDE IR AND GRAVITY (I.E. MICROGRAVITY HYPERGRAVITY) WHICH HAVE BEEN SUGGESTED TO ALTER A VARIETY OF BIOLOGICAL PROCESSES INCLUDING ENDOCRINE SYSTEMS (SUB-TOPIC AB1-B). THE CENTRAL HYPOTHESIS OF THIS PROPOSAL IS THAT IR AND MICROGRAVITY ALTER EPIGENETICALLY ENDOCRINE REGULATION OF DDR AND CELL CYCLE CONTROL IN MAMMARY EPITHELIAL CELLS. THE RATIONALE FOR THIS HYPOTHESIS STEMS FROM OUR OWN AND PUBLISHED EVIDENCE THAT 1) THE ESTROGEN RECEPTOR-ALPHA (ERALPHA) ACTIVATES EPIGENETICALLY THE BRCA1 GENE WHICH PARTICIPATES WITH OTHER DDR FACTORS (EG. ATAXIA TELANGIECTASIA MUTATED ATM ATAXIA TELANGIECTASIA AND RAD-3 RELATED ATR) IN REGULATION OF DNA REPAIR DURING THE S AND G2 PHASES OF THE CELL CYCLE AND 2) CONVERSELY ACTIVATION OF THE ARYL HYDROCARBON RECEPTOR (AHR) PREVENTS ERALPHA-DEPENDENT ACTIVATION OF BRCA1. BECAUSE IR AND MICROGRAVITY REGULATE AHR ACTIVITY WE WILL TEST IF IR MICROGRAVITY AND THEIR COMBINATION ALTER EPIGENETICALLY THE DDR AND CELL CYCLE REGULATION. SPECIFIC AIMS. SPECIFIC AIMS OF THIS NSB PROJECT ARE TO INVESTIGATE IN HUMAN MAMMARY EPITHELIAL CELLS UNDER ENDOCRINE STIMULATION THE EPIGENETIC EFFECTS OF IR (AIM 1) AND MICROGRAVITY ALONE OR IN COMBINATION WITH IR (AIM 2) ON EXPRESSION OF GENES INVOLVED IN DDR AND CELL CYCLE REGULATION. OUTLINE OF THE PLAN TO ACCOMPLISH THE SPECIFIC AIMS. AIM 1. HUMAN MAMMARY EPITHELIAL CELLS (HMEC MCF10A) WILL BE CULTURED IN 2D TISSUE CULTURE DISHES IN THE PRESENCE OF ESTROGEN TO SIMULATE THE PHYSIOLOGICAL EXPOSURE UNDER NORMAL GRAVITY OF MAMMARY EPITHELIAL CELLS TO OVARIAN ESTROGENS. CELLS WILL BE EXPOSED FOR VARIOUS LENGTHS OF TIME (I.E. SHORT- 1 2 AND 3 DAYS MEDIUM- 6 12 18 DAYS AND LONG-TERM 24 36 AND 48 DAYS) TO 3 DOSES (0.5 1.0 AND 2.0 MSV/DAY) OF IR. AIM 2. MAMMARY EPITHELIAL CELLS (HMEC MCF10A) WILL BE CULTURED IN 2D OR TO SIMULATE MICROGRAVITY IN ROTATING WALL VESSEL (RWV) BIOREACTOR 3D CULTURES IN THE PRESENCE OF ESTROGEN WITHOUT OR WITH IR (0.5 1.0 AND 2.0 MSV/DAY) FOR VARIOUS PERIODS OF TIME (I.E. SHORT- MEDIUM AND LONG-TERM). DETERMINATIONS WILL INCLUDE CHANGES IN: (I) MRNA AND PROTEIN EXPRESSION OF BRCA1 ATM ATR ERALPHA AHR AND AHR NUCLEAR TRANSLOCATOR (ARNT) RESPECTIVELY BY RT-PCR AND WESTERN BLOTTING (II) CPG METHYLATION AT BRCA1 ATM AND ATR GENES BY METHYLATION-SPECIFIC RT-PCR (III) LEVELS OF ACETYLATED HISTONE 3 (ACH3) AND TRIMETHYLATED LYSINE 27 AT HISTONE-3 AT THE BRCA1 ATM AND ATR GENES BY CHROMATIN IMMUNOPRECIPITATION (CHIP) ASSAY (IV) RECRUITMENT OF DNA METHYLTRANSFERASE-1 (DNMT1) AND DNMT3A LYSINE-SPECIFIC DEMETHYLASE-1 (LSD1) HISTONE DEACETYLASE-1 (HDAC1) AND PCG ENHANCER OF ZESTE HOMOLOGUE 2 (EZH2) AT THE BRCA1 ATM AND ATR GENES BY CHIP ASSAY (V) GENOME-WIDE PROFILE OF GENES TARGETED BY THE ERALPHA AND AHR BY CHIPSEQUENCING (VI) DNA DOUBLE STRAND BREAKS BY IMMUNO-DETECTION OF GAMMA-H2AX FOCI AND (VII) CELL CYCLE DISTRIBUTION BY FLOW CYTOMETRY.