Source: UNIVERSITY OF NEBRASKA submitted to
EPIGENOMIC SYNERGIES BETWEEN METHYL DONORS AND BIOTIN MAINTAIN GENOME STABILITY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0225394
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Jul 1, 2011
Project End Date
Jun 30, 2016
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF NEBRASKA
(N/A)
LINCOLN,NE 68583
Performing Department
Nutritional & Health Sciences
Non Technical Summary
The vitamins biotin and folate play important roles in the repression of genes. Isolated deficiency of either one of the two vitamins has only moderate effects with regard to abnomrla gene regulation. We hypothesize that biotin and folate have synergistic effects in gene regulation, and that a combined deficiency of biotin and folate leads to aberrant gene regulation that pre-disposes individuals to cancer. Outcomes include the dissemination of our findings at national meetings, publication of peer-reviewed papers, and workforce development in the area of molecular nutrition. Impacts include novel insights into the interaction between diet and cancer and cancer prevention, increased recognition of UNL by peer institutions, and preparation of students and postdocs for future careers in molecular nutrition.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
70260101010100%
Knowledge Area
702 - Requirements and Function of Nutrients and Other Food Components;

Subject Of Investigation
6010 - Individuals;

Field Of Science
1010 - Nutrition and metabolism;
Goals / Objectives
Our long-term goal is to identify diet-dependent epigenomic mechanisms that regulate genes and maintain genome stability. This project has the following specific objectives. OBJECTIVE 1. IDENTIFY AND CHARACTERIZE PHYSICAL INTERACTIONS AMONG THE CHROMATIN PROTEINS HLCS, EHMT-1, DNMT1, AND MeCP2. OBJECTIVE 2. DETERMINE WHETHER FOLATE AND BIOTIN SYNERGIZE IN THE EPIGENOMIC REGULATION OF IMPRINTED GENES AND LTRS, THEREBY MAINTAINING GENOME STABILITY IN HUMAN FIBROBLASTS. OBJECTIVE 3. DETERMINE WHETHER METHYL DONORS AND BIOTIN ACT SYNERGISTICALLY IN THE REPRESSION OF THE AVY LOCUS IN AGOUTI MICE. Outputs: (1) Disseminate finds at least at one national meeting per year. (2) Publish at least 2 peer-reviewed papers per year. (3) Train 5 graduate students and 3 postdocs. (4) Collaborate with other scientists.
Project Methods
Objective 1: Physical interactions among chromatin proteins will be investigated by using yeast-two-hybrid assay, co-immunoprecipitation assays, limited proteolysis assays, and perhaps biomolecular fluorescence complementation assays. Objective 2: Synergistic effects of folate-dependent methylation events and biotinylation of histones will be investigated by using chromatin immunoprecipitation assays/quantitative real-time PCR. Target loci of invetsigation include long-terminal repeats and imprinted genes. Chromosomal abnomrlities will be monitored by Giemsa satining in the cytogenetics core facility at the Medical Center in Omaha. Objective 3: Synergistic effects of folate and biotin will be studied in a whole animal feeding study, using a 3x3 factorial design in the agouti mouse model.

Progress 07/01/11 to 06/30/16

Outputs
Target Audience:1) Fellow scientists 2) Policy makers 3) Legislature 4) University stake holders 5) Students 6) Press Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Since the initiation of this project, six postdoctoral fellows, 17 graduate students, a research technologist, a visiting scientist and 12 undergraduate students have received extensive training in advanced molecular biology techniques and mouse genetics. Those students and postdocs, who have graduated from the program are gainfully employed and/or secured postdoctoral positions at high-profile institutions. How have the results been disseminated to communities of interest?Project discoveries have been disseminated in 53 publications, 36 conference presentations and invited seminars, and four outreach activities (newspaper interviews). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Overall: This is the final report for a 5-year Hatch project. The long-term goal of this project was to identify nutritional factors that prevent human disease, e.g., cancer, obesity, and infertility. In our research, we have succeeded in identifying many such factors as described below, and we have discovered a novel class of bioactive compounds in foods: dietary exosomes and their cargos in bovine milk and other foods. This discovery will be the focus of our new Hatch project (under review). This project had a positive impact on change in knowledge and action, and has a large potential to lead to changes in the following conditions: Change in knowledge: In this reporting period alone, results from this project have been disseminated in 53 publications, 36 conference presentations and invited seminars, and four outreach activities (newspaper interviews). Since the initiation of this project, six postdoctoral fellows, 17 graduate students, a research technologist, a visiting scientist and 12 undergraduate students have received extensive training in advanced molecular biology techniques and mouse genetics. Those students and postdocs, who have graduated from the program are gainfully employed and/or secured postdoctoral positions at high-profile institutions; others are still in the program. In this reporting period alone, this research project was the basis for six theses and dissertations. Change in action: A subset of our studies ("bioavailability of dietary microRNAs") has created an interest in the Environmental Protection Agency to assess the extent by which similar compounds (small interfering RNAs) in genetically modified organisms. We will follow up with a grant proposal to NIH NIEHS. Change in condition: This project suggest that some nutrients should not be considered in isolation when assessing possible health benefits such as optimal fertility, optimal immune function and low cancer risk. These observations have not yet let to changes in the development of recommendations for dietary allowances. Collaborations: Current and possible collaborators include Drs. Jiri Adamec, Audrey Atkin, Juan Cui, Brett White and Jennifer Wood (all at the University of Nebraska-Lincoln), and Mark McCann (New Zealand), Chen Liu (UT Southwestern) and Michael Roberts (Auburn). Goal-specific accomplishments: OBJECTIVE 1. IDENTIFY AND CHARACTERIZE PHYSICAL INTERACTIONS AMONG THE CHROMATIN PROTEINS HLCS, EHMT-1, DNMT1, AND MeCP2. We have demonstrated that the proteins HLCS, DNMT1, MeCP2 and N-CoR synergize in vitamin-dependent (biotin, folate) regulation of genes. In our studies we showed that vitamin deficiency causes a loss of repression of long transposable elements, thereby increasing the risk for DNA damage and cancer. In this objective we also discovered a novel class of bioactive food compounds, i.e., exosomes and their cargos in milk and other foods. We have linked a dietary depletion of exosomes with loss of fecundity, changes in the pro-inflammatory immune response, aberrant metabolism of purines and amino acids, impaired spatial memory and resistance to chemically induced seizures, and changes in the gut microbiome. OBJECTIVE 2. DETERMINE WHETHER FOLATE AND BIOTIN SYNERGIZE IN THE EPIGENOMIC REGULATION OF IMPRINTED GENES AND LTRS, THEREBY MAINTAINING GENOME STABILITY IN HUMAN FIBROBLASTS. This objective is closely related to Objective 1, but has a specific focus on "hot spots" (LTRs) for genome instability and cancer risk in humans. We demonstrated that folate and biotin synergize in the repression of LTRs and maintenance of genome stability in human cell cultures. The same pathways are also critical for the regulation of genes that regulate the human immune system. During the course of our studies, we also discovered that the activity of a transcription factor with anti-cancer activity (MBP-1) is enhanced if MBP-1 is covalently biotinylated (see Objective 3). Results from this research will inform decision makers about human biotin requirements for optimal health. OBJECTIVE 3. DETERMINE WHETHER METHYL DONORS AND BIOTIN ACT SYNERGISTICALLY IN THE REPRESSION OF THE AVY LOCUS IN AGOUTI MICE. This objective took the research conducted in objectives 1 and 2 to the whole animal model. We conducted a mouse feeding study and demonstrated that the tumor load increased in mice fed a biotin-depleted diet compared with biotin-sufficient controls. Our studies in Objectives 2 and 3 implicated the binding of the vitamin biotin to the transcription factor MBP-1 in decreased cancer risk. A manuscript regarding MBP-1 is currently in preparation, and a federal grant proposal was submitted. The reviewers of the proposal suggested we demonstrate that biotin deficiency is common in the U.S. population and re-submit the proposal.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Pannier A, Kelly A, Plautz S, Zempleni J. Glucocorticoid cell priming enhances transfection outcomes in adult human mesenchymal stem cells. Mol Ther 24:331-341
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Jati Kusuma R, Manca S, Friemel T, Sukreet S, Nguyen C, Zempleni J. Human vascular endothelial cells transport foreign exosomes from cows milk by endocytosis. Am J Physiol Cell Physiol 310:C800-C807
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Cordonier EL, Jarecke SK, Hollinger FE, Zempleni J. Inhibition of acetyl-CoA carboxylases by soraphen A prevents lipid accumulation and adipocyte differentiation in 3T3-L1 cells. Eur J Pharmacol 780:202-208
  • Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Camara Teixeira D, Cordonier EL, Wijeratne SSK, Huebbe P, Jamin A, Jarecke S, Wiebe M, Zempleni J. A cell death assay for assessing the mitochondrial targeting of proteins
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Zempleni J, Baier SR, Howard KM, Cui J. Gene regulation by dietary microRNAs. In: Nutrients/natural product (nutraceutical) control of metabolic pathways in relation to the metabolic syndrome (Dakshinamurti, K, Zempleni J, guest editors). Can J Physiol Pharmacol 93:1097-1102
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Zempleni J, Aguilar-Lozano A, Sadri M, Manca S, Wu D, Zhou F, Mutai E, Sukreet S. Extracellular vesicles and their RNA cargos in breast milk likely have biological activities in infants. J Nutr
  • Type: Journal Articles Status: Other Year Published: 2016 Citation: Zempleni J. Milk-based RNAs. Nutr Genes (invited review, in preparation)
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Pinto JT, Zempleni J. Nutrition information brief  riboflavin. Adv Nutr 7:973-975
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Cui J, Zhou B, Ross S, Zempleni J. Nutrition, microRNAs and human health. Minireview based on the symposium Nutrition, microRNAs and human health held 5 April 2016 at the ASN Scientific Sessions and Annual Meeting at Experimental Biology 2016 in San Diego, CA. The symposium was sponsored by the American Society for Nutrition (ASN), and the ASN Nutrition Science Council. Adv Nutr
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Zempleni J. Wolf T, Baier SR. Cross-species transfer of dietary exosomes and microRNA cargos: the intestinal transport of bovine milk exosomes is mediated by endocytosis in human colon cells and rat small intestinal cells. Annual meeting of the American Society for Cell Biology. December 12-16, San Diego, CA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Sadri M, Xie F, Wood J, Zempleni J. Dietary depletion of cows milk microRNAs impairs fecundity in mice. Experimental Biology Meeting; San Diego, CA, April 2-6
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Aguilar-Lozano AG, Baier SR, Adamec J, Sadri M, Giraud D, Zempleni J. Depletion of dietary microRNAs from cows milk causes an increase of purine metabolites in human body fluids and mouse livers. Experimental Biology Meeting; San Diego, CA, April 2-6
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Sukreet S, Zhang H, Adamec J, Cui J, Zempleni J. Identification of glycoproteins on the surface of cows milk exosomes that mediate the uptake of exosomes into human colon carcinoma caco-2 cells. Experimental Biology Meeting; San Diego, CA, April 2-6
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Manca S, Giraud D, Zempleni J. Bioavailability and biodistribution of fluorophore-labeled exosomes from cows milk after intravenous and oral administration in C57Bl/6J mice. Experimental Biology Meeting; San Diego, CA, April 2-6
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Zempleni J. Bioactivity of dietary exosomes and microRNA cargos, presented at the symposium titled Nutrition, microRNAs and human health at Experimental Biology 2016, sponsored by ASN, San Diego, CA, April 5th
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Zempleni J. The delivery of functional RNA species by dietary exosomes. W3002 USDA/NIFA multistate group meeting, University of Illinois-Urbana/Champaign, May 24/25
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Manca S, Zempleni J. Delivery of functional RNA cargos by dietary exosomes from cows milk in mice. Keystone symposium Exosomes/Microvesicles: Novel Mechanisms of Cell-Cell communication, June 19-22, Keystone Resort, Keystone, CO


Progress 10/01/14 to 09/30/15

Outputs
Target Audience:1) Fellow scientists 2) Policy makers 3) Legislature 4) University stake holders 5) Students 6) Press Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Five postdoctoral fellows, 16 graduate students, and nine undergraduate students have been trained over the lifespan of this project. They, and a research technologist, received extensive training in advanced molecular biology techniques and mouse genetics. Those students and postdocs, who have graduated from the program are gainfully employed and/or secured postdoctoral positions at high-profile institutions. How have the results been disseminated to communities of interest?Results have been disseminated through 14 publications, eight conference presentations and invited seminars, and four outreach activities (newspaper interviews) in this reporting period. What do you plan to do during the next reporting period to accomplish the goals?In the last year of this project we will 1) focus on publishing manuscripts remaining for the studies described for Objectives 1 - 3, and 2) continue to expand our new line of research of dietary microRNAs in the context of nutrition and optimal human health.

Impacts
What was accomplished under these goals? The long-term goal of this project is to identify nutritional factors that prevent human disease, e.g., cancer, obesity, and infertility. We are now nearing completion of this 5-year project. In our research, we have succeeded in identifying many such factors, including but not limited to those described below for Objectives 1 -3. This project had a positive impact on change in knowledge and action, and has a large potential to lead to changes in condition: Change in knowledge: In this reporting period alone, results from this project have been disseminated in 14 publications, eight conference presentations and invited seminars, and four outreach activities (newspaper interviews). Since the initiation of this project, five postdoctoral fellows, 16 graduate students, a research technologist, and nine undergraduate students have received extensive training in advanced molecular biology techniques and mouse genetics. Those students and postdocs, who have graduated from the program are gainfully employed and/or secured postdoctoral positions at high-profile institutions; others are still in the program. In this reporting period alone, this research project was the basis for six theses and dissertations. Change in action: A subset of our studies ("bioavailability of dietary microRNAs") has created an interest in the Environmental Protection Agency to assess the extent by which similar compounds (small interfering RNAs) in genetically modified organisms. We will follow up with a grant proposal to the NIFA Biological Risk Assessment Group. Change in condition: This project suggest that some nutrients should not be considered in isolation when assessing possible health benefits such as optimal fertility, optimal immune function and low cancer risk. These observations have not yet let to changes in the development of recommendations for dietary allowances. Specifics of individual objectives are discussed below: OBJECTIVE 1. IDENTIFY AND CHARACTERIZE PHYSICAL INTERACTIONS AMONG THE CHROMATIN PROTEINS HLCS, EHMT-1, DNMT1, AND MeCP2. We have expanded the scientific knowledge base and created an opportunity for change in condition as follows. We used molecular biology techniques (yeast-two-hybrid assays, co-immunoprecipitation, limited proteolysis assays, and split luciferase assays) to demonstrate that the proteins HLCS, EHMT-1, DNMT1, and MeCP2 interact physically in cell cultures and in vitro. These proteins depend on optimal supply with the vitamins biotin or folate for optimal function. By pairing the above tools with gene expression analysis, we demonstrated that the interaction among these four proteins is essential for the regulation of human genes, low cancer risk, and optimal immune function. These studies were carried out by postdoctoral fellows (Y. Li, M. Singh) doctoral students (D. Liu,, J. Xue) and the program director. Results from this research resulted in two publications in this reporting period (to close out Objective 1) and will inform decision makers about human biotin requirements for optimal health. We have expanded this line of research through our paradigm-shifting discovery that humans absorb genetic materials (microRNAs, which are somewhat similar to DNA) from foods and that these dietary microRNAs regulate human genes for optimal fecundity, optimal bone mineralization, normal metabolism of DNA building blocks (purines), and normal lipid metabolism. In this research we conducted human and animal feeding studies, cell culture experiments, analysis of milk samples, and microRNA depletion studies, followed by sample analysis using qRT-PCR, reporter gene assays, western blots, mass spectrometry, and RNA sequencing to demonstrate that dietary microRNAs are a novel class of bioactive compounds in foods. This ground-breaking discovery will be the future focus of our laboratory. This research has already resulted in three peer-reviewed articles, one paper (under revision), two letters to the editor, seven conference presentations, one invited seminar, and four newspaper interviews. OBJECTIVE 2. DETERMINE WHETHER FOLATE AND BIOTIN SYNERGIZE IN THE EPIGENOMIC REGULATION OF IMPRINTED GENES AND LTRS, THEREBY MAINTAINING GENOME STABILITY IN HUMAN FIBROBLASTS. This objective is closely related to Objective 1, but has a specific focus on "hot spots" (LTRs) for genome instability and cancer risk in humans. Using qRT-PCR, reporter gene assays, mass spectrometry, and western blots we demonstrated that folate and biotin synergize in the repression of LTRs and maintenance of genome stability in human cell cultures. The same pathways are also critical for the regulation of genes that regulate the human immune system. This research was carried out by a postdoctoral fellow (Y. Li), a master's student (J. Xie), doctoral students (Y. Ching Chew, D. Liu, J. Xue) and the program director. Throughout the course of this study, we also discovered that the activity of a transcription factor with anti-cancer activity (MBP-1) is enhanced if MBP-1 is covalently biotinylated (see Objective 3). Results from this research will inform decision makers about human biotin requirements for optimal health. OBJECTIVE 3. DETERMINE WHETHER METHYL DONORS AND BIOTIN ACT SYNERGISTICALLY IN THE REPRESSION OF THE AVY LOCUS IN AGOUTI MICE. This objective takes the research conducted in objectives 1 and 2 to the whole animal model. We conducted a mouse feeding study and demonstrated that the tumor load increased in mice fed a biotin-depleted diet compared with biotin-sufficient controls. Laboratory techniques included qRT-PCR, western blots, reporter genes, and mass spectrometry; tumor development was assessed by visual inspection. Our studies in Objectives 2 and 3 implicated the binding of the vitamin biotin to the transcription factor MBP-1 in decreased cancer risk. A manuscript regarding MBP-1 is currently in preparation, and a federal grant proposal was submitted. The reviewers of the proposal suggested we demonstrate that biotin deficiency is common in the U.S. population and re-submit the proposal. We just completed collecting samples from human cancer patients on chemotherapy to demonstrate that therapy impairs biotin studies (analysis ongoing). These studies were conducted by the program director, a master's student (J. Xie), a doctoral student (Y. Ching Chew), a technologist (G. Giraud), and a collaborator (Dr. J. West).

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Howard KM, Jati Kusuma R, Baier SR, Friemel T, Markham L, Vanamala J, Zempleni J. Loss of miRNAs during processing and storage of cows (Bos taurus) milk. J Agr Food Chem 63:588-592
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Wolf T, Baier SR, Zempleni J. The Intestinal Transport of bovine milk exosomes is mediated by endocytosis in human colon carcinoma Caco-2 cells and rat small intestinal IEC-6 cells. J Nutr 145:2201-2206
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Zempleni J, Baier SR, Hirschi K. Diet-responsive microRNAs are likely exogenous [letter to the editor] J Biol Chem 289:25197
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2015 Citation: Cordonier EL*, Adjam R*, Camara Teixeira D, Onur S, Zbasnik R, Read PE, D�ring F, Schlegel VL, Zempleni J. (*contributed equally). Resveratrol compounds inhibit human holocarboxylase synthetase and cause a lean phenotype in Drosophila melanogaster. J Nutr Biochem
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2015 Citation: Chiang K, Shu J, Zempleni J, Cui J. Dietary MicroRNA Database (DMD): an archive database and analytic tool for food-borne microRNAs. PLoS ONE
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2015 Citation: Shu J, Chiang K, Zempleni J, Cui J. Computational characterization of exogenous microRNAs that can be transferred into human circulation. PLoS ONE
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2015 Citation: Pannier A, Kelly A, Plautz S, Zempleni J. Glucocorticoid cell priming enhances transfection outcomes in adult human mesenchymal stem cells. Mol Ther
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Baier SR, Nguyen C, Xie F, Wood JR, Zempleni J. MicroRNAs are absorbed in biologically meaningful amounts from nutritionally relevant doses of cows milk and affect gene expression in peripheral blood mononuclear cells, HEK-293 kidney cell cultures, and mouse livers. J Nutr 144:1495-1500
  • Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Camara Teixeira D, Cordonier EL, Wijeratne SSK, Huebbe P, Jamin A, Jarecke S, Wiebe M, Zempleni J. A cell death assay for assessing the mitochondrial targeting of proteins
  • Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Jati Kusuma R, Friemel T, Zempleni J. Human vascular endothelial cells transport foreign exosomes from cows milk by endocytosis
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2015 Citation: Zempleni J, Baier SR, Howard KM, Cui J. Gene regulation by dietary microRNAs. In: Nutrients/natural product (nutraceutical) control of metabolic pathways in relation to the metabolic syndrome (Dakshinamurti, K, Zempleni J, guest editors). Can J Physiol Pharmacol
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Zempleni J, Baier SR, Cui J, Wolf T. Bovine microRNAs are bioavailable and affect gene expression in humans and mice. American Society for Exosomes and Microvesicles, Asilomar Conference Center, CA, October 10-13
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Zempleni J, Baier SR, Cui J, Wolf T. Bovine microRNAs are bioavailable and affect gene expression in humans and mice. American Society for Cell Biology, Philadelphia, PA, October 6-10, 2014. Mol. Biol. Cell 25: 3987; doi:10.1091/mbc.E14-10-1437
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Baier SR, Nguyen C, Xie F, Wood JR, Zempleni J. Reply to Witwer: Mammals absorb microRNAs from milk. J Nutr 144:1881
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Liu D, Zempleni J. Low activity of LSD1 elicits a pro-inflammatory gene expression profile in riboflavin-deficient human T lymphoma Jurkat cells. Genes Nutr 9:422
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Sittiwong W, Cordonier EL, Zempleni J, Dussault P. ?-Keto and ?-hydroxyphosphonate analogs of biotin-5'-AMP are inhibitors of holocarboxylase synthetase. Bioorg Med Chem Lett 15:5568-5571
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Wolf T, Baier SR, Zempleni J. Transport of microRNA-containing, milk-borne exosomes by human colon carcinoma caco-2 cells. Experimental Biology Meeting; Boston, MA, March 28- April 1
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Zempleni J, Howard KM, Cui J, Shu J, Baier SR. Humans absorb dietary microRNAs from chicken eggs, and the postprandial increase of plasma microRNAs includes a microRNA that humans cannot synthesize endogenously. Meeting of the International Society for Extracellular Vesicles, April 23-26, Washington DC
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Zempleni J, Sadri M, Baier SR, Xie F, Wood J. Depletion of dietary microRNAs from cows milk decreases fecundity in mice. 18th International Conference of FFC - 6th International Symposium of ASFFBC, Functional and Medical Foods for Chronic Diseases: Bioactive Compounds and Biomarkers, September 15-16, Harvard Medical School, Boston, MA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Zempleni J. Invited presentation titled Bovine microRNAs are bioavailable and affect gene expression in humans and mice, Oregon State University, Corvallis, OR. May 15th
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Cordonier EL, Jarecke S, Hollinger F, Zempleni J. Inhibition of acetyl-CoA carboxylase activity prevents adipocyte differentiation in 3T3-L1 cells. Experimental Biology Meeting; Boston, MA, March 28- April 1
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Kusuma Jati R, Friemel T, Zempleni J. Human endothelial cells transport bovine extracellular vesicles. Experimental Biology Meeting; Boston, MA, March 28- April 1
  • Type: Other Status: Awaiting Publication Year Published: 2015 Citation: Interview with a freelance journalist (Dr. Hubertus Breuer) for an article on dietary microRNAs, to be published in the German newspaper Die Suddeutsche Zeitung
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Baier SR, Howard KM, Cui J, Shu J, Zempleni J. MicroRNAs in chicken eggs are bioavailable in healthy adults and can modulate mRNA expression in peripheral blood mononuclear cells. Experimental Biology Meeting; Boston, MA, March 28- April 1
  • Type: Other Status: Published Year Published: 2015 Citation: Interview with the news magazine Spiegel regarding microRNAs in milk: Lebensmittel: macht Milch krank? [in German]; http://www.spiegel.de/gesundheit/ernaehrung/milch-ist-sie-gesund-oder-ungesund-a-1048735.html
  • Type: Other Status: Published Year Published: 2015 Citation: Interview with a freelance journalist (Dr. Hubertus Breuer) for an article on dietary microRNAs, published in the Austrian newspaper Profil: "Weissmalerei Profil, 42:110-112
  • Type: Other Status: Other Year Published: 2015 Citation: Interview with a freelance journalist (Dr. Hubertus Breuer) for an article on dietary microRNAs, to be published in the Swiss newspaper Die Schweizer Sonntagszeitung
  • Type: Theses/Dissertations Status: Published Year Published: 2015 Citation: Daniel Camara Teixeira: Developing novel research tools for biotin research
  • Type: Theses/Dissertations Status: Published Year Published: 2015 Citation: Scott Baier: Bioactivity of cows milk microRNA in humans
  • Type: Theses/Dissertations Status: Published Year Published: 2015 Citation: Elizabeth Cordonier: Bioactive compounds in grape products
  • Type: Theses/Dissertations Status: Published Year Published: 2015 Citation: Rio Jati Kusuma: Mechansism of milk exosome transport of milk exosomes in human vascular endothelial cells
  • Type: Theses/Dissertations Status: Published Year Published: 2015 Citation: Katherine Howard: Biological activity of chicken egg microRNAs in humans
  • Type: Theses/Dissertations Status: Published Year Published: 2015 Citation: Tovah Wolf: Mechansism of milk exosome transport of milk exosomes in human and rat intestinal cells


Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Policy makers and federal officials Faculty Industry partners Graduate and undergraduate students General public Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Four postdoctoral fellows, ten graduate students, and seven undergraduate students have been trained over the lifespan of this project. How have the results been disseminated to communities of interest? Eleven peer-reviewed papers have been published and submitted during this reporting period. Research findings from the project have been disseminated through 15 presentations at the local, national, and international level. Research findings were disseminated through a local radio show (station KFOR Lincoln). What do you plan to do during the next reporting period to accomplish the goals? We will focus on our discovery that milk-borne microRNAs have biological activity in humans. We will address the following objectives. 1) Determine whether dietary microRNAs are conditionally essential nutrients in humans. 2) Assess the phenotypes of microRNA depletion. 3) Discover and validate markers of microRNA status. 4) Assess the biological activity of microRNAs in foods other than milk. 5) Assess the effects of microRNA insufficiency on bone mineralization in infants.

Impacts
What was accomplished under these goals? Impact: This project has created novel insights into how a vitamin, biotin, and proteins in biotin metabolism regulate human genes and decrease cancer risk. Briefly, we have demonstarted that biotin interacts with another vitamin, folate, and a variety of human proteins to stabilize regions in the human genome that would otherwise increase the risk for causing cancer. For example, when mice were fed a biotin-deficient diet, the number of tumors in mice increased by 100% compared with biotin-sufficient mice. The findings from this project are of particular importance for pregnant women, because aboyut 50% of pregnant women might be marginally biotin deficient. Objective 1: We have shown that holocarboxylase synthetase (HLCS) interacts physically with other chromatin proteins (HDAC, N-CoR, EHMT-1, MeCP2, and DNMT1), thereby mediating the repression of genes. A new HDAC splicing variant was discovered. Objective 2: We have shown that the vitamin biotin synergizes with methyl donors in the repression of LTRs, thereby increasing genome stability. The regulation of genes in the immune system, however, is more complicated and includes additional layers of gene regulation. Objective 3: Feeding studies in agouti mice have been completed and produced effects consistent with the notion that biotin interacts with methyl donors in the repression of the Avy locus. In addition we have discovered a novel class of bioactive food compounds, namely microRNAs in cow’s milk, which affect human gene expression. Outcomes: Training -- Four postdoctoral fellows, ten graduate students, and seven undergraduate students have been trained over the lifespan of this project. Students graduating from this project moved on to pursue productive careers in medical schools, graduate schools, and industry (100% placement rate). Collaborations -- New project-related collaborations have been intiated with Drs. Jiri Adamec (Nebraska), Juan Cui (Nebraska), John West (Nebraska), and Jairam Vanamala (Pennsylvania). Publications and disemination -- Eleven peer-reviewed papers have been published and submitted during this reporting period. Research findings from the project have been disseminated through 15 presentations at the local, national, and international level. Research findings were disseminated through a local radio show (station KFOR Lincoln), which contributed to an increase in knowledge among fellow scietists, graduate and undergraduate students, and the lay public. Healthy nutrition -- This project provides an incentive for healthy nutrition in pregnancy and its findings are currently tested in a cohort of cancer patients.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Xue J, Zhou J, Zempleni J. Holocaboxylase synthetase catalyzes biotinylation of heat shock protein 72, thereby inducing RANTES expression in HEK293 cells. Am J Physiol Cell Physiol 305:C1240-C1245
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Baier SR, Zbasnik R, Schlegel V, Zempleni J. Off target effects of sulforaphane include the de-repression of long-terminal repeats through histone acetylation . J Nutr Biochem 25:665-668
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Liu D, Zempleni J. Transcriptional regulation of the albumin gene depends on the removal of histone methylation marks by the FAD-dependent monoamine oxidase LSD1 in HepG2 human hepatocarcinoma cells. J Nutr 144:997-1001
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2014 Citation: Li Y, Malkaram SA, Zhou J, Zempleni J. Lysine biotinylation and methionine oxidation in the heat shock protein HSP60 synergize in the elimination of reactive oxygen species in human cell cultures. J Nutr Biochem
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Liu D, Zempleni J. Holocarboxylase synthetase interacts physically with the nuclear receptor corepressor, histone deacetylase 1, and a novel splicing variant of histone deacteylase 1 to repress repeats. Biochem J 461:477-486
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Baier SR, Nguyen C, Xie F, Wood JR, Zempleni J. MicroRNAs are absorbed in biologically meaningful amounts from nutritionally relevant doses of cows milk and affect gene expression in peripheral blood mononuclear cells, HEK-293 kidney cell cultures, and mouse livers. J Nutr 144:1495-1500
  • Type: Other Status: Awaiting Publication Year Published: 2014 Citation: Baier SR, Nguyen C, Xie F, Wood JR, Zempleni J. Reply to Witwer: Mammals absorb microRNAs from milk. J Nutr (in press) [Letter to the editor]
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Liu D, Zempleni J. Low activity of LSD1 elicits a pro-inflammatory gene expression profile in riboflavin-deficient human T lymphoma Jurkat cells. Genes & Nutrition 9:422
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Zempleni J, Liu D. Teixeira Camara D, Cordonier EL. Novel roles of holocarboxylase synthetase in gene regulation and intermediary metabolism. Nutr Rev 72:369-376
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Romagnolo DF, Zempleni J, Selmin OI. Nuclear receptors and epigenetic regulation: opportunities for nutritional targeting and disease prevention. Adv Nutr 5:373-385
  • Type: Journal Articles Status: Under Review Year Published: 2014 Citation: Zempleni J, Baier SR, Cui J. Gene regulation by dietary microRNAs. In: Nutrients/natural product (nutraceutical) control of metabolic pathways in relation to the metabolic syndrome (Dakshinamurti, K, Zempleni J, guest editors). Can J Physiol Pharmacol (invited review)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Cordonier, EL, Adjam R, Teixeira Camara D, Onur S, Zbasnik R, D�ring F, Schlegel VL, Zempleni J. Resveratrol compounds are potent inhibitors of human holocarboxylase synthetase and cause a lean phenotype in Drosophila melanogaster brummer mutants. Experimental Biology Meeting; San Diego, CA, April 26-30
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Baier SR, Zempleni J. MicroRNAs in bovine milk are bioavailable in healthy adults and down-regulate reporter gene activity in human kidney HEK-293 cell cultures. Experimental Biology Meeting; San Diego, CA, April 26-30
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Teixeira DC, Cordonier EL, Wijeratne SSK, Huebbe P, Jamin A, Jarecke S, Wiebe M, Zempleni J. A cell death assay for assessing the mitochondrial targeting of proteins. Experimental Biology Meeting; San Diego, CA, April 26-30
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Zempleni J. Keynote speaker Annual Meeting of the German Society for Nutrition (Deutsche Gesellschaft f�r Ern�hrung) Bioinformatics approaches to characterize the regulation of nutritionally relevant genes Paderborn, Germany, March 13
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Sittiwong, W, Cordonier EL, Zinniel D, Zempleni J, Barletta RG, Dussault PH. Investigation of Biological Activity of Analogs of Biotin-5?-AMP 247th ACS National Meeting and Exposition, March 16-20, Dallas, TX
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Zempleni J. Invited presentation titled A tale of three stories: holocarboxylase synthetase gene repression complexes, milk-borne microRNAs, and mitochondrial acetyl-CoA carboxylase 2 in the Bortree seminar series in the Department of Veterinary and Biomedical Sciences at Pennsylvania State University, University Park, PA. April 9th
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Friemel T, Kusuma Jati R, Zempleni J. Development of a simple and high-throughput screening method for an anti-obesity compound from the gut metabolome: the importance of mitochondrial docking of acetyl-CoA carboxylase (ACC)-2. The 124th Annual Meeting of the Nebraska Academy of Sciences, Nebraska Wesleyan University, Lincoln, NE April 11
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Zempleni J. Invited presentation titled A tale of three stories: holocarboxylase synthetase gene repression complexes, milk-borne microRNAs, and mitochondrial acetyl-CoA carboxylase 2, Omaha VA Hospital seminar series, Omaha, NE. April 18th
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Zempleni J. Invited presentation titled A tale of three stories: holocarboxylase synthetase gene repression complexes, milk-borne microRNAs, and mitochondrial acetyl-CoA carboxylase 2 as part of the annual W-3002 Multistate group meeting; Purdue University, Lafayette, IN. May 29th
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Baier SR, Zempleni J. MicroRNAs in bovine milk are bioavailable in healthy adults and down-regulate reporter gene activity in human kidney HEK-293 cell cultures. Nebraska Gateway to Nutrigenomics Retreat. Lincoln, NE, June 9th
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Chiang K, Cui, J, Zempleni J. Distinguishing mitochondrial encoded mitochondrions from nuclear encoded mitochondrions. Nebraska Gateway to Nutrigenomics Retreat. Lincoln, NE, June 9th
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Cordonier EL, Adjam R, Camara Teixeira D, Onur S, Zbasnik R, D�ring F, Schlegel VL, Zempleni J. Resveratrol compounds are potent inhibitors of human holocarboxylase synthetase and cause a lean phenotype in Drosophila melanogaster brummer mutants. Nebraska Gateway to Nutrigenomics Retreat. Lincoln, NE, June 9th
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Jati Kusuma R, Friemel T, Fernando S, Zempleni J. Screening the gut metabolome for compounds that prevent acetyl-CoA (ACC)-2 anchoring in mitochondria, causing a lean phenotype. Nebraska Gateway to Nutrigenomics Retreat. Lincoln, NE, June 9th
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Camara Teixeira D, Cordonier, EL, Wijeratne SSK, Huebbe P, Jamin A, Jarecke S, Wiebe M, Zempleni J. A cell death assay for assessing the mitochondrial targeting of proteins. Nebraska Gateway to Nutrigenomics Retreat. Lincoln, NE, June 9th
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Wolf T, Baier SR, Zempleni J. Transport of microRNA-containing, milk-borne exosomes by human colon carcinoma caco-2 cells. Nebraska Gateway to Nutrigenomics Retreat. Lincoln, NE, June 9th


Progress 10/01/12 to 09/30/13

Outputs
Target Audience: Policy makers and federal officials Faculty Industry partners Graduate and undergraduate students General public Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Four postdoctoral fellows, seven graduate students, and 5 undergraduate students have been trained. How have the results been disseminated to communities of interest? Ten peer-reviewed papers have been published and submitted. Research findings from the project have been disseminated through 15 presentations at the local, national, and international level. What do you plan to do during the next reporting period to accomplish the goals? We will expand the research in cell signaling and gene regulation to (1) include food-borne microRNAs, (2) assess natural and synthetic compounds that alter the anchoring of acetyl-CoA carboxylase in the outer mitochondrial membrane, (3) characterize physical interactions among HLCS, histone deacetylases, and the nuclear co-repressor N-CoR, and (4) assess the role of the biotin in activation of the transcription factor and tumor suppressor MBP-1.

Impacts
What was accomplished under these goals? Objective 1: We have shown that holocarboxylase synthetase (HLCS) interacts physically with other chromatin proteins (EHMT-1, MeCP2, and DNMT1), thereby mediating the repression of genes. Objective 2: WE have shown that the vitamin biotin synergizes with methyl donors in the repression of LTRs, thereby increasing genome stability. Th regulation of genes in the immune system, however, is more complicated and includes additional layers of gene regulation. Objective 3: Feeding studies in agouti mice have been completed and produced effects consistent with the notion that biotin interacts with methyl donors in the repression of the Avy locus. This project included collaborations with more than 10 scientists that can be linked to publications, in addition to numerous other collaborations. See below for training and disseminatrion.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Xue J, Wijeratne SSK, Zempleni J. Holocarboxylase synthetase synergizes with methyl CpG binding protein 2 and DNA methyl transferase 1 in the transcriptional repression of long terminal repeats. Epigenetics 8:504-511
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Li Y, Hassan YI, Moriyama H, Zempleni J. Holocarboxylase synthetase interacts physically with euchromatic histone-lysine N-methyltransferase, linking histone biotinylation with methylation events. J Nutr Biochem 24:1446-1452
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Xia M, Malkaram SA, Zempleni J. Three promoters regulate the transcriptional activity of the human holocarboxylase synthetase gene. J. Nutr Biochem 24: 1963-1969
  • Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Xue J, Zhou J, Zempleni J. Holocaboxylase synthetase catalyzes biotinylation of heat shock protein 72, thereby inducing RANTES expression in HEK293 cells. Am J Physiol Cell Physiol
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Xue J, Zempleni J. Epigenetic synergies among biotin, folate, and holocarboxylase synthetase in the regulation of pro-inflammatory cytokines and repeats. Scand J Immunol 78: 419-425
  • Type: Journal Articles Status: Under Review Year Published: 2013 Citation: Baier SR, Zbasnik R, Schlegel V, Zempleni J. Off target effects of sulforaphane include the de-repression of long-terminal repeats through histone acetylation events
  • Type: Journal Articles Status: Under Review Year Published: 2013 Citation: Li Y, Malkaram SA, Zhou J, Zempleni J. Lysine biotinylation and methionine oxidation in the heat shock protein HSP60 synergize in the elimination of reactive oxygen species in human cell cultures
  • Type: Book Chapters Status: Published Year Published: 2012 Citation: Zempleni J, Wijeratne SSK, Kuroishi T. Chapter 23: Biotin. In: Present Knowledge in Nutrition. Erdman JW Jr (ed.), 10th edition. International Life Sciences Institute, Washington, DC, pp. 587-609
  • Type: Book Chapters Status: Published Year Published: 2013 Citation: Zempleni J, Cordonier EL, Baier SR, Xue J. Vitamins, Bioactive Food Compounds, and Histone Modifications. In: Handbook of Vitamins. Zempleni J, Suttie JW, Gregory JF III, Stover PJ (eds), 5th edition. Taylor and Francis, Inc., Boca Raton, FL
  • Type: Book Chapters Status: Under Review Year Published: 2013 Citation: Zempleni J, Barshop B, Cordonier EL, Baier SR. Disorders of biotin metabolism. In: The Online Metabolic & Molecular Bases of Inherited Disease. Editors: Valle, Baudet, Vogelstein, Kinzler, Antonarakis, Ballabio, Scriver (Emeritus), Childs (Emeritus), Sly (Emeritus), Bunz (Parts Editor), Gibson (Parts Editor), Mitchell (Parts Editor). McGraw Hill.
  • Type: Journal Articles Status: Under Review Year Published: 2013 Citation: Zempleni J, Liu D. Teixeira Camara D, Cordonier EL. Novel roles of holocarboxylase synthetase in gene regulation and intermediary metabolism.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Baier SR, Schlegel VL, Zempleni J. Off Target Effects of Dietary Sulforaphane. College of Education and Human Sciences Research Fair; Lincoln, NE, November 2
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Camara D, Malkaram SA, Zempleni J. Enrichment of meiotic recombination hotspot sequences by avidin capture technology. Experimental Biology Meeting; Boston, MA, April 23
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Xue J, Zempleni J. Epigenetic synergies between methyl donors and biotin in gene repression are mediated by holocarboxylase synthetase (HLCS). Experimental Biology Meeting; Boston, MA, April 23
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Baier SR. Zbasnik R, Schlegel VL, Zempleni J. Dietary sulforaphane elicits off-target effects at loci coding for long terminal repeats in lymphocytes from healthy adults and in IMR-90 fibroblast cultures, possibly impairing genome stability. Experimental Biology Meeting; Boston, MA, April 23
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Zhou J, Wijeratne SSK, Zempleni J. Biotinylation of the c-myc promoter binding protein MBP-1 decreases c-myc expression in mammary carcinoma MCF-7 cells. Experimental Biology Meeting; Boston, MA, April 21
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Cordonier EL, Teixeira Camara D, Han Z, Pannier AK, Zempleni J. Acetyl-CoA carboxylases are checkpoints in adipocyte differentiation. Experimental Biology Meeting; Boston, MA, April 21
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Kelly AM, Han ZJ, Zempleni J, Pannier AK. Enhancing Nonviral Gene Delivery to Human Mesenchymal Stem Cells through Upregulation of the Glucocorticoid Receptor. Biomedical Engineering Society Annual Meeting, Seattle, WA, September 26-28
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Camara D, Malkaram SA, Zempleni J. Enrichment of meiotic recombination hotspot sequences by avidin capture technology. Nebraska Gateway to Nutrigenomics Retreat. Lincoln, NE, May 13
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Cordonier EL, Teixeira Camara D, Han Z, Pannier AK, Zempleni J. Acetyl-CoA carboxylases are checkpoints in adipocyte differentiation. Nebraska Gateway to Nutrigenomics Retreat. Lincoln, NE, May 13
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Xue J, Zhou J, Wijeratne SSK, Zempleni J. Holocarboxylase synthetase catalyzes the covalent binding of biotin to lysine residues in the inducible heat shock protein 72. Nebraska Gateway to Nutrigenomics Retreat. Lincoln, NE, May 13
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Baier SR. Zbasnik R, Schlegel VL, Zempleni J. Dietary sulforaphane elicits off-target effects at loci coding for long terminal repeats in lymphocytes from healthy adults and in IMR-90 fibroblast cultures, possibly impairing genome stability. Nebraska Gateway to Nutrigenomics Retreat. Lincoln, NE, May 13
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Zhou J, Wijeratne SSK, Zempleni J. Biotinylation of the c-myc promoter binding protein MBP-1 decreases c-myc expression in mammary carcinoma MCF-7 cells. Nebraska Gateway to Nutrigenomics Retreat. Lincoln, NE, May 13
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Zempleni J. Roles of biotin and holocarboxylase synthetase in disease prevention. W2002 Multistate Meeting at the University of Nebraska-Lincoln, Lincoln, NE, June 3
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Zempleni J. Epigenetic mechanisms of gene regulation by holocarboxylase synthetase. Invited presentation at the symposium titled Nutrigneomics and Personalized Foods held by the Korea Food Research Institute, Seoul, South Korea, October 25
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Wang Q-W, Xue J, Zempleni J. Holocarboxylase Synthetase Catalyzes Biotinylation of Lysine Residues in Enolase-1. University of Nebraska-Lincoln Undergraduate Research Symposium. August 7
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Eng WK, Giraud D, Schlegel VL, Wang D, Lee BH, Zempleni J. Identification and assessment of markers of biotin status in healthy adults. Br J Nutr 110:321-329
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Singh MP, Wijeratne SSK, Zempleni J. Biotinylation of lysine 16 in histone H4 contributes toward nucleosome condensation. Arch Biochem Biophys 529:105-111
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Camara Teixeira D, Malkaram SA, Zempleni J. Enrichment of meiotic recombination hotspot sequences by avidin capture technology. Gene 516:101-106
  • Type: Book Chapters Status: Published Year Published: 2013 Citation: Zempleni J, Liu D, Xue J. Nutrition, histone epigenetic marks, and disease. In: Epigenomics in Health and Disease. Jirtle, RL, Tyson F (eds.), Springer, Heidelberg, Germany, pp. 197-217
  • Type: Book Chapters Status: Published Year Published: 2013 Citation: Zempleni J, Liu D, Camara Teixeira D, Singh MP. Mechanisms of gene transcriptional regulation through biotin and biotin-binding proteins in mammals. In: Vitamin-binding Proteins  Their Functional Consequences. Dakshinamurti D, Dakshinamurti S (eds.), Taylor & Francis, Boca Raton, FL, pp. 219-228
  • Type: Books Status: Published Year Published: 2013 Citation: Handbook of Vitamins. Zempleni J, Suttie JW, Gregory JF III, Stover PJ (eds), 5th edition. Taylor and Francis, Inc., Boca Raton, FL


Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: Discoveries have been disseminated at the Experimental Biology meeting in San Diego, CA (4/2012), at the NIFA Multistate meeting in Fort Collins (6/2013), in 11 full-length research publications, 4 book chapters, 4 review articles, 6 meeting presentations by my students and postdocs, and 14 invited presentations at the local and international level. One student (Wei K. Eng) has graduated with a M.S. degree and has secured a position in a dietetic intership program in Marietta, GA. She is now pursuing a professional degree as a registered dietitian. PARTICIPANTS: Wei Kay ENG, M.S. student Elizabeth Cordonier, Ph.D. student No collaborators in the W2002 group. Collaborators from outside the W2002 group include the following faculty: Pat Dussault, Chemistry Department, University of Nebraska-Lincoln Joel Eissenberg, Department of Biochemistry, St. Louis University School of Medicine Larry Harshman, School of Biological Sciences, University of Nebraska-Lincoln Donald M. Mock, M.D., Ph.D., Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences Hideaki Moriyama, Chemistry Department, University of Nebraska-Lincoln Angela Pannier, Department of Biological Systems Engineering, University of Nebraska-Lincoln Vicki Schlegel, Department of Food Science and Technology, University of Nebraska-Lincoln John West, Department of Microbiology and Immunology at the University of Oklahoma Health Sciences Center, Oklahoma City, OK Dong Wang, Department of Statistics, University of Nebraska-Lincoln TARGET AUDIENCES: Policy makers and federal officials Faculty Industry partners Graduate and undergraduate students General public PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
One student has graduated from the program. Three postdocs, 5 doctoral students, 1 undergraduate student, and one visiting student were partially supported through this grant. The peer recognition of NIFA and the Agricultural Research Station at the University of Nebraska-Lincoln have been increased through publications in journals and books, and through seminars.

Publications

  • Zempleni J, Cordonier EL, Baier SR, Xue J. (2012) Vitamins, Bioactive Food Compounds, and Histone Modifications. In: Handbook of Vitamins. Zempleni J, Suttie JW, Gregory JF III, Stover PJ (eds), 4th edition. Taylor and Francis, Inc., Boca Raton, FL, 2013 (submitted)
  • Malkaram SA, Hassan YI, Zempleni J. Online tools for bioinformatics analyses in nutrition sciences. Adv Nutr 3:654-665, 2012
  • Xia M, Malkaram SA, Zempleni J. Identification of three promoters in the human holocarboxylase synthetase (HCS) gene. Abstract 416/C298 (647.1) Experimental Biology Meeting; San Diego, CA, 12:45 - 1:45 p.m., April 22, 2012
  • Wijeratne SSK, Malkaram SA, Pabian MD, Granatowicz AD, Zempleni J. Identification of biotin- and holocarboxylase synthetase-dependent microRNAs in human fibroblasts. Abstract C300 (647.3) Experimental Biology Meeting; San Diego, CA, 12:45 - 1:45 p.m., April 22, 2012
  • Pratap Singh M, Zempleni J. Biotinylation of K16 in histone H4 causes chromatin condensation. Oral presenttation in minisymposium "Nutrition and Epigenetics" (Chairs: Zempleni J, Ross SA), Experimental Biology Meeting; San Diego, CA, 9:00 - 9:15 a.m., April 22, 2012
  • Liu D, Zempleni J. Holocarboxylase synthetase (HLCS) interacts physically with nuclear corepressor (N-CoR) and histone deacetylases (HDACs) to mediate gene repression. Oral presenttation in minisymposium "Nutrition and Epigenetics" (Chairs: Zempleni J, Ross SA), Experimental Biology Meeting; San Diego, CA, 8:45 - 9:00 a.m., April 22, 2012
  • Singh D, Pannier AK, Zempleni J. Identification of holocarboxylase synthetase chromatin binding sites using the DamID technology. Anal Biochem 413:55-59, 2011
  • Bao B, Wijeratne SSK, Rodriguez-Melendez R, Zempleni J. Human holocarboxylase synthetase with a start site at methionine-58 is the predominant nuclear variant of this protein and has catalytic activity. Biochem Biophys Res Commun 412:115-120, 2011
  • Zhou J, Wang D, Schlegel V, and Zempleni J. Development of an internet based system for modeling biotin metabolism using Bayesian networks. Comp Methods Progr Biomed 104:254-259, 2011
  • Kuroishi T, Rios-Avila L, Pestinger V, Wijeratne SSK, Zempleni J. Biotinylation is a natural, albeit rare, modification of human histones Mol Genet Metabol 104:537-545, 2011
  • Esaki S, Malkaram SA, Zempleni J. Effects of single nucleotide polymorphisms in the human holocarboxylase synthetase gene on enzyme catalysis. Eur J Hum Genet 20:428-433, 2012
  • Bao B, Rodriguez-Melendez R, Zempleni J. Cytosine methylation in miR-153 gene promoters increases the expression of holocarboxylase synthetase, thereby increasing the abundance of histone H4 biotinylation marks in HEK-293 human kidney cells. J Nutr Biochem 23:635-639, 2011
  • Rios-Avila L, Pestinger V, Wijeratne SSK, Zempleni J. K16-biotinylated histone H4 is overrepresented in repeat regions and participates in the repression of transcriptionally competent genes in human Jurkat lymphoid cells. J Nutr Biochem 23:1559-1564, 2012
  • Eng WK, Giraud D, Schlegel VL, Wang D, Lee BH, Zempleni J. (2012) Identification and assessment of markers of biotin status in healthy adults. Br J Nutr (in press)
  • Singh MP, Wijeratne SSK, Zempleni J. (2012) Biotinylation of lysine 16 in histone H4 contributes toward nucleosome condensation. Arch Biochem Biophys (in press)
  • Li Y, Hassan YI, Moriyama H, Zempleni J. (2012) Holocarboxylase synthetase interacts physically with euchromatic histone-lysine N-methyltransferase, linking histone biotinylation with methylation events (submitted)
  • Camara Teixeira D, Malkaram SA, Zempleni J. (2012) Enrichment of meiotic recombination hotspot sequences by avidin capture technology (submitted)
  • Zempleni J, Liu D, Xue J. (2012) Nutrition, histone epigenetic marks, and disease. In: Epigenomics in Health and Disease. Jirtle, RL, Tyson F (eds.), Springer, Heidelberg, Germany (in press)
  • Zempleni, J, Eng WK, Singh MP, Baier SR. The chemistry and biochemistry of biotin. In: Food and Nutritional Components in Focus. Preedy VR (ed.), Royal Society of Chemistry, London, U.K., 2012, pp. 146-157
  • Zempleni J, Liu D, Camara Teixeira D, Singh MP. (2012) Mechanisms of gene transcriptional regulation through biotin and biotin-binding proteins in mammals. In: Vitamin-binding Proteins - Their Functional Consequences. Dakshinamurti D, Dakshinamurti S (eds.), Taylor & Francis, Boca Raton, FL (invited manuscript, in press)
  • Cordonier EL, Kasputis T, Mills JD, Han Z, Pannier AK, Zempleni J. Changes in the carboxylase profile are associated with early and late differentiation stages of osteoblast and adipocytes from human mesenchymal stem cells. Abstract C153 (1018.1) Experimental Biology Meeting; San Diego, CA, 12:45 - 1:45 p.m., April 24, 2012
  • Eng WK, Schlegel VL, Wang D, Zempleni J. Development of an outpatient biotin feeding protocol for studies of biotin requirements in adults. Abstract C155 (1018.3) Experimental Biology Meeting; San Diego, CA, 12:45 - 1:45 p.m., April 24, 2012


Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: (1) Results have been disseminated at national meetings, e.g., Experimental Biology. In addition, seminars have been presented at various research institutions and to the lay public in the state of Nebraska and at both the national and international level. (2) Unique reagents have been shared free of charge with qualified investigators in the U.S. (3) A student has graduated with M.S. degrees from the University of Nebraska-Lincoln. PARTICIPANTS: Subhashinee Wijeratne (UNL), Rocio Rodriguez-Melendez (UNL), Baolong Bao (UNL), Zhongji Han (UNL), Shingo Esaki (UNL), Jing Xue (UNL), Dandan Liu (UNL), Scott Baier (UNL), Elizabeth Cordonier (UNL), Daniel Camara Teixeira (UNL), Wei Kay Eng (UNL), Luisa Rios (UNL), Valerie Pestinger (UNL), Toshinobu Kuroishi (UNL), Mahendra Pratap Singh (UNL), Yong Li (UNL), David Giraud (UNL), Gaganpreet Kaur Mall (UNL), Kate Roehrs (UNL), Andrew Granatowicz (UNL), Michael Pabian (UNL), Angela Pannier (UNL), Don Mock (Little Rock, AR), John West (Oklahoma), Joel Eissenberg (Saint Louis), Yie-Hwa Chang (St. Louis), Dong Wang (UNL), Vicki Schlegel (UNL), Gloria Borgstahl (UNMC), Yuri Lyubchenko (UNMC). TARGET AUDIENCES: Scientists, policy makers, graduate and undergraduate students, publishers, funding agencies, lay public. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Outcomes: (1) Resources from this project paid for supplies to complete one M.S. thesis and continues to support four postdoctoral fellows, four Ph.D. students, one M.S. student, and two technicians. (2) Resources from this project helped to generate numerous publications as described below. Impacts: (1) Developed workforce in the molecular sciences area. (2) Enhanced recognition by peer institutions. (3) Enhanced the competitiveness for additional external funding. (4) Increased the scientific knowledge base in nutrition.

Publications

  • Pestinger V, Wijeratne SSK, Rodriguez-Melendez R, Zempleni J. The histone biotinylation marks H3K9bio, H3K18bio, and H4K8bio are enriched in repeat regions and participate in the repression of transcriptionally competent genes in human primary fibroblasts and Jurkat lymphoblastoma cells. J Nutr Biochem 22:328-333, 2011
  • Bao B, Pestinger V, Hassan YI, Borgstahl GEO, Kolar C, Zempleni J. Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18. J Nutr Biochem 22:470-475, 2011
  • Filenko NA, Kolar C, West JT, Hassan YI, Borgstahl GEO, Zempleni J, Lyubchenko YL. The role of histone H4 biotinylation in the structure and dynamics of nucleosomes. PLoS ONE 6:e16299, 2011
  • Rios-Avila L, Prince SA, Wijeratne SSK, Zempleni J. A 96-well plate assay for high-throughput analysis of holocarboxylase synthetase activity. Clin Chim Acta 412:735-739, 2011
  • Singh D, Pannier AK, Zempleni J. Identification of holocarboxylase synthetase chromatin binding sites using the DamID technology. Anal Biochem 413:55-59, 2011
  • Bao B, Rodriguez-Melendez R, Zempleni J. Cytosine methylation in miR-153 gene promoters increases the expression of holocarboxylase synthetase, thereby increasing the abundance of histone H4 biotinylation marks in HEK-293 human kidney cells. J Nutr Biochem (in press), 2012
  • Bao B, Wijeratne SSK, Rodriguez-Melendez R, Zempleni J. Human holocarboxylase synthetase with a start site at methionine-58 is the predominant nuclear variant of this protein and has catalytic activity. Biochemical and Biophysical Research Communications 412:115-120, 2011
  • Zhou J, Wang D, Schlegel V, and Zempleni J. Development of an internet based system for modeling biotin metabolism using Bayesian networks. Comp Methods Progr Biomed (in press), 2012
  • Kuroishi T, Rios-Avila L, Pestinger V, Wijeratne SSK, Zempleni J. Biotinylation is a natural, albeit rare, modification of human histones Mol Genet Metabol (in press), 2012
  • Esaki S, Malkaram SA, Zempleni J. Effects of single nucleotide polymorphisms in the human holocarboxylase synthetase gene on enzyme catalysis. Eur J Hum Genet (in press), 2012
  • Wijeratne SSK, Zempleni J. Identification of HCS-interacting proteins through the CytoTrapTM two-hybrid system. Nebraska Gateway for Nutrigenomics Retreat. Lincoln, NE, February 28th, 2011.
  • Kuroishi T, Zempleni J. Creation of holocarboxylase synthetase knockdown murine primary fibroblasts. Nebraska Gateway for Nutrigenomics Retreat. Lincoln, NE, February 28th, 2011.
  • Pratap Singh M, Zempleni J. Effects of biotinylation of lysine-16 in histone H4 on nucleosomal assembly. Nebraska Gateway for Nutrigenomics Retreat. Lincoln, NE, February 28th, 2011.
  • Rios-Avila L, Pestinger V, Wijeratne SSK, Zempleni J. K16-biotinylated histone H4 is overrepresented in repeat regions and participates in the repression of transcriptionally competent genes in human Jurkat lymphoid cells. J Nutr Biochem (in press), 2012
  • Xue J, Zempleni J. Epigenetic synergies between methylation of cytosines and biotinylation of histones in gene repression. Abstract 249 (597.7) Experimental Biology Meeting; Washington, DC, April 10, 2011
  • Esaki S, Zempleni J. Effects of single nucleotide polymorphisms in the human holocarboxylase synthetase gene on catalytic activity. Abstract 291 (782.7) Experimental Biology Meeting; Washington, DC, April 11, 2011
  • Zempleni J. Enhancing nutrigenomics research in Auckland. University of Auckland, New Zealand, February 3, 2011.
  • Eng WK, Giraud D, Zempleni J. Development of an outpatient biotin feeding protocol for studies of biotin biology in adults. Nebraska Gateway for Nutrigenomics Retreat. Lincoln, NE, February 28th, 2011.
  • Teixeira D, Malkaram SA, Zempleni J. Detection and enrichment of a common DNA sequence associated with human genome instability. Nebraska Gateway for Nutrigenomics Retreat. Lincoln, NE, February 28th, 2011.
  • Malkaram SA, Wijeratne SSK, Zempleni J. High-throughput ChIP-seq and RNA-seq investigation of epigenetic regulation of gene expression by biotin. Nebraska Gateway for Nutrigenomics Retreat. Lincoln, NE, February 28th, 2011.
  • Li Y, Zempleni J. Holocarboxylase synthetase interacts with euchromatic histone-lysine N-methyltransferase 1, linking histone biotinylation to histone methylation. Nebraska Gateway for Nutrigenomics Retreat. Lincoln, NE, February 28th, 2011.
  • Xue J, Zempleni J. Epigenetic synergies between methylation of cytosines and biotinylation of histones in gene repression. Nebraska Gateway for Nutrigenomics Retreat. Lincoln, NE, February 28th, 2011.
  • Xue J, Zempleni J. Effects of single nucleotide polymorphisms in the human holocarboxylase synthetase gene on catalytic activity. Nebraska Gateway for Nutrigenomics Retreat. Lincoln, NE, February 28th, 2011.
  • Zempleni J. Biotin-dependent epigenetic mechanisms contributing to genome stability. University of Vienna, Austria, June 16, 2011.
  • Zempleni J, Li Y, Xue J, Cordonier EL. The role of holocarboxylase synthetase in genome stability is mediated partly by epigenomic synergies between methylation and biotinylation events. Epigenetics 6:892-894, 2011
  • Zempleni J, Camara Teixeira D, Kuroishi T, Cordonier EL, Baier S. Biotin requirements for DNA damage prevention. Mutat Res (in press), 2012
  • Zempleni J, Kuroishi T. Nutrition information brief - biotin. Adv Nutr 2011 (in press), 2012