Source: UNIVERSITY OF MISSOURI submitted to
NATIONAL ANIMAL GENOME RESEARCH PROGRAM
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0231800
Grant No.
(N/A)
Project No.
MO-MSAS0002
Proposal No.
(N/A)
Multistate No.
NRSP-_OLD8
Program Code
(N/A)
Project Start Date
Oct 1, 2012
Project End Date
Sep 30, 2013
Grant Year
(N/A)
Project Director
Prather, RA, S..
Recipient Organization
UNIVERSITY OF MISSOURI
(N/A)
COLUMBIA,MO 65211
Performing Department
Animal Sciences
Non Technical Summary
The 3-dimensional structure of the chromatin affects gene expression. One well characterized epigenetic mark that affects chromatin structure and gene expression is that of a methyl group that is attached to the 5' position on a cytosine that is adjacent to a guanine in a string of DNA (5mC). Recently it has been shown that another intermediary in the process of demethylation is 5-hydroxymethylcytosine (5hmC). The 5hmC is present in early embryos and may have a profound influence on gene expression and subsequent development. There is about a 30% loss of conceptuses during the first month of development in most mammals. Our goal is to characterize the relative abundance of 5mC and 5hmC in early embryos in an attempt to identify a cause for some of these losses. The techniques that are currently available have not been applied to samples that are limiting, such as the early pig embryo. So here we will use pre-existing techniques and attempt to make them more sensitive so that very limiting samples can be used and accurate measurements of the abundance of 5hmC and mC in specific genomic regions can be made.
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
30335101050100%
Knowledge Area
303 - Genetic Improvement of Animals;

Subject Of Investigation
3510 - Swine, live animal;

Field Of Science
1050 - Developmental biology;
Goals / Objectives
Create shared genomic tools and reagents and sequence information to enhance the understanding and discovery of genetic mechanisms affecting traits of interest. Facilitate the development and sharing of animal populations and the collection and analysis of new, unique and interesting phenotypes. Develop, integrate and implement bioinformatics resources to support the discovery of genetic mechanisms that underlie traits of interest.
Project Methods
Our focus will be on Objective 1, Specific Aim iv) Development of experimental tools and reagents for transcriptional, proteomic and metabolomic analysis. We have recently brought a methylated DNA chromatin immuno precipitation/high throughput sequencing (MeDIP-seq) technique into the laboratory and used it on somatic donor cells for somatic cell nuclear transfer (Whyte et al unpublished). While this technique was highly effective for separating and sequencing DNA that was methylated, two other applications are needed. The first is to increase the sensitivity so that very limiting samples can be used, such as in the case of an early embryo, and the second is to apply the technology to 5-hydroxymethylcytosine. Hypothesis: The 5-hydroxymethylcytosine is an epigenetic mark distinct from 5-methylcytosine in porcine preimplantation embryos. Specific Aim: To explore the role of 5-hydroxymethylcytosine in DNA demethylation during porcine early embryogenesis. Method: 1. Methylated DNA immunoprecipitation (MeDIP) and hydroxymethylated DNA mmunoprecipitation (hMeDIP) by using small amounts of genomic DNA (microMeDIP and microhMeDIP). Since 5-hydroxymethylcytosine (5hmC) is enriched in embryonic stem (ES) cells, we will use genomic DNA (gDNA) from mouse ES cells to establish MeDIP and hMeDIP protocols. The input gDNA will range from 1X106 to 100 cells. Results will be compared to ensure that it is consistent among different DNA inputs. Carrier DNA will be added to improve the recovery if less than 1,000 cells are used. The OCT3/4 gene locus will be used to validate microMeDIP and microhMeDIP protocols. 2. The profiles 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in OCT4 (POU5F1) and NANOG gene loci in porcine gametes and preimplantation embryos will be determined by . microMeDIP and microhMeDIP. Various locations of OCT3/4 and NANOG loci (promoter and gene body) will be assessed for the relative enrichment of 5mC and 5hmC. The dynamic ratios of 5mC and 5hmC will be calculated and compared among different developmental stages. 3. The profiles of 5-hydroxymethylcytosine (5hmC) and 5mC in CpG island (CGI). Two CpG island regions upstream of SOX2 and CDX2 loci will be selected. The relative enrichment of 5mC and 5hmC will be compared along different developmental stages. 4. The profiles of 5hmC and 5mC in repeat elements. Two repeat elements: LINE1 and centromeric repeat will be selected and quantitative PCR will be performed by using DNA pulled down by microMeDIP and microhMeDIP methods. The relative enrichment of 5mC and 5hmC will be compared along different developmental stages. 5. The profiles of 5hmC and 5mC in imprinted control regions of IGF2/H19 gene loci. The differentially methylated regions (DMRs) in the ICR of IGF2/H19 usually show allele-specific DNA methylation. However, it is unknown whether 5hmC is involved in the maintenance of the allele-specific DNA methylation in imprinted IGF2/H19 loci. Two DMRs will be chosen to see whether 5hmC is enriched to protect from DNA methylation in porcine preimplantation embryos.

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

Outputs
Target Audience: Research scientists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Mingtao Zhao was able to complete a chapter of his PhD dissertation. How have the results been disseminated to communities of interest? So far an abstract has been published at the 2014 Plant and Animal Genome Meeting, and a manuscript submitted to a journal. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? DNA modifications, such as methylation and hydroxymethylation, are pivotal players in modulating gene expression, genomic imprinting, X-chromosome inactivation and silencing repetitive sequences during embryonic development. Aberrant DNA modifications lead to embryonic and postnatal abnormalities, and serious human diseases, such as cancer. Comprehensive genome-wide DNA methylation and hydroxymethylation studies provide a way to thoroughly understand normal development and to identify potential epigenetic mutations in human diseases. Here we established a working protocol for methylated DNA immunoprecipitation combined with next-generation sequencing (MeDIP-seq) for low starting amounts of genomic DNA. By using spike-in control DNA sets with standard cytosine, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), we demonstrate the preferential binding of antibodies to 5mC and 5hmC, respectively. MeDIP-PCRs successfully targeted highly methylated genomic loci with starting genomic DNA as low as 1 ng. The enrichment efficiency declined for constant spiked-in controls but increased for endogenous methylated region. A MeDIP-seq library was constructed starting with 1 ng DNA with the majority of fragments between 250 bp and 600 bp. The MeDIP-seq reads showed higher quality than the input control. However, after preprocessed by Cutadapt, MeDIP (97.53%) and Input (94.98%) reads showed comparable alignment rates. SeqMonk visualization tools indicated MeDIP-Seq reads were less uniformly distributed across the genome than Input reads. Several commonly known unmethylated and methylated genomic loci showed consistent methylation patterns in the MeDIP-seq data. Thus we provide proof-of-principle that MeDIP-seq technology is feasible to profile genome-wide DNA methylation in minute DNA samples, such as oocytes, early embryos and human biopsies.

Publications

  • Type: Journal Articles Status: Submitted Year Published: 2014 Citation: Zhao, M., J.J. Whyte, G.M. Hopkins, M.D. Kirk, R.S. Prather. Methylated DNA immunoprecipitation (MeDIP) by using low amounts of genomic DNA.
  • Type: Theses/Dissertations Status: Published Year Published: 2013 Citation: Ming-Tao Zhao. 2013. PhD Dissertation. Stem cells and DNA methylation reprogramming in pigs.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Prather, R.S., M.T. Zhao. 2014. Methylated DNA immunoprecipitation-Seq with low input DNA content. XXII Plant and Animal Genome meeting. San Diego, CA, Jan 10.