Abstract
Somatic cell nuclear transfer (SCNT) in mammalian cloning currently remains inefficient. Incomplete or erroneous epigenetic reprogramming of specialized donor somatic nuclear and resulting aberrant gene expression during development of cloned embryos is commonly believed as the main reason that causes the low efficiency of SCNT. Use of small molecular reprogramming modifiers to assist the somatic nucleus to mimic naturally occurring DNA methylation and chromatin remodeling in nucleus of fertilization-derived zygotes, has been widely attempted to improve cloning efficiency. However, impacts of these small modifiers on gene-specific methylation dynamics and their potential effects on methylation of imprinted gene have rarely been traced. Here, we attempted two relatively novel DNMT1 inhibitor (DNMTi) and histone deacetylase inhibitor (HDACi), scriptaid and RG108, and demonstrated their effects on dynamics of gene-specific DNA methylation and transcription of porcine SCNT embryos. We found that scriptaid and RG108 had synergetic effects on rescuing the disrupted methylation imprint of H19 during SCNT at least partially by repression over-expressed MBD3 in eight-cell cloned embryos. Furthermore, we firstly identified a differential methylation regions (DMRs) at 5' flanking regions of XIST gene and found that scriptaid alone and its combination with RG108 modify the dynamics of both transcription and DNA methylation levels in cloned embryos, by different manners. Additionally, we found that scriptaid alone and its combination with RG108 can significantly promote the transcription of NANOG in cloned embryos and enhance their pre-implantation developmental capacity. Our results would contribute to uncovering the epigenetic reprogramming mechanisms underlying the effects of assisted small molecules on improvement of mammalian cloning efficiency.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Biomarkers / metabolism
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Blastocyst / cytology
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Blastocyst / drug effects
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Blastocyst / metabolism
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Cellular Reprogramming / drug effects*
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Cellular Reprogramming / genetics
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DNA (Cytosine-5-)-Methyltransferase 1
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DNA (Cytosine-5-)-Methyltransferases / antagonists & inhibitors
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DNA (Cytosine-5-)-Methyltransferases / genetics*
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DNA (Cytosine-5-)-Methyltransferases / metabolism
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DNA Methylation / drug effects
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Drug Synergism
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Embryo, Mammalian
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Enzyme Inhibitors / pharmacology*
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Epigenesis, Genetic / drug effects*
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Female
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Histone Deacetylases / genetics*
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Histone Deacetylases / metabolism
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Homeodomain Proteins / genetics
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Homeodomain Proteins / metabolism
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Hydroxylamines / pharmacology*
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Indoles / pharmacology*
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Nuclear Transfer Techniques
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Phthalimides
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Propionates / pharmacology*
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Quinolines / pharmacology*
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RNA, Long Noncoding / genetics
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RNA, Long Noncoding / metabolism
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Swine
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Transcription, Genetic / drug effects
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Tryptophan / analogs & derivatives
Substances
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Biomarkers
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Enzyme Inhibitors
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Homeodomain Proteins
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Hydroxylamines
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Indoles
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Phthalimides
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Propionates
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Quinolines
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RG108
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RNA, Long Noncoding
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XIST non-coding RNA
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scriptaid
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Tryptophan
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DNA (Cytosine-5-)-Methyltransferase 1
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DNA (Cytosine-5-)-Methyltransferases
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Histone Deacetylases
Grants and funding
This study was supported by a grant from the National High Technology Research and Development Program of China (“863” Program, grant number: 2011AA100304), and a grant from the National Science and Technology Major Project of the Ministry of Science and Technology of China (grant number: 2011ZX08006004), and a grant from Department of Science and Technology of Guangdong (grant number: 2011A020901001), and a grant from Guangdong Science and Technology Project (grant number: 2011A020102003), and a grant from Department of Science and Technology of Guangdong (2008A024200012). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.