Chromatin profiling and state predictions reveal insights into epigenetic regulation during early porcine development.

Background: Given their physiological similarities to humans, pigs are increasingly used as model organisms in human-oriented biomedical studies. Additionally, their value to animal agriculture across the globe has led to the development of numerous studies to investigate how to improve livestock welfare and production efficiency. As such, pigs are uniquely poised as compelling models that can yield findings with potential implications in both human and animal contexts.

Chromatin remodeling in mammalian embryos.

The mammalian embryo undergoes a dramatic amount of epigenetic remodeling during the first week of development. In this review, we discuss several epigenetic changes that happen over the course of cleavage development, focusing on covalent marks (e.g.

ARID1A, a component of SWI/SNF chromatin remodeling complexes, is required for porcine embryo development.

Mammalian embryos undergo dramatic epigenetic remodeling that can have a profound impact on both gene transcription and overall embryo developmental competence. Members of the SWI/SNF (Switch/Sucrose non-fermentable) family of chromatin-remodeling complexes reposition nucleosomes and alter transcription factor accessibility. These large, multi-protein complexes possess an SNF2-type ATPase (either SMARCA4 or SMARCA2) as their core catalytic subunit, and are directed to specific loci by associated subunits.

Dynamic changes in nuclear import of a nuclear localisation signal-bearing substrate in 8-cell stage porcine embryos.

Coordinated intracellular trafficking is critically important for proper timing of major cellular events during embryogenesis. Nuclear import mediated by the karyopherin α/β (importin α/β) heterodimer is perhaps the best characterised nuclear trafficking system in eukaryotic cells. Seven karyopherin α subtypes have been identified in the domestic pig, and although each karyopherin α subtype transports proteins bearing classical nuclear localisation signals (NLSs), individual karyopherin α subtypes have been shown to preferentially transport specific cargoes.

Mother's exercise during pregnancy programmes vasomotor function in adult offspring.

The intrauterine environment is influenced by maternal behaviour and programmes atherosclerotic disease susceptibility in offspring. The aim of this investigation was to test the hypothesis that mothers' exercise during pregnancy improves endothelial function in 3-, 5- and 9-month-old porcine offspring. The pregnant sows in the exercise group ran for an average of 39.

The polycomb group protein EED varies in its ability to access the nucleus in porcine oocytes and cleavage stage embryos.

Chromatin-modifying complexes serve essential functions during mammalian embryonic development. Polycomb group proteins EED, SUZ12, and EZH2 have been shown to mediate methylation of the lysine 27 residue of histone protein H3 (H3K27), an epigenetic mark that is linked with transcriptional repression. H3K27 trimethylation has been shown to be present on chromatin in mature porcine oocytes, pronuclear and 2-cell stage embryos, with H3K27 trimethylation decreasing at the 4-cell stage and not detectable in blastocyst stage embryos.

IVMBIX-01294, an inhibitor of the histone methyltransferase EHMT2, disrupts histone H3 lysine 9 (H3K9) dimethylation in the cleavage-stage porcine embryo.

Global patterns of histone methylation are remodelled during cleavage development. Of the five histone methyltransferases known to mediate methylation of the lysine 9 residue of histone H3 (H3K9), euchromatic histone-lysine N-methyltransferase 2 (EHMT2; also known as G9a) has been shown to be a primary mediator of H3K9 dimethylation; BIX-01294 has been shown to be a specific inhibitor of EHMT2. The objective of the present study was to determine the effect of BIX-01294 treatment on global H3K9 dimethylation in porcine embryos.

Identification of karyopherin α1 and α7 interacting proteins in porcine tissue.

Specialized trafficking systems in eukaryotic cells serve a critical role in partitioning intracellular proteins between the nucleus and cytoplasm. Cytoplasmic proteins (including chromatin remodeling enzymes and transcription factors) must gain access to the nucleus to exert their functions to properly program fundamental cellular events ranging from cell cycle progression to gene transcription. Knowing that nuclear import mediated by members of the karyopherin α family of transport receptors plays a critical role in regulating development and differentiation, we wanted to determine the identity of proteins that are trafficked by this karyopherin α pathway.

KPNA7, an oocyte- and embryo-specific karyopherin α subtype, is required for porcine embryo development.

Coordinated partitioning of intracellular cargoes between nuclear and cytoplasmic compartments is critical for cell survival and differentiation. The karyopherin α/β heterodimer functions to import cytoplasmic proteins that possess classical nuclear localisation signals into the nucleus. Seven karyopherinαsubtypes have been identified in mammals.

Differential developmental requirements for individual histone H3K9 methyltransferases in cleavage-stage porcine embryos.

Dimethylated H3K9 is a heritable epigenetic mark that is closely linked with transcriptional silencing and known to undergo global remodelling during cleavage development. Five mammalian histone methyltransferases (HMTases), namely Suv39H1, Suv39H2, SetDB1, EHMT1 and EHMT2, have been shown to mediate the methylation of H3K9. The aim of the present study was to determine the developmental requirements of these HMTases during cleavage development in porcine embryos.

Global H3K9 dimethylation status is not affected by transcription, translation, or DNA replication in porcine zygotes.

Methylation of the lysine 9 residue of histone H3 (H3K9) is linked to transcriptional repression. The observed structure of chromatin in porcine and murine embryos is different with regard to H3K9 dimethylation status, leading to our hypothesis that the intracellular mechanisms responsible for H3K9 methylation would also differ between these two species. The objectives of this study were: (1) to determine the extent that DNA, mRNA, and protein synthesis serve in maintaining the asymmetrical distribution of dimethylated H3K9 in porcine zygotes, (2) determine the extent to which the intracellular localization of individual pronuclei correlated with H3K9 dimethylation status, and (3) to determine the abundance of transcripts encoding the histone methyltransferases, with H3K9 methylation activity, in porcine oocytes and embryos.

Differential remodeling of mono- and trimethylated H3K27 during porcine embryo development.

Histone methylation plays an important role in regulating chromatin structure and gene expression. Methylation of the lysine residue 27 of histone H3 (H3K27) is an epigenetic mark that is closely linked with transcriptional repression; global patterns of H3K27 methylation undergo dramatic changes during cleavage development in the mouse. The aim of this study was to characterize the H3K27 methylation pattern in cleavage stage porcine embryos obtained either by in vivo or in vitro fertilization or parthenogenetic activation and to determine the expression patterns of EED, EZH2, and SUZ12 (regulators of H3K27 methylation).

STIM1 regulates store-operated Ca2+ entry in oocytes.

The single transmembrane-spanning Ca(2+)-binding protein, STIM1, has been proposed to function as a Ca(2+) sensor that links the endoplasmic reticulum to the activation of store-operated Ca(2+) channels. In this study, the presence, subcellular localization and function of STIM1 in store-operated Ca(2+) entry in oocytes was investigated using the pig as a model. Cloning and sequence analysis revealed the presence of porcine STIM1 with a coding sequence of 2058 bp.

Gene expression and development of early pig embryos produced by serial nuclear transfer.

During nuclear transfer, reprogramming makes the donor nucleus capable of directing development of the reconstructed embryo. In most cases reprogramming is incomplete, which leads to abnormal expression of early embryonic genes and subsequently, to reduced developmental potential. In the present study, we monitored the expression of Oct4, Nanog, and Sox2 in cloned porcine embryos and evaluated whether serial nuclear transfer, the transfer of nuclei of cloned embryos into enucleated oocytes, has the potential to provide a more complete reprogramming of the donor genome.

Manipulation of SMARCA2 and SMARCA4 transcript levels in porcine embryos differentially alters development and expression of SMARCA1, SOX2, NANOG, and EIF1.

Epigenetic reprogramming plays a pivotal role during embryogenesis, including both covalent and non-covalent modifications to chromatin. In this study, we investigated the role of SNF2 chromatin remodeling ATPases (SMARCA2 (previously known as BRAHMA), SMARCA4 (previously known as BRG1), SMARCA5 (previously known as SNF2H), SMARCA1 (previously known as SNF2L), CHD3, and CHD5) during porcine preimplantation embryonic development. Transcript levels for these ATPases change dynamically throughout development.

Expression of eukaryotic elongation initiation factor 1A differentially marks zygotic genome activation in biparental and parthenogenetic porcine embryos and correlates with in vitro developmental potential.

Zygotic genome activation (ZGA) is a major event during cleavage development. In vitro manipulation of mammalian embryos (including embryo culture) can result in developmental arrest around the time of ZGA. Eukaryotic elongation initiation factor 1A (eIF1A) has been used as a marker for ZGA in some mammalian species.

In vitro and in vivo derived porcine embryos possess similar, but not identical, patterns of Oct4, Nanog, and Sox2 mRNA expression during cleavage development.

In vitro culture conditions stress the cleavage stage mammalian embryo and can contribute to reduced developmental potential of cultured embryos. One process that may be altered during embryo culture is the establishment and maintenance of pluripotency. Pluripotency is largely controlled by three genes: Oct4, Nanog, and Sox2.

Developmental capacity of porcine nuclear transfer embryos correlate with levels of chromatin-remodeling transcripts in donor cells.

Somatic cell nuclear transfer (SCNT) still retains important limitations. Impaired epigenetic reprogramming is considered responsible for altered gene expression and developmental failure in SCNT-derived embryos. After nuclear transfer the donor cell nucleus undergoes extensive changes in gene expression that involve epigenetic modifications and chromatin remodeling.

Developmental arrest induced in cleavage stage porcine embryos following microinjection of mRNA encoding Brahma (Smarca 2), a chromatin remodeling protein.

Smarca 2 (Brahma) and Smarca 4 (Brahma related gene 1, BRG1) alternatively occupy the catalytic site of SWI/SNF chromatin remodeling complexes. Mammalian embryos undergo a dramatic amount of epigenetic remodeling during cleavage development, which plays key roles in regulating both gene transcription and the developmental potential of the embryo. In order to understand how the epigenetic state of cleavage stage embryos is regulated, it is important to identify the factors that mediate epigenetic changes during cleavage development.

The SNF2 domain protein family in higher vertebrates displays dynamic expression patterns in Xenopus laevis embryos.

All eukaryotes share a common nuclear infrastructure, in which DNA is packaged into nucleosomal chromatin. Its functional states, in particular the accessibility of the chromatin fiber to trans-acting factors, are determined by two classes of evolutionarily conserved enzymes, i.e.

Cleavage stage porcine embryos may have differing developmental requirements for karyopherins alpha2 and alpha3.

Numerous cellular proteins are able to localize to the nucleus due to the fact that they possess a nuclear localization signal (NLS) in their amino acid sequence. Nuclear localization sequences recognized by the importin alpha/beta heterodimer are found in cellular proteins capable of performing many diverse functions, ranging from chromatin remodeling to cell cycle regulation. Evidence has been presented that suggests individual importin alpha homologues are present at varying levels in different adult tissues.

CRM1-mediated nuclear export is present during porcine embryogenesis, but is not required for early cleavage.

Regulated movement of cellular factors between the cytoplasm and nucleus is required for fundamental cellular processes ranging from cell cycle control to transcriptional regulation. CRM1 is a nuclear export factor whose function is to actively transport nuclear cargos that bear nuclear export sequences to the cytoplasm. Because CRM1 likely plays a role in the intracellular regulation of many cellular processes, we set out to characterize CRM1 function during early mammalian embryogenesis.